Advances in Applied Microbiology, Volume 37

ADVANCES

IN

Applied Microbiology VOLUME 37

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ADVANCES IN

Applied Microbiology Edited by

SAUL L. NEIDLEMAN Vacaville, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 37

Academic Press, Inc. Harcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

Volume 37 of Advances in Applied Microbiology is sadly dedicated to Dr. Frank K. Higson, University of California, Riverside, who passed away during the preparation of this volume to which h e made two scholarly contributions. This book is printed on acid-free paper. @

Copyright 0 1992 by ACADEMIC PRESS, INC. All Rights Reserved.

N o part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-4311

United Kingdom Edition published by

Academic Press Limited 24-28 Oval Road. London NWl 7DX Library of Congress Catalog Number: 59.13823 International Standard Book Number: 0- 12-002637-6 PRINTED IN T H E UNITED STATES OF AMERICA 92 93

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CONTENTS

Microbial Degradation of Nitroaromatic Compounds

FRANK K. HIGSON I. Overview .......................................................... 11. Introduction ....................................................... 111. Microbial Reduction of the Nitro Group . . . . . . . IV. Removal of the Nitro Group ......................................... V. Nitroaromatic Growth Substrates .................................... VI. Conclusions . . . . . . . . . . . References .........................................................

1 1 3 6 7 14 14

An Evaluation of Bacterial Standards and Disinfection Practices Used for the Assessment and Treatment of Stormwater

MARIEL. O’SHEAAND RICHARDFIELD I. Introduction

.......................................................

11. Bacterial Criteria Development-A Historical Perspective .............. 111. Stormwater Quality and Its Relationship to Human Disease Potential

IV. Disinfection ....................................................... V. Conclusions and Recommendations .................................. References .........................................................

...

21 22 26 31 34 36

Haloperoxidases: Their Properties and Their Use in Organic Synthesis

M. C. R. FRANSSEN AND H. C. VAN DER PLAS ...............................

I. Introduction . . . . .

11. Sources and Struct ....................................... 111. Reactions ..........................................................

IV. Reaction Mechanisms ............................................... V. Conclusions and Prospects ............................... References . . . . . . . . . . . . . . ..................................

41 43

53 82

90 92

Medicinal Benefits of the Mushroom Ganoderma

s. c. JONG AND J. M. BIRMINGHAM I. Introduction .................................. 11. Chemical Composition ........................ V

.......... ..........

101 102

vi

CONTENTS

111. Medicinal Properties

...............................................

IV. Patented Products and Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions ....................................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

108 121 125 12 7

Microbial Degradation of Biphenyl and Its Derivatives FRANK

K. HIGSON

I. Overview . . .

........

135

IV. Polychlorinated Biphenyls .......................................... V. Growth of Bacteria on Polychlorinated Biphenyls and Coculture Systems . . . . . . . . . . . . . . . . . . VI. Anaerobic Degradation of Polychlorinated B VII. Polychlorinated Biphenyl Bioremediation Trials ........ VIII. Degradation of Other Biphenyl Derivatives IX. Plasmids Encoding the Degradation of Biphenyl and Polychlorinated Biophenyls .................... X. Chromosomal Genes for the Degradation of Biphenyl and Polychlorinated Biphenyls . . . . . . .......................... XI. Fungal and Cyanobacterial Metabolism of Biphenyl .

139

11. Introduction

............................

145

149 150

References . .

The Sensitivity of Biocatalysts to Hydrodynamic Shear Stress

ALESPROKOP AND

RAKESH

K. BAJPAI

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Cell Architecture and Its Relationship to Hydrodynamic Shear S 111. Fluid Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV. V. VI. VII.

Methods of Assessing Shear Sensitivity .............................. Sensitivity of Biocatalysts to Hydrodynamic Stress .................... Summary and Outlook . . . .................................... Nomenclature . . . . . . . . ........................... References .................................................

166 166 170 187 190 219 225 226

Bipotentialities of the Basidiomacromycetes SOMASUNDARAM ~ J A R A T H N A M ,MYSORENANJARAJURS SHASHIREKHA, AND ZAKIABANO I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biology and Cultivated Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

234 236

vii

CONTENTS 111. Chemistry and Biomedical Values of Fruiting Bodies IV. Potential Lignocellulosic Substrates for Bioconversion

V. VI. VII. VIII. IX.

..

.......

Biotransformation of Lignocellulosic Wastes .......................... Changes in the Growth Substrates during Degradation Applications and Implications of Spent Substrate ... Applications of Functions of Fruiting BodieslMycelium ............... Conclusions ................................ References ...........................................

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

250 270

329 336 340

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Microbial Degradation of Nitroaromatic Compounds FRANK K. HIGSON Department of Soil and Environmental Sciences University of California, Riverside Riverside, California 92521 I. Overview 11. Introduction 111. Microbial Reduction of the Nitro Group

IV. Removal of the Nitro Group V. Nitroaromatic Growth Substrates A. Nitrobenzene B. Nitrophenols C. Chloronitrophenols D. Nitroanilines E. Nitrobenzoates F. 1,3-Dinitrobenzene G. 2,4,6-Trinitrotoluene VI. Conclusions References

I. Overview

Nitroaromatics are produced on a massive scale in the manufacture of dyes, plastics, and explosives. Their discharge in wastewater and application as pesticides have broadened their environmental impact and called for solutions for remediation of these toxic compounds. The use of microorganisms to transform or eliminate nitroaromatics has been proposed in effluent treatment and land reclamation. While microbial strains utilizing nitrobenzoates or nitrophenols were isolated from contaminated sources several decades ago, microbial action on 2,4,6-trinitrotoluene (TNT) was largely limited to nitro group reduction and formation of azoxy derivatives which may complex with humus. However, recent work has identified organisms capable of using TNT as the sole carbon and nitrogen source. II. Introduction

Although compounds bearing a nitro substituent are synthesized by microorganisms (Bush etal., 1951; Hirata etal., 1954; Cooke, 1955) and the bacterial degradation of chloramphenicol was reported to generate 4-nitromandelate, 4-nitrobenzyl alcohol, and 4-nitrobenzoate (Lingens et al., 1966),by far the greatest current producer of nitroaromatics is the 1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 37 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved

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FRANK K. HIGSON

chemical industry. Nitrobenzene, nitrotoluenes, nitrophenols, and nitrobenzoates are used in the manufacture of pesticides, dyes, explosives, polyurethane foams, elastomers, and industrial solvents. The antibiotic chloramphenicol and the tranquilizer nitrazepam are examples of drugs whose primary action depends on the presence of aromatic nitro groups. The insecticides parathion and paraoxon are derived from 4-nitrophenol; a class of herbicides that provides broad-spectrum weed control in cotton and soybeans and includes Treflan, is based on 4-trifluoromethyl-2,6-dinitroaniline. 2,4,6-Trinitrotoluene (TNT) has been used extensively in explosives since 1902 and current world annual production is around 2 million pounds (Hartter, 1985). Nitrobiphenyls are important as plasticizers for cellulose acetate and polystyrene, as textile fungicides and wood preservatives, and in the synthesis of dyes (Masse et al., 1985). Nitroanilines and their derivatives occur in wastewater from the dye and pharmaceutical industries and in soils as metabolites from microbial degradation of certain herbicides (Laanio et al., 1973; Golab et a]., 1979). Soil and groundwater contamination by TNT has resulted from munitions manufacture, loading, assembling, and packing (Haas and von Loew, 1986). The practice during shell loading operations has been to discharge hot water saturated with residual explosive (100 ppm) into holding lagoons and allow TNT to pass gradually into local streams. Wastewater from dye production makes a considerable input of nitrotoluenes into the environment. As much as 19 million pounds of nitrobenzene are reportedly discharged into natural waters (von Loew et al., 1989). Studies of Rhine water indicated nitrotoluenes present at concentrations up to 18 ppb (U.S. Environmental Protection Agency, 1978). Nitroaromatics are also present in combustion emissions and airborne particulate matter (Meijers et a]., 1976; Pitts et al., 1982; Schuetzle, 1983).

TNT is toxic to freshwater unicellular algae (Selenastrum capricornutum, Microcystis aeruginosa, ChIamydomonas reinhardtiif, tidepool copepods (Tigriopus californicus), and oyster larvae (Crassostrea gigas) at concentrations as low as 2.5 ppm and is a frameshift mutagen to Salmonella typhimurium (Won et al., 1976; Wang et al., 1980). Hudock and Gring (1970) and Smock et al. (1976) found it to be toxic to fathead minnows (Pimephales promelas) and bluegills (Lepomis macrochirus). Most fungi, yeasts, actinomycetes, and gram-positive bacteria showed severely limited growth in the presence of 50 ppm TNT (Nay et al., 1974). Deaths from toxic hepatitis and aplastic anemia caused by TNT exposure were significant during the world wars [20] and subclinical effects of TNT exposure affecting survival of erythrocytes, liver func-

DEGRADATION OF NITROAROMATICS

3

tion, and the lens of the eye have been described (McConnell and Flinn, 1946). Methemoglobinemia, cyanosis, anemia, and jaundice were reported in man as a result of exposure to dinitrotoluene in the workplace (Hathaway, 1985), and a dose-dependent increase in hepatocellular carcinoma was observed in rats fed technical grade dinitrotoluene (McGee et al., 1942). 1,3-Dinitrobenzene is toxic to humans following occupational exposure (Clark and Paul, 1935; Chemical Industry Institute of Toxicology, 1979), and to fish (Ishihara et al., 1976) and several bacterial and fungal species (Higgins, 1958; Wentsel et al., 1979). It can be generated from the munitions by-product 2,4-dinitrotoluene through photoconversion (Bringman and Keuhn, 1976). Nitrated polycyclic aromatic hydrocarbons such as 1-nitropyrene, which have been detected in carbon black toners (Kitchens et al., 1978), and nitrobiphenyls (Rosenkranz et al., 1980) are mutagenic (McCann et a]., 1975; Schuetzle, 1983) owing to their conversion to N-hydroxyarylamines by mammalian enzymes such as microsomal cytochrome P-450 reductase (Mermelstein et al., 1981). There are bacterial nitroreductases that can also activate nitrated polycyclics to potent mutagens (Harada and Omura, 1980). The U.S. Environmental Protection Agency’s list of 129 priority pollutants includes seven nitroaromatics: nitrobenzene, 2,4- and 2,6-dinitrotoluene, 2- and 4-nitrophenol, 2,4-dinitrophenol, and 4,6-dinitro-2-methylphenol (McCoy et a]., 1981). Bioremediation has been proposed for a number of recalcitrant compounds, including polychlorinated biphenyls (Keith and Telliard, 1979), and a microbial approach might also be appropriate for sites contaminated with nitroaromatics.

I l l . Microbial Reduction of the Nitro Group The microbial reduction of the nitro substituent has been established for several classes of nitroaromatics. A cyanide-sensitive NADH-dependent conversion of nitrobenzoate to aminobenzoate was displayed by growing cultures and cell-free extracts of a Nocardia sp. and a strain of Pseudomonas fluorescens (Furukawa, 1982). Enzyme preparations of the strict anaerobe Veillonella alkalescens catalyzed reduction of 30 mono-, di-, and trinitroaromatics by hydrogen in a three-step process, via R-NO and R-NHOH (Cartwright and Cain, 1959). McCormick et al. (1976) demonstrated formation of aminonitrotoluenes and 3-azoxy compounds from 2,4-dinitrotoluene by the fungus Mucrosporium. Reduction of 2,6-dinitro-4-(trifluoromethyl)aniline,from which several herbicides are derived, was reported (McCormick et al., 1978) for a Streptomyces isolated from soil. Naumova et al. (1986) observed se-

4

FRANK K. HIGSON

quential reduction of the nitro groups of TNT by aerobically growing Escherichia coli; aminodinitrotoluenes represented 70% of nitroaryl losses at the end of the exponential phase. Azoxy derivatives were again generated by coupling reactions. More recently, Wenzhong et al. (1987) investigated the reductase in Citrobacter freundii that degraded TNT aerobically. The K, was estimated at 0.05 mM and the optimum pH and temperature were 7.2 and 30°C, respectively; the addition of 1,3- and 1,4-dinitrobenzenes and 4nitro- and 2,4-dinitrophenols each approximately halved the rate of TNT clearance. When Parrish (1977) screened 190 fungi representing 98 genera for the ability to transform TNT, 183 were active in partial reduction, but surprisingly few (five organisms) were able to transform 2,4-dinitrotoluene. Bielaszczyk et al. (1967) found aerobic reduction of 4-chloronitrobenzene by organisms such as an Arthrobacter sp. obtained from contaminated soil. A basidiomycetous yeast of the genus Rhodosporidium was shown by Corbett and Corbett (1981) to transform 4-chloronitrobenzene by a reductive pathway (Fig. 1).In addition to producing 4-chloroaniline, the intermediate hydroxylamine was proposed to undergo a Bamberger rearrangement, in which the hydroxyl group migrated from N to C. This generated 2-amino-5-chlorophenol and 4-aminophenol by, respectively, ortho and para attack. Acetylation of these amino compounds was reported, but no azo or azoxy derivatives, perhaps because these workers avoided the solvent extraction and vacuum evaporation stages which favor the production of these metabolites by bimolecular reactions. Reduction of 4-chloronitrobenzene was also observed by Russel (1980) in Azotobacter agilis, the amino group then being subjected to acetylation or propionylation. An unusual replacement of the parachlorine of 2,4-dichloronitrobenzene with a methylthio group by Mucor javavicus was reported by Tahara et al. (1981). Hallas and Alexander (1983) reported reduction of nitrobenzene, dinitrobenzenes, nitrotoluenes, and nitrobenzoates in sewage effluent both in the presence and absence of oxygen; gas chromatographicmass spectroscopic (GC-MS) analysis indicated the formation of acetanilide and 2-methylquinoline from the intermediate aniline and 2-methylbenzimidazole from Z-nitroaniline (Fig. 2). That these multiple-ring teratogenic compounds are not simply artifacts of high-temperature-mediated ring closure during analysis is suggested by the formation of benzimidazoles from dinitroaniline herbicides in soil (Kearney et a]., 1976). Uchimura (1987) showed that polypeptone supplementation enhanced dinitrotoluene reduction by seawater microorganisms at a rate dependent on the configuration of the nitro groups.

5

DEGRADATION OF NITROAROMATICS NHOH

N-0

Cl

Cl

I

I1

@\ 111 CI

/

HO 0

J

YHCOCH,

I I1

OH

Vlll

VI NHCOCH,

/

CI

IV

NHCOCH,

@

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OH

IX

CI

V FIG. 1. The degradation of 4-chloronitrobenzene (I) by Rhodosporidium sp. (Corbett and Corbett, 1981) with the generation of nitroso (11) and hydroxylamine (111) intermediates. Also shown are 4-chloroaniline (IV), 4-chloroacetanilide (V), and their 2-hydroxy derivatives (VI, VII), 4-aminophenol (VIII), 4-hydroxyacetanilide (IX), and a hydroxamic acid metabolite (X), perhaps produced by acetylation of 111.

McCormick and co-workers (1985) showed reduction of Z-nitrodiphenylmethane, a by-product of ball powder production that is discharged from manufacturing plants in waste effluents, and the formation of annelated structures (N-phenylbenzimidazole and phenazine) by sewage cultures, especially under anaerobic conditions. A reduction of the fungicide pentachloronitrobenzene by Streptomyces aureofaciens was reported by Chacko et al. (1966), while Nakanishi and Oku (1969) identified pentachloromethylthiobenzene, pentachlorothiophenol, and bispentachlorophenyl disulfide as additional metabolites from the culture broth of Fusariurn oxysporum. E. coli isolated from human intestine reduced dinitrotoluenes to aminonitrotoluenes via the hydroxylamino compounds (Mori et a]., 1984), at a rate dependent on the position of the nitro group relative to the methyl. The intestinal microflora may therefore be involved in induction of methemoglobinemia or cancer (Reddy et al., 1976).

6

FRANK K. HIGSON

&NO2

NO,

NHCOCH,

___)

PcH - &NO2

&N: N HCOC H

NH

FIG. 2. Products of the metabolism of mono- and dinitroaromatic compounds in sewage (Hallas and Alexander, 1983). The nitro group is reduced, and acetylated and annelated structures are additionally produced.

IV. Removal of the Nitro Group

Masse et al. (1985) observed growth of gram-negative strain B206 on 4-nitrobiphenyl, and Takase et al. (1986) reported growth of Pseudomonas cruciviae S93B1 on 2- and 3-nitrobiphenyls. In both cases, nitrobenzoate accumulates unused in pure culture. The utilization of singlering nitroaromatics, however, normally requires removal of the nitro group and two systems have been described that accomplish this. One uses a nitroreductase to generate an amine and then ammonium.

DEGRADATION OF NITROAROMATICS

7

In the other, nitrite is liberated in an oxidative reaction. The alternatives coexisted in the strain of Pseudomonas putida isolated by Zeyer and Kearney (1984), which utilized 2-nitrophenol by the formation of catechol and nitrite, and %nitrophenol with the release of ammonium. While some exposure to nitroaromatic compounds has occurred over a time span that would allow the evolution of microbial degradation systems, the application of nitroaromatic pesticides such as parathion (O,O-diethyl-O-4-nitrophenyl phosphorothioate) and the discharge of nitrobenzene derivatives from manufacturing plants have much increased the selection pressure for the emergence of competent strains. The situation is analogous to the shift from a low-level exposure to biosynthetic haloaromatics such as 2,4-dichlorophenol isolated from a soil fungus (Ando et al., 1970) or brominated phenols produced by red algae (Suida and de Bernardis, 1973) to a widespread distribution as pesticides, solvents, surfactants, and as a result of water prechlorination. Microbes have been observed to degrade partially (cometabolize) these xenobiotics (such as polychlorinated biphenyls), while growing on another substrate, when enzymes involved in major pathways (such as biphenyl degradation) display a relaxed specificity (Slater and Bull, 1982).

Nitroreduction is presumably carried out by enzymes recruited from normal metabolism, for nitroreductases were identified in liver (Egami and Itahishi, 1951), Neurospora crassa (Little, 1951), and peas (Little, 1957) having no prior contact with environmental nitro compounds. Westfall (1943) found that even TNT underwent single reduction by a succinate dehydrogenase preparation from beef heart. An example of a nitroaromatic-degrading strain derived from a source that had received considerable anthropogenic pesticide exposure was presented by Siddaramappa and co-workers (Siddaramappa et al., 1973). A strain of Pseudomonas sp. was isolated from Indian soil that had been repeatedly sprayed with parathion. The organism hydrolyzed the pesticide and then released nitrite from the 4-nitrophenol produced. The following section looks at a number of nitroaromatic series for which microbial mineralization has been demonstrated. V. Nitroaromatic Growth Substrates A. NITROBENZENE

Moore (1949) described two strains of Nocardia that grew on nitrobenzene (and also aniline, nicotinate, and pyridine) as the sole carbon and nitrogen source.

8

FRANK K. HIGSON

B. NITROPHENOLS Simpson and Evans (1953) isolated strains of microbes from sewage that could use either 2- or 4-nitrophenol but not both; nitrite was released and the organisms were induced, respectively, to form catechol and hydroquinone. A strain of Arthrobacter isolated by Gunderson and Jensen (1956) grew on the herbicide 3,5-dinitro-2-methylphenol as the sole carbon and nitrogen source, nitrite being detected in their culture. The soil pseudomonad isolated by Tewfik and Evans (1966) grew on the herbicide by an initial reduction to form 3-amino-5-nitro-z-methylphenol, in turn giving rise to 3-methyl-5-aminocatecho1, rather than liberating nitrite. The strain of Raymond and Alexander (1971) grew on 4-nitrophenol with the liberation of nitrite and cometabolized the meta isomer to nitrohydroquinone. Sudhakar-Barik et al. (1976) showed mineralization of 4-nitro[l4C]phenol by a Pseudomonas sp. with the formation of labeled carbon dioxide. Spain et al. (1979) obtained an enzyme preparation from a Moraxella sp. isolated from sewage by 4nitrophenol enrichment that oxidized the growth substrate to hydroquinone and nitrite. Activity was dependent on NAD(P)H and oxygen and stimulated by the addition of FAD. Experiments with I8O2 showed that the incoming hydroxyl group was derived from molecular oxygen. Zeyer and Kearney (1984) obtained a cell-free nitrophenol-degradation system only for the ortho isomer from their soil isolate of Pseudomonas putida, which grew on 2- and 3-nitrophenols by different mechanisms. C. CHLORONITROPHENOLS An alternative to enrichment developed by Knackmuss and co-workers is the in vivo assembly of partial catabolic sequences to create a complete pathway. This approach was employed (Bruhn et al., 1988) in the transfer of haloaromatic-degrading sequences from chlorobenzoatedegraders to Pseudomonas sp. N31, a strain expressing a nitrophenol oxygenase that allows the organism to use 4-chloro-2-nitrophenol as its sole nitrogen source by the liberation of nitrite. In the transconjugant, the 4-chlorocatechol that normally accumulates from 4-chloro-2-nitrophenol was consumed, so the latter then acted as both carbon and nitrogen source. D. NITROANILINES

Zeyer and Kearney (1983) were the first to describe an organism that utilized a nitroaniline. Pseudomonas P6 grew slowly on 4-nitroaniline (but not the 2- or 3- isomers] as sole carbon source; nitroaniline degra-

DEGRADATION OF NITROAROMATICS

Y

dation was much enhanced by the addition of yeast extract. They failed to identify metabolites by HPLC but, by analogy to the degradation of aniline (a Nocardia sp. forms catechol; Bachofer et al., 1975) and 3chloroaniline [which is converted to 4-chlorocatechol by strains of Pseudomonas multivorans (Reber et al., 1979) and Alcaligenes faecalis (Surovtseva et al., 1980)], a nitrocatechol route was proposed.

E. NITROBENZOATES Cain (1958) obtained the first nitrobenzoate-degraders from soil and polluted streams. Nocardia opaca grew on Z-nitrobenzoate and N. erythropolis on 4-nitrobenzoate as sole carbon and nitrogen sources; 3nitrobenzoate competitively inhibited growth on either substrate. After enrichments lasting 2 years, the Nocardia sp. M1 was obtained that grew on the meta isomer (Cain, 1966). 4-Nitrocatechol was isolated from cultures of N. erythropolis growing on 4-nitrobenzoate, and 4hydroxybenzoate transiently accumulated under conditions of restricted aeration. 3-Hydroxybenzoate was found in cultures of strain M1 growing on 3-nitrobenzoate. Cells from both cultures were induced for protocatechuate oxidation. A scheme was presented (Fig. 3) in which the nitro group was either replaced by a hydroxyl and a second hydroxyl added in a second step, or 4-nitrobenzoate was acted on by a dioxygenase to produce the nitrocatechol. The pathway of Z-nitrobenzoate degradation was not clarified, but, from inhibition studies, did not appear to involve anthranilate (Cain, 1958). Ke et al. (1959) obtained a Flavobacterium growing on 2-nitrobenzoate as sole carbon and nitrogen source; cells were simultaneously induced for 2-nitroso- and 2-hydroxylaminobenzoate but not anthranilate, suggesting that the nitrobenzoate was only partially reduced. F. 1,3-DINITROBENZENE

Tennessee River water taken downstream from a munitions production facility yielded a nonaxenic culture growing on 1,%dinitrobenzene as sole carbon source (Mitchell and Dennis, 1982); the organisms were not concomitantly induced for the oxidation of 1,2- or 1,4-dinitrobenzenes, 1,3,5-trinitrobenzene, or 3,5-dinitroaniline, which also occur in munitions discharges.

G.

2,4,&TRINITROTOLUENE

Whereas nitrophenol-degraders are rather readily obtained from enrichments, the three nitro groups in TNT make it difficult for micro-

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E

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4

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DEGRADATION OF NITROAROMATICS

11

organisms to utilize it. The bulky substituents present steric constraints and reduce the electron density of the aromatic ring (March, 1985), impeding the electrophilic attack of oxygenases. Moreover, reduction to amino groups (described above) allows secondary abiotic coupling reactions which generate biologically inert azoxy and polymeric compounds. Growth of pure cultures on TNT was independently described in 1974 by two groups. Gram-negative rods (mainly pseudomonads) were isolated by Traxler et al. (1974) from sediments, sewage, and boiler plant effluentsby enrichment on 100 ppm TNT; some used TNT as the sole source of nitrogen as well as carbon. Uniformly labeled TNT was cleaved to W O , and nitrite; the low yield (0.8 to 1.2%)of radioactive carbon dioxide may have been due to heterotrophic fixation, since 2% of the activity of sodium [14C]bicarbonatewas taken up by the cells when they were growing on unlabeled TNT. Nitroaromatic compounds did not appear to accumulate in their cultures from thin layer chromatography (TLC) studies. . found three pseudomonads using an 80 ppm TNT Won et ~ l (1974) enrichment procedure; the strains consumed TNT much more readily in the presence of glucose and another nitrogen source. Reduced intermediates were detected by TLC. Later, extended studies by other groups (Spanggord et aI., 1975; Carpenter et al., 1978; Hoffsommer et al., 1978) were unable to repeat the isolation of TNT-utilizers. Work by Neumeier et al. (1989) has been more promising: they isolated strains in TNT and dinitrotoluene enrichments from munition plant soil and activated carbon that grew well on TNT. Strain 14 used TNT as its sole carbon and nitrogen source, although degradation proceeded more quickly in the presence of a secondary nitrogen source. GC-MS data indicated the 2-or 4-nitro group was reduced during TNT metabolism (Haas and von Loew, 1986). Recent studies with 14CO, liberation from labeled TNT by Unkefer and Unkefer (personal communication, Los Alamos Natl. Lab., Los AIamos, New Mexico) have also suggested the presence of ring-cleaving TNT-degraders in contaminated soil. Fernando et al. (1990) have reported TNT degradation by Phanerochaete chrysosporium, a wood-rotting fungus that not only degrades the refractory biopolymer lignin by means of an extracellular hydrogen peroxide/peroxidase radical-generating system but also mineralizes DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], polycyclic aromatic hydrocarbons, chloroanilines, and polychlorinated biphenyls (Arjmand and Sandermann, 1985; Eaton, 1985; Bumpus and Aust, 1987; Bumpus, 1989). The fungus shows real promise in site decon-

12

FRANK K. HIGSON

tamination. At an initial concentration of 1.3 mg/ml, 35% of labeled TNT was degraded to 14C0, in liquid culture over 18 days. The addition of glucose 1 2 days after addition of TNT did not stimulate mineralization and, after 18 days of incubation with TNT, only about 3.3% of the initial TNT could be recovered. The fungus also displayed a slow but sustained production of 14C0, from labeled TNT adsorbed on an agricultural silt loam soil supplemented with ground corncobs such that 6% mineralization was recorded after 30 days. Polar metabolites were found to occur in both systems. When the concentration of TNT in cultures was adjusted to contamination levels that might be found in the environment (0.1 g/liter water and 10 g/kg soil), mineralization studies showed that 20 and 18% of the initial TNT was converted to 14C02in liquid culture and soil, respectively; about 85% of the TNT disappeared in both cases. Since TNT is not lethal to the fungus at effluent and waste site concentrations, white rot fungus may well provide an economical and convenient alternative to physical cleanup processes. The fate of [I4C]TNTin an activated sludge system was investigated by Carpenter et al. (1978), who found no remaining TNT after 5 days: radioactivity did not appear as 14C0, but was about equally divided between the floc and the supernatant. From a comparison of infrared spectra of reactor precipitates and a model compound synthesized from TNT transformation products and lipid precursors, TNT transformation products were found to have formed polyamide-type macromolecular structures with cell components. Kaplan and Kaplan (1982a) showed a gradual binding of products of TNT reduction (Fig. 4) to humus fractions under simulated composting conditions (55°C);their conjugation may prove important in immobilizing the metabolites, known to be toxic and mutagenic (Ellis et al., 1978; Dilley et al., 1979). The coupling resembles the cross-linkage reported by Bollag and co-workers (1980, 1983) between anilines or 2,4-dichlorophenol to phenolic humus constituents. The turnover times of the bound residues are unknown and the effects of stresses such as moisture, pH, and temperature should be investigated to assess the potential for recontamination by these complexes. Another proposed treatment, the complexation of TNT by certain amino surfactants under alkaline conditions to form a water-insoluble, nonexplosive precipitate (Okamoto and Wang, 1977; Croce and Okamato, 1978) which can be separated from the effluent and incinerated or used as landfill, presents problems. Mutagenicity studies indicated that these complexes were more potent mutagens than TNT (Kaplan and Kaplan, 1982b), and soilleaching studies showed that the in situ immobilization of TNT would

13

DEGRADATION OF NITROAROMATICS

-

2-OHA

2A -

/

OZN&.-

02N+NH2

1 \""

-

2,4' Az

NO2

0

1

02NFNo2-

-

4-OHA

NHOH

-

4,C A r

I

0

FIG. 4. Transformation scheme for TNT in compost [Kaplan and Kaplan, 1982a). Compounds that are boxed were identified in solvent extracts. TNT derivatives are abbreviated 2- and 4-OHA (2- and 4-hydroxylamino), 2A and 4A (2- and 4-amino), and 2,4DA and 2,6DA (2,4- and 2,6-diamino). 2,4'Az is 2',4,6,6'-tetranitro-2, 4'-azoxytoluene and 4,4' Az is 4,4',6,6'-tetranitro-2,2'-azoxytohene.

14

FRANK K . HIGSON

not be feasible due to the large quantities of surfactant required and the inability of the surfactant treatment to complex reduced metabolites (Kaplan and Kaplan, 1982~).

VI. Conclusions Aromatics bearing a single nitro function and an activating substituent such as a hydroxyl or carboxyl are rather readily degraded by soil microorganisms but TNT, sterically hindered and electronically deactivated, is much less amenable to microbial oxidation. Research is currently under way to identify bacteria able to use TNT as the sole carbon and nitrogen source, for such strains would be at a selective advantage in contaminated sites. There would be a need, however, to maximize ring cleavage since reduced metabolites could prove more toxic than the original explosive. The degradation of nitroaromatics as a result of an in vivo assemblage of pathway elements, by means of filter mating, multiple chemostat recombination (Kroeckel and Focht, 19871, or gene cloning (Bruhn et a]., 1988),might well accomplish reactions not exhibited by naturally occurring phenotypes. The versatile white rot fungus also merits further attention in nitroaromatic degradation. ACKNOWLEDGMENTS

I thank Dr. Dennis D. Focht (UCR], Dr. Steven D. Aust (Utah State University, Logan,

UT],Dr. Neil G. McCormick (U.S. Army research and development labs., Natick, MA], Dr. R. W. Traxler (Dept. of Nutrition and Food Science, University of Rhode Island], and Dr.

Patricia Unkefer [Los Alamos) for information relating to this review. I am also grateful to Mr. Chunkeun Lim for translation of a Japanese publication. The work was funded by a grant from the U.S.Environmental Protection Agency.

REFERENCES Ando, K., Kato, A., and Suzuki, S. (1970).Isolation of 2,4-dichlorophenol from a soil fungus and its biological significance. Biochem. Biophys. Res. Comrnun. 39. 11041111.

Arjmand, M., and Sandermann, H. (1985).Mineralization of chloroaniline/lignin conjugates and of free chloroanilines by the white rot fungus Phanerochaete chrysosporium. J. Agric. Food Chem. 33, 1055-1060. Bachofer, R.,Lingens, F., and Schaefer, W. (1975).Conversion of aniline into pyrocatechol by a Nocardio sp.: Incorporation of oxygen-18. FEBS Lett. 50, 288-290. Bielaszczyk, E.. Czerwinska, E., Janko, Z., Kotarski, A., Kowalik, R., Kwiatkowski, M., and Zoledziowska, J. (1967).Aerobic reduction of some nitrochloro-substituted benzene compounds by microorganisms. Acta Microbiol. Pol. 16, 243-248. Bollag, J.-M. Liv, S. Y., and Minard, R. D. (1980). Cross-coupling of phenolic humus constituents and 2.4-dichlorophenol. Soil Sci. SOC.Am. J. 44, 52-56.

DEGRADATION OF NITROAROMATICS

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Bollag, J.-M., Minard, R. D., and Liv, S. Y. (1983). Cross-linkage between anilines and phenolic humus constituents. Environ. Sci. Technol. 17, 72-80. Bringman, G., and Keuhn, R. (1976). Comparative results of the damaging effects of water pollutants against bacteria (Pseudomonas putida) and blue-green algae (Microcystis aeruginosa). Wasser-Abwasser 117,410-413. Bruhn, C., Bayly, R. C., and Knackmuss, H.-J. (1988). The in vivo construction of 4chloro-2-nitrophenol assimilatory bacteria. Arch. Microbiol. 150, 171-1 77. Bumpus, 1. A. (1989). Biodegradation of polycyclic aromatic hydrocarbons by Phonerochaete chrysosporium. Appl. Environ. Microbiol. 55, 154-158. Bumpus, J. A., and Aust, S. A. (1987). Biodegradation of DDT (l,l,l-trichloro-2,2bis(4-ch1orophenyl)ethane)by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59, 2001-2008. Bush, M. T., Touster, O., and Brockman, J. E. (1951). The production of P-nitropropionate by a strain of Aspergillus flavus. J. Biol. Chem. 188, 685-693. Cain, R. B. (1958). The microbial metabolism of nitroaromatic compounds. 1. Gen. Microbiol. 19, 1-14. Cain, R. B. (1966). Utilization of anthranilic and nitrobenzoic acids by Nocardia opaca and a flavobacterium. J. Gen. Microbiol. 42, 219-235. Carpenter, D. F., McCormick, N. G., Cornell, J. H., and Kaplan, A. M. (1978). Microbial transformation of [14C]-labelled 2,4,6-trinitrotoluene in an activated sludge system. Appl. Environ. Microbiol. 35, 949-954. Cartwright, N. J., and Cain, R. B. (1959). Bacterial degradation of the nitrobenzoic acids. 2. Reduction of the nitro group. Biochem. I. 73, 305-314. Chacko, C. I., Lockwood, J. L., and Zabik, M. (1966). Chlorinated hydrocarbon pesticides: degradation by microbes. Science 154, 893-895. Chemical Industry Institute of Toxicology (1979). “A Twenty-Four Month Study in Fisher-344 Rats Given Dinitrotoluene,” Interim Rep., Docket No. 327N8. Research Triangle Park, North Carolina. Clark, B. B., and Paul, W. D. (1935). Acute methemoglobinemia following exposure to meta-dinitrobenzene and meta-nitroaniline. J. Iowa State Med. SOC. 25, 449-450. Cooke, A. R. (1955). The toxic constituent of Indigofera endecaphylla. Arch. Biochem. Biophys. 55, 114-120. Corbett, M. D., and Corbett, B. R. (1981).Metabolism of 4-chloronitrobenzene by the yeast Rhodosporidium sp. Appl. Environ. Microbiol. 41,942-949. Croce, M., and Okamoto, Y. (1978). Cationic micellar catalysis of the aqueous alkaline and 1,3,5,7-tetraaza-1,3,5,7hydrolyses of 1,3,5-triaza-1,3,5-trinitrocyclohexane tetranitrocyclooctane. I, Org. Chem. 44, 2100-2103. Dilley, J. V., Tyson, C. A., and Newell, G. W. (1979). “Mammalian Toxicological Evaluation of TNT Wastewaters. VIII. Acute and Subacute Mammalian Toxicity of Condensate Water.” SRI Int., Menlo Park, California. Eaton, D. C. (1985). Mineralization of polychlorinated biphenyls by Phanerochaete chrysosporium, a ligninolytic fungus. Enzyme Microb. Technol. 7, 194-196. Egami, F., and Itahishi, H. (1951). Enzymatic decomposition of nitromethane by liver homogenates. J. Biochem. (Tokyo) 38, 329-333. Ellis, H. V., Hodgson, J. R., Huang, S. W., Halfpap, L. M., Helton, D. O., Anderson, B. S., van Goethem, D. L., and Lee, C. C. (1978). “Mammalian Toxicity of Munitions Compounds: Phase I. Acute Irritation, Dermal Sensitization, Disposition and Metabolism and Ames Tests of Additional Compounds,” Project No. 3900-B. Midwest Res. Inst., Kansas City, Missouri. Fernando, T., Bumpus, j. A., and Aust, S . A. (1990). Biodegradation of TNT (2,4,6-tri-

16

FRANK K. HIGSON nitrotoluene) by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 56,16661671.

Furukawa, K. (1982).Microbial degradation of polychlorinated biphenyls. In “Biodegradation and Detoxification of Environmental Pollutants” (A. M. Chakrabarty, ed.), pp. 33-57. CRC Press, Boca Raton, Florida. Golab, T., Althaus, W. A., and Wooten, H. L. (1979).Fate of [”TI trifluralin in soil. J. Agric. Food Chem. 27, 163-179. Gunderson, K., and Jensen, H. L. (1956).A soil bacterium decomposing organic nitrocompounds. Acta Agric. Scand. 6, 100-114. Haas, R., and von Loew, E. (1986).Grundwasserbelastung durch eine Altlast. Die Folgen einer ehemaligen Sprengstoffproduktion fuer die heutig Trinkwasergewinnung. Forum Staedte-Hyg. 37, 33-43. Hallas, L. E., and Alexander, M. (1983).Microbial transformation of nitroaromatic compounds in sewage effluent. Appl. Environ. Microbiol. 45, 1234-1241. Harada, N., and Omura, T. (1980).Participation of cytochrome P450 in the reduction of nitro compounds by rat liver microsomes. J. Biochem. [TokyoJ 87,1539-1554. Hartter, D. R. (1985).The use and importance of nitroaromatic chemicals in the chemical industry. In “Toxicity of Nitroaromatic Compounds” (D. E. Rickert, ed.), Chemical Industry Institute of Toxicology Series, pp. 1-14. Hemisphere, Washington, D.C. Hathaway, J. A. (1985).Subclinical effects of trinitrotoluene: A review of epidemiological studies. In “Toxicity of Nitroaromatic Compounds” (D. E. Rickert, ed.), Chemical Industry Institute of Toxicology Series. Hemisphere, Washington, D.C. Higgins, E. S. (1958).Inhibition of Aspergillus niger growth by m-dinitrobenzene and its reversal by amino acids. Proc. Soc. Exp. Biol. Med. 99, 527-530. Hirata, Y.,Okuhara, K., and Naito, T. (1954).A toxic nitro compound from Streptomyces thioluteus. Nature [London) 173, 1101. Hoffsommer, J. C., Kaplan, L. A., Glover, D. J., Kubose, D. A., Dickenson, C., Goya, H., Kayser, E. G., Grove, C. L., and Sitzman, M. E. (1978).Biodegradability of TNT: A 3year pilot study. NSWLIWOL PR 77-136. Naval Surface Weapons Laboratory, Silver Springs, Maryland. Hudock, G. A., and Gring, D. M. (1970).“Biological Effects of Trinitrotoluene,” Contract No. N00164-69-CO822, p. 74.Nav. Environ. Health Cent. Ishihara, N., Kanaya, A., and Ikeda, M. (1976).m-Dinitrobenzene intoxication due to skin absorption. Int. Arch. Occup. Environ. Health 36, 161-168. Kaplan, D. L.,and Kaplan, A. M. (1982aJ. Thermophilic biotransformations of 2,4,6trinitrotoluene under simulated composting conditions. Appl. Environ. Microbiol. 44,757-760. Kaplan, D. L., and Kaplan, A.M. (1982b).Mutagenicity of 2,4,6-trinitrotoluene-surfactant complexes. Bull. Environ. Contam. Toxicol. 28, 33-38. Kaplan, D. L., and Kaplan, A. M. (1982~). 2,4,6-Trinitrotoluene-surfactantcomplexes: Decomposition, mutagenicity and soil leaching studies. Environ. Sci. Technol. 16, 566-571.

Ke, Y.-H., Gee, L. L., and Durham, N. N. (1959).Mechanism involved in the metabolism of nitrophenyl-carboxylic acid compounds by microorganisms. J. Bacteriol. 77, 593598.

Kearney, P.C., Plimmer, J. R., Wheeler, W. B., and Kontson, A. (1976).Persistence and metabolism of dinitroaniline herbicides in soils. Pestic. Biochem. Physiol. 6, 229238.

Keith, L. H., and Telliard, W. A. (1979).Priority pollutants. I. A perspective view. Environ. Sci. Technol. 13, 416-423. Kitchens, V. F., Harward, W. E., Lauter, D. M., Wentsel, R. S., and Valentine, R. S. (1978).

DEGRADATION OF NITROAROMATICS

17

“Preliminary Problem Definition Study of 48 Munitions Related Chemicals. I. Explosives Related Chemicals,” Contract No. DAMD 17-77-C7057.Atlantic Res. Corp., Alexandria, Virginia. Klausmeier, R. E., Osmon, J. L., and Walls, D. R. (1974). The effect of trinitrotoluene on microorganisms. Dev. Ind. Microbiol. 15, 309-317. Kroeckel, L., and Focht, D. D. (1987). Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event. Appl. Environ. Microbiol. 53, 2470-2475. Laanio, T. L., Kearney, P. C., and Kaufman, D. D. (1973). Microbial metabolism of dinitramine. Pestic. Biochem. Physiol. 3, 271-277. Lingens, F., Eberhardt, H., and Oltmanns, 0. (1966). Mikrobieller Abbau des Chloramphenicols. Biochem. Biophys. Acta 130, 345-354. Little, H. N. (1951). Oxidation of nitroethane by extracts from Neurospora. J. Biol. Chem. 193, 347-358. Little, H. N. (1957). Oxidation of 2-nitropropane by extracts of pea plants. J. Biol. Chem. 229, 231-238. Lusby, R. W., Oliver, J. E., and Kearney, P. C. (1980). Metabolism of 2,6-dinitro-4-(trifluoromethyl)benzenamineby a Streptomyces isolated from soil. J. Agric. Food Chem. 28, 641-644. March, J. (1985). “Advanced Organic Chemistry: Reactions, Mechanisms and Structure,” 3rd Ed. Wiley, New York. Masse, R., Badr, M., Ayotte, C., and Sylvestre, M. (1985). Gas chromatographic-mass spectrometric characterization of bacterial metabolites of +nitrobiphenyl formed in gram-negative strain B206. Toxicol. Environ. Chem. 10, 225-246. McCann, J. E., Choi, E., Yamasak, E., and Amer, B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72, 5135-5139. McConnell, W. J., and Flinn, R. H. (1946). Summary of twenty-two trinitrotoluene fatalities in World War 11. J. Ind. Hyg. Toxicol. 28, 76-86. McCormick, N. G., Feeherry, F. E., and Levinson, H. S. (1976).Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds. Appl. Environ. Microbiol. 31,949-958. McCormick, N. G., Cornell, J. H., and Kaplan, A. M. (1978). Identification of biotransformation products from 2,4-dinitrotoluene. Appl. Environ. Microbiol. 35, 945-948. McCormick, N. G., Peltonen, T. D., and Kaplan, A. M. (1985). “Biotransformation of Wastewater Constituents from Ball Powder Production,” Tech. Rep. TR-85/050 U.S. Army, Natick, Massachusetts. McCoy, E. C., Rosenkranz, H. S., and Mermelstein, R. (1981).Evidence for the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens. Environ. Mutagen. 3, 421-427. McGee, L. C., McCausland, A., Plume, C. A., and Marlett, N. C. (1942). Metabolic disturbances in workers exposed to dinitrotoluene. Am. J. Dig. Dis. 9, 329-332. Meijers, A. P., and van der Leer, R. C. (1976). The occurrence of organic micropollutants in the River Rhine and the River Maas in 1974. Water Res. 10, 597-604. Mermelstein, R., Kiriazides, D. K., Butler, M., McCoy, E. C., and Rosenkranz, H. S. (1981). The extraordinary mutagenicity of nitropyrenes in bacteria. Mutat. Res. 89,187-196. Mitchell, W. R., and Dennis, W. H. (1982). Biodegradation of 1,3-dinitrobenzene. J. Environ. Sci. Health, Part A A17, 837-853. Moore, F. W. (1949). The utilization of pyridine by microorganisms. J. Gem Microbiol. 3, 143-147. Mori, M., Miyahara, T., Hasegawa, Y., Kudo, Y., and Kozuka, H. (19841. Metabolism of

ia

FRANK K. HIGSON

dinitrotoluene isomers by Escherichia coli isolated from human intestine. Chem. Pharm. Bull. 32, 4070-4075. Nakanishi, T., and Oku, H. 11969). Metabolism and accumulation of pentachloronitrobenzene by phytopathogenic fungi in relation to selective toxicity. Phytopathology 59, 1761-1762. Naumova, R. P., Ofitzerov, E. N., Belousova, T. O., Selivanovskaya, S. Y., and Khisamutdinova, L. (1986). The pathways of 2,4,6-trinitrotoluene biotransformation. rzv. Akad. Nauk SSSR, Ser. Biol. pp. 448-455. (In Russ.) Nay, M. W., Randall, C. W., and King, P. H. (1974). Biological treatability of trinitrotoluene manufacturing wastewater. J. Water Pollut. Control Fed. 46, 485-497. Neurneier, W., Haas, R., and von Loew, E. (1989). Mikrobieller Abbau von Nitroaromaten aus einer ehemaligen Sprengstoffproduktion. Teil 1. Abbau von 2,4,6-Trinitrotoluol (TNT). Forum Staedte-Hyg. 40, 32-37. Okarnoto, Y., and Wang, J. Y. (1977). Micellar effects on the reaction of 2,4,6-trinitrotoluene with amines. 1. Org. Chem. 42, 1261-1262. Parrish, F. W. (1977). Fungal transformation of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene. Appl. Environ. Microbiol. 34, 232-233. Pitts, J. N., Lokensgard, D. M., Harger, W. P., Fisher, T. S . ,Meija, V., Schuler, J. J., Scorziell, G. M., and Katzenstein, Y. A. (1982). Identification and direct activities of B-nitrobenzo(a)pyrene, 9-nitroanthracene, 1-nitropyrene and 5H-phenanthro[4,5-bcd]pyran-5-one. Mutat. Res. 103, 241-249. Raymond, D. G. M., and Alexander, M. (1971). Microbial metabolism and cometabolism of nitrophenols. Pestic. Biochem. Physiol. 1, 123-130. Reber, H., Helm, V., and Karanth, N. G. K. (1979).Comparative studies on the metabolism of aniline and chloroanilines by Pseudomonas multivorans strain Anl. Eur. J. Appl. Microbiol. Biotechnol. 7, 181-189. Reddy, B. G., Pohl, L. R., and Krishna, G . (1976). The requirement of the gut flora in nitrobenzene-induced methemoglobinemia in rats. Biochem. Pharmacol. 25, 11191122. Rosenkranz, H. S., McCoy, E. C., Sanders, D. R., Butler, M., Kiriazides, D. K., and Mermelstein, R. (1980). Nitropyrenes: Isolation, identification and reduction of mutagenic impurities in carbon black and toners. Science 209, 1039-1043. Russel, S. (1980). Microbiological transformations in aniline and its chlorinated derivatives. C.A. 92, 1524. Schuetzle, D.(1983). Sampling of vehicle emissions for chemical analysis and biological testing. Environ. Health Perspect. 47, 65-80. Siddaramappa, R., Rajaram, K. P., and Sethunathan, N. (1973). Degradation of parathion by bacteria isolated from a flooded soil. Appl. Microbiol. 26, 846-849. Simpson, J. R., and Evans, W. C. (1953). The metabolism of nitrophenols by certain bacteria. Biochem. J. 55, xxiv. Slater, J. H., and Bull, A. T. (1982). Environmental microbiology: Biodegradation. Philos. Trans. R. SOC.London, Ser. B 297, 575-597. Smock, L. A., Stoneburner, D. L., and Clark, J. R. (1976). The toxic effects of trinitrotoluene (TNT) and its primary degradation products on two species of algae and the fathead minnow. Water Res. 10,537-543. Spain, J. C., Wyss, O., and Gibson, D. T. (1979). Enzymatic oxidation of p-nitrophenol. Biochem. Biophys. Res. Commun. 88, 634-641. Spanggord, R. J., Mabey, W. R., Chou, T. W., and Smith, J. H. (1975). Environmental fate of selected nitroaromatic compounds in the aquatic environment. In “Toxicity of Nitroaromatic Compounds” (D. E. Rickert, ed.), Chemical Industry Institute of Toxicology Series, pp. 15-34. Hemisphere, Washington, D.C.

DEGRADATION OF NITROAROMATICS

19

Sudhakar-Barik, Siddaramappa, R., and Sethunathan, N. (1976). Metabolism of nitrophenols by bacteria isolated from parathion-amended soils. Antonie van Leeuwenhoek 42, 461-470. Suida, J. F., and de Bernardis, J. F. (1973). Naturally-occurring halogenated organic compounds. Lloydia 36, 107-143. Surovtseva, E. G . , Volnova, A. I., and Shatskaya, T. Y. (1980). Degradation of monochlorosubstituted anilines by Alcaligenes faecalis. Mikrobiologiya 49, 38-42. Tahara, S., Hafsah, Z., Ono, A,, Asaishi, E., and Mizutani, J. (1981). Metabolites of 2,4dichloro-1-nitrobenzene by Mucor javanicus. Agric. Biol. Chem. 45, 2253-2258. Takase, I., Omori, T., and Minoda, Y. (1986). Microbial degradation products from biphenyl-related compounds. Agric. Biol. Chem. 50, 681-686. Tewfik, M. S., and Evans, W. C. (1966). The metabolism of 3,5-dinitro-o-cresol (DNOC) by soil microorganisms. Biochem. J. 99, 31P-32P. Traxler, R. W., Wood, E., and Delaney, J. M. (1974). Bacterial degradation of alpha-TNT. Dev. Ind. Microbiol. 16, 71-76. Uchimura, Y.(1987). Biodegradability of dinitrotoluenes in seawater. Kogai to Taisaku 23, 1379-1384. (In Jpn.) US.Environmental Protection Agency (1978). “Nitrobenzene,” Initial rep. of TSCA Interagency Test. Comm. to administrator, EPA 560-10-781001. Washington, D.C. von Loew, E., Kaminski, L., Neumeier, W., Haas, R., and Steinbach, K. (1989). Mikrobieller Abbau von Nitroaromaten aus einer ehemaligen Sprengstoffproduktion. Teil2. Migration und mikrobielle Metabolisierung von 2,4,6-trinitrotohol im Grundwasser. Forum Staedte-Hyg. 40, 347-349. Wang, C. Y., Lee, M. S., King, C. M., and Warner, P. 0. (1980). Evidence for nitroaromatics as direct-acting mutagens of airborne particles. Chemosphere 9, 83-87. Wentsel, R. S., Hyde, R. G., Jones, W. G., Wilkinson, M. J., Howard, W. E.. and Kitchens, J. F. (1979). “Problem Definition Study on 1,3-Dinitrobenzene, 1,3,5-Trinitrobenzene and di-N-Propyl Adipate,” Final Rep. No. 49-5730-08, Contract No. DAMD 17-77C7057. Atlantic Res. Corp., Alexandria, Virginia. Wenzhong, L., Ping, Y., and Uanxi, Y. (1987). Properties of TNT-degrading enzymes in intact cells of Citrobacter freundii. ACTA Microbiol Sin. 27, 257-263. Westfall, B. B. (1943). The reduction of symmetrical trinitrotoluene by a succinic dehydrogenase preparation. J. Pharmacol. Exp. Ther. 79, 23-26. Won, W. D., Heckly, R. J., Glover, D. J., and Hoffsommer,J. C. (1947). Metabolic disposition of 2,4,6-trinitrotoluene. Appl. Microbiol. 27, 513-516. Won, W. D., di Salvo, L. H., and Ng, J. (1976). Toxicity and mutagenicity of 2,4,6-trinitrotoluene and its microbial metabolites. Appl. Environ. Microbiol. 31, 576-580. Zeyer, J., and Kearney, P. C. (1983). Microbial metabolism of 14C-nitroanilines to 14ccarbon dioxide. J. Agric. Food Chem. 31, 304-308. Zeyer, J., and Kearney, P. C. (1984). Degradation of o-nitrophenol and m-nitrophenol by a Pseudomonas putida. J. Agric. Food Chem. 32, 238-242.

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An Evaluation of Bacterial Standards and Disinfection Practices Used for the Assessment and Treatment of Stormwater MARIEL. O’SHEAAND RICHARDFIELD Storm and Combined Sewer Overflow Pollution Control Program office of Research and Development U S . Environmental Protection Agency Edison, New Jersey 08837

I. Introduction 11. Bacterial Criteria Development-A

Historical Perspective 111. Stormwater Quality and Its Relationship to Human Disease Potential IV. Disinfection A. Chemical Disinfection B. Alternative Disinfection Techniques V. Conclusions and Recommendations References

I. Introduction

Storm-generated flows occur on both an intermittent and a random basis. During and after rainfall, these flows exhibit highly varying intensities over short periods of time with respect to both pollutant and microorganism quality and hydraulic quantity. In general, a sewer or channel can flow from completely dry to a thousand times the steadystate flow conditions associated with sanitary (domestic) wastewater. The characteristics of stormwater also vary according to the manner in which the stormwater is routed to the receiving water. Storm-generated discharges entering a receiving water body can originate from separate storm drainage systems, from combined sewers carrying a mixture of sanitary wastewater and stormwater (combined wastewater), or from sanitary sewers inappropriately or illicitly cross-connected to separate storm sewers. In addition, receiving waters can contain discharges from both separate storm-drainage and combined sewer systems from urban and/or nonurban land areas. In view of the many and varying factors which dictate the pollutant and microbial content of stormwater and/or the receiving waters, the adaptation of existing analytical and disinfection methods to evaluate and treat these microorganisms has proved difficult if not ineffective [I]. 21 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 37 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in a n y form reserved.

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RICHARD FIELD AND MARIE L. O’SHEA

For the control of microorganisms in storm flows, two basic needs have arisen [2]: First, it is necessary to determine a storm flow’s human pathogen content and pathogenicity along with the relationships of the pathogens to certain indicators. In stormwater studies, total coliform (TC), fecal coliform (FC), and sometimes fecal streptococcus (FS) remain the traditional indicators of human pathogens. However, these indicators and their recommended limiting values have been adopted out of their routine use in potable water and sanitary wastewater analysis. Their appropriateness for the analysis of stormwater remains questionable, particularly where stormwater does not enter the receiving water mixed with sanitary wastewater. For stormwater uncontaminated by sanitary wastewater, traditional fecal indicator levels may misrepresent the disease-causing potential of the stormwater, resulting in the premature closure of beaches and the unwarranted adoption of costly disinfection and control measures. In addition, a significant portion of swimming-related illnesses involve infections of the skin, ear, eye, and upper respiratory tract associated with exposure to nonenteric pathogens, e.g., staphylococcus, Pseudomonas aeruginosa, Klebsiella, and adenoviruses [3-61. Risks of this nature cannot be estimated using FC densities alone. In general, criteria based solely on TC or FC densities inadequately represent the actual human-disease contraction potential, i.e., pathogenicity of a storm flow and its receiving water, causing a misguided concern over some disease hazards and the neglect of others. Epidemiological studies are severely lacking which specifically address the human pathogen potential of receiving waters fed by the stormwater runoff of various watershed types. Second, for stormwater discharges which pose serious health hazards, e.g., storm flows from combined sewers or from storm sewers containing a significant number of sanitary cross-connections, disinfection requirements and procedures should be designed to accommodate the unique characteristics of these flows. For example, the high volumes and flow rates of stormwater require the development of high-rate disinfection systems to save on large tankage or dosage requirements, while the highly varying qualitative and quantitative characters of these flows require flexible facility design and operational techniques in order to prevent unnecessary and costly disinfection expenditures. il. Bacterial Criteria Development-A

Historical Perspective

It has long been recognized that water can be a medium for pathogenic bacteria, fungi, protozoa, and viruses and that the source of many

EVALUATION OF THE TREATMENT OF STORMWATER

23

of these disease-causing microorganisms is fecal contamination. Yet, the difficulty and expense associated with the isolation and measurement of pathogenic microorganisms have resulted in the development of methods to monitor certain indicator organisms, i.e., microorganisms indicative of the presence of fecal contamination. Bacteria of the TC group became the generally accepted indicator for fecal pollution despite the fact that many of the bacteria in this group were known to be of a nonfecal origin. Total coliform bacteria are gram-negative, nonspore-forming, and lactose-fermenting bacilli which produce gas within 48 hours at 35°C [7]. These characteristics allow for delineation of the TC group, which includes many different genera, e.g., Escherichia coli, Citrobacter, Klebsiella, and Enterobacter. Because the latter three genera are rarely associated with enteric wastes, attempts were made to narrow the scope of the TC assay to the detection of those organisms which originate solely from fecal wastes. The FC test, an elevated temperature (44.5"C) procedure used with modified media, was thus developed and became the preferred indicator assay for fecal pollution. The FC test selects primarily for Klebsiella and E. coli with infrequent positive results for other genera. However, E. coli is the only member of the FC group that is a consistent inhabitant of the intestinal tract of humans and other warm-blooded animals [8]. Thus, although the FC test is an improvement over the original TC test, it is still not specific to enteric bacteria in general and human-enteric bacteria in particular. The most widely used bacteriological criterion in the United States today is the maximum recommended density of 200 FC organisms/100 ml of sample [9]. However, as a brief review of its adoption will illustrate, this criterion is not supported by either epidemiological or pathogenic-contact evidence. Studies of gastrointestinal (GI) illness in swimmers in the early 1950s found that TC densities of between 2300 and 2400 organisms/100 ml caused a significantly higher incidence of symptoms [6,10].Later, as FC became the favored indicator for sanitary wastewater, early TC data collected on the Ohio River was reevaluated to determine a FC/TC ratio of approximately 0.18 [ll]. This ratio, plus a safety factor of 0.5, was applied to the TC densities (2300-2400 organisms/100 ml) known to produce health effects and an average criterion of 200 FC organisms/100 ml was generated [ l l ] . This value was believed to provide bathers adequate protection from pathogenic contamination and was recommended by the U.S. Public Health Service in 1968 [9]. In 1973, a U.S. Environmental Protection Agency (EPA) publication [12] cited studies by Geldreich [13] and Geldreich and Bordner [14]

24

RICHARD FIELD AND MARIE L. O’SHEA

which correlated the occurrence of Salmonella with FC densities. These studies found that the frequency of Salmonella detection increased sharply at FC densities above 200 organisms/100 ml, and reached a 97.6% detection maximum when FC densities exceeded 2000/100 ml [13]. On the basis of this and other data, the EPA suggested a limit of 2000 FC organisms/100 ml for raw surface water sources of public (potable) water supplies but could not recommend use of the FC index as the sole criterion for recreational waters due to the “paucity of valid epidemiological data” [12]. The report further stated that the FC index, if it is to be used as an index of the sanitary cleanliness of recreational waters, should be used only in conjunction with other evaluative parameters of water quality such as sanitary surveys, other biological indices of pollution, and chemical analyses of water. [12]

In a 1976 report [15], the EPA reinforced the original 1968 criterion of 200 FC organisms/100 ml [9] for recreational waters despite numerous criticisms of its deficiencies [16 191. The 1976 report acknowledged that epidemiological evidence to support the criterion was lacking but concluded that FC levels remained the best measure of microbiological water quality because of problems associated with the detection of other indicators or pathogenic microorganisms. Thus, despite the absence of epidemiological evidence-or an acceptable alternative indicator-TC and FC criteria were adopted and enforced throughout the country. More recently, advances in microorganism isolation and identification have permitted researchers to study the relationship between swimming-associated illnesses and specific taxa of the FC group. In the early 1980s, the EPA conducted two such studies of both marine and fresh waters which aimed to determine the relationship between GI swimming disorders and the bathing-water densities of FC, enterococci, and E. coli [20,21]. Each study used regression and correlation analysis to compare the strength of association of the various indicator bacteria to GI illness, thereby providing both an epidemiological rationale for the suggested criteria and the flexibility to consider other levels of risk. On the basis of the correlation data, the EPA marine study concluded that enterococci would be superior to E. coli as an indicator of fecal pollution at ocean beaches [21], while the statistics generated in the freshwater study indicated that either enterococci or E. coli would be a suitable indicator for freshwater-bathing quality [ZO]. The results of

EVALUATION OF THE TREATMENT OF STORMWATER

25

these studies also revealed that, due to differences in the die-off rate of indicator bacteria in fresh water and seawater, equivalent enterococci densities led to illness rates among swimmers in marine waters approximately three times greater than that observed among freshwater bathers. As stated in the freshwater study, this suggests criteria developed for fresh waters would be inappropriate if similarly applied to marine waters [ZO]: The significance of these findings is that a single water quality criterion for seawater and freshwater has been effectively eliminated from consideration, and therefore a separate criterion should be used for each type of bathing water.

Although the freshwater study found that both enterococci and E. coli densities displayed an excellent relationship to GI illness rates, E. coli exhibited the higher correlation coefficient and a lower standard error 1201. Additional factors favoring E. coli as the indicator of choice for freshwater bathing quality included (1) its often higher density than enterococci both in human feces [22] and sanitary-wastewater effluent [23], and (2) its apparent hardiness in fresh water, relative to that of enterococci [24]. The results of both studies clearly confirmed that the rate of GI illness increased with fecal contamination. However, in statistically evaluating the relationship between FC densities and GI disorders, both studies found that FC densities were unrelated to swimming-associated gastroenteritis [20,21]. Data from other studies were consistent with these findings [25,26]. The implication of these results was best summarized in the freshwater report [ZO]: Bacteria from sources other than the gastrointestinal tract of man and other warm-blooded animals, which fit the definition of fecal coliforrn . . . are present at densities high enough to sufficiently eliminate the usefulness of fecal coliforms as an indicator of fecal contamination of surface waters.

A 1986 EPA publication on water quality criteria addressed the limitations associated with the use of TC and FC indicators in the measurement of bathing-water quality and recommended that states “begin the transition process to the new [E. coli and enterococci] indicators” [27]. The preceding chronology provides the background and rationale for current regulations regarding microbial standards for recreational waters. In general, descriptions of adverse public-health impacts resulting from the discharge of sanitary wastewater without prior treatment have gradually evolved from simple mathematical correlations to the EPA’s current risk-assessment approach. However, despite the lack of correla-

26

RICHARD FIELD AND MARIE L. O’SHEA

tion between TC and FC levels and swimming-related illnesses, and the 1986 EPA recommendation for the adoption of new recreational waterquality criteria, many states still retain the TC and FC criteria first recommended in 1968. In the search for a more accurate determination of the nature of the pollution source and thus a measure of the human-disease potential of the receiving waters, several indicator relationships and/or microbial detection methods for pathogenic bacteria or human enteric viruses have been examined: FCiFS ratios [28]; P. aeruginosaiFC ratios [29]; Clostridium perfringens [30] and its relation to FC densities [26]; fecal sterols, e.g., epicoprostanol and coprostanol [31,32]; species-specific bacteriophages, e.g., RNA coliphages [33], Bacteroides fragilis phages [34,35]; and some species of the genus Bifidobacteria [30,36]. Investigation and evaluation of several of these alternative indicators have shown them to either fall short of the list of requirements commonly cited for indicators [37,38], or possess only limited usefulness. Recent methods allowing the direct detection of waterborne pathogens include gene probes and polymerase chain reaction (PCR) techniques. PCR and DNA probe methods have already been used in the rapid detection and enumeration of coliform bacteria, E. coli, and Shigella spp. [39-411; Salmonella spp. [42]; and Giardia 1431; it is anticipated that these methods will eventually be applied to the direct detection of human enteric viruses [44]. Ill. Stormwater Quality and Its Relationship to Human Disease Potential

Despite ongoing research on alternative indicators, the common bacterial indicators for recreational waters remain TC and FC. For receiving waters contaminated by sanitary wastewater alone or in combination with stormwater (CSO), the choice of either FC or TC densities as an indicator of pathogens may be a satisfactory one. For separate storm-drainage systems and streams that are not separated from the sources of human-fecal contamination, e.g., sanitary wastewater, the results of microbiological analyses suggest that these waters can and do present a potential health hazard. Some of the disease-causing microorganisms isolated from stormwater runoff and urban streams include enteroviruses (e.g., poliovirus, Coxsackie B virus, and Echovirus) [37] and bacteria in the form of P. aeruginosa, Staphylococcus aureus, and Salmonella organisms [37,45-471. As an example, Table I summarizes the results of an extensive microbial analysis of separate storm sewers (containing varying extents of sanitary wastewater inflows) in Baltimore, Maryland.

TABLE I ~ E O M E T R I CMEAN I)ENSITIES OF SELECTED PATHOGENS AND INDICATOR MICROORGANISMS IN STORMWATEHU

Sampling

statinn

Enterovirus PFU/lU liters

Stoney Runb Glen Avenuer Howard Parkd Jones Fallsc Bush Streete

190

Northwoode

170

75 280

30 6.9

Tc FC: Salmonella sp. P. aeruginosa Staph. flureus MPN(100 ml MPN/100 rnl MPN/10 lilers MPN!10 lilers MPN!100 nil (xlO4) [ x 103) 30 24 140 25 30

5.7

1300 3300 5200

12

6600 2000

14 36 40 120

590

12

~ ~ F r uOlivieri m st ul. (19771 [ref. 371. bThree sanitary bleeders (intentional sanitary sewage overflows from interceptors). .One sanitary bleeder. Combined sewer. 'Storm only.

4.8

24 120 29 38

3.8

19 ni 450 120 83

6.9

FS no.llO0 tnl

Enterococci no.llOO ml

(X104)

(xi041

4.1

66 24 28

56

5

1.4 21 5.9 8.7

12 2.1

28

RICHARD FIELD AND MARIE L. O’SHEA

In stormwater flows where pathogen concentrations were significant and could not be correlated to storm events or soil populations, the most frequently cited sources of the contamination were sanitarywastewater line leaks, interceptor diversions, or intentional cross-connections into the storm-drainage system [37,45,46], i.e., a lack of total separation from sanitary-wastewater sources. The Baltimore study determined that the frequency of pathogenic contamination could be directly related to the extent of sanitary-wastewater diversions or number of direct sanitary-wastewater connections into the stormwater system [371.

The analyses of dry-weather base flows in separate stormwater drainage systems can often determine the extent of contamination by sanitary wastewater via illicit or inadvertent cross-connections [48]. As an example, a Toronto, Canada survey found that dry-weather-base flows in the separate stormwater-drainage system exhibited statistically similar FC populations to those observed in stormwater runoff [47], implying the presence of a continuous microbial pollutant source. Despite evidence of pathogenic contamination of stormwater, it has been argued that the presence of these pathogens in stormwater does not, in fact, constitute a significant health hazard [37]. This argument cites the low densities of pathogenic microorganisms observed in urban storm runoff, the further dilution of these flows upon reaching recreational waters, and the large infective doses of bacteria such as selected species of Salmonella (105 organisms) in concluding that any threat to swimmers should be small, “since prodigious swallowing of water would be required in order to increase the risk of enteric disease” [37]. Unfortunately, the evidence of low densities coupled with high infective doses cannot minimize the health hazard of pathogens such as P. aeruginosa, Salmonella typhosa, Shigella, or enteroviruses that either do not require ingestion for infection, or require very low infective doses. However, due in part to past difficulties in the isolation and quantification of some of these species, particularly at the low densities normally observed in storm and receiving waters, there has been little study of their correlation with swimming-associated illnesses. For example, several studies have found large (103-i04 organisms/100 ml) populations of P. aeruginosa (PA) in urban streams and stormwater runoff [37,45,47]. PA/FC ratios ranged over three orders of magnitudes (from 0.01 to >20), indicating that FC populations were poorly related to the density of this pathogen. The predominance of P. aeruginosa in stormwater coupled with its association with diseases transmitted through water contact, e.g., skin and ear infections, signifies its potential importance in evaluating the health hazard of waters

EVALUATION OF THE TREATMENT OF STORMWATER

29

receiving storm runoff. However, studies which have attempted to correlate its densities to illness rates have reported only its poor relation to acute GI distress [21] or total illness rates [3,4]. Little information is currently available regarding its correlation to body contact illnesses due to stormwater exposure, despite the suggested greater risk associated with this mode of transmission. In 1977 it was estimated that 14.4% of urban areas containing 25.2% of the urban population was served by combined sewers [49]. These percentages have since declined due to the ongoing development of suburban communities which are either served by separate storm sewers or are unsewered, and the lessening of combined sewer construction. It has been well established that the bacteria isolated in stormwater runoff are predominantly from nonhuman sources [45,50]. Thus, for receiving waters accepting separate stormwater inflows, a reliance on TC or FC indicators to determine bathing water quality may prove ineffective due to the inability of this method to distinguish human from nonhuman, and possibly nondisease-causing, sources (e.g., vegetation, soil, and animals) [2,3,28,37,51,52]. Several studies have isolated animal-associated enteric viruses and bacteria that can be transmitted to humans, e.g., Yersinia, Cryptosporidium, and Salmonella, in stormwater or surface waters in urban, rural, and agricultural watersheds, indicating that the disease-causing potential of these sources cannot be neglected [50,53-561. However, to date, few epidemiological studies have attempted to correlate incidences of GI or total illness with FC densities arising primarily from nonhuman sources, e.g., stormwater runoff uncontaminated by sanitary wastewater. Such studies, undertaken for a variety of watershed types, are necessary to ensure that the continued reliance on coliform indicators to determine water-quality criteria for stormwater-receiving recreational waters does not erroneously hinder their recreational usage. To date, only one well-documented study has been conducted which has addressed diseases which may result from direct contact with bathing waters whose sole source was rainwater runoff from a (forested) watershed [57]. This study used epidemiological data to compare the health status of swimmers utilizing the waters during wet-weather periods with that of nonswimmers. The study site was located in a semirural community and consisted of a 3-acre freshwater pond with no known source of human fecal contamination. During a 49-day period, water samples were collected three times daily and analyzed for E. coli, enterococci, P. aeruginosa, staphylococci, and FC. Data on rainfall, bather density, and the occurrence of GI illness among the monitored families were also collected.

30

RICHARD FIELD AND MARIE L. O'SHEA TABLE I1

ASSOCIATION BETWEEN CASESOF GI ILLNESS AND VARIOUS MONITORED PARAMETERS" ~

~~~~

Monitored relative parameters

High values

Illnessesb

Low values

Illnessesb

Relative riskr

Rainfalld Enterococcir E. colic FCc Bathers Staphylococci?

20.2 220 275 280 '53 '45

29 35 29 34 29 31

<0.2 <20 <75

14 18 21 20 6 12

2.1 1.9 1.4 1.7 4.8 2.6

High groups

Low groups

<80

153

<45

"From Calderon et al. (1992)[ref. 571. "Number of illnesses per 1000 person-days. <,Thefactor by which an individual would be that much more likely to become ill as a result of swimming under these conditions, than if they did not swim at all. dInches per day. CDensity per 100 ml.

Monitoring results indicated that the geometric mean densities of E. coli and FC were over two times greater on rain days than on nonrain days, while for enterococci the density ratio for raidnomain days was four. These three fecal-related indicators also exhibited significant correlation with each other, i.e., when one increased in density, the other two also increased. No correlation was observed between indicator bacteria levels and bather density. Conversely, staphylococci densities were related to bather density but not to any of the fecal indicator bacteria or to rainfall. Health data were analyzed by pairing each swimmer illness with the indicator density associated with the day of exposure and then segregating these illnesses into two groups based on high and low parameter densities (Table 11). GI illness was observed to be strongly associated with swimming, but illnesses appeared randomly dispersed in the high and low indicator groups, suggesting that no association exists between GI illness and high fecal indicator bacteria densities. However, a significant association was observed between swimming-associated illnesses and high densities of staphylococci or high densities of bathers. The authors concluded that reported illnesses were probably due to agents transmitted from swimmer to swimmer, and were not related to pollution discharged into the pond during wet weather. The high densities of the three fecal indicators, which could be correlated with daily rainfall levels, were attributed to the presence of warm-blooded animals in the wooded areas surrounding the swimming pond [57].

EVALUATION OF THE TREATMENT OF STORMWATER

31

The results of this study are consistent with an earlier work which documented GI and total illness among 8400 swimmers and nonswimmers at 10 freshwater beaches in Ontario using total staphylococci, fecal streptococci, fecal coliform, heterotrophic bacteria, and P. aeruginosa as indicators [3,4]. The findings indicated total staphylococci densities possessed the strongest dose-response relationship and proved to be the most consistent indicator of total illness as well as eye and skin disease. IV. Disinfection

Disinfection requirements and associated facilities in the United States were established and designed to protect waters used for recreational, shellfishing, and potable supply purposes by controlling TC or FC bacteria at various levels. For the treatment of municipal wastewater, the regulations are often expressed as a combination of technologybased definitions (e.g., dosage-contact time) and water quality-based criteria (e.g., coliform density) and may be applied on a continuous or seasonal basis. However, storm-generated flows containing human-fecal contamination require a different disinfection approach since these flows are intermittent, often high-rate, and normally display wide seasonal and intra-/interstorm variations in quantity, temperature, and pollutant and bacterial characteristics. These unique characteristics of storm-generated flows necessitate the adoption of cost-effective, highrate disinfection practices and the use of disinfection facilities that can be adaptable to both intermittent use and varying dosage requirements [ 58,591, Several factors determine the overall effectiveness of disinfection by chemical addition or alternative methods. These include, but are not limited to: (11the nature and concentration/intensity of both the disinfectant and the products formed in the water after reaction with it; (2) the condition of the water, e.g., its suspended solids and chemical characteristics, temperature, and pH; (3) the contact time between the disinfecting agent and the pathogen; (4) the mixing intensity imparted to the water; and (5) the nature and density of pathogens and their resistance to inactivation by the disinfectant used [60]. In general, lowering the pH or increasing the disinfectant concentration, the water temperature, the mixing intensity, or the contact time will increase the effectiveness of a chemical disinfectant. High-rate disinfection, i.e., decreased disinfectant-contact time, can be achieved through the use of one or more of these practices alone or in combination with each other 1581.

32

RICHARD FIELD AND MARIE L. O’SHEA

A. CHEMICAL DISINFECTION Due to their low cost, ease of use, and germicidal properties, elemental chlorine and chlorine compounds have traditionally been the most widely used chemical disinfectants. Other chemical agents which have similar or greater oxidizing potentials (but not necessarily germicidal effectiveness) include the free halogens fluorine [F2),bromine (BrJ, and iodine (I2); ozone (OJ; hydrogen peroxide (H,O,); and potassium permanganate (KMnO,). Early disinfection practices were generally confined to the treatment of sanitary wastewater and potable water after it passed through a treatment facility. These facilities normally employed relatively large chlorine-contact tanks and achieved bacterial and viral kill by the addition of elemental chlorine (Cl,), or, later, sodium hypochlorite (NaOCl), at dosages of 30-100 lb/Mgal and residual concentrations of 0.8-1.8 mg/liter for assured kill [61]. Stormwater and combined sewer overflows (CSO) were generally not treated in a comparable manner due to the assumption that disinfection of dry-weather flow provided effective protection to the receiving waters. This assumption was challenged in the 1960s by several studies which specifically addressed the treatment and disinfection of stormwater and CSO, and which recognized that large variations in flows were the principal problems to be overcome in its effective chlorination [62-641. Throughout the 1970s, EPA research efforts were underway to determine the unique disinfection needs of stormwater and CSO [65-691. Several of these studies employed the screening technique of Microstraining to remove and/or fragment particulate and organic matter containing bacteria [66-691. By reducing particulate size, the number of bacteria and viruses occluded within larger particulates-and consequently shielded from chemical attack-could be minimized. Coliform reductions across the microstrainer were found to be minor; however, it was confirmed that microstrained effluent was more amenable to disinfection, exhibiting a lower C1, demand and requiring shorter detention times. These studies also investigated the importance of maximizing mixing intensity within the disinfection chamber in order to ensure dispersion of the added disinfectant and increase the number of collisions between the bacteria and disinfectant. Increased mixing intensity was shown to be achievable either statically by baffles, corrugatednarrow pathways, or helical vanes; or dynamically by moving impellers. In general, the utilization of such high-rate-mixing techniques within the chlorine contact chamber ensured plug flow conditions, full residence times, and high liquid velocity gradients.

EVALUATION OF THE TREATMENT OF STORMWATER

33

Early microstrainer studies determined that simultaneous disinfection of CSO influent and microstrained effluent (with initial C1, concentrations of 5 mg/liter for 4 . 2 5 minutes and 2.6 mg/liter for 3.25 minutes, respectively) could meet or exceed the current FC and TC criteria of 2 0 0 FC/100 ml and 1000 TC/lOO ml [70]. Later studies determined that sequential, or two-stage, addition of C1, followed by chlorine dioxide (C10,) enhanced disinfection beyond the expected effects for sequential addition of either C1, or C10, alone 171-731. This phenomenon is attributed to the regeneration of the more potent disinfectant, ClO,, through the oxidation of residual chlorite ion (ClO,-) (from the reduction of ClO,, in wastewater) by free C1, [73]. It was determined that a 25 mg/liter initial concentration of C1, followed in 15-30 seconds by 12 mglliter of ClO, reduced TC in simulated CSO to target levels of 1000 TC/100 ml in 2 minutes [73].

B . ALTERNATIVE DISINFECTION ~CHNIQUES Since the 1970s there has been a growing awareness of the adverse environmental impacts associated with many of the chemical reaction products of continuous chlorination [ 74-77]. The chemical species of concern include free available chlorine [i.e., hypochlorous acid (HOCI), hypochlorite ion (OCl-)], chloramines [products of the reaction of C1, with ammonia (NH,)], and other halogenated organic and inorganic compounds. Many treatment facilities, faced with increasingly restrictive residual chlorine requirements, now employ reducing agents or other dechlorination techniques in order to minimize environmental damage. However, general efforts to reduce the chemical demands and contact times which are necessitated by the dechlorination procedure have also fostered a strong interest in alternative disinfection technologies. One disinfection technique which promises short detention times and the absence of toxic reaction products is that of disinfection by ultraviolet (UV) light-irradiation [ 78-82]. This technique works on the principle that all microorganisms which contain nucleic acids are susceptible to damage through the absorption of radiation in the UV energy range. However, the exact extent of damage, mutation, or death will depend on the resistance of an organism to radiation penetration which will in turn depend on several factors, including cell wall composition and thickness [60]. UV-dosage requirements for achievement of target indicator concentrations depend on several parameters, including the frequency and intensity of the UV radiation, the number and configuration of the UV

34

RICHARD FIELD AND MARIE L. O’SHEA

lamps, the distance between the wastewater and the lamp surface, the chamber turbulence, and the wastewater’s absorption coefficient and its exposure time to UV. Early studies of the UV-disinfection process utilizing the maximum UV-exposure levels then available indicated the limitations of the process. In general, disinfection of effluents containing high concentrations of protective suspended solids andlor organics proved largely ineffective [78]. The high absorption of UV radiation by these substances served to attenuate the available UV energy, resulting in a reduction of its depth of penetration into the wastewater [78]. Consequently, organisms contained within large particulates experienced little or no irradiation due to the complete absorption of the radiation by the outer, protective layer. UV-radiation doses have since been increased through improvements in system design and available equipment. However, coliform reductions remain highly dependent on water quality, restricting the practical use of UV-irradiation to the treatment of secondary or tertiary effluent [81-841. However, this disinfection technique, with its absence of toxic residuals and contact times on the order of seconds rather than minutes, would be a desirable choice for the treatment of the high flow rates associated with stormwater and CSO. New York City’s Department of Environmental Protection (DEP) is currently evaluating a large-scale pilot plant proposal which would investigate the primary treatment efficiencies of CSO by improved UV-irradiation techniques. The newly developed system would employ higher pressure lamps which emit higher intensity radiation and a broader spectrum of UV wavelengths [85]. Laboratory evaluation of a higher pressure lamp has demonstrated that its germicidal effectiveness is approximately 10 times that of a conventional low-pressure lamp [ 8 6 ] , effectively offsetting the lo-fold increase in the number of lamps which would normally be required for the disinfection of CSO at efficiencies currently being met for treated effluents. Still more recent experiments have compared the germicidal effectiveness of modulated UV light with that of nonmodulated UV radiation of the same intensity and exposure times. After exposure to pulsed radiation, viable bacterial populations were reported to number approximately 100-fold less than the populations observed after similar exposure to UV light which lacked modulation 1871. V. Conclusions and Recommendations

The EPA’s current emphasis on water-use attainability and risk assessment warrants the reevaluation of existing disinfection require-

EVALUATION OF THE TREATMENT OF STORMWATER

35

ments and bacteriological criteria. Information relating incidence of disease in recreational-water users to instream densities of various indicator microorganisms indicates that current criteria may be inappropriate for correctly assessing the risk to the public health. This is especially true for those receiving waters complicated by the influence of storm-induced inflows. Water quality and disinfection criteria based on human disease contraction potentials of pathogenic microorganisms are clearly needed for stormwater as well as CSO. To develop the former, regulatory agencies must address the problems of choosing the most appropriate indicator(s) and establishing acceptable limiting levels. In order to be most effective, microbial criteria should be derived from direct pathogen and epidemiological analyses for relating risk to a given level of protection. Where sources other than sanitary-wastewater flows, e.g., stormwater runoff, enter bathing waters the current criteria expressed in terms of FC organisms do not have such a basis. The results of a limited number of epidemiological studies strongly suggest that TC or FC indicators cannot be used to assess accurately the pathogenicity of recreational waters receiving stormwater from uncontaminated separate storm sewers or surface water runoff. Additional epidemiological case studies, from a variety of watershed areas, are needed in order to determine the true health risks associated with stormwater contact and to establish correlations between alternative bacterial or viral indicators and total illness rates. Since epidemiological studies of recreational waters receiving mixed sanitary and stormwater inflows will prove statistically difficult to interpret, it is further recommended that such studies focus on recreational waters in subdrainage systems which have been well characterized and whose background flows do not show evidence of the influx of sanitary sewage or other human waste sources. Since the predominant bather-associated risk has been reported to be infections of the skin, ear, eye, and/or upper respiratory system, epidemiological data are also required on the presence of certain nonenteric pathogens. The adoption of multiple indicators (e.g., enteric and nonenteric bacteria) or alternative fecal indicators whose densities can be better correlated with nonenteric infections may be necessary to provide a more accurate estimate of the total health risk associated with stormwater contact. For storm sewers which contain evidence of human-fecal contamination, and thereby indicate the presence of illicit or inappropriate crossconnections, a carefully planned strategy should be adopted for identifying and eliminating these sanitary-wastewater sources 132,331. In stormwater outfalls where cross-connections are too numerous or too

36

RICHARD FIELD AND MARIE L. O’SHEA

costly to be corrected, it may be advisable to deal with the separate storm sewer system as, in fact, a combined sewer system [79]. Where the long-range goal of a cross-connection or CSO elimination project includes the expansion of a water body’s recreational usage, the adoption of new water-quality criteria based on epidemiological data and a complete survey of receiving water inflows should be simultaneously investigated. In general, if little consideration is given to the normally high wet-weather bacterial densities associated with stormwater, then costly programs aimed at eliminating human-fecal contamination or its sources may prove ineffective for achieving the ultimate goal of swimmable waters. REFERENCES 1. Field, R., and Struzeski, Jr., E. J. (1972). Management and Control of Combined Sewer

Overflows. J. Water PoJlut. Control Fed. 44(7), 1393. 2. Field, R. (1976). “Microorganisms in Urban Stormwater-A

U.S. Environmental Protection Agency Program Overview.” Proc. Workshop on Microorganisms in Urban Stormwater, USEPA Report No. EPA-600-2-76-244 (NTIS No. PB-263-030), Nov. 1976. 3. Seyfried, P. L., Tobin, R. S., Brown, N. E., and Ness, P. F. (1985). A Prospective Study of Swimming-Related Illness: I. Swimming-Associated Health Risk. Am. J. Public Health 75(9), 1068. 4. Seyfried, P. L., Tobin, R. S., Brown, N. E., and Ness, P. F. (1985). A Prospective Study of Swimming-Related Illness: Morbidity and the Microbiological Quality of Water. Am. J. Public Health 75(9), 1071. 6.Stevenson, A. H. (1953). Studies of Bathing Water Quality and Health. J. Am Public Health Assoc. 43, 529. 7. “Standard Methods for the Examination of Water and Wastewater,” (L. S. Clesceri, A. E. Greenberg, and R. R. Trussell, eds.), 17th Ed. Am. Public Health Assoc., Washington, D.C., 1989. 8. Dufour, A. P. (1977). Escherichia coli: The Fecal Coliform. In “Bacterial Indicators/Health Hazards Associated with Water” (A. W. Hoadley and B. J. Dutka, eds.), p. 48. ASTM, Philadelphia, PA. 9. National Tech. Advisory Committee, (1968). “Water Quality Criteria.” Report to the Federal Water Pollution Control Administration, U.S. Dept. of Inter., Washington, D.C. 10.Smith, R. S., Woolsey, T. D., and Stevenson, A. H. (1951). “Bathing Water Quality and Health-1-Great Lakes. U.S. Public Health Service, Cincinnati, Ohio. 11. Geldreich, E. E. (1966). “Sanitary Significance of Fecal Coliforms in the Environment.” Water Pollut. Control Res. Ser. No. WP-20-3, Government Printing Office, Washington, D.C. 12. Natl. Acad. Sci., Natl. Acad. Eng. (1973). “Water Quality Criteria, 1972.” USEPA Report No. EPA-R3-73-003, Washington, D.C. 13. Geldreich, E. E. (1970). Applying Bacteriological Parameters to Recreational Water Quality. J. Am. Water Works Assoc. 62, 113. 14. Geldreich, E. E., and Bordner, R. H. (1971). Fecal Contamination of Fruits and Vegetables during Cultivation and Processing for Market-A Review. 1. Milk Food Technol. 34(4), 184.

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37

15. “Quality Criteria for Water.” USEPA Report No. EPA-44019-76-023 (NTIS No. PB-83-25994), Washington, D.C. 1976. 16. Henderson, J. M. (1968). Enteric Disease Criteria for Recreational Waters. J. San. Eng. Div. 94,1253. 17. Moore, B. (1975). The Case against Microbial Standards for Bathing Beaches. In “Int. Symp. Discharge of Sewage from Sea Outfalls” (A. H. L. Gameson, ed.), p. 103. Pergamon Press, London. 18.Cabelli, V. J., Levin, M. A,, Dufour, A. P., and McCabe, L. J. (1975). The Development of Criteria for Recreational Waters. In Int. Symp. Discharge of Sewage from Sea Outfalls” (A. H. Gameson, ed.), p. 63. Pergamon Press, London, 1975. 19. Foster, D. H., Hanes, N. B., and Lord, S. M., Jr. (1971). A Critical Examination of Bathing Beach Standards. J. Water Pollut. Control Fed. 43, 2229. 20. Dufour, A. P. (1984). “Health Effects Criteria for Fresh Recreational Waters.” USEPA Report No. EPA-600/1-84-004, Washington, D.C. 21. Cabelli, V. J. (1983). “Health Effects Criteria for Marine Recreational Waters.” USEPA Report No.. EPA-600/1-80-031, Washington, D.C. 22. Slanetz, L., and Bartley, C. H. (1957). Numbers of Enterococci in Water, Sewage and Feces Determined by the Membrane Filter Technique with improved Medium. J. Bacteriol. 74, 591. 23. Miescier, J., and Cabelli, V. J. (1982). Enterococci and Other Microbial Indicators in Municipal Wastewater Effluents. J. Water Pollut. Control Fed. 54, 1599. 24. Hanes, N., and Fragloa. R. (1976). Effect of Seawater Concentration on the Survival of Indicator Bacteria. J. Water Pollut. Control Fed. 39, 97. 25. Dutka, B. J. (1973). Coliforms Are an Inadequate Index for Water Quality. J. Environ. Health 36, 39. 26. Fujioka, R. S., and Shizumura, L. K. (1985). Clostridium perfringens, a Reliable Indicator of Stream Water Quality. J. Water Pollut. Control Fed. 57, 986. 27. DuFour, A., and Ballentine, R. (1986). “Ambient Water Quality Criteria for Bacteria.” USEPA Report NO. EPA-44015-84-002, (NTIS PB86-158045), Washington, D.C. Jan. 1986. 28. Geldreich, E. E., and Kenner, B. A. (1969). Concepts of Fecal Streptococci in Stream Pollution. J. Water Pollut. Control Fed. 41(8), R336. 29. Cabelli, V. J., Kennedy, H., and Levin, M. A. (1976). Pseudomonas aeruginosa-Fecal Coliform Relationships in Estuarine and Fresh Recreational Waters. J. Water Pollut. ControJ Fed. 48(2), 367. 30. Cabelli. V. J. (1978). Obligate Anaerobic Bacterial Indicators. In “Indicators of Viruses in Water and Food” (G. Berg, ed.), p. 171. Ann Arbor Science, Ann Arbor, Michigan. 31. Eganhouse, R. P. (1988). Use of Molecular Markers for the Detection of Municipal Sewage Sludge at Sea. Marine Environ. Res. 25(1), 1. 32. Walker, R. W., Wun, C. K., and Litsky, W. (1982). Coprostanol as an Indicator of Fecal Pollution. Crit. Rev. Environ. Control 12, 91. 33. Kott, Y . , Roze, N., Sperber, S., and Betzer, N. (1974). Bacteriophages as Viral Pollution Indicators. Water Aes. 8, 165. 34. Jofre, J., Bosch, A., Lucena, F., Girones, R., and Tartera, C. (1986). Evaluation of Bacteroides fragilis Bacteriophages as Indicators of the Virological Quality of Water. Water Sci. Technol. 18, 167. 35. Tartera, C., Lucena, F., and Jofre, J. (1989). Human Origin of Bacteroides fragilis Bacteriophages Present in the Environment. Appl. Environ. Microbiol. 55(10), 2696. 36. Resnick, I. G., and Levin, M. A. (1981). Assessment of Bifidobacteria as Indicators of Human Fecal Pollution. Appl. Environ. Microbiol. 42(3),433.

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RICHARD FIELD AND MARIE L. O’SHEA

37. Olivieri, V. P., Kruse, C. W., Kawata, K., and Smith, J. E. (1977). Microorganisms in Urban Stormwater. USEPA Report No. EPA-600/2-77-087 (NTIS No. PB-272245). 38. Vivian, C. M. G. (1986). Tracers of Sewage Sludge in the Marine Environment: A Review. Sci. Total Environ. 53, 5-40. 39. Bej, A. K., Steffan. R. J., DiCesare, J . * Haff, L.. and Atlas, R. M. (1990). Detection of Coliform Bacteria in Water by Polymerase Chain Reaction and Gene Probes. Appl. Environ. Microbiol. 56(2), 307. 40. Bej, A. K., DiCesare, J., Haff, L., and Atlas, R. M. (1991).Detection of Escherichia coli and Shigella spp. in Water by Using the Polymerase Chain Reaction and Gene Probes for uid. AppJ. Environ. Microbiol. 57, 1013. 41. Bej. A. K., McCarty, S. C., and Atlas, R. M. (1991).Detection of Coliform Bacteria and Escherichia coli by Multiplex Polymerase Chain Reaction: Comparison with Defined Substrate and Plating Methods for Water Quality Monitoring. Appl. Environ. Microbiol. 57(8), 2429. 42. Knight, I. T., Shults, S., Kaspar, C. W., and Colwell, R. R. (1990).Direct Detection of Salmonella spp. in Estuaries by Using a DNA Probe. Appl. Enrriron. Microbiol. 56(4), 1059. 43. Abbaszadegan, M., Gerba, C. P., and Rose, J. B. (19911. Detection of Giardia Cysts with a cDNA Probe and Applications to Water Samples. Appl. Environ. Microbiol. 57,927. 44. Francis, J., and Ellis, L. (1991).“Alternative lndicators of Human Fecal Contamination of Stormwater.” Ongoing Urban Waste Management and Research Center, University of New Orleans Project No. 92-A-010. 45. Qureshi, A. A., and Dutka, B. J. (1979). Microbiological Studies on the Quality of Urban Stormwater Runoff in Southern Ontario, Canada. Water Res. 13, 977. 46. Davis, E. M. (1979). “Maximum Utilization of Water Resources in a Planned Community-Bacterial Characteristics of Stormwater in Developing Rural Areas.” USEPA Report No. EPA-600/2-79-050f (NTIS No. PB 80-129 091). 47. Pitt, R., and McLean, J. (1986). “Toronto Area Watershed Management Strategy Study; Humher River Pilot Watershed Project. Final Report.” The Ontario Ministry of the Environment, Toronto, Ontario. 48. Pitt, R., Lalor, M., O’Shea, M., and Field, R. (1991). “USEPA’s Manual of Practice for the Investigation and Control of Cross-Connection Pollution into Storm-Drainage Systems.” Proc. Int. Conf. Integrated Stormwater Management, Singapore, July 1113. 1991. 49. Sullivan, R. H., Manning, M. J.. Heaney. J. P., Huher, W. C.. Medina. M. A , , Jr.,Nix, S. 1.. and Hasan, S. M. (1977). “Nationwide Evaluation of Combined Sewer Overflows and Urban Stormwater Discharges. Volume 1: Executive Summary.” USEPA Report No. EPA-60012-77-064a (NTIS No. PB 273-133). Sept. 1977. 50. Geldreich, E. E., Best, L. C., Kenner, I3. A,, and Van Donsel, D. J. (1968). The Bacteriological Aspects of Stormwater Pollution. J. Water Pollut. Control Fed. 40111),1861. 51. Field. R., and Turkeltaub, R. (1981).Urban Runoff Receiving Water Impacts: Program Overview. J. Environ. Eng. Div.. Am. Soc. Civil Eng., 107(EE1), 83. 52. Rivera, S. C.. Hazen, T. C., and Toranzos, G. A. (19881. Isolation of Fecal Coliforms from Pristine Sites in a Tropical Rain Forest. Appl. Environ. Microbiol. 54(2), 513. 53. Geldreich, E. E. (1978). Bacterial Populations and Indicator Concepts in Feces, Sewage, Stormwater and Solid Wastes. In “Indicators of Viruses in Water and Food” [G. Berg, ed.), p. 51. Ann Arbor Science, Ann Arbor, MI. 54. Fukushima, H., and Gomyoda, M. (1991). Intestinal Carriage of Yersinia pseudotuberculosis by Wild Birds and Mammals in Japan. Apof. Environ. Microbiol. 57(4), 1152.

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39

55. Madore, M. S., Rose, J. B., Gerba, C. P., Arrowood, M. j., and Sterling, C R. (1987). Occurrence of Cryptosporidium Oocysts in Sewage Effluents and Selected Surface Waters. J. Parisitol. 73, 702. 56. Pitt, R. (1983). “Urban Bacteria Sources and Control by Street Cleaning in the Lower Rideau River Watershed.” Rideau River Stormwater Management Study Technical Report, Ministry of the Environment, Ontario, August 1983. 57. Calderon, R. L., Mood, E. W., and Dufour, A. P. (1991). Health Effects of Swimmers and Nonpoint Sources of Contaminated Water. Int. 1. Environ. Health, 1 , 21--23. 58. Weisman, D. A,, and Field, R. (1981). “A Planning and Design Guidebook for Combined Sewer Overflow Control and Treatment.” USEPA Report No. EPA-60012-82-08, Sept. 1981. 59. Field, R. (1986). State-of-the-Art Update on Combined Sewer Overflow Control. CRC Crit. Rev. Environ. Control 16(2), 147. 60. Weber, W. Jr. (1972). “Physiochemical Processes for Water Quality Control.” Wiley, New York. 61. Steffensen, S. W., and Nash, N. (1967). Hypochlorination of Wastewater Effluents in New York City. J. Water Pollut. Control Fed. 39(8), 1381. 6 2 . Dunbar, D. D., and Henry, J. G. F. (1966). Pollution Control Measures for Stormwaters and Combined Sewer Overflows. 1. Water Pollut. Control Fed. 38(1), 9. 63. Camp, T. R. (1961). Chlorination of Mixed Sewage and Storm Water. J. San. Eng. Div., Proc. Am. Soc. Civil Eng. 87, 1. 64. Evans, F. L., Geldreich, E. E., Weibel, S. R., and Robeck, G. G. (1968). Treatment of Urban Stormwater Runoff. 1. Water Pollut. Control Fed. 40(5), R162. 65. Dow Chemical Co. “Chemical Treatment of Combined Sewer Overflows.” USEPA Report No. 11023FDB09170 (NTIS No. PB-199-070), Sept. 1970. 66. Cochrane Division, Crane Co. “Microstraining and Disinfection of Combined Sewer Overflows.” IJSEPA Report No. 11023EV006170 (NTIS No. PB-195-6741BA), June 1970. 67. Glover, G. E., and Herbert, G. R. (1973). “Microstraining and Disinfection of Combined Sewer Overflows-Phase 11.” USEPA Report No. EPA-R2-73-124 (NTIS NO. PB-219-879), Jan. 1973. 68. Maher, M. B. (1974). “Microstraining and Disinfection of Combined Sewer OverflowsPhase 111.” USEPA Report No. EPA-67012-74-049 (NTIS No. PB-235-771), Aug. 1974. 69. Diaper, E. W. J., and Glover, G. E. (1971). Microstraining of Combined Sewer Overflows. J. Water Pollut. Control Fed. 43(10), 2101. 70. Glover, G. E. (1973). “High-Rate Disinfection of Combined Sewer Overflow.” Combined Sewer Overflow Seminar Papers, USEPA Report No. 67012-73-077 (NTIS No, PB-231-836), Nov. 1973. 71. Tifft, E. C., Moffa, P. E., and Richardson, S. L. (1976). “The Enhancement of High-Rate Disinfection by the Sequential Addition of Chlorine and Chlorine Dioxide.” Proc. Workshop on Microorganisms in Urban Stormwater, USEPA Report No. EPA600-2-76-244 (NTIS NO. PB-263-0301, NOV.1976. 72. Drehwing, F. J., Oliver, A. J., MacArthur, D. A., and Moffa, P. E. (1979). “Disinfection/Treatment of Combined Sewer Overflows.” USEPA Report No. EPA60012-79-134 (NTIS NO. PB-80-113-459). Aug. 1979. 73. Moffa, P. E., Tifft, E. C., Richardson, S. L., and Smith. J. E. (1975). “Bench-Scale HighRate Disinfection of Combined Sewer Overflows with Chlorine and Chlorine Dioxide.” USEPA Report No. EPA-67Oi2-75-021 (NTIS No. PB-242-296), April 1975. 74. Peicuch, P. J. (1974). The Chlorine Controversy. J. Water Pollut. Control Fed. 46(12), 2637.

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RICHARD FIELD AND MARIE L. O’SHEA

75. Zillich, J. A. (1972). Toxicity of Combined Chlorine Residuals to Freshwater Fish. J. Water PolJut. Controf Fed. 44(2), 212. 76. Brungs, W. A. (1973). Effects of Residual Chlorine on Aquatic Life. J. Water Pollut. Control Fed. 45(10), 2180. 77. Ward, R. W., and DeGraeve, G. M. (1978). Residual Toxicity of Several Disinfectants in Domestic Wastewater. J. Water Pollut. Control Fed. 50 46. 78. Roeber, J. A., and Hoot, F. M. (1975). “Ultraviolet Disinfection of Activated Sludge Effluent Discharging to Shellfish Waters.” USEPA Report No. EPA-60012-75-060, Dec. 1975. 79. Oliver, B. G., and Carey, J. H. (1976). Ultraviolet Disinfection: An Alternative to Chlorination. I. Water Pollut. Control Fed. 48(11), 2619. 80.Severin, B. F. (1980).Disinfection of Municipal Wastewater Effluents with Ultraviolet Light. J. Water Pollut. Control Fed. 52(7), 2007. 81. Petrasek, A. C., Wolf, H. W., Esmond, S. E., and Andrews, D. C. (1980). “Ultraviolet Disinfection of Municipal Wastewater Effluents.” USEPA Report No. EPA600/2-80-102, Aug. 1980. 82. Venosa, A. D. (1983). Current State-of-the-Art of Wastewater Disinfection. J. Water Pollut. Control Fed. 55(5), 457. 8 3 . Scheible, 0. K., and Bassell, C. D. (1981). “Ultraviolet Disinfection of a Secondary Wastewater Treatment Plant Effluent.” USEPA Report No. EPA-60012-81-152 (NTIS No. PB-81-242125), Aug. 1981. 84. Scheible, 0. K., and Forndran, A. (1985). “UV Disinfection of Secondary Effluent and CSO Wastewaters.” USEPA Report No. EPA-600/2-86-005 (NTIS No. PB86-1451823, Dec. 1985. 85. Scheible, 0. K., Smith, R., and Cairns, W. (1991). “The Application of Ultraviolet Disinfection to Combined Sewer Overflows.” Position paper, prepared for the New York City Department of Environmental Protection, June 1991. 86. Whitby, G. E., and Engler, F. (1988). “A Preliminary Study to Determine the Feasibility of Medium Pressure Lamps for Disinfecting Low Quality Wastewaters.” Prepared for the Research Management Office, Ontario Ministry of the Environment, RAC Project No. 380C, Toronto, Ontario, September 1988. 87. Bank, H. L., John, J., Schmehl, M. K., and Dratch, R. J. (1990). Bactericidal Effectiveness of Modulated UV Light. Appl. Environ. Microbiol. 56(12) 3888.

Haloperoxidases: Their Properties and Their Use in Organic Synthesis M. C. R.

FRANSSEN AND

H. C. VAN

DER

PLAS

Department of Organic Chemistry Wageningen Agricultural University Wageningen, The Netherlands

I. Introduction 11. Sources and Structures A. New Properties of Enzymes from Known Sources B. Enzymes from New Sources 111. Reactions A. Assay Methods B. Reactions with Alkenes C. Reactions with Aromatic Compounds D. Reactions with Heterocyclic Compounds E. Reactions with Amines F. Miscellaneous Substrates G. Reactions Using Immobilized Enzymes IV. Reaction Mechanisms A. Heme-Containing Enzymes B. Vanadium-Containing Enzymes C. Others V. Conclusions and Prospects References

I. Introduction

Haloperoxidases are enzymes which are capable of halogenating a variety of organic compounds using hydrogen peroxide and halide ions as substrates. The general overall reaction is given below: AH

+ HzOZ

f

H+

+ X-

+ AX

+ ZHz0

(1)

where AH is organic substrate; X is chloride, bromide, or iodide ion; AX is halogenated product. The enzymes are called chloro-, bromo-, or iodoperoxidases, depending on the smallest halide ion they can oxidize. Some 30 years ago, haloperoxidases were thought to be unique enzymes: their halogenating capacities were unusual and they were considered to be rare. Nowadays, haloperoxidases are known from almost 100 sources, including mammals, birds, plants, algae, molds, and bacteria, clearly showing their wide occurrence. Haloperoxidases are unique reagents, but their reaction mechanism 41 ADVANCES IN APPLIED MICROBIOLOC;Y, VOLUME 37 Copyrighl 0 1992 by Academic Press, Inc. All rights of reproduction in anv form reserved.

42

M. C. R. FRANSSEN AND H. C. VAN DER PLAS O2

E

n

z

e

F

>

+ H202

T+ >Em *+ Compound 1

H20

E + >zn

+ A-X + HzO

FIG. 1. The reaction mechanism of heme-containing haloperoxidases. The protein part of the enzyme is represented as Enz; the heme group is depicted as FeX+ (x = 3 or 4) in the center of an ellipse, for reasons of clarity. In Compound I, the iron ion has a 4 f oxidation state and the porphyrin ring is oxidized to a radical cationic species.

is not fully understood. The first haloperoxidases that were isolated contained a heme molecule (protoporphyrin IX) as the prosthetic group. Figure 1 shows the reaction sequence in simplified form. In the first step, hydrogen peroxide is bound to the ferric ion and by hydrogen b o d i n g to certain amino acid residues of the protein backbone; the iron ion and the heme group are oxidized with simultaneous cleavage of the hydrogen peroxide molecule. Then, a halide ion binds with subsequent reduction of the iron ion and the heme group, after which the organic substrate is halogenated. There are also two groups of nonheme haloperoxidases; recently, their mode of action was unraveled. The natural function of these enzymes is far from clear. They seem to be involved in the defense mechanism of their hosts; it is known that a lot of halogen-containing compounds are physiologically more active than their nonhalogenated counterparts. Halometabolites have the same broad occurrence as the haloperoxidases, but are especially abundant in sea organisms, in which they sometimes make up 20% of the dry weight. Some algae produce halometabolites which make them unedible to predators. However, some sea hares (Aplysia) prefer these algae as their food and become unedible themselves. The only halometabolites that have been found in mammalian tissues are iodotyrosine and derivatives thereof, which are formed by the action of thyroid peroxidase (an iodoperoxidase). The other mammalian

HALOPEROXIDASES

43

haloperoxidases act in a different way. It has been found that the white blood cell enzymes myeloperoxidase and eosinophil peroxidase produce the reactive species HOCl and lo,, both of which are poisonous to microorganisms. Lactoperoxidase, which is found in milk and saliva, produces HOBr and oxidizes thiocyanate (SCN- ) to the antimicrobial agent hypothiocyanate (OSCN- ). In short, haloperoxidases play a much more important role in nature than thought in the past. All aspects of biohalogenation have been extensively covered in the book by Neidleman and Geigert (1986). The purpose of the present review is to give an update of this book, with emphasis on the reaction mechanisms of the enzymes and the possible applications of haloperoxidases in organic synthesis. We strictly confine ourselves to the process of halogenation; other well-known activities like “classical” peroxidase and catalase are not discussed in this review. The isolation and structure elucidation of new halometabolites are included in several other reviews (Engvild, 1986; Faulkner, 1986, 1990) and are not discussed here either. II. Sources and Structures

Although in this review, as mentioned above, we concentrate on the organic reactions carried out with haloperoxidases, the basic research on enzyme structures, isolation procedures, and new sources of haloperoxidases also deserve attention. A distinction is made between those enzymes that have been described before (“enzymes from known sources”] and enzymes from new sources. A. NEW PROPERTIES

OF

ENZYMESFROM KNOWN SOURCES

In the past 5 years, much effort has been put into the perfection of the isolation techniques of useful haloperoxidases. This section reviews the literature on these new isolation techniques and the progress that has been made in the elucidation of the structure of chloro- and bromoperoxidases from known sources. I . Chloroperoxidases

An important improvement to the isolation procedure of the chloroperoxidase from the mold Caldariomyces fumago was developed by Conzalez-Vergara et al. (1985). They found that the sticky black pigment which is formed during the biosynthesis of this enzyme can be selectively precipitated by polyethylene glycol. The pH has to be monitored very carefully during the whole isolation process: incuba-

44

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

tion at pH 7.0 or higher for 2 hours at 4°C leads to complete inactivation of the enzyme. Another production method uses a roller bottle, in which the mold sticks to the wall (Blanke et al., 1989). In batch culture this roller bottle system is active for more than 200 days, producing about 500 mg of enzyme per liter of culture medium. When this system is used as a continuous unit, production values reached 700 mg of enzyrnelliter during about 70 days. An additional advantage is that pigment formation is minimal using this technique. The biosynthesis of this pigment is stimulated by the malt extract that is used in the inoculation. Since the reactor used has to be charged only once, the concentrations of this malt extract and the pigment remain low. The optimum culture medium for C. furnago contains fructose as the carbon source, since it has been found that fructose induces high chloroperoxidase mRNA levels (Axley et al., 1986). The enzyme can also be produced by C. furnago cells which are immobilized in calcium alginate. Using this immobilized system, in an airlift loop reactor 30-50 mg of enzyme per liter of medium could be obtained. In batch culture this figure rose to 50-100 mglliter. Normal growth conditions were found to be necessary for enzyme production (Carmichael et al., 1986). C. furnago can also be induced to pelletformation, in which case immobilization is no longer needed. With this system a production of 280 2 80 mg of enzyme/liter could be obtained in batch culture, and 1.2 2 0.1 mg/hour during 8 days in an airlift loop fermentor (Carmichael and Pickard, 1989). Several articles discuss the nature of the fifth axial ligand of the heme iron in the chloroperoxidase from C. fumago. EXAFS spectroscopy (Dawson et a]., 1986),resonance Raman studies (Bangcharoenpaurpong et a]., 1986), UV-spectroscopy, and determination of binding kinetics and spin states of chloroperoxidase containing different external ligands (Sono et al., 1986) were used to enhance understanding of this matter. All studies point to the presence of a sulfur ion derived from cysteine as the fifth ligand. However, previous studies had shown that the enzyme contains only two cysteine residues which are both involved in a disulfide bridge. This enigma was finally solved when the chloroperoxidase gene was cloned and its DNA sequence determined. It appears that chloroperoxidase possesses three cysteine residues, which enables sulfur ligation to the heme iron (Fang et al., 1986).It had already been known for a long time that, although chloroperoxidase strongly resembles cytochrome P-450 in almost all spectral properties and also in the environment of the heme, the catalytic reactions of the enzymes are totally different (Sono et al., 1986; Dawson and Sono, 1987). Because of its strong similarity with a comparable region in

HALOPEROXIDASES

45

cytochrome P-450it was thought that the fifth ligand of iron in chloroperoxidase would be Cys-87 (Fang et al., 1986; Kenigsberg et al., 1987). After 5,5’-dithiobis(2-nitrobenzoicacid) (DTNB)-blocking of the free sulfide and isolation of the blocked peptide fragment, the ligating residue turned out to be Cys-29 (Blanke and Hager, 1988). The conclusion seems justified that, although the direct environment of the heme may resemble cytochrome P-450,the polypeptide chains and the active sites are totally different (Dawson and Sono, 1987; Blanke and Hager, 1988). The active pocket of chloroperoxidase rather looks like horseradish peroxidase, with a large number of polar amino acid residues (Dawson and Sono, 1987). The sixth ligand of the heme iron in the chloroperoxidase from C. fumago is still not known with certainty. In an attempt to shed more light on this matter, Blanke and Hager (1990) studied the modification of the enzyme by the histidine-specific reagent diethyl pyrocarbonate. They found that the enzyme lost activity in a time-dependent manner during this process, which lead them to the conclusion that a vital histidine residue was affected, presumably His-38. The authors suggest that this residue is the sixth axial ligand in chloroperoxidase. The chloroperoxidase from C. fumago is a mixture of three isoenzymes which differ in their sugar contents. The glycosylated positions are Asn-12 and Asn-216 (Kenigsberg et aI., 1987). Attempts to crystallize the protein have so far been unsuccessful because of heterogeneity in the sugar parts of the enzyme (Fang et a]., 1986). Labat and Meunier (1990) have investigated various iron and manganese porphyrin complexes as mimics for the chloroperoxidase from C. fumago. When dimedone (5,5-dimethyl-1,3-cyclohexanedione) was used as a substrate, the products were the corresponding 2-chloro and 2-hydroxy derivatives. meso-Tetrakis(3,5-disulfonatomesityl)porphyrinato manganese (11) complexed with a polyvinylpyrrolidine polymer gave the highest chloro/hydroxy ratio at pH 3.0, whereas iron complexes gave only the hydroxy compound. Reactions rates were not reported. A remarkable article appeared on the heme-containing haloperoxidase from the green alga PeniciIIus capitatus (Manthey and Hager, 1989). This enzyme, originally described as a bromoperoxidase at pH 6.0, upon closer consideration appeared to be a chloroperoxidase at pH 4.0.It was proved that the enzyme converts the standard substrate monochlorodimedone (see below) into dichlorodimedone. Its chlorinating activity, however, is low compared to the rate of bromination at pH 6.0. Myeloperoxidase is a chloroperoxidase of mammalian origin. Both canine (Fenna, 1987) and human myeloperoxidase (Sutton et a]., 1988)

46

M. C. R. FRANSSEN AND H. C. VAN DEK PLAS

have been crystallized and appear to be similar in structure. Both are composed of heavy and light chains in a 2 : 2 ratio and the molecular weight of the chains are, respectively, 57,000 and 10,500. The sugar chains are attached to the heavy (a)chain. There are two heme groups per enzyme molecule which are not identical in the case of canine myeloperoxidase. Both halves of the enzyme (aP-aP) are linked through a disulfide bridge. Detailed X-ray studies of these enzymes are on their way. The nature of the catalytic center of myeloperoxidase has been studied by means of NOE-spectroscopy (Dugad et aJ., 1990). The prosthetic group of the enzyme is, most probably, a hemin group in which the methyl group at C-8 is replaced by a methylthiol group. This unusual porphyrin derivative was first found in lactoperoxidase (see below). Eosinophil peroxidase is a chloroperoxidase which is, just like the previous one, found in certain white blood cells. The human gene of this enzyme has been located and cloned (Sakamaki et al., 1989). 2 . Bromoperoxidases

About 50 bromoperoxidases were described by Neidleman and Geigert (1986) in their book. Only a few of these enzvmes have been the subject of further studies. Most articles are devoted to the interesting new class of nonheme bromoperoxidases, with the enzyme from the brown alga AscophyJJum nodosum as the main representative. Its isolation procedure was improved considerably by application of calcium chloride to precipitate the viscous alginate which makes u p a large part of the alga (De Boer et al., 1986b).It was shown that the occurrence of bromoperoxidase in A. nodosum is confined to the receptacles, and that they contain two different bromoperoxidases. One enzyme (BPO I) was found within the thallus, the other one (BPO 11) on the thallus surface. Their amino acid compositions, their immunological behavior, and their vanadium content are comparable but their molecular weights differ slightly. It appears that their sugar contents are nonidentical. BPO I, the one with the lowest molecular weight, is a little more stable toward organic solvents and heat (Krenn et a]., 1989b). The sedimentation coefficient of BPO I was shown to be 6.96s, which indicates a very compact molecule when compared with its molecular weight of 97,000. The enzyme contains about 16 cysteine residues, all of which are involved in disulfide bridges. Circular dichroism studies of this enzyme showed that about 74% of its amino acids are involved in a-helices, an exceptionally high percentage (Tromp et al., 1990). It has been shown unequivocally that vanadium is an essential ele-

HALOPEROXIDASES

47

merit for the brominating activity of this bromoperoxidase: when the

enzyme was dialyzed against EDTA all activity was lost, but could be fully restored by the addition of vanadium (V) ions. No other metal ion was able to restore activity. About one vanadium ion was present per enzyme molecule (De Boer et al., 1986b). The dissociation constant of vanadate is 55 nM at pH 8.5 (Tromp et a]., 3990). Several studies have been performed to determine the chemical environment of the vanadium ion in the active site of the enzyme. Using EPR (De Boer et al., 1988b),EXAFS (Arber et al., 1989),XANES (Hormes et al., 1988), electron spin echo (De Boer et a]., 1988a), and 51V-NMR (Rehder et al., 1987), it was established that the vanadium ion is in the center of a distorted octahedron with oxygen atoms and a few nitrogen atoms as ligands. One of the oxygens can be represented as a V=O group; the others may be derived from water, tyrosine, or carboxylate. The imidazole group of histidine is possibly responsible for the nitrogen-containing ligand(s). Two research groups have synthesized simple inorganic models for the enzyme (Hormes et al., 1988; Sakurai and Tsuchiya, 1990). In both studies, the strongest spectral similarity with bromoperoxidase was obtained when oxalate was used as a ligand. as a Hormes et al. (1988) proposed (NH,),[VO(0,)(0,C--C0,)2-].H,0, model for the bromoperoxidase-H,O, complex. The bromoperoxidase from A. nodosum has the interesting property that it is very stable in aqueous and organic media. For example, the brominating activity was not affected when the enzyme was kept under turnover conditions for a period of 3 weeks. When stored at room temperature in organic solvents such as acetone, methanol, ethanol [present up to 60% (v/v)],and 1-propanol [40% (v/v)],the enzyme was stable for more than 1 month (De Boer et al., 1987b). The enzyme is not affected by mixtures of 1-butanol, ethanol, and water (De Boer et ul., 1987a), relatively high concentrations of HOBr (De Boer et al., 1987a; Everett el al., 1990a), 4 M guanidine, or 4% sodium dodecyl sulfate (SDS) (Tromp et d.,1990). The very high stability of this bromoperoxidase was explained by its compact structure, its high number of disulfide bridges, its high a-helix content, and the absence of a heme group which is known to be vulnerable (Tromp et a]., 1990). Recently, just like in the case of the heme enzyme from Penicillus capitatus, some evidence was obtained that the bromoperoxidase from A. nodosum has chlorinating activity (Soedjak and Butler, 1990b). When the enzyme was incubated with chloride it was able to convert monochlorodimedone into dichlorodimedone (see Section III), although the rate of chlorination is 5 0 0 X lower than the rate of brornination of this substrate. The K, value for chloride is 344 mM at pH 5.0,

48

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

which indicates that the enzymatic affinity for chloride is very low. This explains why the chlorinating ability of this bromoperoxidase had not been observed before. The bromoperoxidase from the bacterium Streptornyces phaeochromogenes has been purified and its molecular structure has been studied (Van Pee and Lingens, 1985). The enzyme is a dimer of two identical subunits, each of which has a molecular weight of 72,500. The enzyme has one ferriprotoporphyrin IX as a prosthetic group per enzyme molecule. More recently, a “known” enzyme has shown bromoperoxidase activity: the ligninase of the white-rot fungus Phanerochaete chrysosporiurn (Renganathan et al., 1987). This discovery was not unexpected, since it was shown earlier that this enzyme is a peroxidase and not an oxidase (Harvey et al., 1985; Schoemaker et al., 1985) and that its heme active site is comparable to those of haloperoxidases (Kuila et al., 1985). For an excellent review on the isolation and structure of the human enzyme lactoperoxidase (milk, saliva, tears), the reader is referred to Paul and Ohlsson (1985). The prosthetic group of this enzyme has been isolated by Nichol et al. (1987). Its structure appears to be unusual: it is a hemin group in which the methyl group at C-8 is replaced by a methylthiol group. Presumably, this group is covalently linked to the apoprotein via a disulfide bridge. The cDNAs of bovine and human lactoperoxidase have been cloned (Dull et al., 1990). 3. lodoperoxidases

New information about iodoperoxidases from known sources has been very limited during the past 5 years. In the case of horseradish peroxidase, more insight into its structure has been obtained by various spectroscopic and genetic studies, but the implications for its halogenating behavior were not discussed (see, e.g., Thanabal et al., 1987). A few porphyrin derivatives were found to be excellent mimics of one-electron oxidation reactions catalyzed by horseradish peroxidase; again, the implications for halogenation were not discussed (Ci and Wang, 1990). The gene and protein sequences of human and porcine thyroid peroxidase have been determined (Kimura et a]., 1987; Magnusson et al., 1987). In conclusion, concerning iodoperoxidases, to our knowledge nothing has been published in the past few years which has significantly changed views on the halogenating action of these enzymes. New sources of iodoperoxidases have not been reported recently.

49

HALOPEROXIDASES

B. ENZYMESFROM NEWSOURCES In the past 5 years a host of new haloperoxidases from various sources have been detected, purified, and characterized. We confine ourselves here to a brief description of newly detected chloroperoxTABLE I

NEWSOURCES OF HALOPEROXIDASES Halogenating activity, type of organism

Species

Ref.

~~

Chloroperoxidase bacterium Bromoperoxidase marine snail Rhodophyta [red algae)

Chlorophyta (green algae)

Phaeophyta (brown algae)

Lichen Bacteria

Pseudomonas pyrrocinia

Wiesner et al. (1986)

Murex trunculus Alotocladia modesta Amphirao ephedraea Amphirao misakiensis Amphirao zonata Bossiella cretocea Calliarthon yessoense Corallina officinalis Corallina pilulifera Gelidium amansii Hypnea chararoides Neodilsea yendoana Bostrycia tenella Gelidiello acerosa Mastophora rosea Galaxaura fastigata Iania decussata-dichotoma Ceramium rubrurn Corallina vancouveriensis Codium fragile Chlorodesmis comosa Codium repens Halimedo incrassata Laminaria saccharina Fucus spiralis Fucus serratus Fucus vesiculosus Pelvetia canaliculata Chorda filum Xanthoria parietina Streptomyces aureofaciens Streptomyces griseus Streptomyces venezuelae

Jannun and Coe (1987) Yamada et al. (1985b)

Itoh et al. (1987d)

Krenn et al. (1987) Everett et al. (1990b) Yamada et al. (1985a) Itoh et al. (1987d)

De Boer et ol. (1986a)

Plat et al. (1987) Van Pee et al. (1987) Zeiner et al. (1988) Knoch et al. (1989)

50

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

idases and bromoperoxidases; for details on the purification procedures the reader is referred to the articles cited. A discussion on the prosthetic group of new nonheme, nonvanadium enzymes is presented in Section IV,C. K, values for hydrogen peroxide and halide ions, though important and intrinsic properties of haloperoxidases, are not discussed here. Their magnitudes are strongly pH-dependent, hampering a direct comparison of these values. A list of new sources of haloperoxidases is given in Table 1. 1. Chloroperoxidases

The only company that has tried to commercialize haloperoxidases thus far is Cetus (Emeryville, CA). The results of an extensive screening program for nonheme haloperoxidases which are resistant to H,O, and HOCl was published in a Canadian patent (Hunter et al., 1986). One hundred and twelve different dematiaceous hyphomycetes were screened and 32 of them proved to have high chloroperoxidase activity. A list of these molds was given in the book of Neidleman and Geigert (1986). The enzyme from Curvularia inaequalis was partially purified and characterized (see also Liu et al., 1987). The properties of this and other haloperoxidases from new sources are listed in Table 11. As can be seen from Table 11, the chloroperoxidase from Curvularia inaequalis is indeed very stable. It can be incubated with 200 mM H,O, for 25 hours without any loss of activity. For comparison, the heme-containing chloroperoxidase from C. fumago is completely inactivated in 2 minutes under these conditions (Liu et al., 1987). Also, the resistance against HOCl is remarkable: after incubation for 2 minutes in the presence of 0.5 mM HOCl the chloroperoxidase from Curvularia inaequalis still has 75% of its residual activity, whereas in the case of the enzyme from C. fumago there is only 2% left. The prosthetic group of the enzyme is unknown. This very interesting enzyme should certainly be studied in more detail. The bacterium Pseudomonas pyrrocinia produces two different haloperoxidases: a heme-containing bromoperoxidase and a nonheme, nonvanadium chloroperoxidase (Wiesner et al., 1986). The chloroperoxidase was purified and characterized (Wiesner et a)., 1988). Its prosthetic group is still unknown. The most remarkable feature of this enzyme is its ability to convert indole into 7-chloroindole and its inability to chlorinate monochlorodirnedone, the standard substrate for all chloroperoxidases (see Sections II1,A and II1,E for structures and comments). The P. pyrrocinia chloroperoxidase is thermostable: it has its optimum activity at 60°C and is stable at this temperature for 2 hours

TABLE I1 PROPERTIES OF NEWHALOPEROXIDASES THATHAVEBEEN PURIFIED Source Curvulario inoequalis Pseudomonas pyrrocinia Murex trunculus Amphirao ephedroea Ceromium rubrum Corallina ojficinolis Corallina pilulifero Laminaria saccharina Xanthoria parietina Pseudomonos pyrrocinia Streplomyces aureofaciens

Type of organism

Halogenating activity0

Molecular weight

Subunit composition

Hyphomycete (mold) Bacterium

CPO

240,000

5.0

60

Liu et al. (1987)

64,000

Unknown, nonheme

4.0-4.5

4.2

Wiesner et 01. (1988)

Carnivorous marine snail Rhodophyta Ired algae) Rhodophyta (red algae) Rhodophyta (red algae) Rhodophyta (red algae) Phaeophyta (brown algae) Lichen Bacterium

BPO

Not determined

Tetramer, identical subunits Dimer, identical subunits Not determined

Unknown, nonheme

CPO

Not determined

4.0-7.4

35 (maximally)

Jannun and Coe (1987)

npo

Not determined

Not determined

Not determined

5.5 (4.0-7.5)

0.83 (crude extract)

Yamada et 01. (1985b)

BPO

58,000

Vanadium

7.4 (6.0-8.3)

6.5

Krenn et al. (1987)

BPO

64,000

Monomer (or tetramer?) Monomer

Vanadium

6.0

130C

BPO

790.000

Vanadiumd

6.0 (5-11)

26.3

Yu and Whittaker (1989) Itoh et al. (1986)

npo

108,000

Vanadium

6.0-6.5

134

De Boer et ol. (1986a)

BPO BPO

65,000 155,000

Vanadium Heme

5.5 4.5-5.5

79

Plat et al. (1987) Wiesner et 01. (1985)

Bacterium

BPO

92,500

Monomer Dimer, identical subunits Trimer

Unknown, nonheme

4.5

2.1

Dimer, identical subunits Dimer, identical subunits Dimer, identical subunits Trimer, identical subunits Dimer. identical subunits

Unknown, no heme or vanadium Unknown, nonheme

4.3

65

Van Pee et al. (1987; Van P6e (199Oa) Krenn et al. (1988)

4.5

1.6

Zeiner et ol. (1988)

Unknown, nonheme

Not determined

2.8

Unknown, nonheme

4.0-4.5

1.1

Heme

4.75

1.8

5

20,000

Streptomyces venezuelae

Bacterium

Bacterium

Optimum pH

Specific activityb

Ref.

66,000

65,000 Slreplomyces griseus

Dodecamer, identical subunits up, 64,000 and

Prosthetic group

BPO (BPO l a )

70,000 i- 10.000

BPO (BPO I b )

90,000 t 10,000

BPO (BPO 3)

90,000

BPO

127,000

?

10,000

C P O , Chloroperoxidase; BPO, bromoperoxidase. bDefined as micromoles of monochlorodimedone that is brominated per milligram of enzyme per minute. CJ. W. Whittaker (personal communication). dKrenn et al. (1989a).

3.2

Knoch et 01. (1989)

52

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

(Wiesner et al., 19881. The enzyme is cloned and expressed in Escherichia coli (Wolfframm et al., 1988). 2. Bromoperoxidases

Bromoperoxidases appear to be very abundant in marine organisms. The carnivorous snail Murex trunculus produces 6,6'-dibromoindigo and should therefore possess a haloperoxidase. The enzyme was indeed detected and partially purified by Jannun and Coe (1987). Its prosthetic group was not determined. Yamada and co-workers (1985b) surveyed 42 coralline algae which occur in the Japanese sea. Twelve of them contained bromoperoxidase activity. The enzyme from Corallina pilulifera (Rhodophyta, red alga) was purified (Itoh et al., 1985). The authors found that it does not contain any heme or flavin. They believed that iron ions were involved in the reaction mechanism of the enzyme (Itoh et al., 1986, 1987a),but later experiments showed that vanadium (V) is the prosthetic group of this enzyme (Krenn et al., 1989a). Presumably, the first authors did not find any vanadium in their preparation because it was lost during the isolation procedure. In general, there is a lot of interest in these vanadium-containing haloperoxidases, which are a new class of enzymes. Vanadium bromoperoxidases were found in Ceramium rubrum (Rhodophyta; Krenn et al., 1987), Corallina officinalis (Rhodophyta; Yu and Whittaker, 19891, Corallina vancouveriensis (Rhodophyta; Everett et al., 1990a], Laminaria saccharina, Fucus spiralis, Fucus serratus, Fucus vesiculosus, Pelvetia canaliculata, Chorda filum (Phaeophyta, brown algae; De Boer et al., 1986a), and the lichen Xanthoria parietina (Plat et al., 1987). For a comparison of structural and immunological properties of these enzymes, the reader is referred to the articles of Wever et al. (1988),Itoh et al. (1987d), and Krenn et al., (1989a).Vanadium-containing bromoperoxidases are much more stable toward hydrogen peroxide, hypohalous acids, organic solvents, and high temperatures than their heme congeners. The vanadium ion is relatively loosely bound and has about the same environment as the vanadium ion in the bromoperoxidase from Ascophyllum nodosum (see Section II,A,Z). Lichens are a completely new source for haloperoxidases, although this discovery is not unexpected: lichens are symbioses of algae and molds, and in both types of organisms haloperoxidases have been detected. The enzyme from X . parietina is extremely thermostable, which is presumably due to the natural habitat of the lichen. It grows on stones in coastal areas, which may get very warm in summer. The K, value for bromide for this enzyme is also remarkable, since it is only 28 pM at pH

HALOPEROXIDASES

53

5.5. In conclusion, vanadium seems to be an essential element for a large group of haloperoxidases, although these enzymes are not unique in this respect since a nitrogenase has been found which also has vanadium as a prosthetic group (for a short review see Wever et al., 1987). Two articles are devoted to the isolation and characterization of a nonheme, nonvanadium bromoperoxidase in the bacterium Streptomyces aureofaciens (Van Pee et al., 1987; Krenn et al., 1988). The enzyme is as thermostable as the vanadium enzymes (Krenn et a]., 1988; Van Pee et aI., 1987). Its gene has been cloned and expressed in Streptomyces lividans in a very efficient way, leading to production values of 180 mg of enzymelliter (Van Pee, 1988). It should be mentioned that S. aureofaciens also produces a heme-containing bromoperoxidase, but this was not studied further (Van Pee et al., 1987). The bacterium Streptomyces griseus has four different bromoperoxidases, one heme (BPO 2) and three nonheme enzymes (BPO l a , Ib, and 3). BPO 2 remains present as an impurity during the isolation of the nonheme enzymes, but can be easily removed by a brief heat treatment. This shows once more that nonheme haloperoxidases are much more thermostable than their heme congeners! The three nonheme enzymes differ in their kinetic properties and in their thermostability (Zeiner et a]., 1983).

Two additional heme-containing bromoperoxidases were purified from the bacteria Pseudomonas pyrrocinia (Wiesner et a]., 1985) and Streptomyces venezuelae (Knoch et al., 1989). Ill. Reactions

A large variety of synthetic reactions using haloperoxidases have been described during the past 5 years. A survey is given below; the reactions are classified according to the type of substrate used. Yields are given whenever mentioned in the references. Underlined figures refer to yields of isolated products. Immobilization procedures and reactions using immobilized haloperoxidases are grouped as a separate subsection (Section IT1,G). A. ASSAYMETHODS Most articles which are devoted to the isolation and characterization of haloperoxidases use monochlorodimedone (Z-chloro-5,5-dimethylcyclohexane-1,3-dione 1;see Scheme 1)as standard substrate to determine enzyme activity. This compound is converted into its 2,2-dichloro (Za) or 2-bromo-2-chloro derivative (2b) upon chlorination or bromina-

Go- ooo

0 H

CI

1

x

CI

2a (X = C1) b (X = Br)

4a (X=Cl)

b (X=Br)

OH

OH

3

~&Q -OH

X

\ I

OH

5a (X=CI) b (X=Br) SCHEME 1. These reactions are catalyzed by almost all haloperoxidases

55

HALOPEROXIDASES

tion, respectively. In general, it is a useful reagent because all haloperoxidases accept it as a substrate (see, however, Section II1,E) and the reaction can be easily monitored by UV-spectroscopy. However, there is some evidence that the bromination product of monochlorodimedone is not stable and gives ring-opened nonhalogenated decomposition products (Itoh et a]., 1988). Whether this affects the measurement of enzymatic activity is unclear. Serious problems can be expected in the case of enzymes that have a strong peroxidase activity. All heme-containing haloperoxidases show this activity, converting organic compounds into dimers or dehydrogenated products via oneelectron oxidations. The reaction equations look like the following: ENZ

+ HZO2+ Compound

Compound I

+ BH,

--j

Compound I1

Compound I1 + BH2 + ENZ 2 BH. 2

+

I

+ BH.

(BH),

BH*+ BH,

+B

(2)

+ BH-

(3) (4)

(5) (6)

where ENZ is enzyme, BH, is substrate, BH* is product radical, (BH), is dimeric product, B is dehydrogenated product, Compound I is doubly oxidized enzyme, and Compound I1 is oxidized enzyme. In the first step the enzyme is oxidized by hydrogen peroxide in the normal way, producing a species that is two oxidation states above the level of native enzyme [Compound I, Eq. (21; see Section IV,A for an extended discussion on reaction mechanisms]. This species can then react with one-electron acceptors (such as guaiacol and other aromatics], resulting in the formation of an enzyme intermediate which is one oxidation state above native level [Compound 11, Eq. (3)]. This can in its turn be reduced to the native enzyme in a very slow step [Eq. (4)]. The radicals produced may combine to a dimer [Eq. ( 5 ) ] or exchange hydrogen radicals [Eq. (611. This reaction sequence, well known for horseradish peroxidase (an iodoperoxidase), may be a complicating sidereaction in the halogenation of 1 since it has been found that 1 is a one-electron acceptor too (Kettle and Winterbourn, 1988). In case of enzymes with high classical peroxidase activity, like myeloperoxidase, burst kinetics are observed, due to the accumulation of Compound 11. The problem can be circumvented by the addition of ascorbic or uric acid to the reaction mixture; these compounds reduce Compound I1 quickly to native enzyme. Nonheme enzymes in general do not give any problems because they do not have this classical peroxidase activity.

56

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

A very convenient assay method for haloperoxidases uses the dye phenol red (phenolsulfonphthalein, 3; see Scheme 1). On bromination, this compound is converted into its 3',3",5',5"-tetrabromo derivative 5b (De Boer et al., 1987b). Compound 4 can also be used in chlorination assays, as was shown recently for the bromoperoxidase from A. nodosurn; the 3',3"-dichloro compound 4a is an intermediate in this reaction (Soedjak and Butler, 1990b). The formation of 5 can be monitored colorimetrically, is quantitative, and is very sensitive and extremely well suited for the detection of haloperoxidase activity in tissues, cells, or crude extracts (Hunter et al., 1986). The method can also be applied in water-organic solvent mixtures (De Boer et al., 1987b; Wiesner et al., 1990). B. REACTIONSWITH ALKENES

Neidleman and co-workers have shown that haloperoxidases are able to convert alkenes into halohydrins or dihalides. These studies have been extended to the chloroperoxidase from Curvularia inaequalis (Hunter et al., 1986; Liu et al., 1987). This enzyme reacts in a manner comparable to the chloroperoxidase from Caldariornyces fumago, converting allylchloride (6; see Scheme 2) into a mixture of 1,3-dichloropropanol-2 ( 7 ) and 2,3-dichloropropanol-1 [ 8 ) in low yield. Similarly, ally1 alcohol (10)gives a mixture of the halohydrins 8,11,and 12 (Hunter et al., 1986). In this case, seawater was used as the source of chloride ions. When the medium is spiked with bromide ions, a mixture of brominated and chlorinated propanols is obtained. Compounds 7 and 8 were applied for the synthesis of epichlorohydrin 19); 11 and 12 afforded glycidol (13).The reactions can be carried out either with purified enzyme or with immobilized whole cells. The latter system has the advantage that higher pH values can be used during the reactions, causing the spontaneous dehydration of the halohydrins into epoxides. The products 8 and 11 have chiral carbon atoms, but since the authors state that the enzyme produces HOCl as the active chlorinating species these compounds will be formed as a 1: 1 mixture of both stereoisomers. Wiesner et al. (1990) reported that the chloroperoxidase from Pseudornonas pyrrocinia in the presence of bromide ions converts 10 into a mixture of 14 and 15. Yamada and co-workers (1985a) have investigated the halogenation of trans-cinnamic acid and some of its derivatives by the chloroperoxidase from C. furnago (see Scheme 3). On bromination, trans-Cinnamic acid (16)gave the expected bromohydrin 17 in moderate yield. The byproduct trans-1-bromo-2-phenylethene (18)was assumed to be formed

57

HALOPEROXIDASES

7

6

9

8

16% (ratio 5456)

mOH dOH OH m CI CI L + OH m OH CI +OHCI

9

10

b

I

13

1

OH Br Br

14

+

I

OH OH Br

15

SCHEME2. a, Chloroperoxidase from Curvularia inaequalis, H 2 0 2 , C1-; b, chloroperoxidase from Pseudomonas pyrrocinia, H2O2,Br ~.

through the nonenzymatic decarboxylation and simultaneous dehydration of 17. This minor product became the major product when an electron-donating group was attached to the benzene ring. Thus, trans-3-(4-hydroxyphenyl)propenoic acid (19)gave the bromo or chloro alkenes 20a and 20b, respectively, in low yield. These alkenes can be substrates for the enzyme itself, as was shown in the halogenation of trans-3-(4-methoxyphenyl)propenoicacid (211.The major product, 22, was presumably formed in a three-step process: (1)enzymatic bromination of 21 giving the bromohydrin; (2) nonenzymatic decarboxylation of the bromohydrin giving a monobrominated alkene; (3) enzymatic bromination of this alkene, yielding the dibromo compound 22. The formation of the second product 23 can be visualized through the enzymatic conversion of 21 into its bromohydrin, and nonenzymatic substitution of the bromide ion by water (presumably in an S,2 manner). trans-3-(3,4-Dimethoxyphenyl)propenoic acid (24)reacts in a compara-

Q

a,c,d

H

H'

+

O ! - Y - C O O H

c=c

c=c

OH Br 'COOH

17 (rat.-erythro)

16

63%

Q H-

HO

\Br

H'

18

Ho\

a or b

H ,

c=c\

I

H

c=c\ COOH

H

X

20a X = Br 0%) 20b X = C1m)

19 CH30,

4

c=c

I

H

2CH30ea-i-Br

0; -;

CH30

-COOH

OH Br

I

\

+

22 (racemic)

COOH

OH OH

a

23 (racemic) 24%

21 CH,O

H

c=c\

I

H

a

CH30

CH30

CH30&--!--".r

I

+ C H 3 0 a ! - ! - C OI O IH

/

OH Br

25 (racemic)

OH Br

12%

26 (racemic)

CH30

24

+

CH30 fB;G;$$

a

27 (racemic) CH30

-

qC1

CH30

b

24

8%

COOH

CH30&!-!-CI

I

+

I

OH CI

CH30

H ,

c=c

racemic,

H/ 28

CH30

+

@cH;H

H'

3%

'Cl

14%

c=c 'Cl

SCHEME 3. a, Chloroperoxidase from Caldariomyces fumago, H,O,, Br - ; b, chloroperoxidase from C. fumago, Hz02, C1- ; C, lignin peroxidase, H,O,, Br- (no yields were given for the reactions with this enzyme): d , bromoperoxidase form Corallina pilulifera, H,O,, Br- (no yields were given for the reactions with this enzyme).

59

HALOPEROXIDASES

30

29

+ several other minor products

CH,O H

c=c\ I

n

31

C2H50

OH Br

cn, 32

SCHEME 3. (continued)

ble way, although there are some minor differences between the bromination and the chlorination reactions. Because of the increased electron density of the benzene ring, electrophilic aromatic substitution becomes another reaction path for these substrates (see structures 27 and 28). All compounds are formed as racemic mixtures. It should be noted that spontaneous decarboxylation after enzymatic halogenation had already been observed in the early days of haloperoxidase research during the chloroperoxidase-mediated chlorination of 3-ketoadipic acid (Shaw and Hager, 1959). Cinnamic acid and its derivatives were also used as substrates in research on the halogenating capabilities of the lignin peroxidase from the white-rot fungus Phanaerochaete chrysosporium (Renganathan et a]., 1987). The enzyme appeared to be a bromoperoxidase. Compound 16 was converted into its bromohydrin 17, whereas 24 gave several products on incubation with this enzyme. The major products were 25, its precursor 29, and an unidentified compound. Several minor products were also formed, including 26 and the aldehyde 30. Interestingly, ring-brominated compounds like 27 which are produced by the chloroperoxidase from C. fumago were not obtained in these studies. Finally, l-(4-ethoxy-3-methoxyphenyl)propene(31)reacted with lignin peroxidase giving the bromohydrin 32 as the only product. No yields were given. In addition, it was reported that the bromoperoxidase from the brown alga A.nodosum converts 19 into a monobromo compound (De

60

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

H ,

c=c\

I

H

a

OH Br

CH,OH

rac., erythro

33

a

o T - C H 2 B r

C=CH2 H

39%

OH

rac.

69%

34

36

SCHEME 4.

270/0

a, Bromoperoxidase from Corallina pilulifera, H202, Brr .

Boer and Wever, 1988). Its structure has not been proved, although it seems reasonable to assume that this product is the bromo alkene 20a. Some alkenes, including 16,were again used in studies on the substrate specificity of the bromoperoxidase from the red algae Corallina pilulifera (Itoh et al., 1988). On incubation with hydrogen peroxide, bromide ions, and this enzyme, 222 mg of 16 gave 40 mg of a mixture of 17 and 18. trans-Cinnamyl alcohol (33),styrene (34),cyclohexene (35), and cis-propene-1-phosphonic acid (36)gave their corresponding bromohydrins (see Scheme 4). Yields were low, with exception of the bromination product of styrene, where the yield obtained was 69%. Fukuzawa and co-workers devoted several articles to the biosynthesis of the marine bromoether laurencin (37a;see Scheme 5) and its derivatives. Laurencin is a major constituent of the red alga Laurencia nipponica and some sea hares (Aplysia). The biosynthesis of 37a is thought to occur via a haloperoxidase-catalyzed cyclization. In model studies, Fukuzawa et al. (1990a) found that, on incubation with lactoperoxidase, the alkenediols 38 and 40 cyclized to give bromotetrahydrofurans 39 and 41 and 42,respectively. Furthermore, the alkenediol 43 yielded octahydrodeacetyl laurencin (44).In a second article, the

61

HALOPEROXIDASES

L

Br

a

OH

-

M

4

5H

OH

38

39 (8.8)

+ bromohydnns(12.3%) + 38 (726)

40

41

W)

Br

+ 42 OH

6H (m

a

5H t

Br

bromohydnns (18.6%)

+ 40 (64.7%)

0.yOH

= /

43

44 (I&%)

+ bromohydrins (17.5%) + (544’0)

OH

37a

45

(0.73)

+ unknown cyclic ether (0.91%) + bromohydrins (28%) + (B)

SCHEME 5. a, Lactoperoxidase, HZOZ.Br- ; b, thyroid follicles (containing thyroid peroxidase).

62

M. C. R. FRANSSEN AND H. C. VAN DER PLAS a 37b

t

il 46

Br

/" 49

48

-%

40%.(a) no yield given (b) 51

0

-

SCHEME 5 . (continued)

final proof for the biosynthetic route leading to 37 was given: highly unsaturated (3E,GR,7R)-Iaurediol(45) gave cyclic ether 37b on incubation with lactoperoxidase. Chemical acetylation gave laurencin (Fukuzawa et al., 1 9 9 0 ~ )The . cyclic bromoether laureoxanyne (46) is a minor constituent of L. nipponica. Fukuzawa et a]., (1990a) found that

HALOPEROXIDASES

63

deacetyllaurencin (37b)gives 46 on incubation with lactoperoxidase, proving that the biosynthesis of 46 can be haloperoxidase-mediated too. Other products obtained were 47 (a regioisomer of 46),bromohydrins, and starting material. The authors provided a mechanism which explains the occurrence of the metabolites laureoxanyne (46),notoryne (48), and laurefucin (49)from the common precursor 37b (see Scheme 5). Although the yields in all the above-mentioned reactions are low and a nonalgal enzyme was used, the results clearly show that a haloperoxidase is involved in the biosynthesis of these halogenated compounds. Bromoperoxidase activity was detected in L. nipponica, but the purification of the enzyme(s) proved to be difficult (Fukuzawa et ai., 1990a). In the thyroid gland iodide ions are covalently linked to organic compounds through the action of thyroid peroxidase, an iodoperoxidase. The hormone thyroxine is one of the compounds formed in this way, but there are also iodinated substances involved in the control of proliferation of thyroid cells. One of them is, most probably the Siodolactone from arachidonic acid (51;see Scheme 5). Dugrillon et al. (1990) have shown in model studies that arachidonic acid (50) indeed yields 51 with a 40% yield on incubation with lactoperoxidase, hydrogen peroxide, and iodide ions. Thyroid follicles themselves were also able to catalyze this reaction, although in this case no yield was given.

c. REACTIONS WITH AROMATIC COMPOUNDS Haloperoxidases produce electrophilic halide species which normally do not react with benzene rings unless they are activated by one or more electron-donating groups. However, according to Wiesner et al. (1990),the chloroperoxidase from Pseudomonas pyrrocinia reacts with benzene. The product(s) was not characterized. Compounds like phenol, anisole, and their derivatives are known to be good substrates for these enzymes. In the course of a study on biological production of pollutants such as halophenols, Wannstedt et 01. (1990) investigated the ability of the chloroperoxidase from the mold C. fumago to chlorinate phenol, cresol, and several chlorinated phenols. The results are depicted in Table 111. As can be seen in Table 111, chloroperoxidase gives the expected products: chlorine atoms are always introduced ortho or para with respect to the hydroxyl group, with a preference toward the para position because of steric hindrance of the ortho position. Although the yields obtained are low, the results show that chlorinated phenols which are found in the environment are not only man-made pollutants but may also originate from biological halogenation of phenols. As

64

M. C. R. FRANSSEN AND H. C. VAN DER PLAS TABLE 111

REACTIONPRODUCTS OBTAINED FROM CHLOROPEROXIDASE-INDUCED CHLORINATION OF PHENOLIC SUBSTRATES ~

Substrate

~~

Phenolic product

Phenol

2-Chloro 4-Chloro 2-Chlorophenol 2,4-Dichloro 2,6-Dichloro 2,4,6-Trichloro 4-Chlorophenol 2,4-Dichloro 2,4-Dichlorophenol 2,4,6-Trichloro 2,6-Dichlorophenol 2,4,6-Trichloro 2,3,5-TrichlorophenoI 2,3,4,5-Tetrachloro 2,3,5,6-Tetrachloro 2,3,4,5-Tetrachlorophenol Pentachloro 2,3,5,6-Tetrachlorophenol Pentachloro 2-Methylphenol (0-cresol) 2-Methyl-4-chloro 2-Methyl-6-chloro 3-Methylphenol (rn-cresol) 3-Methyl-4-chloro 5-Methyl-2-chloro 3-Methyl-2-chloro 4-Methylphenol ( p-cresol) 4-Methyl-2-chloro

Product formed. Ob

25

Trace Trace Trace 5 9-12

12-15 15 5 5 2

38 0

33 15 0 0

oAs percentage of the converted substrate bToo low to be quantified.

Table I11 shows, the chlorination of tetrachlorophenols is very slow. This implicates that pentachlorophenol will hardly occur in unpolluted nature. Thus, its presence among other halogenated aromatics in a soil or water sample indicates with great certainty that the halogenated material originates from man-made pollution and not from biological sources. The group of Yamada in Kyoto, Japan, has performed a large screening program for haloperoxidase activity in coralline algae. The most active were Corallina pilulifera, Corallina officinalis, and Amphirao zonata. All of the bromoperoxidases from these sources were able to convert phenol (52; see Scheme 6) and o-hydroxymethylphenol (54) into 2,4,6-tribromophenol (53). No yields were given, nor an explanation for the remarkable cleavage of the hydroxymethyl side chain during the bromination of 54. Other bromoperoxidases that catalyzed these reactions originated from the algae Amphirao ephedraea, Amphirao misakiensis, Bossiella cretacea, Cailiarthon yessoense, and Alatocladia modesta (Yamada et al., 1985b). The chloroperoxidase from P. pyrrocinia and the bromoperoxidase from Streptomyces aureofaciens also halogenate phenol, but the products were not identified (Wiesner et al., 1990).

65

HALOPEROXIDASES

I Er

52

54

53

& b&+ OH

OH

OH

X

58

59a (X=Br) (X=CI) 60

SCHEME 6. a, Bromopeioxidase from various coralline algae, H,OZ, Br-; b, &loroperoxidase from Caldariomyces fumago, H 2 0 2 , C1- ; c, chloroperoxidase from C. fumago, H,O,, Br-.

In the course of a search for the natural substrates of the chloroperoxidase from C. fumago, we found that tyrosol ( 5 5 ) was chlorinated by this enzyme in vitro giving its 3-chloro derivative 56 and its 3,5-dichloro derivative 57. The latter compound was only formed in very small amounts. All three of these compounds were present in the culture medium of this mold, but it is not known if the reaction described above also occurs in vivo (Franssen et al., 1988b). The halogenation of resorcinol ( 5 8 ) using immobilized enzymes is discussed in Section II1,G. Everett et aJ. (1990a) found that phloroglucinol (60) was a substrate for the bromoperoxidases from the brown alga A. nodosum and the red alga CoraJlina vancouveriensis. The product(s) was not identified. Itoh et al. (1988) reported that the vanadium bromoperoxidase from Corallina piJuJifera converts anisole (61; see Scheme 7) into a mixture of its o-bromo and p-bromo derivatives (62 and 63, respectively). The

66

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

61

62

63

3% (ratio 0.021:l)

68

67

66

100%

SCHEME 7. a, Bromoperoxidase from Corallina pilulifera, H202,Br ; b, lignin peroxidase from Phanaerochaete chrysosporium, H,O,, Br- . ~

ortho-para ratio was 0.021, due to steric hindrance at the ortho position. The isolated yield was only 3%; the authors filed a patent (It0 et a)., 1987). 1-Methoxynaphthalene (64) gave the 4-bromo derivative 65 on incubation with the same enzyme (Itoh et a]., 1988). The lignin peroxidase from Phanaerochaete chrysosporium quantitatively converts veratryl alcohol (66) into a mixture of the bromo alcohol 67 and the bromo aldehyde 68 (Renganathan et al., 1987), the alcohol being the major product. Some more examples of halogenation reactions of aromatic systems are depicted in Schemes 1 and 3.

D.

REACTIONS WITH

HETEROCYCLIC COMPOUNDS

Heterocyclic systems were, beyond any doubt, the most popular substrates for haloperoxidases during the past 5 years. More than 20 articles were devoted to reactions with these compounds in this period.

H 69

70

H

+cl% H

H

H

71

72

b

71

73

+

73

Br

H

74

N

H

X

vBrwBr 75a (X=Cl) b (X=Br)

-Brqv + B r + Br

d,e

Br

Br

76

77

81

Br

78

a: R=H

80

Br %8r

Br

79

b: R=CH,

82

83

SCHEME 8 . a, Chloroperoxidase from Pseudomonas pyrrocinia, HZOz,C1-; b, chloroperoxidase from P. pyrrocinia, HZOz, Br - ; c, bromoperoxidase from P. pyrrocinia, bromoperoxidase l a and 3 from Streptomyces griseus, and bromoperoxidase from Streptamyces aureofaciens, HzOz, Br- ; d , chloroperoxidase from P. pyrrocinia and bromoperoxidase from S. aureofaciens, HzO,, Br-; R = H; e, bromoperoxidase from Streptomyces venezuelae, HzOz, Br-; R = CH3.

68

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

Pyrrole (69; see Scheme 8) is very reactive toward electrophilic reagents and most of its halogenated derivatives are unstable. Hence, it was not unexpected that pyrrole reacted with the chloroperoxidase from C. fumago and that the products could not be isolated (Franssen et a]., 1987a). Exciting research has been performed on substituted pyrroles. The bacterium Pseudomonas pyrrocinia produces the fungicide pyrrolnitrin (70;see Scheme 8) and several studies have been devoted to its biosynthesis. Clearly, a chlorinating enzyme must be involved, but initially only a bromoperoxidase was found in this bacterium (Wiesner et a]., 1985). Continuous research led in 1986 to the discovery of a chloroperoxidase (Wiesner et a]., 1986) which chlorinated indole (see below) but was not able to chlorinate the “standard substrate” monochlorodimedone!! This result clearly shows that caution has to be exercis.ed in screening programs for haloperoxidases: enzymes may be overlooked because the correct substrate was not offered. Remarkably, the chloroperoxidase from P. pyrrocinia brominates monochlorodimedone efficiently (Wiesner et al., 1986). One of the natural substrates of this enzyme is presumably 3-(2-amino-3-chlorophenyl)pyrrole(71).On incubation with the enzyme, this compound gave the 3-chloro derivative 72, together with a small amount of the corresponding bromo compound 73. The latter compound will arise from a contamination with bromide in the chloride salt used. When only bromide ions were provided, the dibromo compound 74 was also formed (Wiesner et al., 1990). In principle, these reactions are not regioselective, although it is remarkable that the activated benzene ring in 71 is not halogenated. This may indicate some selectivity in the enzyme. Pyrrolnitrin itself (70)is also a substrate for this enzyme and gives the corresponding 2halo derivatives (75;Wiesner et al., 1990). This reaction was also catalyzed by the heme-containing bromoperoxidase from this organism (Wiesner et al., 19851, the bromoperoxidases “ l a ” and “3” from Streptomyces griseus (Zeiner et a]., 1988),the bromoperoxidase from Streptomyces aureofaciens, and “other bacterial bromoperoxidases” (Wiesner et al., 1990).Another pyrrole derivative that was investigated is 2-(3,5-dibromo-2-methoxyphenyl)pyrrole (76a)and its 1-methyl derivative (76b).The chloroperoxidase from P. pyrrocinia and the bromoperoxidase from S. aureofaciens and S. venezuelae gave mixtures of mono-, di-, and tribrominated products (77a,78a,and 79a;Wiesner et a]., 1990; Knoch et al., 1989). The chloroperoxidase from P. pyrrocinia converts indole (80) into its 7-chloro derivative 81 (Wiesner et al., 1986, 1990). This is a highly interesting reaction, as the heterocyclic ring in indole has the highest

69

HALOPEROXIDASES

COOH

N H

H

84

71

N

N

72

70

SCHEME 9. a, Chloroperoxidase from Pseudomonas pyrrocinia, H,02, C1-.

electron density and should normally be attacked in halogenation reactions instead of the benzene ring (Remers, 1972). Hence, this reaction is the first unambiguous example of a regiospecific halogenation reaction that is carried out by a haloperoxidase. This regiospecificity was not observed in the bromination of 80 by this enzyme and the bromoperoxidase from S. aureofaciens. These reactions yielded a monobromo and a dibromo compound (82and 83,respectively), in which at least one of the bromine atoms is located in the heterocyclic ring. The results obtained with the chloroperoxidase from P. pyrrocinia led van Pee (1990b) to a proposal for the biosynthesis of pyrrolnitrin (70) starting from tryptophan (see Scheme 9). The indole nucleus is first chlorinated in the 7-position and then cleaved oxidatively leading to 2carboxy-4-(~-amino-3-chloro)-phenylpyrrole (84).This compound decarboxylates, giving 71,which is chlorinated again yielding the 3-chloro derivative 72.Pyrrolnitrin (70)is obtained through oxidation of the

70

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

85a R,,R,=H

b R,=CH3, R,=H c R,=H, R,=CH3

86a X=C1, R,,R,=H (conditions a, 68%; conditions b, 75%i n case of chloroperoxidase) b X=C1, R,=CH-,, R,=H (conditions a, 83%) c X=CI, R,=H, R2=CH3(conditions a, 91%) d X=Br, R,,R,=H (conditions b, 14%in case of brornoperoxidase) e X=I, R,,R,=H (conditions b)

CI

N"

N+

I

H

9Oa R,=CH3, R,=H, R,=COOCH3 b R,=CH3, R,=OH, R,=COOCH, c RI=CH3, R,=COOCH,, R?=OH d R,=CHO, R,=H, R,=COOCH,

Yla R,=CH,, R,=H, R,=COOCH, (75-80%) b R,=CH,, R,=OH, R,=COOCH, (25%) c R,=CH3, R2=COOCH3,R3=OH (35%)

SCHEME 10. a, Chloroperoxidase from Caldariomyces fumago, HzOz, C1-; b, chloroperoxidase from C. fumago or bromoperoxidase from Corallina pilulifera, H,Oz, X - ; c, myeloperoxidase, H,O,, C1- .

amino group. All compounds have been detected in bacterial cells, except 7-chlorotryptophan. However, in vitro chlorination of tryptophan by the chloroperoxidase from P. pyrrocinia has not been investigated yet, so the mechanism still contains some uncertainties. Pyrazole (85a; see Scheme 10) is less electron-rich than pyrrole and

HALOPEROXIDASES

71

is less reactive; its halogenated derivatives are more stable. We have studied the chlorination of 85a and the two methyl derivatives 85b and 85c by the chloroperoxidase from C. fumago and found that they were converted into their 4-chloro derivatives (86) in good yields. UV-spectroscopy revealed that the first step of the reaction is presumably the enzymatic formation of the 4H-pyrazolium ion 87 which rapidly rearranges into the final product in a nonenzymatic fashion. The yields obtained are very good compared to nonenzymatic procedures (Franssen et a]., 1987a). Itoh et al. (1987b) also described the halogenation of 85a with this enzyme and the bromoperoxidase from Corallina pilulifera, although the yield is only 7%. Bromination and iodination of 85a gave 86d and 86e, respectively, in equally low yields. Uetrecht and Zahid (1988) have shown that the toxicity of the heterocyclic drug phenytoin (88; see Scheme 10) may be due to in 17ivo chlorination by myeloperoxidase. The product obtained in vitro was the N,N’-dichloro derivative 89. This is a reactive compound which couples quickly to tryptophan, albumin, and DNA. Even such large heterocycles as chlorophylls can be substrates for the chloroperoxidase from C. fumago, as was shown by Senge and Senger (1989). Chlorophyll a (90a; see Scheme 10) was converted into its 20chloro derivative 91a in high yield. 13-Hydroxychlorophyl1a (90b) and its CIS-epimer (9Oc) also gave their corresponding 20-chloro derivatives, 91b and 91c, although in low yield. Chlorophyll b (god) reacted to give an unknown product with a 20% yield. This reaction looks regiospecific, but normal chlorinating agents also react at the 20-position (Woodward and Scaric, 1961; Hynninen and Lotjonen, 1981).However, the enzymatic route is favored because loss of metal ion (phaeophytinization) is minimized. A study on the reactivity of two pyridine derivatives toward the chloroperoxidase from C. fumago was published by us (Franssen et al., 1987a). 2-Aminopyridine (92; see Scheme 11) gave 2-amino-3-chloropyridine (93) but with a yield of only 18%. Again this reaction looks regiospecific, but it appeared that, at pH 2.7, the optimum pH for this enzyme, HOCl also attacks at position 3. The 5,7-dihalo compounds 95a and 95b were obtained when 8-hydroxyquinoline (94) was incubated with the enzyme. The dibromo derivative was isolated in good yield. Barbituric acid (96a; see Scheme 1 2 ) and its derivatives have been extensively studied as substrates for haloperoxidases. The chloroperoxidase from C. fumago converted 96a and its 1-methyl and 1,3-dimethyl congeners (96b and 96c, respectively) via their 5-chloro derivatives (97a-c) into the 5,5-dichloro compounds (98a-c) with excellent yields.

72

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

93 (18%)

92

X

I

OH 94

OH 95a X=CI (conditions a) b X=Br (79%, conditions b)

SCHEME 11. a, Chloroperoxidase from Caldariornyces furnago, H202, CI-; b, chloroperoxidase from C. furnago, Hz02, X -.

Also, the 5-phenyl derivatives (100a,b) reacted smoothly, giving the 5chloro compounds (10la,b) (Franssen and van der Plas, 1987). The intermediate 5-monochlorobarbituric acids could not be obtained in this way because 96a-c and 97a-c react with equal velocity with the enzyme. Interruption of the reaction at 50% conversion therefore always leads to a mixture of products. This problem was circumvented by the use of an bioelectrochemical system containing immobilized enzyme; it is discussed in Section II1,G. The bromoperoxidase from the brown alga A. nodosum reacted with 96a-c in a way that was comparable to the chloroperoxidase from C. fumago, yielding the dibromobarbituric acids 98d-f via the monobromo compounds 97d-f with good yields (Franssen et a]., 1988a). Interestingly, 5-chlorobarbituric acid (97a) gave a compound with two different halogen atoms attached to the same carbon atom (99). 5-Phenylbarbituric acid (100a), its 1-methyl derivative (lOOb),and some compounds in which the benzene ring was substituted (100~-f)smoothly gave the corresponding 5-bromo derivatives 10lc-h (Franssen et a]., 1988a; H. Kamphuis et al., unpublished observations). In contrast to the chlorination reactions, it was possible in these reactions to isolate the intermediate 5-monobromo compounds (97d-f). On closer consideration, it appeared that the dibromo compounds were brominating agents themselves, and as a result thereof 98 + 96+ 2 97 was a competing reaction. This explains why, at 50% conversion, the reaction mixture exclusively contains 97 (Franssen et aI., 1988a). Another algal bromoperoxidase, namely the one from L. saccharina, was also able to convert 96a-c into 98d-f and loob into l o l d (E. W. J. Mosmuller et al., unpublished observations).

73

HALOPEROXIDASES 0

96a RI,R2=H b R,=CH3, R,=H c R,,R,=CH,

97a R,,R,=H, X=CI b R,=CH,, R2=H, X=CI c R,,R,=CH,, X=CI d R,,R,=H, X=Br e R,=CH3, R,=H, X=Br f Rl,R2=CH3, X=Br

0

b

98a R,,R2=H, X=CI (99%,conditions a) b RIzCH3, R2=H, X=C1 (98%, conditions a) c R,,R2=CH3, X=CI (94%, conditions a) d R,,R2=H, X=Br (>95%,conditions b; conditions c; *) e R,=CH3, R2=H, X=Br (295%, conditions b; conditions c) f R,,R,=CH,, X=Br (>95%,conditions b; conditions c)

H

99

HN

R,

lOOa R,,R,=H b R,=CH,, R2=H c Rl=H, R2=C1 d R,=H, R,=F e R,=H, R2=CH, f Rl=H, R,=OCH,

Rl

lOla R,,Rz=H, X=CI (98%, conditions a) b RI=CH,, R,=H, X=CI (98%, conditions a) c Rl,R2=H, X=Br (>95%.conditions b) d R,=CH3, R2=H, X=Br (>95%.conditions b; conditions c) e R,=H, R,=CI, X=Br (conditions b) f Rl=H, R*=F, X=Br (conditions b) g R,=H, R2=CH3, X=Br (conditions b) h R,=H, R,=OCH,, X=Br (conditions b)

SCHEME 12. a, Chloroperoxidase from Caldariomyces fumago, H,O,, C1-; b, bromoperoxidase from Ascophyllum nadosum, HZOZ, Br - ; c, bromoperoxidase from Laminaria saccharina, HZOz,Br- ; d, chloroperoxidase from Pseudomonas pyrrocinia, HzO,, Br-; *, in the case of conditions d only 97d was obtained.

The enzyme-mediated halogenation of some nucleic bases was studied by Itoh et al. (1987b) and Itahara and Ide (1987). Both research groups used the chloroperoxidase from C. fumago for the conversion of uracil (102a; see Scheme 13) into its 5-chloro derivative [103a;no yield given (Itoh et a].); 7% of 103a plus 70% of 102a (Itahara and Ide)] and its 5-bromo derivative [103b;11% (Itoh eta].); 41% of 103b with 34% of

74

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

a,b,c,d,e 0 R

R

a,

102a R=H b R=2-deoxyribose

103a R=H, X=CI conditions a) b R=H, X=Br conditions b; conditions d) c R=2-deoxyribose, X=Br (5%.conditions b) d R=H, X=I (conditions c and e)

105

104

R

106a R=H b R=ribose

".i_-? Hod A

HzN

(m,conditions b; conditions d and f)

107a R=H b R=ribose

(m,conditions b; conditions d)

b

N

OH OH

1OY

H2N

108a R=H b R=ribose

0 A H N y N; > . :

111

112

OH OH

110

SCHEME 13. a, Chloroperoxidase from Caldariomyces fumago, H202, CI-; b, chloroperoxidase from C. fumago, H,OZ, B r - ; c, chloroperoxidase from C. fumago, H Z 0 2 .I - ; d, bromoperoxidase from Corallina pilulifera, H,02, B r r ; e, bromoperoxidase from Co. pilulifera, H z 0 2 , I-; f, bromoperoxidases from Fucus distichus and Macrocystis pyrifera, CHJOOOH, Br - .

102a (Itahara and Ide)]. According to Itoh et al., 2'-deoxyuridine (102b) gave the 5-bromo product 103c in only 5% yield. 5-Iodouracil (103d) could also be obtained in this way. The same authors showed that the bromoperoxidase from the red alga Corallina pilulifera also catalyzed the production of 103b and 103d (Itoh et al., 1 9 8 7 ~ ) .

HALOPEROXIDASES

75

Thymine (104; see Scheme 13),the 5-methyl congener of uracil, can be converted into the bromohydrin 105 by the chloroperoxidase from C. fumago, according to Itoh et al. (1987b). This result is in contrast to those of Itahara and Ide (1987),who could not get any product from this reaction. The bromoperoxidase from Corallina pilulifera when reacting with 104 gave a different product which, however, could not be identified (Itoh et a]., 1987b). Cytosine (106a) could be chlorinated or brominated with the above-mentioned enzymes, giving the 5-halo derivatives 107a and 107b. The yield of the latter compound was 55% (Itoh et al.) or 41% (Itahara and Ide) using chloroperoxidase. Cytidine (l06b) was brominated by the above-mentioned enzymes; chloroperoxidase gave 68% of the product 107b (Itoh et al., 1987b). The vanadium-containing bromoperoxidases from the brown algae Fucus distichus and Macrocystis pyrifera also convert 106a into 107a (Soedjak and Butler, 1990a). The attempts of Itoh and co-workers (1987b) to halogenate purines were not very successful. On chlorination, adenine (108a) and adenosine (l08b) gave unstable products which returned to starting material after a few hours; guanosine (109) completely decomposed under these conditions. Bromination of 109 by the chloroperoxidase from C. fumago gave the 8-bromo compound 110. The yield was not reported. To complete this section, it should be mentioned that the bromoperoxidase from Corallina pilulifera converts thiophene (111) into its 2-bromo derivative (112; Itoh et al., 1988). E. REACTIONS

WITH

AMINES

The reaction of haloperoxidases with amino acids, which has been known for a long time (Zgliczynski et al., 1971), gives as initial products N-halo and N,N-dihalo derivatives, which are stable in the case of chlorine (Weil and Morris, 1949) and labile in the case of bromine (Kanofski, 1989). This result explains why amino acids such as valine and alanine are converted into nitriles and aldehydes on incubation with the bromoperoxidases of the green alga Penicillus capitatus and the red alga Bonnemaisonia hamifera (Nieder and Hager, 1985). The reaction sequence is depicted in Scheme 14. The N-bromo intermediate 113 is either brominated again to give the N-dibromo compound 116 or loses carbon dioxide and hydrogen bromide to form the imines 114. These compounds hydrolyze in the aqueous system to give aldehydes (115).The dibromide 116 in its turn undergoes the same reaction, yielding the bromoimine 117 which gives a nitrile (118) on spontaneous elimination of hydrogen bromide. In the case of valine [R = CH,-

76

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

R-CH-NH, I

COOH

a

R-CH-NHBr COOH

113

//. -HBr

R-CH=NH

\\\

R-CH-NBr, I

114

COOH

116

1

-co, -HBr

R-CH=O

R --CH=NaI

115

117

R-CEN

118

SCHEME 14. a, Bromoperoxidases from Penicillus capitatus or Bonnemaisonia hamifera, H,02, C1-; b, disproportionation (2RNHBr + RNH2 + RNBr2).

CH(CH,)J, 115 and 117 are formed quantitatively in a ratio of about 1 : 2 (Nieder and Hager, 1985). An alternative to the above-mentioned pathway involves oxidative cleavage of the N-bromo bond in 113 by hydrogen peroxide, resulting in the formation of and the unbrominated amino acid (Kanofski, 1989). When peracetic acid is used as a substrate this reaction is not possible, and therefore Soedjak and Butler (1990a) were able to detect the monobromo derivatives of several amines during incubation with the bromoperoxidases from Fucus distichus and Macrocystis pyrifera. The amines used were taurine (119;see Scheme 15), Tris (120),CAPS0 (121),TES (1221,HEPES (123),and MOPS (124).Taurine (119)can also be chlorinated to give its N-chloro derivative by the bromoperoxidase (!) from A. nodosum (Soedjak and Butler, 1990b). N-Chlorination is also involved in the oxidative degradation of 1,4diazabicyclo[2,2,2]octane (125;see Scheme 15) by the chloroperox-

77

HALOPEROXIDASES

119

120

121

HOH,C, HOHZC >C -NH-CH,CH,SO,H HOH,C

HOCH,CH,-N

122

/-7

N-CH,CH,SOSH

W

123

n N-CH,CH,CH,SO~H

0

W

124

125

CI

126

SCHEME 15. a, Chloroperoxidase from Caidariomyces fumago, H202. C1- . The compounds 119-124 are converted into their N-monobromo derivatives by the bromoperoxidases from Fucus distichus and Macrocystis pyrifera, H2O2, and Br-. Compound 119 gave its N-monochloro derivative on incubation with the “bromoperoxidase” from Ascophyllum nodosum, HZO,, and C1- .

idase from C. fumago. The reaction sequence involves N-chlorination, homolytic splitting of the chlorine-nitrogen bond, disproportion of the radical resulting in the cleavage of the central carbon-cartion bond, hydrolysis of the imine formed, and chlorination of the resulting secondary amine. Only the first and the last steps are enzymatic. The final product is N,N-dichloropiperazine (126)(Say0 et al., 1988).

F. MISCELLANEOUS SUBSTRATES

In this subsection, some reactions are mentioned which do not fit in the previous subsections.

78

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

127

128

xs A N

0

H

H

N 5 H

129

0

130

SCHEME 16. a, Chloroperoxidase from Penicillus capitatus, H202, CI-; X bromoperoxidase from Ascophyllum nodosum, H,OZ, Br-; X = Br.

=

C1; b,

Penicillin G (127;see Scheme 16)and ampicillin (128)are substrates for the chloroperoxidase from P. pyrrocinia. Penicillin G (127)could be chlorinated and brominated, whereas 128 could only be brominated by this enzyme. The structures of the product(s) were not elucidated. The bromoperoxidase from S . aureofaciens was also able to brominate 127 and 128,giving unknown products (Wiesner et ai., 1990). Thiouracil (129)is oxidized to its dimer 130 by various haloperoxidases. It has been found that the “chloroperoxidase” from Penicillus capitatus is also able to catalyze this reaction (Manthey and Hager, 1989). Chloride or bromide ions are necessary, so this reaction is presumably not a simple oxidation but a halogenation of the sulfur atom of 129 followed by a substitution of the halide by another molecule of 129 (see Scheme 17). The same reaction was reported for the bromoperoxidase from the brown alga A. nodosum (De Boer and Wever, 1988). To complete this overview, it should be mentioned that several haloperoxidases catalyze the oxidation of iodide into the triiodide anion. A few examples include the chloroperoxidases from the bacteria Curvularia inaequalis (Hunter et al., 1986) and P. pyrrocinia (Wiesner et al., 1988) and the bromoperoxidases from the brown algae Laminaria saccharina, Fucus spiralis, Fucus serratus, Fucus vesiculosus, Pelvetia canaliculata, and Chorda filum (De Boer et al., 1986a).

HALOPEROXIDASES

79

Millard (1988) describes the radioiodination of plasma membranes from protoplasts of Arabidopsis thaliana with lactoperoxidase. The author found that 4.6% of all radioactivity ended up in the lipid fraction and 3.0% in proteins. The most active lipid fractions contained monogalactosyldiacylglycerol (23.9%), digalactosyldiacylglycerol (15.0%), and chlorophyll b (13.7%).Details on structures of products were not given.

G. REACTIONS USING IMMOBILIZED ENZYMES Although most reactions with haloperoxidases are carried out using free enzymes, for large-scale conversion it is profitable to use immobilized enzymes. Immobilization enables continuous conversion of substrates as well as easy recovery and reuse of the biocatalyst. Immobilization is, in principle, every method that confines the biocatalyst to a restricted area. The methods that have been applied to haloperoxidases during the past 5 years are adsorption to a solid matrix, covalent binding to a solid matrix, entrapment in fibers and polymers, and entrapment in reverse micelles. The results discussed in this section are grouped per enzyme. The immobilization of the chloroperoxidase from C. fumago was studied in detail by us (Franssen, 1987). Several different methods were used, including adsorption to neutral and ionic supports, covalent binding to solid supports, and entrapment in calcium alginate. The only technique which gave yields above 40% (based on specific activity) was entrapment in a cross-linked gel of poly(acry1amide-co-Nacryloxysuccinimide) as described by Pollak et al. (1980). The chlorination of 94 (see Scheme 11) could be carried out in a continuous system with this chloroperoxidase immobilized by Pollak’s method (E. W. J. Mosmuller et a1., unpublished observations). Another good method was described by Kadima and Pickard (1990). These authors bound the chloroperoxidase from C. fumago ionically to aminopropyl-ControlledPore-Glass, and then cross-linked the enzyme with glutaraldehyde. The yield was 36% on a specific activity basis. In a paper published in 1984 (Laane et a]., 1984), we described the simultaneous production of gluconolactone and 5-monochlorobarbituric acid (97a; see Scheme 12) using a bioelectrochemical fuel cell. An improved system was also published by us (Laane et al., 1986) in which a slightly different approach was used. In this so-called bioelectrochemical system, the electric current was applied externally by means of a potentiostat and the enzyme and the electrode processes are separated. The complete system is depicted in Fig. 2. It consists of three

80

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

Electrolytic cell

Hollow fiber

membrane reactor

I 1

11 I 1 I1 II

I1 I1 I 1 I1 I 1 I1 I 1

I1 I 1 I1 11 11 11 I 1 II

I

I 1

I 1

I 1 I 1 I 1 11

(

I1

11 I1

11 I1 11 I1 I) I

uuu

I - Chloroperoxidase stock FIG. 2 . Schematic representation of the bioelectrolytical system which is used for the continuous production of 5-chlorobarbituric acid (97a) by the chloroperoxidase from Caldariomyces furnago (Laane et al., 1986).

interconnected units: (1) an electrolytical cell that oxidizes water to oxygen at the anode and reduces oxygen to hydrogen peroxide and 98a to 97a at the cathode using electricity as energy source: (2) a reactor containing chloroperoxidase, immobilized by entrapment into hollow fibers; the electrolytically produced hydrogen peroxide is utilized here for the halogenation of 96a, giving a mixture of 97a and 98a; and (3) an anion exchanger that scavenges the 97a produced in the membrane reactor and in the anode compartment. The product can be recovered from the column by applying a salt gradient. In this small model system more than 50 mg of pure 97a was produced in 24 hours (9670yield). The halogenation of monochlorodimedone (1;see Scheme 1) by haloperoxidases entrapped into reversed micelles was studied. It appeared that the chloroperoxidase from C. fumago rapidly halogenates 1 into 2 (X = Br, C1) in reversed micelles composed of cetyltrimethylammonium halide (CTAX; X = Br or CI), pentanol, octane, and a small . enzyme amount of aqueous buffer (Franssen et al., 1987b, 1 9 8 8 ~ )The and the substrate hydrogen peroxide are confined to the aqueous phase, whereas the organic substrate is located in the micellar interphase. The CTAX serves a dual function: (1)as a surfactant (it stabilizes the re-

HALOPEROXIDASES

81

versed micelle), and (2) as a supplier of the halide substrate. The initial reaction rates obtained are about twice as high as in water, but the inactivation of the enzyme by hydrogen peroxide is quite rapid. Oeberg (1987) has found that CTA' ions stabilize Br; ions, and since it is speculated that chloroperoxidase produces Br; [see Section IV,A), this might explain the high initial rate of bromination. The bromoperoxidase from the brown alga A. nodosum is about twice as active in this micellar system as in water. In addition, this enzyme is much more stable in this system than the fungal chloroperoxidase (Franssen et aI., 1987b). Resorcinol (58;see Scheme 6) is converted into its 4-bromo or 4chloro derivative (59a and 59b,respectively) by the chloroperoxidase from C. fumago in reversed micelles (Franssen et al., 1978b, 1 9 8 8 ~ ) . The reversed micelles are composed of cetyltrimethylammonium halide (CTAX; X = Br or Cl), pentanol, octane, and aqueous buffer. The rate of conversion of 58 is 2.5 times higher in this system than in water. It has been reported that the bromoperoxidase from A. nodosum can be covalently immobilized to Sepharose with a 44% yield [A. Berg et a]., unpublished observations) using the method of Kohn and Wilchek (1982). However, binding this bromoperoxidase by absorption to a hydrophobic matrix like octylamine-modified Sepharose is a more effective procedure. This method afforded yields of around 72% (M. Jacobs et al., unpublished observations). The chloroperoxidase from Pseudomonas pyrrocinia was used for the continuous synthesis of 5-monobromobarbituric acid (97d; see Scheme 12) from 96a in a fixed-bed reactor. The enzyme was covalently immobilized on the polymeric carrier Eupergit C (Wiesner et cd., 1989). The bromoperoxidase from the red alga C o r a l h a piluliferci was immobilized by several methods: adsorption to hydrophobic surfaces, entrapment in natural and synthetic gels, covalent attachment to supports containing epoxide groups, and adsorption to ionic supports. The latter method appeared to be the best, giving yields of 72437% of immobilized enzyme when DEAE-Cellulofine was used as the support. This system was used for the continuous production of 5-bromouracil[103b; see Scheme 13; Itoh et al., 1 9 8 7 ~ ) . IV. Reaction Mechanisms

Knowledge of reaction mechanisms of haloperoxidases has been gathered from various points of view. Biochemists have developed models of the way in which the enzymes function, based on kinetic measurements and data obtained by spectroscopic techniques. (Bio)-

82

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

organic chemists have tried to fit their results, obtained by careful investigation and interpretation of the composition of the enzymatic reaction mixtures, into a working model. This working model enables chemists to predict the outcome of haloperoxidase reactions with new substrates. Both disciplines have produced important contributions to the elucidation of the reaction mechanisms of the enzymes. A compilation of recent results, arranged according to the prosthetic group of the enzymes, is given below. ENZYMES A. HEME-CONTAINING

The reaction mechanisms of heme-containing haloperoxidases, although they were the first to be detected, are still the subject of some dispute. Two alternative pathways are advanced concerning the following steps (see Fig. 3): route I, suggesting that the organic substrate (AH) binds to the enzyme to form a ternary complex which splits into the native enzyme, the organic product, and a molecule of water; and/or route 11, in which Compound I decomposes to native enzyme and a molecule of hypohalous acid/molecular halogenhihalide ion. In the latter case, these inorganic species, which are in equilibrium with each other, are the active halogenating agents in the reaction. For bromination reactions, there is little doubt that the reaction proceeds via hypobromous acid/molecular bromine/tribromide [see for the chloroperoxidase from C. fumago, Yamada et al, 1985a; Itoh et al., 1987b; see for lignin peroxidase, Renganathan et a ] . , 1987; see for the bromoperoxidase from the green alga Penicillus capitatus, Manthey and Hager, 1989). Iodination reactions are presumed to proceed similarly via molecular iodine or triiodide. Concerning chlorination reactions, it is known that the mammalian enzymes myeloperoxidase and eosinophil peroxidase produce HOCl as the active species (Harrison and Schulz, 1976; Buys et al., 1984).In the case of the chloroperoxidase (former bromoperoxidase) from the green alga Penicillus capitatus, the reaction mechanism could not be established due to experimental difficulties [Manthey and Hager, 1989).Concerning the chloroperoxidase from the mold C. fumago, it is far from clear whether route I or route II is favored. Several articles deal with this topic, from both points of view; a survey is given below. Dunford et al. (1987) have used stopped-flow, rapid-scan techniques to study the reaction of Compound I and HOCl with chloride and monochlorodimedone, respectively (1;see Scheme 1).They had to apply these advanced methods because Compound I and all subsequent intermediates are unstable and have very short lifetimes. They could deduce from

83

HALOPEROXIDASES

ENZ

O2

eT> H202

ENZ

t HZO

Compound I

/ ,

ew-

CompoundEOX

native enzyme

A-X

ENZ-+T>

+

A-x

native enzyme

FIG. 3. The reaction mechanism of heme-containing haloperoxidases. ‘The protein part of the enzyme is represented by ENZ, the heme group is depicted as Fex+ (x = 3 or 4)in the center of an ellipse, for reasons of clarity. In compound I, the iron ion has a 4 + oxidation state and the porphyrin ring is oxidized to a radical cationic species. Covalent bonds which are generally accepted in the literature are indicated by solid lines between the atoms; if there is any doubt, dashed lines are used. A-H, Organic substrate; A-X, halogenated product. (After Franssen and van der Plas, 1987.)

their experimental data that Compound EOX really exists, but the species was so unstable that its specific UV-absorptions were never seen. Their kinetic studies, combined with previous results (Champion et al., 1973; Lambeir and Dunford, 1983) strongly favored route I, although route I1 could not be completely excluded. Other routes involving molecular chlorine or radical species were insignificant.

84

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

Libby et al. (1989) performed competition reactions with Compound I as the enzyme intermediate and catechol (131; see Scheme 17) and chloride as substrates. The authors nicely found that the two substrates competed for the same binding site on Compound I, again proving the (transient?) existence of Compound EOX. Although the results confirm the validity of route I, in our opinion route I1 cannot be excluded. We observed, by evaluating the kinetic data of the chlorination of monochlorodimedone (1; see Scheme 1) and barbituric acid and its derivatives (96a-c and 100a,b; see Scheme 12), that the differences in conversion rates of the compounds tested were marginal, and that there was no effect of the concentration of the organic substrate on the reaction rates (Franssen and van der Plas, 1987).The results obtained do not allow any decision to be made as to whether route I or I1 is most favored either. The fact that the concentration of the organic substrate has no influence on the rate-determining step does not exclude route 11. The reaction between monochlorodimedone or barbiturates and HOCl is possibly so fast that the first two steps in the enzymatic reaction sequence, which are independent of the organic substrate concentration, determine the overall reaction velocity. The same reasoning is valid for route 1: if step 1 or 2 is much slower than the subsequent steps, the concentration of the organic substrate will not affect the overall reaction velocity. Libby and Rotberg (1990) clearly established that the second step (binding and oxidation of chloride) is the rate-limiting one. This shows the central problem with this enzyme: the crucial step (transfer of halogen to the organic substrate) is so fast that it cannot be measured with any technique. Also, the reaction between HOX and the organic substrate is much too fast, as was found by us during the comparison of enzymatic and HOX-mediated halogenation of !i-(p-X-phenyl) barbituric acids (100~-f;H. Kamphuis et a]., unpublished observations). An article by Itoh et al. (1987b) describes the chlorination of uracil and cytosine (102a and 106a; see Scheme 13) by this chloroperoxidase and by HOCl/CI, under similar conditions. The HPLC chromatograms of the mixtures of products obtained in the two systems are identical, which lead Itoh et al. to the conclusion that this enzyme produces

aoH OH

131

132

133

SCHEME 17. a, Chloroperoxidase from Caldariomyces fumago, tBuOOH. No halide ions were added.

HALOPEROXIDASES

85

HOCl/Cl,. We too found that the products from our enzymatic reactions were identical to those obtained by careful addition of HOCl to the substrates (although the yields were much lower in the latter case). Furthermore, the enzyme-mediated chlorination of 1OOb was not stereoselective. Although it is tempting to conclude from this work that the chloroperoxidase from C. fumago reacts via route 11, generating free HOC1, caution should be exercised, as explained above. There have been published two articles in which a stereospecific reaction of the chloroperoxidase from C. fumago was reported. Colonna et aI. (1988,1990)found that dialkyl sulfides (132;see Scheme 17) gave the corresponding R-sulfoxides 133 when incubated with chloroperoxidase and tert-butyl hydroperoxide. Enantiomeric excess values were 19-92%, depending on the substrate used. Other peroxides, including hydrogen peroxide itself, gave lower enantiomeric excess values. Essential in these reactions is the fact that no chloride ions are present in the reaction mixture. So, this is an example of the “classical” peroxidase reaction. Apparently, in this case, the substrate binds directly to the active site, leading to chiral compounds. This reaction is excellently suited to improving insight into the three-dimensional structure of the active site of this chloroperoxidase. Horseradish peroxidase also catalyzed this reaction, but only racemic mixtures were obtained (Colonna et al., 1990). Summarizing, the reaction mechanism of the chlorination by the chloroperoxidase from C. furnago is still obscure, and more (advanced) kinetic data are needed. Most promising in this respect is the approach of Libby et al. (1989), in which a comparison is made between the chlorination and the “classical” peroxidation (without chloride) of the same molecule. If a sterically hindered derivative of a known substrate is not oxidized without chloride, but is readily halogenated in the presence of chloride, there must be a low-molecular-weight mediator that transfers the chlorine to the substrate. In that case, the evidence for HOCl is beyond any doubt. However, if a series of hindered substrates are neither oxidized nor chlorinated, there is strong evidence for the involvement of the ternary complex (Compound I-halide-organic substrate). At any rate, the chloroperoxidase from C . fumago is a very useful enzyme in organic synthesis. For example, the chlorination of chlorophylls (90a-c; see Scheme 10) occurred smoothly without any loss of metal ions from the porphyrins (phaeophytinization), which is a serious side-reaction in more usual chlorination procedures (Senge and Senger, 1989). The enzymatic chlorination of barbituric acid and its derivatives (96, 97, and 100; see Scheme 1 2 ) gave much better yields than the chemical route (Franssen and van der Plas, 1987). All these features make this enzyme a useful tool in organic synthesis.

86

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

B. VANADIUM-CONTAINING ENZYMES

The mechanism by which vanadium-containing bromoperoxidases brominate organic substrates has been almost completely elucidated. In 1988, we showed that the bromoperoxidase from the brown alga A. nodosum produces HOBr as the active brominating agent (Franssen et al., 1988a). The evidence was obtained by studying the kinetics of the bromination of monochlorodimedone (1)and several barbituric acid derivatives (96a-c, 97a, and 100a,b). The rate of bromination was found to be dependent on the concentration of the organic substrate, resulting in “apparent K, values.” These values, however, were not real Michaelis-Menten kinetic parameters but were simply derived from two competing reactions which have about the same velocity at low concentrations of organic substrate. Both reactions involve enzymatically formed HOBr; the two reactions are given below.

-

HOBr + AH ABr + H,O + H,O HOBr + HzOz +

+ H + + Br

Reaction (7) is the normal bromination of the organic substrate; reaction (8) is a side-reaction which is very fast, especially at neutral pH according to Kanofski (1984). Since, as already stated above, the reaction of hypohalous acid with electron-rich organic acceptor molecules is of about the same velocity as this reaction, this unusual “semi-MichaelisMenten behavior” was obtained. These results were confirmed by Everett et al. (199Ob). A more detailed steady-state kinetic analysis was performed by De Boer and Wever (1988) and their results are depicted in Fig. 4. The exact nature of some enzyme intermediates during the catalytic activity of this bromoperoxidase has yet to be elucidated. De Boer et al.

ti

H+

E . HOBr

H+r!

E . H202

1t-

E + HOBr

OHE . HOBr J = Br-

FIG. 4. The reaction kinetics of vanadium-containing bromoperoxidases, as found for the enzyme from Ascophyffum nodosum (De Boer and Wever, 1988). E, Enzyme; inhibited forms are boxed.

HALOPEROXIDASES

ENZ - V5+ - OOH

ENZ - Vs+ H+

e-

87

ENZ - V5+ .. OBr

H+ HzO

I

4 ENZ-V” + HOBr FIG. 5. Proposal for the enzyme intermediates during bromination reactions of vanadium-containing bromoperoxidases.

ai. (1988b) have found that the native, resting enzyme contains vanadium (V) and that this ion does not undergo any changes in the redox state during catalysis. Since several inorganic vanadium-hydrogen peroxide complexes are known, it is reasonable to assume that in the first reaction step hydrogen peroxide binds covalently to the vanadium ion (De Boer et a]., 1986a) (Fig. 5 ) . In our opinion, the next step is the oxidation of the bromide ion with simultaneous cleavage of the oxygen-oxygen bond. The resulting vanadium-hypobromite complex returns to native enzyme and releases hypobromous acid or an equivalent species which is responsible for all reactions of the enzyme (Wever et aI., 1988). Whether in the first step the hydrogen peroxide molecule enlarges the coordination shell of the vanadium or replaces another ligand is left unresolved for the time being. Evidence for the mechanism described above was obtained by Tromp et al. (1990), who observed that, on addition of hydrogen peroxide to native bromoperoxidase, a small but significant change in the UV-spectrum of the enzyme occurs, which disappears again on the addition of bromide ions. The results discussed above were obtained with the bromoperoxidase from A. nodosum, but there is no reason to assume that other vanadium enzymes will react in a different way. For instance, production of HOBr and lo, was also found in the bromoperoxidase from Coraliina vancouveriensis (Everett et a]., 1990a). Evidence for HOBr was also obtained by Itoh et aI. (1988)in the comparison of ortholpara ratios of bromoanisoles that were gained by the bromination of anisole with HOBr, Br,, or the bromoperoxidase from the red alga Coraliina piiulifera. At any rate, no evidence was found for ternary complexes (bromoenzyme-hydrogen peroxide-organic substrate), so regio- or stereospecific reactions are not to be expected from these kinds of enzymes. Nevertheless, like the chloroperoxidase from C. fumago, these enzymes work in a much smoother way than their inorganic counterparts (Franssen et a]., 1988a).

TABLE IV

METALCONTENTS AND INHIBITORS -

~~~~

OF

SOMENONHEME, NONVANADIUM MICROBIAL HALOPEROXIDASES ~-

~~

Metal contentc Source0

Halogenating activityb

Ti

V

Cr

Fe

Ni

0 . 1 5 0.7

-

Mn

Curvularia inaequalis

CPO

Pseudomonas pyrrocinia Streptomyces aureofaciens

CPO

_ _ _ _

BPO

-

Cu

Inhibitorsd

Zn

Se Pb

F-

C1-

N,- CN- SH DT EDT 0 t h

Ref. Hunter ef QJ. (1986), Liu et al.

2.2

(1987) 0.1

-

-

0.2

- - 0.18

-

-

0.01

0.02

-

-

-

-

+

Wiesner et al. (1988)

-e

-e

-e

0.22

-e

0.2

-e

0.23 0.34

0.12

+

+ - -

f

Krenn et al. (1988)

Van Pee et al. (1987)

a C inoequolis is a mold, the other two are bacteria.

WPO, Chloroperoxidase; BPO, bromoperoxidase. .Numbers refer to the content of this metal, expressed in moles of metal ion per mole of native enzyme. -, Metal is absent; blank, the presence of this metal was not investigated. "umbers refer to K , values in mM [Iso values, in mM, are given in boldface). f , Enzyme is inhibited by this compound; -, enzyme is not inhibited by this compound; blank, the effect of this compound was not tested. SH, Thiol-reducing compounds, e.g., p-mercaptoethanol and dithioerytbreitol; DT, sodium dithionite; EDT, EDTA; Oth, others. .The metal-depleted enzyme could not be reactivated by this element. f Chlorotetracyclin.

HALOPEROXIDASES

89

The mechanism by which the bromoperoxidase from A. nodosum chlorinates monochlorodimedone and phenol red, as observed by Soedjak and Butler (1990b), is still a mystery. More research is needed in order to investigate whether chlorination is an intrinsic property of this enzyme or a side-reaction which only occurs at high chloride concentrations. C. OTHERS The prosthetic group of the enzymes which are discussed in this section is virtually unknown. All enzymes definitely do not contain heme or flavin groups, and presumably most of them do not contain vanadium either. The metal contents of the enzymes, insofar as they have been investigated, are presented in Table IV, together with some data from inhibition studies. As can be deduced from Table IV, iron and zinc are present in all four enzymes, although not always in equimolar amounts. Whether these metals are essential for the catalytic activity of the enzymes is not certain. Their importance can only be determined from reconstitution experiments, but these have either not been performed (Curvularia inaequalis) or were not successful (P. pyrrocinia, S. aureofaciens). Unfortunately, the presence of well-known redox-active metals like molybdenum and cobalt was not investigated at all. It is interesting to see that, like “normal” haloperoxidases, fluoride and cyanide ions are inhibitors of these enzymes. An exception is the bromoperoxidase from S. aureofaciens isolated by Krenn et al. (1988), which is inhibited by fluoride but not by cyanide. The two research groups that have been working on haloperoxidases from this source have isolated two different enzymes, as is clearly visible from the enzyme properties that are shown in Tables I1 and IV. Although some information has been disclosed about metal contents and inhibitors of the enzymes that are mentioned in this section, almost nothing is known about the actual halogenating intermediates in their reactions. Hunter et al. (1986) state that the chloroperoxidase from Curvularia inaequalis produces HOC1, but this was only based on the disputable fact that the products they obtained were identical to those formed by the chloroperoxidase from C. fumago. Nothing is known about the two other enzymes, and this is especially unfortunate for the enzyme from P. pyrrocinia. This enzyme is up to now the only haloperoxidase for which regioselective halogenation reactions have been unambiguously demonstrated.

90

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

V. Conclusions and Prospects

It will be clear from the preceding sections that, since the appearance of the book of Neidleman and Geigert (1986), haloperoxidases have remained the subject of extensive studies. Many enzymes have been detected, in various new organisms. We may safely conclude that haloperoxidases can be found throughout nature and seem to be essential to life. In strong contrast to their importance, not much progress has been made in the unraveling of their natural function. For instance, although a few new halometabolites from the mold Caldariomyces fumago have been reported (Franssen et al., 1988b), the natural substrates of its chloroperoxidase are largely unknown. Furthermore, suppose they were found, the question would remain what benefit the mold gains by halogenating these compounds. Nevertheless, screening for halometabolites can be a valuable tool for the enzymologist. Their structure give at least an indication of the natural substrates of the responsible haloperoxidases, and this aides the search for these enzymes. This is shown perfectly by Wiesner’s work (Wiesner et al., 1986, 1988) on the chloroperoxidase from Pseudomonas pyrrocinia. This bacterium produces, among others, the two chlorinated compounds pyrrolnitrin (70; see Scheme 8) and 7-chloroindole (81).Hence, it must contain a chlorinating enzyme. An enzyme was found which could brominate but not chlorinate monochlorodimedone (1;see Scheme 1). Application of a “biomimetic” substrate (indole, 80) finally revealed that the enzyme found was indeed a chloroperoxidase, but one that does not react with the unnatural substrate I! This approach may very well lead to the detection of new and exciting haloperoxidases in other organisms and to the elucidation of the biosynthesis of halometabolites (Van PBe, 1990b). Roughly, the main sources of haloperoxidases can be divided into two groups: algae and bacteria. Algae produce several heme-containing haloperoxidases and, as has become clear recently, a lot of vanadiumcontaining bromoperoxidases. The heme enzymes are mostly bromoperoxidases which produce molecular bromine and are therefore not of much use to the synthetic organic chemist. However, Manthey and Hager (1989) have shown that an enzyme from Penicillus capitatus, which is bromoperoxidase at neutral pH, can be a chloroperoxidase at low pH. Unfortunately, they were not able to investigate the mechanism of chlorination. It is quite possible that several of the other algal enzymes appear to be chloroperoxidases too on closer examination. Furthermore, although all heme-containing haloperoxidases that have been isolated thus far possess ferriprotoporphyrin IX as the pros-

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thetic group, not all enzymes have the same halogenating abilities: some can chlorinate, while others can only brominate. Since their prosthetic groups are identical, these differences in behavior must be due to dissimilarities in the electronic environment of the heme group. Up to now, the exact nature of these dissimilarities has still not been unraveled. Hopefully, in the future, techniques such as proton NMR will be helpful in this respect (see for preliminary results on myeloperoxidase, Dugad et a]., 1990; on lactoperoxidase, Shiro and Morishima, 1986; on horseradish peroxidase, Thanabal et a]., 1987). The vanadium-containing enzymes are very abundant (Wever et a]., 1987) and are possibly the major source of marine halometabolites. It has been recognized that this enormous wealth of halogenating capacities might be partly responsible for the volatile haloalkanes which are found in the stratosphere and are suspected to destroy the ozone layer (Wever, 1988). It was estimated that the biological production of volatile bromine-containing compounds is about 1 x lo4 ton/year, which is of the same order as the human industrial production of these compounds (5 X lo4 ton/year: Wever et al., 1988). The catalytic action of the vanadium bromoperoxidases involves the formation of a vanadium-hydrogen peroxide complex, which binds a bromide ion followed by the release of hypobromous acid. As a result of that, these enzymes do not have properties that are useful for the synthetic organic chemist; this reaction mechanism practically excludes regio- or stereospecific bromination reactions. This leaves unresolved the occurrence of various marine halometabolites which have their halogen atoms attached to chiral carbon atoms (see, for example, compound 134; Scheme 18). The origin of vicinal chlorobromo compounds has already been described by the group of Neidleman, but the chirality of compounds like 134 is hard to explain without the involvement of stereospecific haloperoxidases (Sakai et a]., 1986). When chirality is already present in the substrate, subsequent enzymatic halogenation reactions can be stereospecific, as is illustrated by the work of Fukuzawa et al. ( 1990a,b,c) on lactoperoxidase-mediated cyclizations of unsaturated alcohols. These results show that more re-

*..

CI

CI

CI

134

SCHEME 18. Isolated from Plocamium sp. (Faulkner, 1986).

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search on the occurrence and the reaction mechanisms of algal haloperoxidases can be fruitful. The detection of chlorinating activity in the bromoperoxidase from Ascophyllum nodosum is very interesting and should be studied in more detail. Perhaps this enzyme is able to chlorinate a special (natural) substrate in a specific way, just like the chloroperoxidase from Pseudomonas pyrrocinia. It has been shown that A. nodosum releases volatile chlorinated compounds into the seawater (Gschwend et al., 1985). Originally, their occurrencewas explained by nucleophilic attack of chloride ions on enzymatically formed brominated compounds; now, direct enzymatic chlorination has to be taken into account too. Further knowledge of the halometabolites of this alga will help to unravel the natural function and substrates of the bromoperoxidase. Concerning the organic substrates used, there are almost no structural limits to them as long as they are relatively electron-rich. This can be illustrated by the fact that simple heterocyclic systems like pyridine or pyrimidine are unreactive toward haloperoxidases (Itoh et al., 1987b), but once some electron-donating substituents are attached they are readily converted by the enzymes. This phenomenon can also be seen in alkenes, since they are halogenated reluctantly unless they are conjugated with electron-rich systems (cf. a benzene ring in the case of cinnamic acid). Although stereoselective halogenations with haloperoxidase are still unknown, the enzymes are useful catalysts because they act in a much smoother way than the conventional halogenating agents. In conclusion, the prospects for haloperoxidase research are still good, particularly in the field of nonheme microbial chloroperoxidases which are stable toward heat and oxidizing chemicals. An intensive screening program by Cetus resulted in the discovery of such an enzyme in the mold Curvularia inaequalis. This result, and particularly the first evidence for a regiospecific enzyme (in P. pyrrocinia), will undoubtedly stimulate fundamental studies on the structures and reaction mechanisms of microbial haloperoxidases. Once large-scale production of these enzymes is advanced, their applications in industrial organic synthesis will follow immediately. REFERENCES Arber, J. M., de Boer, E., Garner, C. D., Hasnain, S., and Wever, R. (1989).Vanadium K-edge X-ray absorption spectroscopy of bromoperoxidase from Ascophyllurn nodosum. Biochemistry 28, 7968-7973. Axley, M. J., Kenigsberg, P., and Hager, L. P. (1986). Fructose induces and glucose represses chloroperoxidase mRNA levels. J. Biol. Chern. 261, 15058-15061.

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Bangcharoenpaurpong, O., Champion, P. M., Hall, K. S., and Hager, L. P. (1986). Resonance Raman studies of isotopically labeled chloroperoxidase. Biochemistry 25, 2374-2378. Blanke, S. R., and Hager, L. P. (1988). Identification of the fifth axial heme ligand of chloroperoxidase. J. Biol. Chem. 263, 18739-18743. Blanke, S. R., and Hager, L. P. (1990). Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue. J. Biol. Chem. 265, 12454-12461. Blanke, S. R., Yi, S., and Hager, L. P. (1989).Development of semi-continuous and continuous flow bioreactors for the high level production of chloroperoxidase. Biotechnol. Lett. 11, 769-774. Buys, J., Wever, R., and Ruitenberg, E. J. (1984). Myeloperoxidase is more efficient than eosinophil peroxidase in the in vitro killing of newborn larvae of Trichinella spiralis. Immunology 51, 601-607. Carmichael, R. D., and Pickard, M. A. (1989). Continuous and batch production of chloroperoxidase by mycelial pellets of Caldariomyces fumago in an airlift fermentor. Appl. Environ. Microbiol. 55, 17-20. Carmichael, R. D., Jones, A., and Pickard, M. A. (1986). Semicontinuous and continuous production of chloroperoxidase by Caldariomyces fumago immobilized in K-carrageenan. Appl. Environ. Microbiol. 51, 276-280. Champion, P. M., Miinck, E., Debrunner, P. G., Hollenberg, P. F., and Hager, L. P. (1973). Mossbauer investigations of chloroperoxidase and its halide complexes. Biochemistry 12, 426-435. Ci, Y.-X., and Wang, F. (1990). Studies on catalytic fluorescence formation with peroxidase-like metallotetrakis-(N-methylpyridiniumyl)porphyrins. Talanta 37, 11331136. Colonna, S., Gaggero, N., Manfredi, A., Casella, L., and Gullotti, M. (1988). Asymmetric oxidation of sulfides, catalyzed by chloroperoxidase. J.C.S. Chem. Commun. pp. 1451-1452. Colonna, S., Gaggero, N., Manfredi, A., Casella, L., Gullotti, M., Carrea, G . , and Pasta, P. (1990). Enantioselective oxidations of sulfides catalyzed by chloroperoxidase. Biochemistry 29, 10465-10468. Dawson, J. H., and Sono, M. (1987). Cytochrome P450 and chloroperoxidase: thiolate ligated heme enzymes. Spectroscopic determination of their active site structures and mechanistic implications of thiolate ligation. Chem. Rev. 87, 1255-1276. Dawson, J. H., Kau, L.-S., Penner-Hahn, J. E., Sono, M., Eble, K. S., Bruce, G. S., Hager, L. P., and Hodgson, K. 0. (1986). Oxygenated cytochrome P450CAM and chloroperoxidase: direct evidence for sulfur ligation trans to dioxygen and structural characterisation using EXAFS spectroscopy. J. Am. Chem. SOC. 108, 8114-8116. De Boer, E., and Wever, R. (1988). The reaction mechanism of the novel vanadiumbromoperoxidase: a steady-state kinetic analysis. J. Biol. Chem. 263, 12326-12332. De Boer, E., Tromp, M. G. M., Plat, H., Krenn, G. E., and Wever, R. (1986a). Vanadium (V) as an essential element for haloperoxidase activity in marine brown algae. Purification and characterisation of a vanadium (V) containing bromoperoxidase from Laminaria saccharina. Biochim. Biophys. Acta 872, 104-115. De Boer, E., van Kooyk, Y., Tromp, M. G. M., Plat, H., and Wever, R. (1986b). Bromoperoxidase from Ascophyllum nodosum: a novel class of enzymes containing vanadium as a prosthetic group? Biochim. Biophys. Acta 869, 48-53. De Boer, E., Plat, H., and Wever, R. (1987a). Algal vanadium (V)-bromoperoxidase, a halogenating enzyme retaining full activity in apolar solvent systems. Stud. Org. Chem. [Amsterdamj 29, 317-322.

94

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

De Boer, E., Plat, H., Tromp, M. G. M., Wever, R., Franssen, M. C. R., van der Plas, H. C., Meijer, E. M., and Schoemaker, H. E. (1987b). Vanadium containing bromoperoxidase, an example of an oxidoreductase with high operational stability in aqueous and organic media. Biotechnol. Bioeng. 30, 607-610. De Boer, E., Keijzers, C. P., Klaassen, A. A. K., Reijerse, E. J., Collison, D., Garner, C. D., and Wever, R. (1988a). 14N-coordination to VO* in reduced vanadium bromoperoxidase, an electron spin echo study. FEBS Lett. 235, 93-97. De Boer, E., Boon, K., and Wever, R. (1988b). Electron paramagnetic resonance studies on conformational states and metal-ion exchange properties of vanadium-bromoperoxidase. Biochemistry 27, 1629-1635. Dugad, L. B., La Mar, G., Lee, H. C., Ikeda-Saito, M., Booth, K. S., and Caughey, W. S. (1990). A Nuclear Overhauser Effect study on the active site of myeloperoxidase. Structural similarity of the prosthetic group to that of lactoperoxidase. J. Biol. Chem. 265, 7173-7179. Dugrillon, A., Bechtner, G . , Uedelhoven, W. M ., Weber, P. C., and Gartner, R. (1990). Evidence that an iodolactone mediates the inhibitory effect of iodide on thyroid cell proliferation but not on adenosine 3’3’-monophosphate formation. Endocrinology (Baltimore] 127, 337-343. Dull, T.J., Uyeda, C., Strosberg, A. D., Nedwin, G., and Seilhamer, J. J. (1990). Molecular cloning of cDNAs encoding bovine and human lactoperoxidase. DNA Cell Biol. 9, 499-509. Dunford, H. B., Lambeir, A. M., Kashem, M. A., and Pickard, M. (1987). On the mechanism of chlorination by chloroperoxidase. Arch. Biochem. Biophys. 252, 292-302. Engvild, K. C. (1986). Chlorine-containing natural compounds in higher plants. Phytochemistry 25, 781-791. Everett, R. R., Kanofski, J. R., and Butler, A. (1990a). Mechanistic investigations of the novel non-heme vanadium bromoperoxidases. Evidence for singlet oxygen production. J. Biol. Chem. 265, 4908-4914. Everett, R. R., Soedjak, H. S., and Butler, A. (1990b). Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination. I. Biol. Chem. 265, 15671-15679. Fang, G. H., Kenigsberg, P., Axley, M. J., Nuell, M., and Hager, L. P. (1986). Cloning and sequencing of chloroperoxidase cDNA. Nucleic Acids Res. 14,8061-8071. Faulkner, D. J. (1986). Marine natural products. Nat. Prod. Rep. 3, 1-33. Faulkner, D. J. (1990). Marine natural products. Nat. Prod. Rep. 7, 269-310. Fenna, R. E. (1987). Crystallization and subunit structure of canine myeloperoxidase. J. MoJ. Biol. 196,919-925. Franssen, M. C. R. (1987). Studies on the use of haloperoxidases in organic synthesis. Ph.D. Thesis, PUDOC, Wageningen, Netherlands. Franssen, M. C. R., and van der Plas, H. C. (1987). The chlorination of barbituric acid and some of its derivatives by chloroperoxidase. Bioorg. Chem. 15,59-70. Franssen, M. C. R., van Boven, H. G., and van der Plas, H. C. (1987a).Enzymatic halogenation of pyrazoles and pyridine derivatives. J. Heterocycl. Chem. 24, 1313-1316. Franssen, M. C. R., Weijnen, J. G. J., Vincken, J. P., Laane, C., and van der Plas, H. C. (1987b). Haloperoxidases in reversed micelles: use in organic synthesis and optimization of the system. Stud. Org. Chem. [Amsterdam) 29, 289-294. Franssen, M. C. R., Jansma, J. D., van der Plas, H. C., de Boer, E., and Wever, R. (1988a). Enzymatic bromination of barbituric acid and some of its derivatives. Bioorg. Chem. 16, 352-363. Franssen, M. C. R., Posthumus, M. A., and van der Plas, H. C. (1988b). New halometabolites of Caldoriomyces fumago. Phytochemistry 27, 1093-1096. +

HALOPEROXIDASES

95

Franssen, M. C. R., Weijnen, J. G. J., Vincken, J. P., Laane, C., and van der Plas, H. C. (1988~).Chloroperoxidase-catalyzed halogenation of apolar compounds using reversed micelles. Biocatalysis 2, 205-216. Fukuzawa, A., Aye, M., Nakamura, M., Tamura, M., and Murai, A. (1990a). Biosynthetic formation of cyclic bromo-ethers initiated by lactoperoxidase. Chem. Lett. pp. 12871290. Fukuzawa, A,, Aye, M., and Murai, A. (1990b). A direct enzymatic synthesis of laurencin from laurediol. Chem. Lett. pp. 1579-1580. Fukuzawa, A,, Aye, M., Nakamura, M., Tamura, M., and Murai, A. (1990~).Structure elucidation of laureoxanyne, a new nonisoprenoid CI5 enyne, using lactoperoxidase. Tetrahedron Lett., 31, 4895-4898. Gonzalez-Vergara, E., Ales, D. C., and Goff, H. M. (1985). A simple, rapid, high yield isolation and purification procedure for chloroperoxidase isoenzymes. Prep. Biochem. 15, 335-348. Gschwend, P. M., MacFarlane, J. K., and Newman, K. A. (1985). Volatile organic halogenated organic compounds released to seawater from temperate marine macroalgae. Science 227, 1033-1035. Harrison, J. E., and Schulz, J. (1976). Studies on the chlorinating activity of myeloperoxidase. J. Biol. Chem. 251, 1371-1374. Harvey, P. J., Schoemaker, H. E., Bowen, R. M., and Palmer, J. M. (1985). Single-electron transfer processes and the reaction mechanism of enzymatic degradation of lignin. FEBS Lett. 183, 13-16. Hormes, J., Kutgens, U., Chauvistre, R., Schreiber, W., Anders, N., Vilter, H., Rehder, D., and Weidemann, C. (1988). Vanadium K-edge absorption spectrum of bromoperoxidase from Ascophyllum nodosum. Biochim. Biophys. Acta 956,293-299. Hunter, J. C., Belt, A., Sotos, L. S . , Fonda, M. E., Geigert, J., and Liu, T. E. (1986). Fungal chloroperoxidase and method. Can. Pat. 3,212,061. Hynninen, P. H., and Lotjonen, S . (1981). Electrophilic substitution at the &methine bridge of pheophorbicide a and a’. Tetrahedron Lett. pp. 1845-1846. Itahara, T., and Ide, N. (1987). Chloroperoxidase-catalyzed halogenation of pyrimidine bases. Chem. Lett. pp. 2311-2312. Ito, S . , Yamada, H., and Izumi, Y. (1987). Enzymic halogenation of organic compounds with bromoperoxidase. Jpn. Pat. 88,196,295; C.A. 110, 191280 (1989). Itoh, N., Izumi, Y., and Yamada, H. (1985). Purification of bromoperoxidase from Corallina pilulifera. Biochem. Biophys. Res. Commun. 131,428-435. Itoh, N., Izumi, Y., and Yamada, H. (1986). Characterization of nonheme type bromoperoxidase in Corallina pilulifera. J. Biol. Chem. 261, 5194-5200. Itoh, N., Izumi, Y., and Yamada, H. (1987a). Characterization of nonheme iron and reaction mechanism of bromoperoxidase in Corallina pilulifera. J. Biol. Chem. 262, 11982-11987. Itoh, N., Izumi, Y., and Yamada, H. (1987b). Haloperoxidase-catalyzed halogenation of nitrogen-containing aromatic heterocycles represented by nucleic bases. Biochemistry 26, 282-289. Itoh, N., Cheng, L. Y.,Izumi, Y.,and Yamada, H. (1987~). Immobilized bromoperoxidase of Corallina pilulifera as a multifunctional halogenating biocatalyst. J. Biotechnol. 5, 29-38. Itoh, N., Hasan, A. K. M. Q.. Izumi, Y., and Yamada, H. (1987d).Immunological properties of bromoperoxidases in Coralline algae. Biochem. Int. 15, 27-33. Itoh, N., Hasan, A. K. M. Q., Izumi, Y., and Yamada, H. (1988). Substrate specificity, regiospecificity and stereospecificity of halogenations reactions catalyzed by nonheme type bromoperoxidase of Corallina pilulifera. Eur. J. Biochem. 172,477-484.

96

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

Jannun, R., and Coe, E. L. (1987). Bromoperoxidase from the marine snail, Murex trunculus. Comp. Biochem. Physiol., B: Comp. Biochem. 88B, 917-922. Kadima, T. A., and Pickard, M. A. (1990). Immobilization of chloroperoxidase on aminopropyl-glass. Appl. Environ. Microbiol. 56, 3473-3477. Kanofski, J. R. (1984). Singlet oxygen production by chloroperoxidase-hydrogen peroxide-halide systems. J. Biol. Chem. 259, 5596-5600. Kanofski, J. R. (1989). Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen. Arch. Biochem. Biophys. 274, 229-234. Kenigsberg, P., Fang, G. H., and Hager, L. P. (1987). Post-translational modifications of chloroperoxidase from Caldariomyces fumago. Arch. Biochem. Biophys. 254, 409415.

Kettle, A. J., and Winterbourn, C. C. (1988). The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon. Biochem. Biophys. Acta 957, 185-191, Kimura, S . , Kotani, T., McBride, 0. W., Umeki, K., Hirai, K., Nakayama, T., and Ohtaki, S. (1987). Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNA’s. Proc. Natl. Acad. Sci. U.S.A. 84, 5555-5559. Knoch, M., van Pbe, K-H., Vining, L. C., and Lingens, F. (1989). Purification, properties and immunological detection of a bromoperoxidase-catalase from Streptomyces venezuelae and from a chloramphenicol-nonproducing mutant. J. Gen. Microbiol. 135, 2493-2502.

Kohn, J., and Wilchek, M. (1982). A new approach (cyano-transfer) for cyanogen bromide activation of Sepharose at neutral pH, which yields activated resins, free of interfering nitrogen derivatives. Biochem. Biophys. Res. Commun. 107, 878-884. Krenn, B. E., Plat, H., and Wever, R. (1987). The bromoperoxidase from the red alga Ceramium rubrum also contains vanadium as a prosthetic group. Biochim. Biophys. Acta 912, 287-291. Krenn, B. E., Plat, H., and Wever, R. (1988). Purification and some characteristics of anonhaem bromoperoxidase from Streptomyces aureofaciens. Biochim. Biophys. Acto 952, 255-260.

Krenn, B. E., Izumi, Y., Yamada, H., and Wever, R. (1989a). A comparison of different (vanadium) bromoperoxidases: the bromoperoxidase from Corallina pilulifera is also a vanadium enzyme. Biochim. Biophys. Acta 998, 63-68. Krenn, B. E., Tromp, M. G. M., and Wever, R. (1989b). The brown alga Ascophyllum nodosum contains two different vanadium bromoperoxidases. J. Biol. Chem. 264, 19287-19292.

Kuila, D., Tien, M., Fee, J. A,, and Ondrias, M. R. (1985). Resonance Raman spectra of extracellular ligninase: evidence for a heme active site similar to those of peroxidases. Biochemistry 24, 3394-3397. Laane, C., Pronk, W., Franssen, M., and Veeger, C. (1984). Use of a bioelectrochemical cell for the synthesis of (bio)chemicals. Enzyme Microb. Technol. 6, 165-168. Laane, C., Weijland, A., and Franssen, M. (1986). Bioelectrosynthesis of halogenated compounds using chloroperoxidase. Enzyme Microb. Technol. 8, 345-348. Labat, G., and Meunier, B. (1990). First example of a chloroperoxidase-type chlorination of dimedone using a supported manganese porphyrin catalyst. 1. Chem. SOC.Chem. Commun. 1414-1416. Lambeir, A. M., and Dunford, H. B. (1983). A steady state kinetic analysis of the reaction of chloroperoxidase with peracetic acid, chloride, and 2-chlorodimedone. 1. Biol. Chem. 258, 13558-13563. Libby, R. D., and Rotberg, N. S. (1990). Compound 1 formation is a partially rate limiting

HALOPEROXIDASES

97

process in chloroperoxidase catalyzed bromination reactions. J. Biol. Chem. 265, 14808-14811.

Libby, R. D., Rotberg, N. S., Emerson, J. T., White, T. C., Yen, G. M., Friedman, S. H., Sun, N. S., and Goldowski, R. (1989).The chloride-activated peroxidation of catechol as a mechanistic probe of chloroperoxidase reactions: competitive activation as evidence for a catalytic chloride binding site on Compound I. J. Biol. Chem. 264,15284-15292. Liu, T. E.,M’Timkulu, T., Geigert, J., Wolf, B., Neidleman, S. L., Silva, D., and HunterCevera, J. C. (1987).Isolation and characterization of a novel nonheme chloroperoxidase. Biochem. Biophys. Res. Commun. 142,329-333. Magnusson, R. P., Gestautas, J., Taurog, A., and Rapoport, B. (1987).Molecular cloning of the structural gene for porcine thyroid peroxidase. J. Biol. Chem. 262,13885-13888. Manthey, J. A., and Hager, L. P. (1989).Characterization of the catalytical properties of bromoperoxidase. Biochemistry 28, 3052-3057. Millard, M. M. (1988).Lactoperoxidase-catalyzed iodination of plasma membrane lipids and proteins in Arabidopsis protoplasts. Plant Cell Physiol. 29, 901-905. Neidleman, S. L.,and Geigert, J. (1986).“Biohalogenation: Principles, Basic Roles and Applications.” Horwood, Chichester, England. Nichol, A. W., Angel, L. A., Moon, T., and Clezy, P. S. (1987).Lactoperoxidase haem, an iron-porphyrin thiol. Biochem. J. 247, 147-150. Nieder, M., and Hager, L. P. (1985).Conversion of &-aminoacids and peptides to nitriles and aldehydes by bromoperoxidase. Arch. Biochern. Biophys. 240, 121-127. Oeberg, L. G. (1987).Cetyltrimethylammonium ion stabilization of tribromide ion from peroxidase reaction. Acta Chem. Scand., Ser. B B41, 422-425. Paul, K. G., and Ohlsson, P. I. (1985).The chemical structure of lactoperoxidase. Immunol. Ser. 27, 15-29. Plat, H., Krenn, B. E., and Wever, R. (1987).The bromoperoxidase from the lichen Xanthorio parietina is a novel vanadium enzyme. Biochem. J. 248, 277-279. Pollak, A., Blumenfeld, H., Wax, M., Baughn, R. L., and Whitesides, G. M. (1980).Enzyme immobilization by condensation copolymerization into cross-linked pol yacrylamide gels. J. Am. Chem. SOC.102, 6324-6336. Rehder, D., Vilter, H., Duch, A., Priebsch, W., and Weidemann, C. (1987).A vanadate (V)dependent peroxidase from pigweed (Ascophyllum nodosum): the 51V NMR study of an unusual enzyme and simple vanadate-peptide systems. Recl. Trav. Chim. PaysBas 106,408. Remers, W. A. (1972).Properties and reactions of indoles. Chem. Heterocycl. Comp. 25, Part 1, 1-226. Renganathan, V.,Miki, K., and Gold, M. H. (1987).Haloperoxidase reactions catalyzed by lignin peroxidase, an extracellular enzyme from the basidiomycete Phanerochaete chrysosporium. Biochemistry 26, 5127-5 132. Sakai, R., Higa, T., Jefford,C. W., and Bernardinelli, G. (1986).The absolute configurations and biogenesis of some new halogenated chamigrenes from the sea hare Aplysia dactylomela. Helv. Chim. Acta 69,91-105. Sakamaki, K., Tomonaga, M., Tsukui, K., and Nagata, S. (1989).Molecular cloning and characterization of a chromosomal gene for human eosiniphil peroxidase. J. Biol. Chern. 264, 16828-16836. Sakurai, H., and Tsuchiya, K. (1990).A biomimetic model for vanadium-containing bromoperoxidase. FEBS Lett. 260, 109-112. Sayo, H., Hosokawa, M., Lee, E., and Kariya, K. (1988).Studies on the ethylhydroperoxide-supported oxidation of 1,4-diazabicyclo[2,2,2]octaneby chloroperoxidase. Chern. Pharm. Bull. 36. 2485-2489.

98

M. C. R. FRANSSEN AND H. C. VAN DER PLAS

Schoemaker, H. E., Harvey, P. J., Bowen, R. M., and Palmer, J. M. (1985). On the mechanism of enzymatic lignin breakdown. FEBS Lett. 183, 7-12. Senge, M., and Senger, H. (1989). Enzymic meso-chlorination of chlorophylls using chloroperoxidase. Biochim. Biophys. Acta 977, 177-186. Shaw, P. D., and Hager, L. P, (1959). An enzymatic chlorination reaction. J. Am. Chem. SOC.81, 1011-1012. Shiro, Y., and Morishima, I. (1986). Structural characterization of lactoperoxidase in the heme environment by proton NMR spectroscopy. Biochemistry 25, 5844-5849. Soedjak, H. S., and Butler, A. (1990a). Characterization of vanadium bromoperoxidase from Macrocystis and Fucus: reactivity of vanadium bromoperoxidase towards acyl and alkyl peroxides and bromination of amines. Biochemistry 29, 7974-7981. Soedjak, H. S., and Butler, A. (1990b). Chlorination catalyzed by vanadium bromoperoxidase. Inorg. Chem. 29, 5015-5017. Sono, M., Dawson, J. H., and Hager, L. P. (1986). Ligand and halide binding properties of chloroperoxidase: peroxidase type active site heme environment with cytochrome P450 type endogenous axial ligand and spectroscopic properties. Biochemistry 25, 347-356.

Sutton, B. J., Little, C., Olsen, R. L., and Willassen, N. P. (1988). Preliminary crystallographic analysis of human myeloperoxidase. J. Mol. Biol. 199, 395-396. Thanabal, V., De Ropp, J. S., and La Mar, G. N. (1987). Identification of the catalytically important amino acid residue resonances in ferric low spin horseradish peroxidase with Nuclear Overhauser Effect measurements. 1. Am. Chem. Sac. 109, 7516-7525. Tromp, M. G. M., Olafsson, G., Krenn, B. E., and Wever, R. (1990).Some structural studies of vanadium bromoperoxidase from Ascophyllum nodosum. Biochim. Biophys. Acta 1040, 192-198. Uetrecht, J,, and Zahid, N. (1988). N-chlorination of phenytoin by myeloperoxidase to a reactive metabolite. Chem. Res. Toxicol. 1, 148-151. Van Pee, K.-H. (1988). Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tu24. 1. Bacteriol. 170, 5890-5894. Van Pee, K.-H. (199Oa). Enzymology and genetics of halogenating enzymes from bacteria. Biocatalysis 4, 1-9. Van Pke, K.-H. (19gOb). Bacterial haloperoxidases and their role in secondary metabolism. Biotech. Adv. 8, 185-205. Van Pee, K.-H., and Lingens, F. (1985).Purification and molecular and catalytic properties of bromoperoxidase from Streptomyces phaeochromogenes. J. Gen. Microbiol. 131, 1911-1916.

Van Pee, K.-H., Sury, G., and Lingens, F. (1987). Purification and properties of a nonheme bromoperoxidase from Streptomyces aureofaciens. Biol. Chem. Hoppe-Seyler 368, 1225-1232.

Wannstedt, C., Rotella, D, and Siuda, J. F. (1990). Chloroperoxidase mediated halogenation of phenols. Bull. Environ. Contam. Toxicol. 44, 282-287. Weil, I., and Morris, J. C. (1949). Kinetic studies on the chloramines. I. The rates of formation of monochloramine, N-chlormethylamine and N-chlordimethylamine. J. Am. Chem. Soc. 71,1664-1671. Wever, R. (1988). Ozone destruction by algae in the arctic atmosphere. Nature (LondonJ 335, 501.

Wever, R., d e Boer, E., Plat, H., and Krenn, B. E. (1987). Vanadium-an element involved in the biosynthesis of halogenated compounds and nitrogen fixation. FEBS Lett. 216, 1-3.

Wever, R., Krenn, B. E.. de Boer, E., Offenberg, H., and Plat, H. (1988). Structure and

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function of vanadium-containing bromoperoxidases. Prog. Clin. Biol. Res. 274,477493.

Wiesner, W., van Pee, K.-H., and Lingens, F. (1985). Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia. Biol. Chem. Hoppe-Seyler 366, 10851091.

Wiesner, W., van PBe, K.-H., and Lingens, F. (1986). Detection of a new chloroperoxidase in Pseudomonas pyrrocinia. FEBS Lett. 209, 321-324. Wiesner, W., van Pee, K.-H., and Lingens, F. (1988). Purification and characterization of a novel bacterial non-heme chloroperoxidase from Pseudomonas pyrrocinia. J. Biol. Chem. 263, 13725-13732. Wiesner, W., Otto, M. K., and Kulbe, K. D. (1989). Continuous bromination of barbituric acid by chloroperoxidase from Pseudomonas pyrrocinia. Proc. DECHEMA Biotechnol. Conf. pp. 275-278; C.A. 113, 170466 (1990). Wiesner, W., Otto, M. K., and Kulbe, K. D. (1990). Bacterial enzymes in halogenation processes. Ann. N.Y. Acad. Sci. 589, 705-711. Wolfframm, C., van Pee, K.-H., and Lingens, F. (1988). Cloning and high-level expression of a chloroperoxidase gene from Pseudomonas pyrrocinia in Escherichia coli. FEBS Lett. 238, 325-328. Woodward, R. D., and Scaric, V. (1961). A new aspect of the chemistry of chlorins. J. Am. Chem. Soc. 83, 4676-4678. Yamada, H., Itoh, N., and Izumi, Y. (1985a). Chloroperoxidase-catalyzed halogenation of trans-cinnamic and its derivatives. J. Biol. Chem. 260, 11962-11969. Yamada, H., Itoh, N., Murakami, S., and Izumi, Y. (1985b). New bromoperoxidase from coralline algae that brominates phenol compounds. Agric. Biol. Chem. 49, 29612967.

Yu, H., and Whittaker, J. W. (1989). Vanadate activation of bromoperoxidase from Cora h a officinalis. Biochem. Biophys. Res. Commun. 160, 87-92. Zeiner, R., van PBe, K.-H., and Lingens, F. (1988).Purification and partial characterization of multiple bromoperoxidases from Streptomyces griseus. J. Gen. Microbiol. 134, 3141-3149.

Zgliczynski, J. M., Stelmaszynska, T., Domanski, J., and Ostrowski, W. (1971). Chloramines as intermediates of oxidation reactions of amino acids by myeloperoxidase. Biochim. Biophys. Acto 235, 419-424.

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Medicinal Benefits of the Mushroom Ganoderma

s. c. JONG AND J. M. BIRMINGHAM Mycology a n d Botany Department American 5 p e Culture CoIlection RockvilIe, Maryland 20852

I. Introduction 11. Chemical Composition 111. Medicinal Properties A. Antitumor Action B. Protein Synthesis, Nucleic Acid Synthesis Enhancer C. Nervous System Regulation D. Liver Protection and Detoxicant Action E. Cardiovascular System Regulation F. Respiratory System Action G . Immunomodulatory Action H. Muscular Dystrophy Studies I. Radiation Protection J. Toxicity Experiments IV. Patented Products and Processes A. Antitumor Preparations B. Liver Function Stimulants C. Hypotensive Preparations D. Hypocholesterolemic Preparations E. Hypoglycemic Preparations F. Chronic Bronchitis Treatment G . Immunomodulatory Agents H. Alzheimer’s Disease Treatment I. Antibiotic-Bacteriolytic Enzyme J. Antimutagen K. Skin Preparation L. Hair Tonics M. Bath Preparation N. Beverages V. Conclusions References

1. Introduction

Since the earliest times, mushrooms have been treated as a special food. They have been widely consumed not only for their taste, texture, and nutritious composition, but also for their claimed therapeutic value. The practice of using fungi as medicines is found in the traditions of many cultures, past and present. The first Chinese book on medicinal 101 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 37 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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substances, the Shen Nung’s Herbal from 2000 years ago, recorded the beneficial effects of various fungi. In his Compendium of Materia Medica, Li Shih-chen of the Ming Dynasty listed more than 20 species of medicinal fungi (Ying et a ] . , 1987). It was not until this century, when antibiotics were obtained from Penicillium, that the medicinal value of fungi first gained worldwide attention. It is now well documented that the major fungal groups produce antibiotic substances, and quite a number have been shown to possess antitumor activities and other pharmacodynamic properties (Jong and Donovick, 1989; Jong et a]., 1991). Basidiomycetes include many of the familiar larger fleshy mushrooms. Most are saprophytes, causing decay of litter, wood, or dung; some are plant pathogens. While the fleshy mushrooms have a reputation for being poisonous, the majority are harmless and several species are edible. The Ganodermataceae family includes about 40 similar species with hard basidiocarps (fruit bodies) in red, white, yellow, purple, or other colors. In Chinese folklore the fruit body of Ganoderma (Lingzhi) has been regarded as a panacea for all types of diseases. This is probably due to its demonstrated efficacy as a popular remedy to treat hepatopathy, chronic hepatitis, nephritis, hypertension, hyperlipemia, arthritis, neurasthenia, insomnia, bronchitis, asthma, gastric ulcer, arteriosclerosis, leukopenia, diabetes, anorexia, mushroom poisoning, and debility due to prolonged illness (Liu et al., 1979a; Liu and Bau, 1980; Chen, 1986; Ying et al., 1987; Kabir et al., 1988; Kimura et al., 1988; Willard, 1990). As the yield of wild Ganoderma is very low, only the rich in old China could afford it. Today, Ganoderma is being produced on a large scale for pharmacological and clinical studies by artificial cultivation and submerged fermentation (Liu et al., 1979a; Zhang, 1980). Species that are used for fermentation purposes are G. lucidum, G. japonicum, G. capense, G. applanatum, G. boninense, and G. tsugae.

I t . Chemical Composition The fruit body of G. lucidum contains ergosterol, fungal lysozyme, and acid protease. Soluble proteins, amino acids, polypeptides, and saccharides have been isolated from the aqueous extract of the sclerotium. The mycelium and the filtrate of deep culture have sterols, lactones, alkaloids, and polysaccharides. Ergosterol, organic acids, glucosamine, and polysaccharides are found in the fruit body of G. japonicum. Adenine, adenosine, uracil, uridine, and D-mannitol have been isolated from the aqueous extract of the mycelia of G. capense (Chen, 1986).

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FIG. 1. Lanostane triterpenoid skeleton (adapted from Lin et al., 1988).

Of particular interest is the fact that Ganoderma is a rich source of bitter triterpenes. Bitter taste has long been associated with some of its therapeutic properties. Currently, about 100 different triterpenoids (Fig. I) are known to occur in G. lucidum alone. The vast majority are ganoderic [C3J and lucidenic (CZ7)acids (Fig. 2) (Nishitoba et al., 1984), but ganodermic acids, ganoderenic acids, lucidone, ganoderal, and ganoderols are also present. The triterpenes can vary from strain to strain and from one growth stage of the fungus to another through side-chain cleavage and skeleton oxidation (Nishitoba et al., 1987a,b). The ganoderic acids can be divided into three types according to their location (Hirotani and Furuya, 1990). Ganoderic acids A, B, and H (type I) were detected only in the fruit body, whereas ganoderic acids R, S , and T (type 111) were the major triterpenoids of the mycelium. Miyahara et al. (1987) found that the triterpene content increased after the appearance of the fruit bodies and was more concentrated in the outer or older sections. Significant amounts of triterpenes can be obtained from both fruit bodies and the mycelial mat of static liquid culture, but the yield from mycelial pellets of shaking liquid culture is very poor. Extraction is usually by means of methanol, ethanol, acetone, chloroform, ether, or a

FIG. 2. C2, and C,, terpenoids (adapted from Nishitoba et al., 1984).

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Me

FIG. 3. Ganoderic acids A (I) and B (11). I: RR1 R3 = H; 11: R = OH, R1 = H, R2R3 =O (Kubota et ol., 1982).

=

0, R2

=

OH,

mixture of these solvents. Depending on whether they contain acidic or nonacidic triterpenoids, the extracts can be further purified according to different methods (Su, 1991). Ganoderic acids A and B from G. Iucidum (Fig. 3) were first described by Kubota et al. (1982),who characterized ganoderic acid A as a novel highly oxidized triterpene bearing a boat-shaped A-ring of lanostane. Toth et al. (1983a,b) isolated ganoderic acids T through Z from G. Iucidum. They determined that T and Z contained the same terminally carboxylated side chain and displayed cytotoxic activity in vitro on hepatoma cells. Much of the investigative work on terpenoid compounds has been conducted by Nishitoba and co-workers. They isolated new lucidenic acids A (Fig. 4), B, and C, ganoderic acids C and J, along with known ganoderic acid B and ganolucidic acid C from G . Iucidum (Nishitoba et al. 1985b,c). The last was unique in having a hydroxyl group at C-29. In

0 Me Me FIG. 4. Lucidenic acids A (I) and D (11). I: R et al., 1985a,b).

= H, R1 =

OH; 11: R = 0,R1 = 0 (Nishitoba

MEDICINAL BENEFITS OF THE MUSHROOM GANODERMA

105

Ac

Me

FIG. 5. Lucidone A (Nishitoba et al., 1985a).

addition, they found two new bitter C,, terpenoids, lucidenic acids D (Fig. 4) and E; a C,, terpenoid, lucidone A (Fig. 5); nonbitter C,, ganoderic acid D; and nonbitter C,, lucidone B (Nishitoba et al., 1985a). The intensity of bitterness was determined as follows: lucidenic acid D > ganoderic acid C > lucidone A > lucidenic acid A %ganoderic acid B > lucidenic acids B, C, E. Nishitoba et al. (1986) then extracted four new terpenoids from the fruit body of G. lucidurn. Ganolucidic acid D, with an allylic alcohol group in the side chain, is a possible intermediate between the mycelial components and terpenoids of the fruit body. Ganoderic acid L, with a hydroxyl on C-20,is a possible precursor of lucidone C. The others were lucidone C and lucidenic acid G, unique in having a hydroxyl group at C-26.They isolated novel ganoderic acids Ma, Mb, Mc, Md, Me, Mf, Mg, Mh, Mi, Mj, and Mk from the mycelial mat of G. lucidum and obtained ganoderic acids W, R, and T (Nishitoba et al., 1987a,b).Moreover, Nishitoba et al. ( 1 9 8 7 ~ )described the structure of the novel minor component ganoderic acids M, N, and 0,ganoderenic acid E, and lucidenic acids H, I, J, K, L, and M, along with identification of ganoderic acids H (previously reported as C or D) and K, compound B9, and lucidenic acid E, (previously reported as E). All were isolated from the fruit body of G. lucidum. Nishitoba et al. (1988a) documented the structure of four novel C,, triterpenoids from the fruit body of G. lucidum: epoxyganoderol A, B, and C , and ganoderal B, along with the known ganoderol B and 6phydroxy-ergosta-4,7,22-trien-3-one and the novel steroid 6n-hydroxyergosta-4,7,22-trien-3-one. The absolute configuration of the a$-epoxy alcohol moiety of the first three was demonstrated. In addition, Nishitoba et al. (1988b) isolated ganoderiols C, D, E, F, G, H, and I, and ganolucidic acid E and determined the configuration at C-23 of ganolucidic acid D. They found some intensely bitter compounds: lu-

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S . C. JONG AND J. M. BIRMINGHAM

Me

FIG. 6. Ganolucidic acids A (I) and B (11). I: RR' al., 1 9 8 6 ~ ) .

=

0; If: R

=

H, R*

=

OH (Kikuchi et

cidenic acid A, ganoderic acid A, ganoderic acid C,, ganoderic acid J, lucidone A, and lucidone C and resolved that the spatial relationship of the hydrophobic methyl groups to the three functional oxygen atoms plays an important role in generating the bitterness (Nishitoba et a]., 1988~). Working with the fruit body of G. lucidurn, Hirotani et al. (1985) isolated known ganoderic acids A and B and elucidated the structure of ganoderic acid C. Kikuchi et al. (1985a,b, 1986a,b,c) extracted the new triterpenoid ganoderic acids C2, D, E, F, G, H, I, and K, lucidenic acids D, E, and F, and ganolucidic acids A and B (Fig. 6) from the dried fruit body of G. lucidurn. Eight terpenoid constituents were isolated from the dried fruit bodies of G. lucidum by Komoda et al. (1985), who identified them as ganoderenic acids A, B (Fig. 7), C, and E, ganoderic acids D, F, and G, and lucidenic acid D. Hirotani and Furuya (1986) reported on the isolation of the ganoderic acid C and two new ganoderic acids E and F from

Me

FIG. 7. Ganoderenic acids A (I] and B (If). I: RR' 11: R = OH, R1 = H, R2R3 = 0 (Komoda et al., 1985).

=

0, R2 = H, R3 = OH;

MEDICINAL BENEFITS OF THE MUSHROOM GANODERMA

107

'Ci0H)Me

0

Me FIG. 8. Ganodermadiol (I) and ganodermatriol (11). I: R al., 1986).

=

Me; 11: R

=

CH20H (Fujita et

G. lucidum. They established that the triterpene acids of the differentiated fruit body, such as ganoderic acids B and D, have the 3P-hydroxyl group, whereas the main triterpene components in the cultured mycelia were 3a-hydroxytriterpene acids, Two new lanostanoids, ganodermadiol and ganodermatriol (Fig. 8), were isolated from the fruit body of G. lucidum by Fujita et al. (1986). while Sat0 et al. (1986) identified ganoderiols A and B (Fig. 9). Hirotani et al. (1987) determined that six new ganoderic acid derivatives, 0, P, Q, K, S, and T, identified as major or minor triterpenoid components from the cultured mycelium of G. lucidurn, have 3a- substitutes. In contrast, the ganoderic acid derivatives from the fruit bodies are 3P-substituted or 8-keto compounds. H

Me FIG. 9. Ganoderiols A (Ij and B (11).I: R' = RZ = R" = R7 = H, R = R3 = R4 = R5 = OH; 11: RR' = 0, R2 = R5 = R6 = OH, R3R4 = unsaturated bond, R7 = H (Sato et al., 1986).

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Nishitoba et al. [1989) also investigated the bitter constituents of the fruit body of G. applanatum. In addition to previously known alnusenone and friedelin, they defined the structure of ganoderenic acids F, G, H, and I, furanoganoderic acid, and ganoderic acid AP and identified ganoderenic acid A and compound B8. Ganoderenic acids G and A and furanoganoderic acid are distinguished by intense bitterness. Ill. Medicinal Properties

Although Chinese medicines have long been used clinically, in most cases the chemical basis for their therapeutic action has not been understood. It is widely held that the pharmacological effect of Lingzhi depends on its color. Actually, Lingzhi is not a single medicine but possesses different properties depending on the stage and species of Ganoderma and the environment in which it has grown. In the past few decades, scientists have conducted extensive research to explore further and possibly exploit the claimed therapeutic effects of Ganoderma. Over 100 reports have been published concerning the chemical constituents of G. lucidum and the physiologically active new compounds isolated from its fruit body and mycelium with the help of chromatography, spectrochemistry, and bioassay systems (Su, 1991). A. ANTITUMOR ACTION High-molecular-weight polysaccharides from the cell walls of fungi have been found to stimulate both nonspecific host resistance and specific immunological reactivity and to exert inhibitory effects against transplantable tumors [Badger, 1984). Much of this experimental work has relied on a bioassay system that normally uses sarcoma 180 in mice based on an allogeneic, tumor-graft rejection mechanism [Whistler et al., 1976). The polysaccharides of Ganoderma comprise one of the major sources of its physiologically active compounds and are extracted from the dried fruit body or separated from the broth of a shaking liquid culture. Analysis of the purified polysaccharide utilizes acid hydrolysis to determine monosaccharide constitution, enzyme hydrolysis to detect anomeric structure, methylation and Smith degradation to elucidate linkage type, and gas chromatography or mass spectrometry with NMR information to ascertain structure [Mizuno and Hazama, 1986). The molecular weight of the polysaccharide is indefinite, but generally ranges from 4 x lo5 to 1 x 106 in the primary structure (Su, 1991),SO it is usually described in terms of the repeat unit.

MEDICINAL BENEFITS OF THE MUSHROOM GANODERMA

109

Sasaki et al. (1971) extracted an antitumor polysaccharide (G-Z)with hot water from G. applanatum and tested it against subcutaneously implanted sarcoma 180. G-Z is considered to be a glucan, as the hydrolysate consisted of a partial mixture of p-(1-+3)? and p-(1-+4)-linkedDglucose residues. Four kinds of polysaccharide preparations from the fruit body of G. lucidum were tested for antitumor activity against sarcoma 180 in mice by Ito et al. (1977). They proved highly effective and nontoxic. The active principle seemed to be a polysaccharide combined with a small amount of protein. Matsumoto et al. (1978) studied the effect of an alcohol-extracted polysaccharide mainly from G. applanatum. They found it ineffectual against Ehrlich's ascites tumors in mice, but it did inhibit the growth of Ehrlich's subcutaneous tumors by 60%. From the effects of the extract on cellular respiratory metabolism, the investigators inferred that the antitumor activity of the polysaccharide might be mediated by its immunological actions and effect on host function. Kim et al. (1980) extracted a dark brown powder from the fruit bodies of G. lucidurn which exhibited antitumor activity against sarcoma 180 in mice. The antitumor fraction consisted of a polysaccharide-protein complex in which 4 monosaccharides and 18 amino acids were identified. Sixty-one polysaccharides were screened by Mizuno et al. (1980) for host-mediated antitumor activity against sarcoma 180 in mice. A p-Dglucan from the fruit body of G. applanatum showed significant tumor inhibition. Usui et al. (1981) isolated water-soluble P-D-glucans from the fruit body of G. applanatum with a low protein content. They determined that the glucans had a backbone of p-(1-+3)-linkedD-glucopyranosyl residues, of which 15% were substituted in position 6 by D-glucopyranose. Solubility in water seemed to play an important role in the antitumor activity, and there was an optimum molecular size for the glucan to be effective. The two polysaccharides had molecular weights of 3.12 x 105 and 1.05 X 10"; the smaller had the greater activity. Later structural investigations (Usui et al., 1983) indicated the glucans were highly branched polysaccharides containing a framework of p-(1 4 3 ) linked D-glucopyranosyl residues, substituted at 0-6 in every third residue with single branches; (1-4) interresidue linkages were absent. A water-soluble antitumor polysaccharide (GL1) with a molecular weight of 4 x 104 was isolated by Miyasaki and Nishijima (1981) from the fruit bodies of G. lucidurn. They determined that GL1 is a branched arabinoxyloglucan that strongly inhibited the growth of sarcoma 180

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S. C. JONG AND 1. M. BlRMINGHAM

solid-type tumors in mice when injected intraperitoneally. The essential structure for the antitumor activity appeared to be a branched glucan core involving p-(1+3), B-(1+4), and p-(1-+6) linkages. Mizuno et al. (1981) obtained antitumor active P-D-glucans, an inactive P-D-glucan, an a-D-glucan, and heterogalactans from fractionation of water-soluble homoglucans isolated from the fruit body of G. applanatum. The three p-o-glucans were composed of a linear P-(1+3)-linked D-glucopyranosyl backbone having a single p-(1-+6)-glucopyranoside branch for every 3-5 p-(1+3)-glucopyranoside linear linkages. Their molecular weights were 1.05 x lo", 3.12 x lo5, and 2.43 x lo3, respectively. The first two markedly inhibited the growth of sarcoma 180 in mice; the third did not show any antitumor activity. Kang et a]., (1981) isolated a protein-bound polysaccharide from G. lucidum composed of galactose, glucose, xylose, and 16 amino acids that suppressed sarcoma 180 in A-strain mice. A water-soluble neutral heteroglycan was extracted by Miyazaki and Nishijima (1982) from the fruit body of G. Iucidum. The polysaccharide was 97% carbohydrate and lacked N and P, with a molecular weight of 3 x lo4. The investigators proposed that the core portion consists of a (1+4)-linked mannopyranosyl main chain, that fucosyl residues are located as terminal positions of the side chains, and that each xylopyranosyl residue is linked directly to mannopyranosyl and fucopyranosyl residues. Mizuno et al. (1982) isolated and characterized the glucans from a hot-water extract of mycelial cells of G. applanatum. The a-glucan fractions had a linear a-(1+4)-glucoside backbone with a branch every 9-12 residues of the backbone through a-(l+6) bonding. They showed little antitumor activity. The P-glucan fractions had a linear p-(1+3)glucoside backbone with a monoglycosyl branch every 1 2 residues of the backbone through P-(l+6) bonding. One fraction showed a marked antitumor activity against sarcoma 180 in mice. The minimum common unit of antitumor active glucans of various basidiomycetes, including G. lucidurn, was determined by Miyazaki (1983) to be a C-6 branched ~-(1+3)-~-glucopyranosyl residue. Branching frequency seemed to be important for antitumor activity. Ukai et al. (1983b) investigated the antitumor activity of a waterinsoluble glucan (G-A) from the fruit bodies of G. japonicum against sarcoma 180 in mice by intraperitoneal administration. The glucan G-A consisted of a main chain of p-(1-+3)-linked D-glucosyl residues with side chains of single D-glucosyl units attached by P-(1+6) linkage to the main chain.

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111

Mizuno et aI. (1984) isolated five fractions from the water-soluble polysaccharide from the fruit body of G. Iucidum: a- and P-glucans, fucogalactan, mannofucogalactan, and acidic P-glucan. The P-glucan and the acidic P-glucan showed antitumor activity in the growth of sarcoma 180 in mice. The former was a P-(l-+3)-~-glucan with P-(136) branching and a molecular weight of 1.05 x lo6. The latter had a molecular weight of 4.50 X lo5 and was composed of D-glucose, Dmannose, D-glucuronic acid, D-galactose, and D-xylose. Several P-D-glucans with different degrees of branching isolated by Sone et al. (1985) from water and alkali extracts of the fruit body of G. Iucidum had host-mediated antitumor activity against sarcoma 180 solid tumor in mice. The purified glucans that were mostly water-insoluble had a backbone of P-( 1+3)-linked D-glucose residues, attached mainly with single D-ghIcOsy1 units at 0-6, and also with a few short P-(l+$)-linked glucosyl units at 0 - 2 positions. However, their degree of branching appeared to differ, depending on the extracted glucan fractions. In addition to the P-glucans, the fruit body contained watersoluble heteropolysaccharides, comprising D-glucose, D-galactose, D-mannose, L (or D)-arabinose, D-xylose, and L-fucose. A branched B-(l-+3)-~-glucan was also isolated from the culture filtrate of G. Iucidum. The extracellular P-D-glucan was less soluble in water after purification, but soluble in dilute alkali. This glucan was found to have essentially the same structure as that of hot-water-extracted polysaccharide from the fruit body. Both showed relatively high growth-inhibition activities against sarcoma 180 solid tumor in mice when administered by successive intraperitoneal injections. The mode of the branching affected the antitumor activity, and the attachment of polyol groups to the (1*3)-linked backbone significantly enhanced its hostmediated antitumor effect. Mizuno and Hazama (1986) conducted studies on four fibrous (noncellulose) polysaccharides isolated from the fruit body of G. lucidurn. They were composed of glucose with small amounts of uronic acid, xylose, and mannose and consisted of P-(1+3)-~-glucanwith 6-(1+6)glucosyl branching, with average molecular weights of 3.3 x 105, 6.0 x lo4, 1.6 X lo5, and 1.1 X lo5, respectively. The polysaccharides demonstrated host-mediated antitumor activity against sarcoma 180 in mice on intraperitoneal administration. Kishida et al. (1988) obtained several branched P-(l+3)-D-glUCanS from the fruit body of G. lucidurn by successive extractions. These glucans contained a backbone of (1+3]-linked D-glucosyl residues attached mainly with single D-glucosyl groups at 0 - 6 and also a few short

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chains of (1-+4)-linked glucose units at 0 - 2 . The dimethyl sulfoxide (DMS0)-extracted P-glucan had somewhat longer side chains of (1-61linked D-glucosyl units than other P-D-glucans. Degrees of branching of the glucans appeared to differ, depending on whether hot water or hot alkali was used for extraction. In addition to the glucans, the fruit body contained water-soluble heteropolysaccharides comprising D-glUCOSe, D-galactose, D-mannose, D-xylose, D (or L)-arabinose, and L-fructose. The hot-alkali and DMSO-extraction residue, probably originating from its innermost core, consisted of chitin, (3-glucan,and a small proportion of peptides. The hot-water-extractable glucan showed relatively high inhibitory activity on the growth of sarcoma 180 solid tumor implanted intraperitoneally in mice. However, the other (3-(1-+3)-D-glucans showed no or lower antitumor activity. Modification of D-glucosyl groups of side chains to polyol groups significantly enhanced its tumorinhibiting activity. Maruyama et al. (1989) tested various fractions of methanol and aqueous extracts of the fruit body of G. lucidum for antitumor activity against solid type of sarcoma 180 by intraperitoneal administration. The methanol fraction had no activity. The aqueous fraction with a molecular weight >1 x lo4 had high inhibitory activity, but that with a lower molecular weight did not. Chairul et al. (1990) isolated and elucidated the structure of two new homolanosteroid carboxyacetylquercinic acid derivatives from wild Ganoderma species of West Java and determined the stereochemistry at the C-17 side chain by X-ray crystallography. The antitumor activity of the neutral acids and their methyl esters was tested using an in vitro assay of Epstein-Barr virus activation. One of the isolates and its ester showed a remarkable inhibitory effect but became toxic at higher concentrations.

B. PROTEINSYNTHESIS, NUCLEICACIDSYNTHESIS ENHANCER Administration of polysaccharide D, from the fruit body of G. lucidum was found to increase serum, liver, and bone marrow protein synthesis in mice (Guan and Cong, 1982). D, also enhanced the incorporation of labeled uridine into liver RNA but not labeled thymidine into liver DNA. In contrast, treatment with the polysaccharide increased both RNA and DNA formation in the bone marrow where B cells are manufactured. The action of the polysaccharide D, lends support to the material basis for the multiple pharmacological activities attributed to Ganoderma.

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C. NERVOUS SYSTEM REGULATION

Since tonics in general may increase nonspecific body resistance, the results of Liu et al. (1979a) imply that the regulatory action of Ganoderma on the nervous system and liver function (considered in the next section) may contribute to the increase of nonspecific resistance. These investigators (Liu et al., 1979a,b) conducted pharmalogical studies on mice using the spores of G. lucidum and the mycelium of G. capense produced by submerged fermentation. Their results indicated the alcohol-water-soluble extracts of the spores and mycelium acted on the central nervous system and were essentially similar. The extracts decreased spontaneous motor activity in mice, prolonged barbital sleeping time, prevented nicotine-induced convulsions, and inhibited pilocarpine-induced salivary secretion. Both preparations also had anticholinergic action. Kasahara and Hikino (1987) found that extracts of G. lucidum show an analgesic activity in mice. The adenosine isolated from the fruit body inhibited central inhibitory-reduced spontaneous motor activity, elevated pain threshold, prolonged the death time induced by caffeine, and relaxed skeletal muscle in mice. D. LIVER-PROTECTION AND DETOXICANT ACTIONS An alcohol-ether-soluble fraction from the mycelium of G. capense produced by submerged fermentation has been shown to protect the liver and enhance its detoxifying function (Liu et al., 1979a,b). The fraction lowered the serum transaminase levels induced by CCl,, promoted the regeneration of the liver in partially hepatectomized mice, and increased the resistance of mice to the toxicity of indomethacin and digitoxin. It also decreased accumulation of triglycerides (Liu et al., 1979~).The action of a spore extract from G. lucidum was similar but weaker. Yu et al. (1981) examined the chemical constituents of the deep-layer fermentation mycelia of G. capense. Four furan derivatives and one water-soluble component, identified as nicotinic acid, were isolated from the mycelium, while the major fatty acids were palmitic, linoleic, and linolenic acids (Yu et al., 1983). Zhang et al. (1986) detected uridine, uracil, adenosine, and adenine in the submerged culture of G. capense. Using galactosamine-induced cytotoxity in primary-cultured rat hepatocytes, Hirotani et al. (1986) observed that ganoderic acids R and S are strongly antihepatotoxic.

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Byun and Kim (1987) investigated the administration of G. lucidum extract and the free radical-scavenging amino acid glutathione in limiting liver damage induced by CC1, in rats as indicated by blood transaminase levels, lipid peroxidation values, and histological findings. They found that concurrent administration of the extract and the acids was more effective than either one alone, particularly against liver necrosis and hepatitis.

E. CARDIOVASCULAR SYSTEM REGULATION 1. Effect on Cardiac Function

Concentrated G. lucidum culture extract has a significant cardiotonic action on isolated frog heart and the pentobarbital sodium-inhibited heart (Chen, 1986). Intraperitoneal injection of the tincture of G. lucidum or the alcohol extract of the mycelia increased contraction amplitude of the in situ rabbit heart, and the hot-alcohol extract produced a cardiotonic effect and bradycardia in anesthetized cats. The polysaccharide fraction of the aqueous solution of G. lucidum also has a significant cardiotonic action. Intravenous injection of the total alkaloids isolated from the culture extract of G. lucidum increased coronary flow and lowered coronary resistance and myocardial oxygen consumption in cats and dogs, which suggest changes in the coronary hemodynamics and myocardial metabolism (Chen, 1986). Coronary dilation was produced by the alcohol extract or crude alkaloids of natural or industrially cultured G. lucidum and of natural G. japonicum perfused into isolated guinea pig hearts. In normal mice, the intragastric administration of the tincture and the aqueous solution of G. lucidum or the oral or intraperitoneal administration of the concentrated culture extract markedly increased tolerance to hypobaric and normobaric hypoxia. 2. Hypotensive Activity

In the course of their research on antihypertensive substances, Morigiwa et al. (1986)found that a 70% methyl alcohol extract of G. lucidum exhibited an inhibitory activity on angiotensin-converting enzyme [ACE) prepared from hog kidney. From this extract they isolated and characterized five new lanostane triterpenes, ganoderal A (Fig. lo), ganoderols A and B, and ganoderic acids K and S, along with five known triterpenes, ganoderic acids Y, F, H, B, and D[C). Eight of the compounds were inhibitory, and ganoderic acid F had the greatest effect. Kabir et al. (1988) studied the effect of a powder prepared from the

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R'

Me

I

Me

Me

FIG. 10. Ganoderal A. R'

=

CHO, R Z = 0 (Morigiwa et ol., 1986).

cultured fruit body of G. lucidurn on the blood pressure of spontaneously hypertensive rats. After a 4-week feeding period, the systolic blood pressure of rats fed G. lucidurn was significantly lower than that of the control, indicating the powder contained some substance which suppresses the elevation of blood pressure. 3. Hypocholesterolernic Activity

To evaluate the effect of Ganoderma on cholesterol metabolism, Kabir et al. (1988)tested the effect of the mushroom powder on the lipid levels of spontaneously hypertensive rats. The plasma total cholesterol level of those fed Ganoderma was significantly lower than that of the control, whereas no significant difference in plasma-free cholesterol, triglyceride, and phospholipid levels was observed between the groups. The total liver cholesterol and triglyceride levels were significantly lower in the Ganoderma-fed rats when compared with the control. There was almost no difference in liver-free cholesterol between the two groups. The lower level in the test animals may be due to the inhibition in cholesterol synthesis and/or acceleration of cholesterol metabolism. While investigating the hypocholesterolemic constituents of G. lucidurn mycelium, Shiao et al. (1987,1988)reported on the isolation of ganodermic acids R, S (Fig. ll), 0, and Q together with previously known ganoderic acid X and ganoderic acid Mf. New triterpenoids were identified as ganodermic acids Ja, Jb, PI, and P2. The same group of investigators (Lin et al., 1988)named three new compounds, ganodermic acids T-N, T-0, and T-Q, and found that ganoderic acid Mf and ganodermic acid T-0 exhibited an inhibitory activity on cholesterol synthesis. Many oxygenated sterols have been found to be potent inhibitors of

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Me FIG. 11. Ganodermic acids R (I) and S (11). I: R = AcO, R1 = H; 11: R (Shiao et al., 1987).

=

H,

R1 =

AcO

sterol synthesis in animal cells in culture, and 7-0x0 or l5a-hydroxy groups may be very important in this regard. Oxygenated lanostanoid triterpenes, including ganoderic acid B and ganoderic acid C with oxygenated groups on both the 7- and 15- positions, have been isolated from G. lucidum. Using rat liver homogenate, Komoda et al. (1989) tested ganoderic acid B and derivatives of ganoderic acids B and C for inhibitory effects on cholesterol biosynthesis from lanosterol or 24,25dihydrolanosterol. One derivative, with no functional group in the side chain and having both 7-Ox0 and 15a-hydroxy groups on the same skeleton, showed potent inhibitory effect. The key placement of oxygen is common to the cholesterol biosynthesis-inhibiting derivatives. Li et al. [1989) examined the antiatherosclerotic potential of the alcohol and water extracts of 20 edible fungi using human cell culture. Ganoderma exhibited both antiatherogenic [prophylactic) and antiatherosclerotic (therapeutic) action, indicating it may be useful in the prevention and treatment of atherosclerosis. 4. Hypoglycemic Activity

Hikino et al. (1985) showed that a water extract of the dried fruit bodies of G. lucidum decreased plasma sugar level in normal and alloxan-induced hyperglycemic mice and isolated two homogeneous glycans, ganoderans A and B, as the active principles. The neutral sugar components were rhamnose, galactose, and glucose for ganoderan A and mannose and glucose for ganoderan B. Ganoderan B also contained galacturonic acid and glucuronic acid as acidic sugar components. The presence of acetoxyl groups in both ganoderans was also indicated. Ganoderan A had little or no peptide moiety; ganoderan B contained a fair amount. By intraperitoneal administration to normal mice, both

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mediated a hypoglycemic effect. Ganoderan A showed stronger activity than ganoderan B. Intraperitoneal administration to alloxan-hyperglycemic mice also reduced plasma glucose level. Tomoda et al. (1986) isolated two hypoglycemic peptidoglycans, ganoderans B and C, from the fruit bodies of G. lucidum. The molecular weights were 7.4 X lo3 and 5.8 X lo3. The presence of p-(1+6)-linked D-glucosyl chains in these ganoderans was unique as compared with the other glucans of the crude drug. When administered intraperitoneally, both reduced blood glucose concentration. Intraperitoneal injection of ganoderan C to alloxan-induced hyperglycemic mice also lowered the blood glucose level. Kimura et aJ. (1988) investigated the effects of water and alcohol extracts of the fruit body of G. lucidurn on blood glucose and insulin levels in rats by the oral glucose tolerance test. The water extract reduced elevation of blood glucose and insulin levels 10 minutes after oral glucose infusion, as compared to control rats, in which the levels were higher. On the other hand, plasma insulin remained at a high level 20 to 60 minutes after oral glucose infusion in rats administered with the water extract, as compared to control rats, where it dropped. Finally, the water extract reduced the elevation of blood glucose without elevating blood insulin after an intravenous infusion of adrenaline and oral infusion of glucose. Hikino et al. (1989) determined that ganoderan B is the major hypoglycemic glycan, and its structure was mainly composed of a p-(1+3)and p-(1+6)-linked D-glucopyranose moiety. Hypoglycemic activity is due to an increase of the plasma insulin level and an acceleration of the glucose metabolism. The enhancement of the glucose utilization is considered to occur not only in the peripheral tissues but also in the liver, as shown in the potentiations of the activities of hepatic key enzymes participating in the carbohydrate metabolisms. Antitumor heteroglycans were examined for hypoglycemic activity by Hikino and Mizuno (1989). Most exhibited hypoglycemic effects whose potencies were rather weak as compared with those of the ganoderans. Furthermore, the potencies of these hypoglycemic effects did not parallel the antitumor effects. Purification of the heteroglycans decreased the hypoglycemic activity, whereas the antitumor activity remained unchanged. 5. Platelet Aggregation Inhibition

In their work, Shimizu et al. (1985) demonstrated for the first time that the water-soluble fraction of the fruit body of G. lucidum suppressed platelet aggregation. The mechanism was not determined, but

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the inhibitor of platelet aggregation was shown to be adenosine, with a content of at least 40 mgi1OOO g of the dried fruit body preparation. Inhibition of platelet aggregation reduces the incidence of blood clots and stroke.

F. RESPIRATORYSYSTEM ACTION Chen (1986) reported that, in albino rats with experimentally induced chronic bronchitis, daily intragastric doses of a concentrated solution containing the mycelia of G. Iucidum induced a regeneration of the bronchial epithelium. In addition, intraperitoneal injection of the alcohol extract of the mycelium or the aqueous extract of G. lucidum had significant antitussive and expectorant actions on induced cough.

G. IMMUNOMODULATORY ACTION Nakashima et al. (1979) demonstrated that prior intraperitoneal or oral administration of a polysaccharide-rich fraction prepared from the fruit body of G. applanatum exerted an enhancing effect on the induction of delayed hypersensitivity to protein antigen, as measured by the foot pad reaction, and expanded the size of T cell memory for the immunoglobulin G (IgG) antibody response. IgG constitutes a major class of antibodies that enhance phagocytosis. It was thought that treatment with the appropriate dose of the extract activated nonspecific amplifier T cells. Kandefer-Szerszen et al. (1979) found that the nucleic acids of three fungi, including G. applanatum, induced small but detectable amounts of an interferon-like substance, as seen in the reduction of the number of vaccinia virus plaques in chick embryo fibroblast tissue culture. When administered intravenously to white mice, it protected them against lethal infection with tick-borne encephalitis virus strain K,. In vivo tests showed that of the three fungi tested, only the RNA from G. applanatum induced a substance showing interferon properties in the spleen of mice. RNA is found in the fruit body but in even higher levels in the mycelium (Kim and Nam, 1984). Xie et al. (1985) determined that a polysaccharide component (BN,C) from Ganoderma enhanced concanavalin A-induced murine T cell proliferation, but a two-agent combination with d-matrine decreased interleukin 2 (IL2) formation. The polysaccharide could inhibit T cell proliferation as well; B cells were less responsive to the combination. A novel protein with mitogenic activity in vitro and immu-

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nomodulating activity in vivo was isolated from the mycelial extract of G. lucidium by Kin0 et al. (1989). The protein Ling-Zhi-8 (LZ-8) was capable of hemagglutinating sheep red blood cells but not human red blood cells. In vivo, LZ-8 prevented the production of a systemic anaphylaxis reaction in mice, if it was administered repeatedly. Reduction of antibody production was the suggested mechanism. Tanaka et al. (1989) determined the complete structure of LZ-8 by the sequencing of 110 amino acid residues. Similarity to the variable region of immunoglobin heavy chain both in its sequence and in its predicted secondary structure was noted. The major biological activities of LZ-8 resembled lectins, with mitogenic capacity toward mouse spleen cells and human peripheral lymphocytes and agglutination of sheep red blood cells in vitro. Neither activity was inhibited by the mono- or dimeric sugars examined, indicating that LZ-8 is not a lectin per se. LZ-8 did not agglutinate human red blood cells and could function as a potent suppressor of bovine serum albumin-induced anaphylaxis in CFW mice in vitro. It appears to be related to an ancestral protein of the immunoglobulin superfamily. 1. Histamine-Release Inhibition Activity Kohda et al. (1985) investigated the inhibitory action of a methanol extract of G. lucidum fruit body on histamine release from rat mast cells induced by compound 48/80 and concanavalin A. The test used was a simple in vitro assay for antiinflammatory or antiallergic drugs. From the physiologically active fraction of the extract, two new triterpenes, named ganoderic acids C and D, were isolated, along with the known triterpenes ganoderic acids A and B. The results indicated that the newly identified compounds had distinct inhibitory activity on histamine release and are at least partly responsible for one of the pharmacological activities of the crude drug. Tasaka et al. (1988aj also identified active ingredients in a chloroform extract from G. lucidum broth which markedly inhibited histamine release from rat peritoneal mast cells induced by A-23187 and compound 48/80. Palmitic acid, stearic acid, oleic acid, and linoleic acid were isolated from the active fractions. It was concluded that one of the effective constituents was oleic acids, which induces membranestabilization in model membrane systems. Using the same method, Tasaka et al. (1988b) extracted cyclooctasulfur from the culture medium. They concluded that a disulfide exchange reaction probably takes place in the cell membrane, decreasing the Ca uptake from the extracellular medium and inhibiting histamine release from mast cells.

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2. Antiinflammatory Activity

Among the fungal polysaccharides tested by Ukai et al. (1983a) in two inflammatory models (carrageenin-induced edema and scald-induced hyperalgesia in hind paw of rats), a glucan from G. japonicum (G-A) was found to inhibit in the former model but not in the latter. None of the fungal polysaccharides tested affected pathways of arachidonic acid metabolism in canine platelets. Their antiinflammatory action is apparently via another mechanism, possibly related to the higher-order structure of the polysaccharide molecule and its molecular weight in addition to the chemical property.

H. MUSCULAR DYSTROPHYSTUDIES Yu and Zhai (1979) used five compounds isolated from the watersoluble portion of the mycelium of G. capense in the treatment of progressive muscular dystrophy, atrophic myotonia, and certain other related diseases with encouraging results. The compounds were identified as adenine, adenosine, uracil, uridine, and D-mannitol. Animal experiments showed that uracil and uridine are capable of lowering the elevated serum aldolase level of mice with experimental myotonia. Hyperaldolasemia has been detected in both progressive muscular dystrophic and hereditary muscular dystrophic animals. The herbicide ~,4-dichlorophenoxyacetic acid (2,4-D) has been used for producing experimental myotonia in animals. Liu et al. (1980) raised the serum aldolase level in mice with 2,4-D and found that G. capense and G. japonicum preparations given intraperitoneally could lower the elevated levels. Uracil and uridine isolated from G. capense mycelium showed the same effect. Zhang (1980) also found uracil and uridine isolated from G. capense effective for impeding the elevation of serum aldolase. Apparently, these two compounds are the active principles of G. capense used in the treatment of muscular dystrophy.

I. RADIATIONPROTECTION Nucleated bone marrow cells of mice can be protected against the effect of radiation-induced micronucleus formation with the polysaccharides extracted from Ganoderma (Chu et al., 1988). The efficacy of the radioprotection was comparable to that of L-cysteine. Where acute radiation sickness in mice was induced by a lethal dose of “OCo, administration of a Ganoderma preparation, given intragastrically for 20 days before irradiation and 20 days after, significantly reduced animal mor-

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tality. Postirradiation application alone did not change the lethal action of 60C0, but it did prolong survival time (Chen, 1986). J. TOXICITY EXPERIMENTS

Various preparations of G. Jucidum reported by Chen (1986) were shown to have a very low toxicity in experiments with mice, rabbits, dogs, and rats. At high doses, the rabbits and dogs became inactive, but no abnormalities were discovered in pathological examinations. Administration to young rats did not affect the growth and development of the animals or produce any abnormalities in liver function, electrocardiogram, and major organs. IV. Patented Products and Processes

Patents contain the most complete and detailed information on the compounds with medicinal effects which have been extracted from Ganoderma. A. ANTITUMOR PREPARATIONS

Many patents have been issued for antitumor products. An anticarcinogen has been obtained from the hot-water extracts of G. boninense and G. tsugae mycelia (Kureha Chem. Ind. Co., 1976). The effective polysaccharides had molecular weights of I x lo5 and contained 63% glucose, 12% galactose, 13% mannose, and 12% xylose. A similar patent was issued for antitumor polysaccharides and their production from an extract of, or the cultured broth of the mycelium of, various basidiomycetes, including G. tsugae (Ohtsuka et a]., 1977). Tivo products effective against sarcoma 180 in mice are an antitumor agent isolated from Ganoderma (Teikoku Chem. Ind. Co., 1982) and an antitumor polysaccharide from G. applanatum (Sankyo Co., 1983). In the latter, the glucans were characterized as f3-1, 3-glucosides in the main chain with one P-1, 6-glucoside linkage for every 1 2 glucose units. A P-glucan ganoderan from the cultivation of Ganoderma hyphae (Hayashibara Biochem. Lab., 1985) is not only anticarcinogenic but also hypoglycemic, hypocholesteremic, and useful as a food thickener, binder, and dough conditioner as well. Hybrid cells with antioncotic activity were obtained by the fusion of Ganoderma lucidum with Pleurotus ostreatus, Lentinus edodes, or Grifola frondosa (Piasu Co., 1986a,b,c). A medium has been developed (Germax, 1986) for use in the produc-

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tion of germanium-containing basidiomycetes mycelia, including that of Ganoderma, that are effective as cytostatics. A composition which contains G. lucidum is active against ascites tumors (Kawano, 1987).

B. LIVER FUNCTION STIMULANTS Ganodosterone and ganoderic acids isolated from G. lucidum have been used as liver function stimulants (Advance Co., 1987). A fermentation product containing P-D-glucan, saponins, and ergosterol derived from culture medium containing Ganoderma, cereals (or potatoes), and beans is a food or beverage additive which has shown significant therapeutic value for patients with liver failure (Asahi Chem. Ind. Co., 198 7b).

C. HYPOTENSIVE PREPARATIONS An antihypertensive agent has been extracted from G. lucidum fruit bodies (Morinaga Milk Ind., 1981).Biologically active compounds from G. lucidum with antiallergic or hypotensive activity have also been obtained from mycelium grown in a solid bagasse (Nagaoka, 1985). An extract from G. lucidum used in a food is capable of suppressing arachidonic acid formation and normalizing elevated blood viscosity (Osaka Pharm. Res. Inst., 1985a). Extracts of Ganoderma have been utilized in a preparation with thrombolytic activity to treat thrombosis and hypertension (Jafuto Co., 1985). A preparation of G. lucidum powder rich in ganoderic acids has been found to inhibit formation of peroxylipid and angiotensin-converting enzyme (Osaka Pharm. Res. Inst., 1986a,b). A nonbitter Ganoderma extract powder has also been used for treatment of hypertension and hyperlipemia (Kyodo Kenko Shizen, 1986). Lanostane derivatives from G. lucidum are antihypertensive in spontaneously hypertensive rats (Asahi Brew. Co., 1987).

D. HYPOCHOLESTEROLEMIC PREPARATIONS The P-glucan mentioned as an antitumor agent also has hypocholesteremic effects (Hayashibara Biochem. Lab., 1985). A fermentation product containing P-D-glucan, saponins, and ergosterol that is derived from culture medium containing Ganoderma and is effective as a liver function stimulant, is also anticholesteremic (Asahi Chem. Ind. Co., 1 9 8 7 ~ ) .

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E. HYPOGLYCEMIC PREPARATIONS A product isolated from G. lucidum that contains ganoderan A and B has demonstrated hypoglycemic activities in mice (Toyo Yakushohu Kogyo Co., 1985). The p-glucan cited as an antitumor agent is also hypoglycemic (Hayashibara Biochem. Lab., 1985). F. CHRONICBRONCHITIS TREATMENT A tasteless and nonbitter extract for the treatment of chronic bronchitis is obtained from Ganoderma by extraction with an organic solvent (Shunyo Yakuhin Hanb, 1986).

G . IMMUNOMODULATORY AGENTS A glycoprotein derived from G. lucidum mycelia acts as an immunosuppressive agent in the treatment of allergic diseases and cell-mediated immune disease (Tsunoo et a]., 1988), while a protein derived from the mycelium of Ganoderma also shows immunosuppressive activity (Meiji Milk Prod. Co., 1990a). An anti-retrovirus drug containing the active ingredient LZ-8 has been derived from the mycelium of Ganoderma (Meiji Milk Prod. Co., 1990b). The agent is composed of mannose, galactose, and hexosamine; does not coagulate human erythrocytes; has immunosuppressive activity; and is effective for disease therapy. A phagocyte-activator has been extracted from Ganoderma mycelium cultured in liquid medium (Sunstar Co., 1986). H. ALZHEIMER’S DISEASETREATMENT

A fermentation product, similar to the food or beverage additive containing G. lucidum that is effective in liver failure, has been found to have a significant therapeutic effect on patients with Alzheimer’s disease (Asahi Chem. Ind. co., 1987a).

I. ANTIBIOTIC-BACTERIOLYTIC ENZYME Fungal lysozyme and an acid protease have been produced from a variety of mushrooms, including G. lucidum (Takeda Chem. Ind., 1969).

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J. ANTIMUTAGEN

An antimutagen glycoprotein has been derived from basidiomycetes such as Ganoderma (Kureha Chem. Ind. Co., 1990). The antimutagen inhibits the manifestation of teratogenicity caused by chemical or physical factors.

K. SKINPREPARATION

A melanin-inhibiting skin preparation containing kojic acid and pharmaceutical natural products includes G. lucidurn (Sansei Pharm. Co., 1989). L. HAIR TONICS

Hair-growing material combines a Ganoderma extract with an extract of a Japanese horseradish, wasabi (Anan Koryo Sangyo, 1984). This material effectively accelerates the growth of hair by providing a nutritive substance to the hair roots. Six patents for hair tonics contain an extract from Ganoderma in combination with other ingredients (Kanebo Co., 1985, 1987a,b,c,d, 1988). One blends chlormadinone acetate and at least one compound selected from a group of agents, including G. lucidum, which accelerates blood circulation and/or stimulates hair follicles. The second contains spironolactone: the third, cyproterone acetate: the fourth, a dithiolane-3-pentanoic acid derivative; the fifth, y-amino-P-hydroxybutyric acid, benzyl nicotinic acid, and/or vitamin E nicotinate: the sixth, cimetidine. Some of the combinations prevent dandruff as well. M. BATH PREPARATION

A bath preparation contains polysaccharides, polypeptides, and thiamine from mushrooms (Nikkei Co., 1986). G. applanatum and G. lucidurn are grown and freeze-dried for use in the preparation. N. BEVERAGES A sake drink has been manufactured using a Ganoderma-flavored or Ganoderma-containing extract (Nishiyama, 1981). Cholane steroids isolated from G. lucidurn are used as hop flavor substitutes in beer and other food additives (Honda and Sakamura, 1985).

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A refreshing medicinal drink free from distasteful flavors is made by culturing Ganoderma in a liquid culture medium containing Chlorella or its extract as the nitrogen source (Kureha Chem. Ind. Co., 1984). When an extract of G. lucidum is added to a rice vinegar health drink, it neutralizes the strong smell and acidity and adds synergistic biological activity (Osaka Pharm. Res. Inst., 1985b). Another vinegar-containing health drink also contains G. Iucidum (Kotobuki Kenkosa Co., 1988). An extract of G. lucidum added to a process for the production of green tea makes the beneficial extract available at a low cost (Ichihara, 1985). A pleasant-tasting dietetic drink is made from the extract of a mixture of a species of Polyporaceae and Indian hydrangea tree (Matoba and Shino, 1985). A powder or extract of G. Iucidum added to a coffee drink gives it laxative properties (Kojien Co., 1987). V. Conclusions

Traditional Chinese medicine has a history of more than 2000 years. While there may be an element of folklore in the use of mushrooms for medicinal purposes, modern research has shown that extracts of Ganoderma can act as metabolic regulators and demonstrate antitumor and immunomodulating activity (Table I). Current screening efforts for chemotherapeutic agents are concentrating on the search for anticancerous agents. Recently, the fruit body and liquid-cultured mycelium of Ganoderma have been reported to contain polysaccharides which inhibit the growth of tumors. Identified as glucans, one of the major constituents in the cell wall of fungi, they appear to increase bodily resistance against the growth of tumors, induce the production of interferon, enhance the immunity function, and kill tumor cells within the body. Their inhibitory effect on tumor growth in experimental animals, particularly sarcoma in white mice, has been the subject of numerous investigations. The role of medicinal mushrooms in metabolic regulation is gaining greater attention. Their use in the development and application of beneficial biological activities offers an advantage in that the active principle is safe and can be tolerated by humans. Cultivation of such mushrooms would provide an adequate supply but is unnecessary if mycelial cultures grown in large-scale fermentations can produce the same active principle. The search for new uses for the traditional medicinal fungi will continue and the public should be educated about their potential value.

TABLE I

MEDICINAL BENEFITSOF THE MUSHROOM Ganoderma Benefit

Compound

Analgesic

Adenosine

Antihepatotoxic

Ganoderic acids R, S Ganosterone P-Glucan G-A Pol ysaccharides

Antiinflammatory Antitumor

Polysaccharide GL-1 Polysaccharide G-Z P-D-Glucans

Cardiotonic Histamine-release inhibitor Hypocholesterolemic

Hypoglycemic Hypotensive

Immunomodulator y

Interferon-inducing, antiviral Neural-muscular restorative Platelet aggregation inhibitor Protein synthesis, nucleic acid synthesis enhancer Radiation protection

0-o-Glucans F-I-lal-P, F-I-la2-P P-D-Glucan G-A Polysaccharide-protein complex Alkaloids Polysaccharides Ganoderic acids C, D Cyclooctasulfur Oleic acid Ganoderic acid B Ganoderic acid Mf Ganodermic acid T-0 Ganoderans A, B Ganoderan C Ganoderol B Ganoderic acids B, D, F, H, K, S , Y Poly saccharides Polysaccharide BN,C Protein LZ-8 RNA

Ref. Shimizu et af. (1985) Kasara and Hikino (1987) Hirotani et al. (1986) Liu et aJ. (1980) Ukai et aJ. (1983b) Ito et al. (1977) Matsumoto et al. (1978) Miyazaki and Nishijima (1981) Sasaki et al. (1971) Sone et al. (1985) Mizuno and Hazama (1986) Kishida et af. (1988) Usui et al. (1983) Ukai et a f . (1983a) Kim et al. (1980) Kang et a f . (1981) Chen (1986) Chen (1986) Kohda et al. (1985) Tasaka et a f . (1988a) Tasaka et al. (1988b) Komoda et a f . (1989) Lin et aJ. (1988) Lin et al. (1988) Hikino et al. (1985) Tomoda et a f . (1986) Morigiwa et aJ. (1986) Nakashima et aJ. (1979) Xie et a]. (1985) Kino et al. (1989) Kandefer-Szerszen et a]. (1979)

Adenosine

Yu and Zhai (1979) Zhang (1980) Shimizu et a f . (1985)

Polysaccharide D,

Guan and Cong (1982)

Polysaccharide

Chu et a f . (1988)

Uridine, uracil

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REFERENCES Advance Co., Ltd. (1987). Isolation of ganodosterone and ganoderic acids as liver function stimulants. Jpn. Pat. 8 7 270,595. Anan Koryo Sangyo (1984). Hair-growing material capable of giving nutritive substance to hair roots. Jpn. Pat. 59184116. Asahi Brew. Co., Ltd. (1987). Extraction of antihypertensive lanostane derivatives from mushrooms. Jpn. Pat. 8 7 67,025. Asahi Chem. Inc. Co., Ltd. (1987a). Fermentation product as food for patients with Alzheimer’s disease. Jpn. Pat. 8 7 234,025. Asahi Chem. Ind. Co., Ltd. (1987b). Fermentation product as food for patients with liver failure. Jpn. Pat. 8 7 234,026. Asahi Chem. Ind. Co. Ltd. ( 1 9 8 7 ~ )Fermentation . product as food additives for patients with hyperlipidemia. Jpn. Pat. 8 7 234,027. Badger, A. M. (1984). Detection of biological response modifiers of natural origin: A review. In “Developments in Industrial Microbiology” (C. H. Nash and L. A. Underkofler, eds.), Vol. 25, pp. 277-291. SIM, Arlington, Virginia. Byun, S. H., and Kim, D. H. (1987). Studies on the concurrent administration of drugs. VII. Effects of concurrent administration of Ganoderma lucidum extract and glutathione on the liver damage induced by carbon tetrachloride in rats. Yakhak Hoechi 31, 133-139.

Chairul, Tokuyama, T., Nishizawa, M., Shiro, M., Tokuda, H., and Hayashi, Y. (1990). Malonate half-esters of homolanostanoid from an Asian Ganoderma fungus. Phytochemistry 29, 923-928. Chen, Q. (1986). Lingzhi. In “Pharmacology and Applications of Chinese Materia Medica” (H.-M. Chang and P. P.-H. But, eds.), Vol. 1, pp. 642-653. World Sci. Publ. co., Singapore. Chu, F., Luo, H., Luo, G., Chen, S., and Liu, Z. (1988). Protection of nucleated bone marrow cells of mice against the effect of radiation-induced micronucleus formation with polysaccharides extracted from Zizhi (Ganoderma). Fushe Fanghu 8, 16-20. Fujita, A., Arisawa, M., Saga, M., Hayashi, T., and Morita, N. (1986). Two new lanostanoids from Ganoderma lucidum. J. Not. Prod. 49, 1122-1125. Germax (1986). Germanium-containing mycelia of Basidiomycetes fungi. Jpn. Pat. 61009280.

Guan, H. C., and Cong, Z. (1982). Effects of Ling zhi polysaccharide D6 on the biosynthesis of nucleic acid and protein and its preliminary analysis. Yaoxue Tongbao 17,177-178.

Hayashibara Biochem. Lab., Inc. (1985). Manufacture of a new P-glucan. Jpn. Pat. 85 188,402.

Hikino, H., and Mizuno, T. (1989). Hypoglycemic actions of some heteroglycans of Ganoderma lucidum fruit bodies. Planta Med. 55, 385. Hikino, H., Konno, C., Mirin, Y., and Hayashi, T. (1985). Oriental medicines. Part 9 1 . Antidiabetes drugs. Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies. Planta Med. 51, 339-340. Hikino. H., Ishiyama, M., Susuki, Y., and Konno, C. (1989). Antidiabetes drugs. Part 33. Validity of Oriental medicines. Part 135. Mechanisms of hypoglycemic activity of ganoderan B: a glycan of Ganoderma lucidum fruit bodies. Planta Med. 55, 423428.

Hirotani, M., and Furuya, T. (1986). Studies on the metabolites of higher fungi. Part 5.

12%

S. C. JONG AND J. M. BIRMINGHAM

Ganoderic acid derivatives, highly oxygenated lanostane-type triterpenoids. from Ganoderma lucidum. Phytochemistry 25, 1189-1193. Hirotani, M., and Furuya, T. (1990).Changes of the triterpenoid patterns during formation of the fruit body in Ganoderma Jucidum. Phytochemistry 29, 3767-3771. Hirotani, M., Furuya, T., and Shiro, M. (1985). Studies on the metabolites of higher fungi. Part 4. A ganoderic acid derivative, a highly oxygenated lanostane-type triterpenoid from Ganoderma lucidum. Phytochemistry 24, 2055-2061. Hirotani, M., Ino, C., Furuya, T., and Shiro, M. (1986). Ganoderic T, S and R, new triterpenoids from the cultured mycelium of Ganoderma lucidum. Chem. Pharm. Bull. 34, 2282-2285. Hirotani, M., Asaka, I., Ino, C., Furuya, T., and Shiro. M. (1987). Ganoderic acid derivafrom Ganoderma lucidum. Phytochemistry tives and ergosta-4,7,22-triene-3,6-dione 26, 2797-2803. Honda, J., and Sakamura, S. (1985). Cholane steroids. Jpn. Pat. 85 258,197. Ichihara, R. (1985). Ganoderma Jucidum tea. Jpn. Pat. 60056924. Ito, H., Naruse, S., and Shimura, K. (1977). Studies on antitumor activity of basidiomycete polysaccharides: VII. Antitumor effect of the polysaccharide preparations from Ganoderma lucidum on mouse Sarcoma 180. Mie Med. I. 26, 147-152. Jafuto Co.,Ltd. (1985). Preparation of Ganoderma extract with thrombolytic activity. Jpn. Pat. 60222423. Jong, S. C., and Donovick, R. (1989). Antitumor and antiviral substances from fungi. Adv. Appl. Microbiol. 34, 183-262. Jong, S.C., Birmingham, J. M., and Pai, S. H. (1991). Immunomodulatory substances of fungal origin. EOS-J. Immunol. Immunopharmacol. 11, 115-122. Kabir, Y., Kimura, S., and Tamura, T. (1988). Dietary effect of Ganoderma lucidum mushroom on blood pressure and lipid levels in spontaneously hypertensive rats (SHR). J. Nutr. Sci. Vitaminol. 34,433-438. Kandefer-Szerszen, M., Kawecki, Z . , and Guz, M. (1979). Fungal nucleic acids as interferon inducers. Acta Microbial. Pol. 28, 277-291. Kanebo Co., Ltd. (1985). Hair tonics containing chlormadinone acetate and blood circulation accelerators. Jpn. Pat. 85 199,810. Kanebo Co., Ltd. (1987a). Hair tonics containing spironolactone and blood-circulation accelerators. Jpn. Pat. 87 103,006. Kanebo Co., Ltd. (1987b). Hair tonics containing cyproterone acetate and blood-circulation accelerators. Jpn. Pat. 87 103,005. . tonic composition. Jpn. Pat. 62175417. Kanebo Co., Ltd. ( 1 9 8 7 ~ )Hair Kanebo Co., Ltd. (1987d). Hair tonic preventing dandruff and hair loss. Jpn. Pat. 62255409. Kanebo Co., Ltd. (1988). Hair tonics containing cimetidine. Jpn. Pat. 88196507. Kang, C. Y., Shim, M. J., Choi, E. C., Lee, Y. N., and Kim, B. K. (1981). Studies on antineoplastic components of Korean basidiomycetes. Mycelial culture and an antineoplastic component of Ganoderma lucidum. Hanguk Saenghwa Hakho Chi 14, 101-112. Kasahara, Y.,and Hikino, H. (1987). Validity of the oriental medicines. Part 122. Central actions of adenosine, a nucleotide of Ganoderma lucidurn. Phytother. Res. 1, 173176. Kawano, G. (1987). Pharmaceutical composition effective against ascites tumor. Jpn. Pat. 62022723. Kikuchi, T., Matsuda, S., Kadota, S., Murai, Y., and Ogita, Z. (1985a). Ganoderic acid D. E,

MEDICINAL BENEFITS OF THE MUSHROOM GANODERMA

129

F, and H and lucidenic acid D, E, F. new triterpenoids from Ganoderrna lucidum. Chem. Phorm. Bull. 33, 2624-2627. Kikuchi, T., Matsuda, S., Kadota, S., Murai, Y., and Ogita, Z. (1985b1. Ganoderic acid G and I and ganolucidic acid A and B, new triterpenoids from Ganoderma lucidum. Chem. Pharm. Bull. 33, 2628-2631. Kikuchi, T., Kanomi. S., Kadota, S., Murai, Y., Tsubono, K. and Ogita, Z. (198621).Constituents of the fungus Ganoderrno lucidum (Fr.) Karst. I. Structures of ganoderic acids CZ, E, I, and K, lucidenic acid F and related compounds. Chem. Pharm. Bull. 34, 3695-3712. Kikuchi, T., Kanomi, S., Murai, Y., Kadota, S., Tsubono, K., and Ogita, Z. (1986b). Constituents of the fungus Ganoderrna lucidum (Fr.) Karst. 11. Structures of ganoderic acids F, G, and H, lucidenic acids D2 and E2, and related compounds. Chem. Pharm. Bull. 34, 4018-4029. Kikuchi, T., Kanomi, S., Murai, Y., Kadota, S., Tsubono, K., and Ogita, Z. (1986~). Constituents of the fungus Ganoderma lucidum (Fr.) Karst. 111. Structures of ganolucidic acids A and B, new lanostane-type triterpenoids. Chern. Pharm. Bull. 34,40304036. Kim, B. K., Chung, H. S., Chung, K. S., and Yang, M. S. (1980). Studies on the antineoplastic components of Korean basidiomycetes. Hanguk Kyunhokhoe Chi 8,107113.

Kim, J. H., and Nam, J. S. (1984). Studies on distribution of the mononucleotides in Ganoderma lucidum. Han’guk Kyunhakhoechi 12, 111-116. Kimura, Y., Okuda, H., and Arichi, S. (1988). Effects of the extracts of Gonoderma lucidum on blood glucose level in rats. Planta Med. 54, 290-294. Kino, K., Yamashita, A . , Yamaoka, K.. Watanabe, J,, Tanaka, S., KO, K., Shimizu, K., and Tsunoo, H. (1989). Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidum. J. Biol. Chem. 264, 472-478. Kishida, E., Okuda, R., Sone, Y., and Misaki, A. (1988). Fractionation structures and antitumor activities of the polysaccharides of Reishi, the fruiting body of Ganoderma lucidum. Osaka-Shiritsu Daigaku Seikatsukagakubu Kiyo 35, 1-10, Kohda, H., Tokumoto, W., Sakamoto, K., Fujii, M., Hirai. Y., Yamasaki, K., Komoda, Y., Nakamura, H., Ishihara, S., and Uchida, M. (1985). The biologically active constituents of Ganoderma Jucidum (Fr.] Karst. Histamine release-inhibitory triterpenes. Chern. Pharrn. Bull. 33, 1367-1374. Kojien, Co., Ltd. (1987). Coffee drink. Jpn. Pat. 87 051,934. Komoda, Y., Nakamura, H., Ishihara, S., Uchida, M., Kohda, H., and Yamasaki, K. (1985). Structures of new terpenoid constituents of Ganodermo lucidurn (Fr.) Karst. (Polyporaceae). Chem. Pharm. Bull. 33, 4829-4835. Komoda, Y., Shimizu, M., Sonoda, Y., and Sato, Y. (1989).Ganoderic acid and its derivatives as cholesterol synthesis inhibitors. Chem. Pharrn. Bull. 37, 531-533. Kotobuki Kenkosha Co., Ltd. (1988).Vinegar-containing health drink. Jpn. Pat. 88 68,069. Kubota, T., Asaka, Y., Miura, I., and Mori, H. (1982). Structures of ganoderic acid A and B, two new lanostane type bitter triterpenes from Ganoderma lucidum (Fr.) Karst. Helv. Chim. Acta 62, 611-619. Kureha Chem. Ind. Co., Ltd. (1976). Anticarcinogen. Jpn. Pat. 76 17,16-Kureha Chem. Ind. CO.,Ltd. (1984). Preparation of Mannentake medicir.. i drink. Jpn. Pat, 59162842. Kureha Chem. Ind. Co., Ltd. (1990). Anti-teratogenicity agent Jpn. Pa;. Kyodo Kenko Shizen (1986). Non-bitter Ganoderma extract powder pr

130

S. C. JONG AND J. M. BIRMINGHAM

or spray-drying Ganoderma extract containing beta-cyclodextrin. ]pn. Pat. 61 069729. Li, H. R., Tertov, V. V., Vasil’ev, A. V., Tutel’yan, V. A,, and Orekhov, A. N. (1989). Antiatherogenic and antiatherosclerotic effects of mushroom extracts revealed in human aortic intima cell culture. Drug Dev. Res. 17, 109-117. Lin, L.-J., Shiao, M.-S., and Yeh, S.-F. (1988). Triterpenes from Ganoderma lucidum. Phytochemistry 27, 2269-2271. Liu, B. and Bau, Y.-S. (1980). “Fungi Pharmacopoeia (Sinica).” Kinoko Co., Oakland, California. Liu, G.-T., Bao, T.-T., Niu, X.-Y., Li, S.-Z., and Song, Z.-Y. (1979a). Some pharmacological actions of the spores of Ganoderma Jucidum and the mycelium of Ganoderma capense (Lloyd) Teng cultivated by submerged fermentation. Chin. Med. J. 92,496-500. Liu, G.-T., Bao, T. T., Wei, H.-L., and Song, 2.-Y. (1979b). Some pharmacological effects of alcoholic extracts of Ganoderma lucidum and G. japonicum Lloyd on mouse liver. Yao Hsueh Hsueh Pa0 14, 284-287. . of the Liu, G.-T., Wang, G.-F., Wei, H.-L., Bao, T.-T., and Song, 2.-Y. ( 1 9 7 9 ~ )Comparison protective actions of dimethylbiphenyldicarboxylate, trans-stilbene, alcoholic extracts of Polyporus japonicus and Ganoderma towards experimental liver injury in mice. Yao Hsueh Hsueh Pa0 14,598-604. Liu, G.-T., Wei, H.-L., Bao, T.-T., and Song, Z.-Y. (1980).Effect of Ganodermas on elevated serum aldolase levels in experimental muscular dystrophy induced by 2,4-dichlorophenoxyacetic acid (2,4-D) in mice. Yao Hsueh Hsueh Pa0 15, 142-146. Maruyama, H., Yamazaki, K., Murofushi, S., Konda, C., and Ikekawa, T. (1989). Antitumor activity of Sarcodon aspratus (Berk.) S. Ito and Ganoderma lucidum (Fr.) Karst. J. Pharmacobiodyn. 12, 118-123. Matoba, T., and Shino, K. (1985). A sanitary drink. U.S. Pat. 4,512,983. Matsumoto, Y., Onishi, M., and Yamagami, K. (1978). An antitumor substance extracted from Polyporaceae. Acta Med. Univ. Kagoshima 20, 209-213. Meiji Milk Prod. Co., Ltd. (1990a). Protein produced by Ganoderma mycelium as immunosupressant. Jpn. Pat. 90 124,899. Meiji Milk Prod. Co., Ltd. (1990b). Anti-retro-virus drugs. Jpn. Pat. 2032026. Miyahara, R.. Yoshimoto, T., and Asawa, K. (1987). Chemical structures and changes of extracts during growth of reishi (Ganoderma lucidurn). Mokuzai Gakkaishi 33,416422. Miyazaki, T. (1983). Relationship between the chemical structure and antitumor activity of basidiomycete glucans. Shinkin to Shinkinsho 24, 95-101. Miyazaki, T., and Nishijima, M. (1981). Studies on fungal polysaccharides. XXVII. Structural examination of a water-soluble, antitumor polysaccharide of Ganoderma lucidurn. Chem. Pharm. Bull. 29, 3611-3616. Miyazaki, T., and Nishijima, M. (1982). Studies on fungal polysaccharides. Part XXXII. Structural examination of an alkali-extracted, water-soluble heteroglycan of the fungus Ganoderma Jucidum. Carbohydr. Res. 109, 290-294. Mizuno, T., and Hazama, T. (1986). Studies on the host-mediated antitumor polysaccharides. X. Fractionation, formolysis and antitumor activity of fibrous polysaccharides (noncellulose) from Reishi, the fruiting body of Ganaderma lucidum. Shizuoka Daigaku Nogakubu Kenkyu Hokoku 36, 77-83. Mizuno, T., Usui, T., Tomoda, M., Shinkai, K., Shimizu, M., Arakawa, M., and Tanaka, M. (1980). Studies on the host-mediated antitumor polysaccharides. 11. Screening test on antitumor activity of various kinds of polysaccharides. Shizuoka Daigaku Nogakubu Kenkyu Hokoku 30, 41-50.

MEDICINAL BENEFITS OF THE MUSHROOM GANODERMA

131

Mizuno, T., Hayashi, K., Arakawa, M., Shinkai, K., Shimizu, M., and Tanaka, M. (1981). Host-mediated antitumor polysaccharides. 111. Fractionation, chemical structure, and antitumor activity of water-soluble homoglucans isolated from kofukisarunokoshikake, the fruit body of Ganoderma applanatum. Shizuoka Daigaku Nogakubu Kenkyu Hokoku 31,49-64. Mizuno, T., Ushiyama, M., Usui, T., Arakawa, M., Shinkai, K., and Shimizu, M. (1982). Studies on the host-mediated antitumor polysaccharides. VI. Isolation and characterization of antitumor active P-D-glucan from mycelial cells of Ganoderma applanatum. Shizuoka Daigaku Nogakubu Kenkyu Hokoku 32,41-58. Mizuno, T., Kato, N., Totsuka, A., Takenaka, K., Shinkai, K., and Shimizu, M. (1984). Fractionation, structural features and antitumor activity of water-soluble polysaccharides from “Reishi”, the fruit body of Ganoderma lucidurn. Nippon Nogei Kagaku Kaishi 58, 871-880. Morigiwa, A., Kitabatake, K., Fujimoto, Y., and Ikekawa, N. (198Sj. Angiotensin converting enzyme-inhibiting triterpenes from Ganoderma lucidum. Chem. Pharm. Bull. 34, 3025-3028. Morinaga Milk Ind., Ltd. (1981). Ganoderma lucidum antihypertensive component. Jpn. Pat. 81 57,801. Nagaoka, H. (1985). An extract of Ganoderma lucidum mycelium and a method for its production. Jpn. Pat. 60149369. Nakashima, S., Umeda, Y., and Kanada, T. (1979). Effects of polysaccharide from Ganoderma applanatum on immune responses. Microbiol. Immunol. 23, 501-513. Nikkei Co., Ltd. (1986). Bath preparations containing mushroom polysaccharides. Jpn. Pat. 86 129,113. Nishitoba, T., Sato, H., Kasai, T., Kawagishi, H., and Sakamura, S. (1984). New bitter CZ7 and Cs0 terpenoids from the fungus Ganoderma Iucidum (Reishi). Agric. Biol. Chem. 48, 2905-2907. Nishitoba, T., Sato, H., and Sakamura, S. (1985a). New terpenoids from Ganoderma lucidum and their bitterness. Agric. Biol. Chem. 49,1547-1549. Nishitoba, T., Sato, H., Kasai, T., Kawagishi, H., and Sakamura, S. (1985b). New bitter CZ7 and CJo terpenoids from the fungus Ganoderma lucidum (Reishi). Agric. Biol. Chem. 49,1793-1798. Nishitoba, T., Sato, H., and Sakamura, S. (1985~). New terpenoids, ganoderic acid J and ganolucidic acid C, from the fungus Ganoderma lucidum. Agric. Biol. Chem. 49, 3637-3638. Nishitoba, T., Sato, H., and Sakamura, S. (1986). New terpenoids, ganolucidic acid D, ganoderic acid L, lucidone C and lucidenic acid G, from the fungus Ganoderma lucidum. Agric. Biol. Chem. 50, 809-811. Nishitoba, T., Sato, H., Shirasu, S., and Sakamura, S. (1987a). Novel triperpenoids from the mycelial mat at the previous stage of fruiting of Ganoderma lucidurn. Agric. Biol. Chem. 51, 619-622. Nishitoba, T., Sato, H., and Sakamura, S. (1987b). Novel mycelial components, ganoderic acid Mg, Mh, Mi, Mj, and Mk, from the fungus Ganoderma lucidurn. Agric. Biol. Chem. 51, 1149-1153. Triterpenoids from the fungus GanoderNishitoba, T., Sato, H., and Sakamura, S . (1987~). ma lucidurn. Phytochemistry 26, 1777-1784. Nishitoba, T., Sato, H., Oda, K., and Sakamura, S. (1988a). Novel triterpenoids and a steroid from the fungus Ganoderma lucidum. Agric. Biol. Chem. 52, 211-216. Nishitoba, T., Oda, K., Sato, H., and Sakamura, S. (1988b). Novel triterpenoids from the fungus Ganoderma lucidurn. Agric. Biol. Chem. 52, 367-372.

132

S. C. JONG AND J. M. BIRMINGHAM

Nishitoba, T., Sato, H., and Sakamura, S. ( 1 9 8 8 ~ )Bitterness . and structure relationship of the triterpenoids from Ganoderma lucidum (Reishi). Agric. BioJ. Chem, 52, 17911795. Nishitoba, T., Goto, S., Sato, H., and Sakamura, S. (1989). Bitter triterpenoids from the fungus Ganoderma applanatum. Phytochemistry 28, 193-197. Nishiyama, S. (1981). Preparation of sake drink containing Ganoderma extracts. Jpn. Pat. 81 61,985. Ohtsuka, S., Ueno, S., Yoshikumi, C., Hirose, F., Ohmura, Y., Wada, T., Fujii, T., and Takahashi, E. (1977). Polysaccharides. U.S. Pat. 4,051,314. Osaka Pharm. Res. Inst. (1985a). Ganoderma lucidurn-containing food capable of suppressing arachidonic acid formation and normalizing elevated blood viscosity. Jpn. Pat. 60034914. Osaka Pharm. Res. Inst. (1985b). Health drink. Jpn. Pat. 60037962. Osaka Pharm. Res. Inst. (1986a). Preparation of therapeutic and prophylactic Gonoderna lucidum rich in ganaderic acids. Ipn. Pat. 86 194,032. Osaka Pharm. Res. Inst. (1986b). Hypertension inhibitor comprising a bracket fungus (Ganoderma lucidum Karsp.). Jpn. Pat. 61267527. Piasu Co., Ltd. (1986a). Hybrid cells with antioncotic activity. Jpn. Pat. 61158778. Piasu Co., Ltd. (1986b). Hybrid cells with a antioncotic activity. Jpn. Pat. 61158779. Piasu Co., Ltd. ( 1 9 8 6 ~ )Hybrid . cells with antioncotic activity. Jpn. Pat. 61158780. Sankyo Co., Ltd. (1983). Antitumor polysaccharides from Ganoderma applanatum. Jpn. Pat. 83 93,702. Sansei Pharm. Co., Ltd. (1989). Melanin-inhibiting skin preparations containing kojic acid (esters) and pharmaceutical natural products. Jpn. Pat 89 83,009. Sasaki, T., Arai, Y., Ikekawa, T., Chihara, G., and Fukuoka, F. (1971). Antitumor polysaccharides from some Polyporaceae, Ganoderma applanatum and Phellinus linteus. Chem. Pharm. Buff. 19, 821-826. Sato, H., Nishitoba, T., Shirasu, S., Oda, K., and Sakamura, S. (1986).Ganoderiol A and B, new triterpenoids from the fungus Ganoderma lucidum (Reishi). Agric. Biol. Chem. 50, 2887-2890. Shiao, M.-S., Lin, L.-J., Yeh, S.-F., and Chou, C . 3 . (1987). Two new triterpenes of the fungus Ganoderma lucidum. J. Nat. Prod. 50, 886-890. Shiao, M.-S., Lin, L.-J., and Yeh, S.-F. (1988). Triterpenes in Ganoderma lucidurn. Phytochemistry 27, 873-875. Shimizu, A , , Yano, T., Saito, Y., and Inada, Y. (1985). Isolation of an inhibitor of platelet aggregation from a fungus, Ganoderma lucidurn. Chern. Pharrn. Bull. 33,3012-3015. Shunyo Yakuhin Hanb (1986). Extraction of tasteless essence of polyporales Ganoderma involves treatment in organic solvent to remove bitter components. Jpn. Pat. 61 030530. Sone, Y . , Okuda, R., Wada, N., Kishida, E., and Misaki, A. (1985). Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderrna Jucidum. Agric. Biol. Chem. 49, 2641-2653. Su, C.-H. (1991). Taxonomy and physiologically active compounds of Ganoderma-A review. Bull. Taipei Med. Coll. 20, 1-16. Sunstar Co., Ltd. (1986). Phagocyte activator for medical use includes Ganoderrna mycelium as active ingredient. Jpn. Pat. 61130235. Takeda Chem. Ind., Ltd. (1969). Bacteriolytic enzyme. Jpn. Pat. 6906623. Tanaka, S., KO, K., Kino, K., Tsuchiya, K., Yamashite, A., Murasugi. A,, Sakuma, S., and Tsunoo, H. (1989). Complete amino acid sequence of an immunomodulatory protein, Ling Zhi-8 (LZ-8). An immunomodulator from a fungus Ganoderma lucidurn, having similarity to immunoglobulin variable regions. 1. Biol. Chem. 264, 16372-16377.

MEDICINAL BENEFITS OF THE MUSHROOM GANODERMA

133

Tasaka, K., Akagi, M., Miyoshi, K., Mio, M., and Makino, T. (1988a). Anti-allergic constituents in the culture medium of Ganoderma lucidum. (I). Inhibitory effect of oleic acid on histamine release. Action Agents 23, 153-156. Tasaka, K., Mio, M., Izushi, K., Akagi, M., and Makino, T. (1988b).Anti-allergic constituents in the culture medium of Ganoderma lucidum. (11). The inhibitory effect of cyclooctasulfur on histamine release. Action Agents 23, 157-160. Teikoku Chem. Ind. Co., Ltd. (1982). Mushroom glycoproteins as neoplasm inhibitors. Jpn. Pat. 82 75,926. Tomoda, M., Gonda, R., Kasahara, Y., and Hikino, H. (1986). Antidiabetes drugs. Glycan structures of ganoderans B and C, hypoglycemic glycans of Ganoderma lucidum fruit bodies. Phytochemistry 25, 2817-2820. Toth, J. O., Luu, B., Beck, J. P., and Ourisson, G. (1983a). Chemistry and biochemistry of oriental drugs. Part IX. Cytotoxic triterpenes from Ganoderma lucidum (Polyporaceae): structures of ganoderic acids U-2. J. Chem. Res. Synop. No. 12, p. 299. Toth, J. O., Luu, B., and Ourisson, G. (1983b). Ganoderic acid T and Z: cytotoxic triterpenes from Ganoderma lucidum (Polyporaceae). Tetrahedon Lett. 24, 1081-1084. Toyo Yakushohu Kogyo CO.,Ltd.; Koshiro, C. and Co., Ltd. (1985).Isolation of polysaccharides from Ganoderma Jucidum as hypoglycemics. Jpn. Pat. 85 184,025. Tsunoo, H., Kino, K., and Yamasita, A. (1988).Glyco-protein from Ganoderma mycelia. Eur. Pat. 288959. Ukai, S., Kiho, T., Hara, C., Kuruma, I., and Tanaka, Y. (1983a). Polysaccharides in fungi. XIV. Anti-inflammatory effect of the polysaccharides from the fruit bodies of several fungi. J. Pharmacobiodyn. 6, 983-990. Ukai, S., Kiho, T., Hara, C., Morita, M., Goto, A., Imaizumi, N., and Hasegawa. Y . (1983b). Polysaccharides in fungi. XIII. Antitumor activity of various polysaccharides isolated from Dictyophor indusiata, Ganoderma japonicum. Cordyceps cicadae, AuricuJaria auricula-judae, and Auricularia species. Chem. Pharm. Bull. 31, 741-744. Usui, T., Iwasaki, Y., Hayashi, K., Mizuno, T., Tanaka, M., Shinkai, K., and Arakawa, M. (1981). Antitumor activity of water-soluble P-D-glucan elaborated by Ganoderma applonatum. Agric. Biol. Chem. 45, 323-326. Usui, T., Iwasaki, Y., Mizuno, T., Tanaka, M., Shinkai, K., and Arakawa, M. (1983). Isolation and characterization of antitumor active P-D-glucans from the fruit bodies of Ganoderma applanatum. Carbohydr. Res. 115, 273-280. Whistler, R. L., Bushway, A. A., Singh, P. P., Nakahara, W., and Tokuzen, R. (1976). Noncytotoxic antitumor polysaccharides. Adv. Carbohydr. Chem. Biochem. 32,235275. Willard, T. (1990). “Reishi Mushroom-Herb of Spiritual Potency and Medical Wonder.” Sylvan Press, Issaquah, Washington. Xie, D., Wu, Q., Zhang, H., Chen, W., Li, R., and He, Y. (1985).Effect of the polysaccharide component BN& from Ganoderma and d-matrine on murine T lymphocytes. Zhonghua Weishengwuxue He Mianyixue Zazhi 5, 8-13. Ying, J., Mao, X., Ma, Q., Zong, Y., and Wen, H. (1987). “Icones of Medicinal Fungi from China.” Science Press, Beijing. Yu, J.-C.,and Zhai, Y.-F. (1979). Studies on the constituents of Gonoderma capense. Part I. Yao Hsueh Hsueh Pa0 14, 374-378. Yu, J., Shen, F., Hou, C., and Yang, S. (1981). Studies on chemical constituents of deeplayer fermentation mycelia of Ganoderma capense (Lloyd) Teng. Part 11. Zhongcaoyao 12, 7-11. Yu, J., Chen, R., and Yao, 2. (1983). Studies on the chemical constituents of Bao Gai Ling Zhi (Ganoderma capense) mycelium by submerged fermentation 111. Zhongcaoyao 14, 438-439.

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Zhang, G . D., Liu, H., and Liang, Y. (1986). Reverse-phase HPLC determination of nucleosides and their bases in the submerged culture of Ganoderma capense. Yaoxue Xuebao 21,35-39. Zhang, J. (1980). Recent achievements of the Institute of Materia Medica on studies on natural products. U S . China Pharmacol. Syrnp. Washington, D.C. (J. J. Burns and P. J. Tsuchitani, eds.), pp. 15-54.

Microbial Degradation of Biphenyl and Its Derivatives FRANKK. HIGSON Department of Soil and Environmental Sciences University of California at Riverside Riverside, California 92521

I. Overview 11. Introduction 111. Metabolic Pathway in Bacteria IV. Polychlorinated Biphenyls V. Growth of Bacteria on Polychlorinated Biphenyls and Coculture Systems VI. Anaerobic Degradation of Polychlorinated Biphenyls VII. Polychlorinated Biphenyl Bioremediation Trials VIII. Degradation of Other Biphenyl Derivatives IX. Plasmids Encoding the Degradation of Biphenyl and Polychlorinated Biphenyls X. Chromosomal Genes for the Degradation of Biphenyl and Polychlorinated Biphenyls XI. Fungal and Cyanobacterial Metabolism of Biphenyl XII. Conclusions References

1. Overview

Pyrolysis of lignin over geological time has provided a selection pressure for microorganisms to develop the potential for biphenyl metabolism, and bacteria that can use biphenyl as sole carbon source are rather readily obtained from enrichments. The pathway of biphenyl degradation in these strains has been examined. Benzoate is formed by the action of a suite of enzymes that may accept certain chlorine substitutions on the biphenyl nucleus. The benzoate dioxygenase, however, is more specific, so that a range of chlorobenzoates are produced by the cometabolism of polychlorinated biphenyls (PCBs). 4-Chlorobiphenyl is the only PCB reported to serve as growth substrate for a naturally occurring strain; multichemostat and mating techniques have, however, succeeded in generating recombinants able to grow on 2- and 3chlorobiphenyl. For the higher PCB congeners, a combination of anaerobic and aerobic processes seems appropriate. The enzymes that convert PCBs to chlorobenzoates have been found to be both plasmid- and chromosomally encoded. The observation of constitutive synthesis of 135 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 37 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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these enzymes from a isolate particularly competent in PCB cometabolism when cloned into Escherichia coli may prove very important if the recombinant is used in soil remediation. Coculture biodegradation of PCBs has been investigated for such congeners as 4,4'-dichlorobiphenyl. Bacteria have been described that utilize biphenyl ether and hydroxylated biphenyls, but a strain that can grow on 3,-4dihydroxybiphenyl has not yet been reported. II. Introduction

The role of microorganisms in maintaining steady-state concentrations of environmental chemicals is well established and these activities constitute the basis for the cycle of carbon in nature. Microbes have been exposed for millions of years to aromatic hydrocarbons, the predominant source being the pyrolysis of organic materials such as the amorphous polymer lignin. Forest fires and anthropogenic combustion of fossil fuels also deliver hydrocarbons into the atmosphere. The ubiquitous distribution of soil bacteria capable of metabolizing organic compounds such as naphthalene, phenol, and cresol was demonstrated by Gray and Thornton (1928). Several groups have identified gram-negative strains that use biphenyl as sole carbon source (Lunt and Evans, 1970; Catelani et al., 1970; Gibson et a]., 1973). Growth on biphenyl by gram-positive strains has also been reported, viz. Arthrobacter simplex BPA (Tittmann and Lingens, 1980) and Arthrobacter sp. B1B (Kohler et al., 1988a).

Ill. Metabolic Pathway in Bacteria Chemical studies by Catelani et al. (1973) and Catelani and Colombi (1974) and the use of a pathway mutant of Beijerinckia sp. by Gibson et al. (1973) indicated that the hydrocarbon was subject to dioxygenase attack to form a dihydrodiol (2,3-dihydroxy-l-phenylcyclohexa-4,6-diene) of cis stereochemistry. Dehydrogenation then yielded 2,3-dihydroxybiphenyl (2,3-HB), which underwent meta-cleavage-as reported for methylcatechols (Bayly and Dagley, 1969; de Frank and Robbins, 1977)-to a yellow compound, Z-hydroxy-6-keto-6-pheFIG. 1. The degradation of biphenyl by pseudomonads (Omori et al., 1988). Throughout, R is a phenyl group. Compounds A through J are, respectively, biphenyl; 2,3-dihydro-2,3-dihydroxybiphenyl;2,3-dihydroxybiphenyI; 2-hydroxy-6-keto-6-phenylhexa2,+dienoic acid (HOPDA); 2-ketopent-4-enoic acid; benzoic acid; 2-methoxy-6-keto6-phenylhexa-2-enoic acid; 2,6-diketo-6-phenylhexanoicacid; y-benzoylbutyraldehyde; y-benzoylbutyric acid.

4 R

A

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FRANK K. HIGSON

nylhexa-2,4-dienoic acid (HOPDA).A hydrolase, purified to a homogeneous state from Pseudomonas cruciviae strain S93B1 (Omori et al., 1986a), converts HOPDA to benzoic acid and a five-carbon fragment, 2ketopent-4-enoic acid (Fig. 1). Omori et al. (1986b) reported three HOPDA reductases in strain S93B1 which used NADPH to reduce the double bonds of the metacleavage product. The isozymes were active on certain other ring-fission products: I and I1 on those from catechol and 3-methoxycatechol, I11 on those from %methyl- and 3-isopropylcatechols. A reduction between carbons 4 and 5 was suggested (Omori et al., 1988) using methylated HOPDA (the 2-methoxy group no longer exhibited ketoenol tautomerism as occurs in HOPDA itself). The identification of ybenzoylbutyrate and y-benzoylbutyraldehyde in extracts of enzyme transformations of HOPDA suggested an alternative pathway by which the meta-cleavage product is metabolized in pseudomonads (Omori et al., 1988). The culture supernatant of strain S93B1 accumulated significant amounts of y-benzoylbutyrate but not benzoate, so that reduction might not be productive. Masse et al. (1984) also detected several trace metabolites in the media of Achromobacter sp. B218 and Bacillus brevis B257 growing on 4-chlorobiphenyl. These minor degradation products represented reduction of one or both double bonds in HOPDA. Biphenyl dioxygenase has proved difficult to prepare, but by analogy with benzene (Axcell and Geary, 1975), toluene (Yeh et al., 1977), and naphthalene (Ensley et al., 1982) dioxygenases, the enzyme is expected to be multicomponent and may contain ferredoxin to shuttle reducing power between NADH dehydrogenase and terminal oxidase components. Dehydrogenases with activity on a range of cis-dihydrodiols, including that formed from biphenyl, were obtained by Pate1 and Gibson (1976) from three pseudomonads and a Nocardia sp. 2,3-HB dioxygenase was partially purified from strain S93B1 (Ishigooka et a]., 1986). The enzymes involved in the conversion of biphenyl to benzoic acid have been shown to exhibit broad specificity. 2,3-HB dioxygenase, for example, is highly active on 3-methyl and 3-isopropylcatechols (Ishigooka et al., 1986). HOPDA hydrolase also acts on the ring-fission product from catechol and %methyl- and 3-isopropylcatechols (Omori et al., 1986b). Moreover, the initial dioxygenase can be active on a number of analogs, so that strains have been isolated from soil and sewage enrichments that can use 4-nitrobiphenyl (Masse et al., 1985),4-chlorobiphenyl (Furukawa et al., 1978a,b),or +methylbiphenyl (Fedorak and Westlake, 1983) in addition to biphenyl The para-substituted benzoate

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normally accumulates in the culture, since the enzyme at this level is more discriminating in its substrate range. IV. Polychlorinated Biphenyls

Polychlorinated biphenyls (PCBs) were formerly extensively used in plastics, carbon paper, cooling systems, and transformers and as fire retardants until their embryotoxicity and possible carcinogenicity led to a worldwide ban on their application. There are 209 possible forms, differing in the number and arrangement of chlorine substituents, and the numbering system of Ballschmiter and Zell (1980) has now been generally adopted for these compounds. The aqueous solubility and volatility decrease considerably with additional substitution (Opperhuizen et al., 1988). Industrial mixtures (Aroclors in the United States or Kaneclors in Japan), produced by the restricted chlorination of biphenyl, contain various combinations of these congeners: the extent of chlorination rises in the order Aroclor 1242 < 1248 < 1260. Their persistence and tendency to bioaccumulate as they ascend the food chain now make them major environmental pollutants (Waid, 1986). Several expensive methods of PCB destruction have been tested. They can be burnt at very high temperatures, yielding hydrogen chloride, but there is a risk of generating small quantities of the much more toxic polychlorinated dibenzodioxins and dibenzofurans (Hutzinger et al., 1985), and dechlorination using alkali metals and naphthalene is restricted to PCBs of high purity. The bacterial degradation of highly chlorinated congeners has been observed via cometabolism, in which an organism transforms a nongrowth compound in the presence of another carbon source (Horvath, 1972). Several groups have isolated strains recognized as especially competent in the cometabolism of recalcitrant Aroclor components (Furukawa et al., 1983; Bedard et al., 1986; Kohler et al., 1988a). The principal route of aerobic PCB degradation in most prokaryotes appears to involve 2,s-dioxygenase attack at an unsubstituted ring or at rings offering at least one pair of adjacent unchlorinated 2,3- (or 5,6-) carbons (Fig. 2). Furukawa (1982) established additional correlations between PCB structure and biodegradability on the basis of work with Acinetobacter sp. P6 and Alcaligenes sp. Y42. Ease of degradation decreases as more chlorines are placed on the biphenyl nucleus; congeners with two ortho chlorines are particularly resistant and 2,6,2',6'tetrachlorobiphenyl is not attacked at all. The presence of an unsubstituted ring enhances degradation and the cleavage generally takes

140

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FRANK K. HIGSON 4

g s / +

2' 3' \

o

,

-2H

-

-

\

to,

\

4'

B

A

D

E

C

F

FIG. 2. The degradation by aerobic bacteria of polychlorinated biphenyl congeners (A).Products B through F are, respectively, the 2,3-dihydro-2,3-dihydroxy derivative: the acid; chloro2,3-dihydroxy compound; 2-hydroxy-6-keto-6-(chlorophenyl)hexa-2-enoic benzoic acid plus 2-ketopent-4-enoic acid; chloroacetophenone (Bedard and Haberl, 1990).

place at the unchlorinated or less chlorinated ring. While these observations were valid for many of the Bedard group's isolates such as Corynebacterium sp. MB1, the strain Alcaligenes eutrophus H850 differed substantially in congener specificity (Bedard et a]., 1987). The strain readily degraded diortho-substituted isomers such as 2,2'-or 2,6,2',5'-PCBs, and highly chlorinated biphenyls bearing a 2,3-dichlorophenyl function were degraded less rapidly than those with a 2,5-dichlorophenyl group. H850 metabolized 2,4,4'- and 2,5,4'-PCBs to 4-chlorobenzoate. Moreover, Pseudomonas sp. LB400 can degrade PCBs that lack adjacent free carbons, such as 2,4,5,2',4',5'-hexachlorobiphenyl (Bopp, 1986).Indeed, four classes of PCB-dioxygenase activity have been proposed in a survey of strains by Bedard and Haberl (1990). Furukawa et al. (1979) reported hydroxylated PCBs as well as chlorobenzoates when strains P6 and Y42 were allowed to metabolize specific congeners. It has been suggested that strain H850 employs a 3,4-dioxygenase in addition to one with 2,3- specificity, since it formed a 3,4-dihydrodiol and a 3,4,3',4'-bi~-dihydrodiolfrom 2,5,2',5'-tetrachlorobiphenyl (Nadim et a]., 1988). A secondary 3,4-acting enzyme was proposed by Masse et al. (1985) in gram-negative strain B206 from gas chromato-

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graphic-mass spectroscopic (GC-MS) identification of trimethylsilylated metabolites of 4-nitrobiphenyl. Polar metabolites were reported from 2,5,2‘- and 2,5,2’,5‘-PCBswhen Carey and Harvey (1978) incubated the I4C-labeled compounds with aerobic seawater; purely from infrared data, a lactone acid was postulated to be formed from the trichlorobiphenyl. Autoclaved or anaerobic seawater failed to show the thin-layer chromatography (TLC)-determined formation of metabolites. . reported novel metabolites, chlorinated acetoBedard et ~ l (1987) phenones (Fig. 2F), when strain H850 acted on certain congeners such as 3,s‘-and 2,4,5,2’,5‘-PCBs.Barton and Crawford (1988) also reported the accumulation of large amounts of 4’-chloroacetophenone in the medium of Pseudomonas sp. MB86 growing on 4-chlorobiphenyl The growth of strain MB86 (obtained from 4-chlorobenzoate enrichment) appeared to be inhibited by the buildup of the ketone, which the authors considered a dead-end metabolite. These ketones are reduced to chlorophenylethanols, or oxidized to chlorophenols in a biological Baeyer-Villiger reaction, by soil bacteria obtained from acetophenone enrichment (Higson and Focht, 1990), and there was also indication of their further metabolism in H850 itself (Bedard et al., 1987). Sylvestre et al. (1982) presented evidence of monohydroxylation of 4-chlorobiphenyl by the bacterium B206. An arene oxide intermediate was proposed, similar to that generated by fungal oxidation of aromatic hydrocarbons (Cerniglia, 1984), and nonenzymatic rearrangement would explain the appearance of two phenols. In this process, medium nitrate could be incorporated into the ring as a nitro group; nitrobiphenyls have been shown to be mutagenic (McCann et al., 1975). Kohler et al. (1988b) found that growing cells of either Acinetobacter sp. P6 or Arthrobacter sp. B1B were more active in degrading Aroclor 1254 components than were resting cells. The biphenyl dioxygenase activity falls off rapidly after harvesting of the cells. V. Growth of Bacteria on Polychlorinated Biphenyls and Coculture Systems

The only PCB reported to allow growth of a naturally occurring microorganism is 4-chlorobiphenyl. Growth on 4-chlorobiphenyl in pure culture usually leaves the chlorobenzoate unmetabolized, but mineralization was indeed reported by Shields et al. (1985) with isolates from a mixed culture obtained from PCB-contaminated river sediments (Kong and Sayler, 1983). Alcaligenes sp. A2, for example, carries a 35-

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MDa plasmid pSS50 that mediates mineralization. A cured derivative failed to act on 4-chlorobiphenyl and return of the plasmid via filter mating restored mineralization activity. A multiple chemostat system of Krockel and Focht (1987) has been shown to generate recombinants that can utilize 2- and 3-chlorobiphenyls as sole carbon source (Higson and Focht, 1989a; Huang, 1988). In these experiments, a biphenyl degrader and a halobenzoate utilizer were taken from separate chemostats onto a column of ceramic beads, where conjugation was increased by the large surface area available. The novel recombinant phenotype was enriched for from a third chemostat into which the column effluent was delivered. The strain Acinetobacter sp. CB15 can use biphenyl, 3-chlorobiphenyl, or 3-chlorobenzoate as sole carbon source (Huang, 1988). Recently, Mokross et al. (1990) transferred the genes for chlorocatechol degradation from Pseudomonas sp. B13 to a biphenyl degrader, Pseudomonas putida BN10. They also succeeded in introducing the BNlO biphenyl-pathway genes into strain B13. Derivatives of the parents first obtained were able to grow in the presence of streptomycin (strR) or nalidixic acid (nalR); in one mating, strains BN10-strR and B13, and in a second making, strains BNlO and B13-nalR were grown together for 24 hours on nutrient agar. A suspension of each cross was then spread on minimal agar supplemented with 3-chlorobenzoate and streptomycin, or biphenyl and nalidixic acid; subsequent transfers yielded two colonies, BN210 (strR) and B131 (nalR), that grew on and dechlorinated 3-chlorobiphenyl. This is in contrast to a drastic drop in viability when BNlO cells were incubated with 3-chlorobiphenyl, owing to the accumulation of 3-chlorocatechol as a toxic product. A temporary accumulation of 3-chlorobenzoate was observed when recombinant strains acted on 3-chlorobiphenyl, but chloride recovery was eventually about 90%. The hybrids degraded the mono- and dichlorobiphenyls of Aroclor 1 2 2 1 more efficiently than parent BN10, with mineralization shown for the 3-chlorobiphenyl component. This strategy looks promising; the outcome, in theory is controlled by the halobenzoate and halocatechol degradative activities of the parents. Total degradation of 4-chlorobiphenyl was achieved in a two-step process by Hiramoto et al. (1989) using Arthrobacter sp. M5 (which transforms 4-chlorobiphenyl to 4-chlorobenzoate) and Pseudomonas aeruginosa 4-CBA (which grows on 4-chlorobenzoate) in the presence of emulsifier P-cyclodextrin at 15 g/liter. The strain 4-CBA produced an inhibitor of the growth of M5; thus, dechlorination was optimized by an initial 4-day stand with M5, followed by inoculation with 4-CBA, which utilized the transformation product. The latter activity was

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much faster than the transformation of 4-chlorobiphenyl by M5, so a sufficient first-step incubation was essential. Adriaens and Focht (1990) set up a continuous aerobic fixed-bed system, filled with polyurethane foam to support a bacterial biofilm, to study coculture degradation of 4,4’-dichlorobiphenyl by strain P6 and Acinetobacter sp. 4CB1 (a 4-chlorobenzoate degrader isolated from PCB-contaminated soil). The reactor was supplied with benzoate as primary substrate, while P6 was induced for PCB degradation by biphenyl vapors in the incoming airstream. The mineralization of 4,4’PCB in this system amounted to 6.5% over 45 days: Adriaens and Focht suggested the value was kept low by the high affinity constant (K,) of 4CB1 for 4-chlorobenzoate when grown on benzoate. Thus, Adriaens et al. (1989) observed that dechlorination of 4,4’-PCB only began in a batch coculture of P6 and 4-CB1 when 4-chlorobenzoate had accumulated to concentrations higher than the K, value. The freshwater consortium LPSlO of Pettigrew et al. (1990) achieved 81% dehalogenation of 4,4’-PCB. It consisted of three strains, Pseudomonas testosteroni as the biphenyl degrader, an Arthrobacter sp. as the 4-chloro benzoate degrader, and Pseudomonas putida, whose role in the consortium was not determined. None of these strains harbored a plasmid or sequences homologous to pSS50 of Shields et al., yet, in chemostat enrichments, LPSlO outcompeted bacterial populations containing this plasmid. VI. Anaerobic Degradation of Polychlorinated Biphenyls

PCBs are lost from the environment by a combination of anaerobic and aerobic activities. Brown et al. (1987a) observed that PCB residues in the aquatic sediments from six PCB spill sites showed changes in congener distribution that indicated the occurrence of reductive dehalogenation. The changes pointed to several distinct populations of microorganisms that modified congeners to different extents. The transformation processes fell into two broad categories: 0,m, p-dechlorinations, with congener reactivities dependent on reduction potential, and m, p-dechlorinations, where molecular shape was more important (Brown et al., 1987b). The PCB acts as an electron sink and chloride is liberated as a by-product; there is no disruption of the carbon skeleton. Similar replacement of chlorine by hydrogen has been reported for yhexachlorocyclohexane (Ohisa and Yamaguchi, 1979) and 4-chlororesorcinol (Fathepure et al., 1987). Thermodynamic calculations with chlorobenzene and hexachlorobenzene (Brown et a]., 1987a) indicate

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that oxidation with the aid of chloroaromatics offers a greater freeenergy gradient than that provided by the other oxidants commonly available to anaerobes, namely, carbon dioxide and sulfate. Positionselective dechlorinations have also been described for anaerobic cultures acting on chlorobenzoates (Suflita et al., 1982) and chlorophenols (Boyd et al., 1983). Prior to the work of Brown and colleagues, the only known route for the environmental destruction of the more heavily chlorinated PCBs was photolysis by near-ultraviolet radiation (Bunce et al., 1978),but PCBs that lie buried in aquatic sediments are obviously inaccessible to sunlight. Indeed, since only the top few centimeters of sediments are generally aerobic, the largest reservoir of PCBs in the river is in an anaerobic environment. Anaerobic bacteria in these sites convert the more thymotoxic higher congeners (Parkinson et al., 1983) to less chlorinated compounds that aerobic organisms at the surface can readily degrade to chlorobenzoates. Frequently, higher congeners are only partially metabolized by aerobes to meta-cleavage compounds (Bedard and Haberl, 1990), if they are attacked at all. Quensen et al. (1990) investigated the anaerobic degradation of Aroclors by inocula prepared from two sediments, one contaminated with Aroclor 1 2 4 2 from the upper Hudson River (HR) and one contaminated with Aroclor 1260 at Silver Lake, P i (SR). Dechlorination of all four main Aroclor mixtures was observ Jthough higher congeners were transformed at a lower rate and to er extent. The SR bacteria exhibited both a shorter lag time a n t rapid dechlorination of Aroclor 1260 than the HR inoculum. nably on account of site ies from PCBs by the HR adaptation. A greater removal of pr xoorganisms of different inoculum suggested that this cor itamination in these sedispecificity. We should conclude 1 lechlorinators, since termiments represented a selection pr imiting factor in anaerobic nal electron acceptors are gene )ility of sediment bacteria to environments (Brown et al., 198 may well be important as a remove chlorines from higher ( s substituted species that are pretreatment of PCB waste, gent msen et al. (1990) suggest that more amenable to aerobic organir m d SL bacteria would best be the complementary specificities ultures. applied in a combination of the tx Rhee et al. (1989) studied the anaerobic disappearance of PCBs from Hudson River sediments subjected to various treatments in the laboratory and from Moreau sediments encapsulated in situ with clay. About 53% of the total PCBs (375 mg/kg sediment dry wt), mainly mono- to pentachlorobiphenyls, were degraded in the biphenyl-amended Hudson River sediments after 7 months; the amendment enhanced the dis-

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appearance of highly chlorinated congeners. Moreau sediments incubated in situ showed little change in congener concentrations, suggesting temperature is an important factor. In contrast to the work of Quensen and co-workers, Rhee et a1. did not observe the accumulation of less-chlorinated congeners as a result of reductive dechlorination of higher ones.

VII. Polychlorinated Biphenyl Bioremediation Trials Decontamination of polluted soil can sometimes be achieved by optimizing physicochemical conditions and introducing suitable “vanguard” organisms that catalyze difficult stages in xer,obiotic transformation, thus enabling other members of the soil microbiota to complete the process (Bewley, 1986), as described for the reclamation of a British gasworks site contaminated with phenols and polycyclic aromatic hydrocarbons (Bewley and Thiele, 1988). The application of strains H850, LB400, and MB1 to PCB--soil formulations has been attempted by Unterman et al. (1988). In their studies, washed, biphenyl-grown cells were added to soil spiked with 50 or 500 ppm Aroclor 1242 or 1254. Extensive removal of PCBs was observed within 3 days: interestingly, H850 and LB400 were more active than MBl, with congeners carrying a 2,5-dichlorophenyl group and MB1 was superior with doubly para-substituted congeners, just as with free PCBs. Addition If a rr~ixedculture was even more effective. Bioremediation of contatn” !d soil from a drag-racing track under simulated in situ conditions .A water, no aeration, 8°C lower temperature) took longer, since cell concentrations and temperatures were less favorable. After repeated inoi illations, however, significant PCB degradation was recorded within a: Jut 8 weeks (Unterman et al., 1988). It is important in these studies . e sure that disappearance of PCBs is due to biological activity ratk i a n physical losses. These processes are disition of the GC profiles: biodegradation retinguished by an P moves specific I ,_ ,em s while physical depletion results in uniform depletion of all corlgeners (adsorption) or of lower congeners (evaporation). The production metabolites points to biodegradation, if these are not detected in deac-cellcontrols. The addition of a nonbiodegradable internal standard such as 2,4,6,2’,4‘-PCBwas routine in the studies of Unterman and co-workers. The investigation of Viney and Bewley (1990) of the degradation of PCB on spiked horticultural sand showed up differences in congener specificity for a range of test isolates, just as observed by Bedard anL’ yo-workers with their strains. Hrunner et al. (1985) shobved that the application of Acinetobacter 7

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FRANK K. HIGSON

sp. P6 together with biphenyl enhanced the mineralization of added [14C]Aro~lor1242 in soil, whereas the addition of strain P6 alone did not. The technique was termed analog enrichment and halogenated products were presumed to be further metabolized by the indigenous soil microflora. The work emphasizes that bioremediation of PCBladen soils may be limited by the expense of biphenyl amendment to allow increase in the PCB-cometabolizer population. Sewage sludge has been demonstrated to increase PCB mineralization in soil, with up to 11%recorded by Fairbanks et al. (1987) over 240 days, although a turnover time for 95% of 500 ppm PCBs was estimated under such conditions to be from 6 to 26 years. Hickey (1990) showed that mineralization of PCBs from a biphenylsupplemented Altamont soil was considerably enhanced by inoculation with halobenzoate-degrading bacteria. P. aeruginosa JB2, which utilizes 2- and 3-chloro, 2,3- and 2,5-dichloro-, and 2,3,5-trichlorobenzoates (Hickey and Focht, 1990), and P. putida P111, which grows on all these substrates plus 4-chlorobenzoate (Hernandez et a]., 1990), had no direct activity on PCBs and yet mineralization amounted to 17.7 and 16.0Y0,as compared to 2% from uninoculated soil. Soil inoculated with the PCB-degrading pseudomonads PB133, UCRl, and UCR2 showed 7.4, 6.0, and 10.3% mineralization, respectively. The strains UCRl and UCR2 were derived from the multiple chemostat system of Krockel and Focht (1987) in which genes from biphenyl- and halobenzoate-utilizing cells can be consolidated into one organism. The construct UCRl used 3-chlorobenzoate and UCR2 used 2-chloroand 2,5-dichlorobenzoates, but neither recombinant grew on the range of halogenated benzoates displayed by strains JB2 or P111. This may have accounted for the lower mineralization-enhancement efficiency of these inoculants relative to the halobenzoate degraders alone. The longer lag preceding the maximum 14C0, evolution rate (5 versus 20 days) and the lower absolute mineralization rates in Hickey’s study were attributed to more organic matter in the test soil. PCBs sorb to soil to an extent dependent on the organic content (Griffinand Chow, 1981) and would have limited accessibility to microbes in a humus-rich soil. In theory, inoculating contaminated soil with both PCB and chlorobenzoate degraders should provide for efficient mineralization. Dual inoculation, however, runs the risk of competition for nutrients such as the benzoate produced by the breakdown of biphenyl; the work of Hickey and Focht suggested that an effective inoculum should deliver a low density of a selected PCB cometabolizer and a higher density of the chlorobenzoate degrader. One factor limiting the removal of a xenobiotic from soil may be the

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inaccessibility to degrading organisms; PCBs sorb well to organic matter and higher congeners have exceedingly low aqueous solubilities, so that emulsifying factors have been proposed to allow desorption from the soil matrix. Rosenberg et al. (1979) have identified a natural emulsifier in Arthrobacter sp. RAG1. Synthetic surfactants such as Triton XlOO or Tensoxid S50 were shown to increase desorption of PCBs from sand (Viney and Bewley, 1990) but also reduced the degradative activity of several isolates, with strain MB1 showing the least effect at the 1%level of Triton. VIII. Degradation of Other Biphenyl Derivatives

Polybrominated biphenyls (PBBs) were used as flame retardants in the early 1970s until their effects on liver metabolism and teratogenicity prompted a complete ban (Kimbrough, 1987). Strains that cometabolize PCBs might be expected to be active on the brominated analogs, but because PBBs are a more limited problem, their degradation has received less attention than PCBs. Strain S93B1 grows on 2-bromobiphenyl with the accumulation of 2-bromobenzoate (Takase et al., 1986). The soil isolates AIcaIigenes sp. KF708 and Pseudomonas paucimobilis KF706 grow on 2-bromo- and 2-nitrobiphenyls as well as biphenyl and diphenylmethane (Furukawa et al., 1989). Pfeifer et al. (1989) reported a strain of Pseudomonas cepacia able to utilize diphenyl ether, a model compound representing the diary1 substructure of hard coals, as sole carbon source and identified a 2,3dihydroxy derivative in the culture fluid. Further metabolism of this intermediate yielded 2-pyrone-6-carboxylic acid (PCA;the lactone of 2hydroxymuconatef and phenol by meta-fission and ether cleavage. A cis-trans tautomerization was presumed to occur in the same step as ring cleavage, without the participation of free intermediates. PCA was a dead-end product, as reported for the degradation of the herbiby cide chloridazon (5-amino-4-chloro-3-phenyl-~-H-pyridazin-~-one) Miiller and Lingens (1980). Wittich et al. (1990) isolated Pseudomonas sp. strain NSS2 that used 3- or 4-carboxybiphenyl ether as sole carbon source. A dioxygenase was proposed to generate a dihydrodiol at the carbon bearing the bridging oxygen and this then decomposed to phenol and protocatechuic acid (Fig. 3). This mechanism is an alternative to the classical monooxygenase attack on the alkyl group of ethers to produce hemiacetals which spontaneously hydrolyze to aldehydes and phenols (Gliick and Lingens, 1988; Jezequel et a]., 1984; Meigs, 1987). Using the 4-carboxybiphenyl ether enrichment isolate Pseudomonas sp. POB310, Engesser et al. (1990) provided evidence for a 1,Z-dioxy-

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FRANK K. HIGSON R

02 + NADH+H+ r

1 NAD' I /R

L

NADH+H' G+

H20 \ NAO+ +

H 0'

TCA C Y C L E

FIG. 3 . The degradation of carboxylated diphenyl ethers by Pseudomonas sp. NSS2 (the 3-isomer has R = COOH, R' = H; the 4-isomer has R = H, R' = COOH) (Wittich et al., 1990).

genase from the cometabolism of fluorinated analogs from which either 4-fluorophenol or 5-fluoroprotocatechuate was accumulated in high yield. Moreover, 4-carboxy-1,2-dihydro-1,2-dihydroxybenzophenone was produced as a dead-end metabolite from 4-carboxybenzophenone. The strain generated exclusively the 2-hydroxy derivative from 4-carboxybiphenyl, as expected to results from dehydration of the 1,2-dihydrodiol, and biologically synthesized 2,3-dihydroxy-4-carboxybiphenyl ether was not transformed substantially by strain POB310. Xanthone, which is a diphenyl ether with an additional carbonyl bridge between the rings, can also serve as sole carbon source for an Arthrobacter sp. and is degraded by 3,4-dioxygenation: a pathway has been presented involving 4-hydroxycoumarin and gentisate (Tomasek and Crawford, 1986). The degradation of hydroxylated biphenyls bas been addressed by Kohler et al. (1988b) and Higson and Focht (1l~I:bj (Fig. 4). Pseudomonads were isolated from sewage enrichments that utilized 2- or 3hydroxybiphenyl as sole carbon source; monohydroxylation in either case generated 2,3-dihydroxybiphenyl, which was meta-cleaved as in

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3-HB

OH 4-HB

2,3-HB

OH

2,3,4‘- HB

149

BENZOIC ACID

OH

4 -HY OROXYBENZOIC ACID

FIG. 4. The degradation of 3- and 4-hydroxybiphenyls (HB) by pseudomonads via to benzoic or 4-hydroxybenzoic acids (Hig~,3-dihydroxy-or 2,3,4’-trihydroxybiphenyls son and Focht, 1990).

the biphenyl pathway. Growth was also seen on 2,2‘- and 3,3’-dihydroxybiphenyls, when 2- and 3-hydroxybenzoates respectively replaced benzoate as intermediate. Growth of Pseudomonas sp. FH23 on 4-hydroxybiphenyl involved dioxygenase attack on the unsubstituted ring and 4-hydroxybenzoate was produced as an intermediate. The strain failed to use 4,4’-dihydroxybiphenyl. Although steric hindrance to the attack of oxygen would be greater and aqueous solubility lower in the doubly substituted compound, nongrowth probably reflected the fact that 3,4-dioxygenation is nonproductive. Thus, no organism has been reported to use 3,4-dihydroxybiphenyl. Another factor here, however, is rapid autoxidation of the phenylcatechol. Strain H850 cometabolizes 3,4-dihydroxybiphenyl to protocatechuic acid (Higson et a]., 1987). The anaerobic conversion of dibenzothiophene to biphenyl has been described for the sulfate-reducing soil isolate Desulfovibrio desulfuricans M6 (Kim et al., 1990). IX. Plasmids Encoding the Degradation of Biphenyl and Polychlorinated Biphenyls

A variety of degradative phenotypes have been attributed to catabolic plasmids (Sayler et a]., 1990). These extrachromosomal elements are

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widespread in nature and an increase within a community has been observed in pollutant-stressed environments (Burton et al., 1982; Hada and Sizemore, 1981; Ogunseitan et al., 1987). Sequence homology between plasmids with the same catabolic phenotype suggests that, in many cases, the structural genes are conserved (Chatterjee and Chakrabarty, 1983; Heinaru et al., 1978). Movement of plasmid genes into the chromosome can occur when the growth substrate is changed [Carney and Leary, 1989). In several cases, the same catabolic plasmid has been isolated from two different genera (Shields et al., 1985; Ghosal et al. 1985) and transmission between species is thought to occur primarily by conjugation, transduction, and transformation (reviewed in Levy and Miller, 1989).Kochetkov et al. (1982) reported a 195-kb transmissible plasmid, pBS241, coding for biphenyl degradation in P. putida BS893. Furukawa and Chakrabarty (1982) found pKFl (78.1 kb) coded for 4-chlorobiphenyl degradation in strain P6; biphenyl-negative segregants (produced by growth in nutrient broth) showed a small change in the size of one of the BamHI restriction fragments. A transmissible 97.5-kb plasmid, pAC21, codes for 4-chlorobiphenyl degradation in a strain of Klebsiella pneumoniae (Kamp and Chakrabarty, 1979). Normally, enterobacteria cannot utilize hydrocarbons and catabolic genes are not expressed when they are received on pseudomonad plasmids. The presence of pAC21 in cells of K. pneumoniae allowed them to express genes on pAC8, a hybrid plasmid coding for toluene and methylbenzoate degradation (Chatterjee and Chakrabarty, 1981). The plasmid pSS50 has been found to code for 4-chlorobiphenyl mineralization in Alcaligenes spp. A2, A20, and A5 and Acinetobacter sp. A8 (Shields et al., 1985).

X. Chromosomal Genes for the Degradation of Biphenyl and Polychlorinated Biphenyls

A gene cluster encoding biphenyl degradation (bph)was cloned from a soil isolate Pseudomonas pseudoalcaligenes KF707 into P. aeruginosa PA01161 (Furukawa and Miyazaki, 1986). Purified genomic DNA from the biphenyl degrader was digested with restriction endonuclease XhoI and ligated to XhoI-digested broad-host-range plasmid pKF330. Streptomycin-resistant transformant colonies were sprayed with 2,3-dihydroxybiphenyl solution; one colony among about 8000 quickly turned yellow (indicating an active meta-cleavage enzyme). This clone (KF257) was grown in Luria broth containing streptomycin, and its hybrid plasmid, designated pMFB1, was found to contain a 7.9kb insert in the unique XhoI site of pKF330. Southern blot experiments

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showed that the insert was derived from KF707 DNA; there was no homology, however, with the plasmid present in KF707. The transformant converted biphenyl to the meta-cleavage product only; the hydrolase gene was missing. A variety of derivatives were constructed from pMFB1: the small SmaI fragment was removed to produce pMFB4, and the derivative pMFB5 was obtained by subcloning this fragment into the unique SmaI site of vector pKF330. Furukawa and Miyazaki found that pMFB4 only allowed biphenyl to be transformed to the dihydrodiol, while pMFB5 only catalyzed the oxidation of 2,3-dihydroxybiphenyl. They therefore proposed the gene order to be bphABC, as for the metabolic pathway, with A the biphenyl dioxygenase, B the dihydrodiol dehydrogenase, and C the 2,3-HB dioxygenase. The SmaI cut had been made within the gene B. Furukawa and Suzuki (1988) investigated the action of strains of P. aeruginosa, containing recombinant plasmids bearing bph genes, on biphenyl and its derivatives with a view to isolating specialty chemicals. 2,3,2',3'-Tetrahydroxybiphenylwas produced from biphenyl, and 2,3-dihydroxy-4'-chlorobiphenyl from 4-chlorobiphenyl by a strain carrying plasmid pMFB6, which allowed expression of bphAB genes. By following the formation of meta-cleavage product in a strain expressing bphABC genes, the investigators showed that a single chlorine, bromine, or methyl did not affect the reaction rate, while hydroxyl or carboxyl substitution markedly reduced activity. There was no activity on 4,4'-dichlorobiphenyl, indicating that there are greater restrictions on the dioxygenase activity in P. pseudoalcaligenes than in strain P6. A series of gram-negative biphenyl degraders isolated from soil in various locations in Japan were analyzed for the presence of the bphABC cluster by Furukawa et al. (1989). Among 15 strains tested, five Pseudomonas strains and one Alcaligenes strain possessed the cluster on the XhoI 7.2-kb fragment described in KF707. The restriction profiles of these bph ABC-XhoI fragments were essentially identical, despite the dissimilarity of the flanking sequences. The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well with the DNA homology. On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1 (isolated from soil in Chicago), lacked genetic and immunological homology with any of the other 15 strains tested. The work suggested that a segment containing the bphABC genes encodes a transfer mechanism. The mobilization of chromosomal xenobioticdegrading genes to other soil pseudomonads is a new idea, although genes for toluene metabolism occur on a transposable element (transposon) on pWW0, a "TOL" plasmid (Tsuda and Ilino, 1987).

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Hayase et al. (1990) used one of the 15 strains above, namely, P. putida KF715, to obtain an entire bphABCD sequence, where D indicates the HOPDA hydrolase gene. A 9.4-kb fragment of XhoI digestion of KF715 chromosomal DNA had been shown to hybridize to the bphABC probe from strain KF707. A XhoI digest of KF715 DNA was ligated to XhoI-digested vector pHSG396 and the mixture used to transform E. coli JMlo9; at first, transformants were selected by the chloramphenicol resistance originating from the vector. When 2,3-dihydroxybiphenyl solution was applied, one colony turned yellow. The transformant harbored a hybrid plasmid, pYH715, which contained a 9.4-kb insert in the unique XhoI site of pHSG396, and the organism could convert 4-chlorobiphenyl to 4-chlorobenzoate. The use of exonuclease I11 to partially digest the insert gave rise to various deletion mutants, a metabolite analysis of which indicated the order of bph genes in KF715 to be ABCD. Mutant cells transformed biphenyl to the dihydrodiol, 2,3-HB, HOPDA, or 4-chlorobenzoate, depending on the size of the deletion. The plasmid pNHF715 was constructed by inserting the 9.4-kb XhoI (bphABCD) fragment into the XhoI site of broad-host-range plasmid pKT230. pNHF715 was introduced into various benzoate utilizers by the triparental mating method, using pRK2013 as a helper plasmid (Hayase et a]., 1990). It rendered three strains-Pseudomonas aeruginosa PA01161, P. putida AC30, and Achromobacter xerosis IF012668-able to assimilate biphenyl as sole carbon source. It will be interesting to see if PCB utilizers can be obtained in the same way by the introduction of biphenyl-degradation genes into organisms that grow on chlorobenzoates. Khan and co-workers (1988; Khan and Walia, 1989) cloned the “cbp” genes coding for the conversion of 4-chlorobiphengl to 4-chlorobenzoate from P. putida OU83 using the endonuclease HindIII and broadhost-range cosmid vector pCP13. The fragments were ligated into the HindIII site of the cosmid and the DNA packaged in vitro for transfection into E. coli. Tetracycline-resistant transformants bearing degradative genes were identified by spraying with 2,3-dihydroxybiphenyl solution: one, strain AC812, had the entire upper pathway. The relative activity of 2,3-HB dioxygenase in crude extracts of E. coli containing different recombinant cosmids varied considerably. Restriction digest analysis indicated the inserts in these cosmids ranged from 6 to 30 kb, and differences could be expected in the expression of regulatory genes in these constructs. The dioxygenase activity in cells bearing pAW6194 only acted on 2,3-dihydroxybiphenyl and its 4’-chloro derivative; the extract from cells carrying another construct, pOH810, additionally

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oxidized catechol and 4-chloro- and 4-methylcatechols. Khan and Walia (1989) also reported that recombinants metabolized 4-chlorobiphenyl and its 2',3'-dihydroxy derivative to benzoate as well as chlorobenzoate, and a dehalogenase was speculated to occur alongside the cbp genes. Mondello (1989) cloned bphABCD genes from Pseudomonas sp. LB400 and found the bph restriction map to be quite different from that for the P. putida KF715 genes. A genomic library of LB400 was constructed using the broad-host-range plasmid pMMB34 and E. coli; of about 1500 recombinant clones tested, five generated a yellow color with 2,3-HB. Two recombinant plasmids encoding the conversion of PCBs to chlorobenzoates were identified and one (pGEM410) was chosen for subcloning experiments which localized bphABCD genes to a 12.4-kb region. The ability of strains of succinate-grown cells of E. coli bearing either bphABCD functions (FM41lo), or bphABC (FM4560) to degrade the components of defined PCB mixtures was compared to that of LB400 grown on succinate or on biphenyl as sole carbon source. The recombinants were highly active on a range of congeners, with the bphABC-positive strain showing much greater activity with doubly para-substituted compounds than succinate-grown (uninduced) LB400. Strain FM4560 also depleted nearly all the components of Aroclor 1242 in a 24-hour resting-cell transformation (Mondello, 1989). The improvement of degradative competence in FM4560 compared to FM4110 might be the result of copy number differences between the cloning vectors used in the two constructs. Plasmids derived from the RSFlOlO replicon (the type present in FM4110) are usually maintained at 15-20 copies per cell, whereas those from pUC vectors (such as the FM4560 plasmid) may be present at 50-60 copies per cell. In conclusion then, we can say that, in situations where it would be impractical to grow an organism on biphenyl or in which other carbon sources would be present, a strain such as FM4560 might be more preferable to the parent LB400 for effecting bioremediation of soils. Yates and Mondello (1989) found strains LB400 and H850 to show strong conservation of restriction sites in the region of DNA encoding PCB metabolism, but no other sequence similarities in the two genomes. The LB400 probe did not indicate homology with several other degraders of lower PCB cometabolic activity. Ahmad ef a]. (1990) cloned the genes of Pseudomonas testosteroni strain B356 specifying the transformation of 4-chlorobiphenyl into 4chlorobenzoate into P. putida KT2440 using the broad-host-range plasmid pPSA842; one hybrid plasmid, pDAl, that encoded these genes was radiolabeled to demonstrate some homology with the PCB-degra-

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dative sequences in bacteria such as LB400 or Alcaligenes sp. A5 obtained from different geographical locations. This close proximity of the genes coding for the conversion of biphenyl to benzoate is similar to the clustering of genes for the metabolism of toluene [Franklin et al., 19811, naphthalene [Cane and Williams, 1986), and chlorobenzoate (Chatterjee and Chakrabarty, 1984; Weisshaar et al., 1987). Recently, Walia et al. (1990) constructed [32P]DNAprobes for PCBdegradative genotypes from PCB-degrading bacteria. Less than 1% of colonies isolated from garden topsoil and >80% of bacteria isolated from contaminated soils showed homology with the radioactive probes, by dot blot analysis. Several organisms showed homology but failed to clear biphenyl when the hydrocarbon was sprayed onto minimal medium. The investigators suggested that the DNA probe technique could be used alongside biodegradation assays in assessing the PCB-degradative capability within soils. XI. Fungal and Cyanobacterial Metabolism of Biphenyl

Fungi monohydroxylate biphenyl, primarily at the para position [Smith and Rosazza, 1974; Dodge et al., 1979). Cerniglia and colleagues observed that half of the biphenyl metabolites formed from Cunninghamella elegans were glucuronide and sulfate conjugates. The ability to metabolize biphenyl is not particularly widespread: of 66 species tested by Smith et al. (19801, only 7 were active hydroxylators, of which Helicostylium piriforme QM6945 was distinctive in selectively hydroxylating biphenyl at the 2-position. A trans-dihydrodiol has been demonstrated as a biphenyl metabolite in rats (Halpaap et al., 1978) but not in fungi: the arene oxide that was supposed to form initially was presumed to be too labile or not a good enough substrate for epoxide hydratase for the dihydrodiol to appear rather than the phenol. The occurrence of an NIH shift during the formation of 4hydroxybiphenyl by Cunninghamella echinulata (Smith et al., 1981) provided strong evidence for the prior formation of biphenyl 3,4-oxide. Schwartz (1981) observed exclusively meta-hydroxylation of biphenyl by Nocardia salmonicolor 21243. The degradation of Aroclor 1242 (but not 1254 or 1260) by resting cells of Saboraud-grown Aspergillus niger was reported by Dmochewitz and Ballschmiter (1988); para-substituents inhibited attack and 4,4’-dichlorination essentially prevented attack by the fungus. The white rot fungus Phanerochaete chrysosporium, which secretes hydrogen peroxide and peroxidases to degrade the recalcitrant amorphous polymer lignin, has been found capable of mineralizing a

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number of persistent xenobiotics (Aust, 1990).Bumpus and Aust (1986) reported a 2.0 and 1.7% mineraIization of 3,4,3',4'-tetrachloro- and 2,4,5,2',4',5'-he~achloro[U-~~C]biphenyl by P. chrysosporium (concentrations used were, respectively, 0.0363 and 0.045 ppm). A much higher degree of mineralization was reported for 0.04 ppm [14C]Aroclor 1242 (20.50/0) and 1254 (18%).The onset and duration of PCB degradation and its diminution when nutrient nitrogen no longer limited growth matched those for ligninolytic activity. In contrast, hexachlorobenzene, known to be recalcitrant in soil (Aust and Bumpus, 1987),was not mineralized by ligninolytic cultures of P. chrysosporium (Bumpus and Aust, 1986),presumably due to complete substitution of the aromatic nucleus. Eaton (1985) reported about 7% mineralization of 0.3 ppm [U14C]Aroclor 1254 by nitrogen-limited cultures of P. chrysosporium over 22 days. Other white rot fungi were found to be less active: Phlebia brevispora released 1.0% 14C0,, FunaJia galJica 0.7%, Coriolus versicolor 0.32%, and Poria cinerescens 0.13%. Moreover, raising the ammonium chloride concentration in the P. chrysosporium medium from 2.2 to 6.6 mM decreased the mineralization to 3% of the PCB added. Eaton found that mineralization commenced on about the third day of incubation and was absent in sterilized or sodium azide-killed controls; there was also no induction period (the fungus did not require acclimation to the Aroclor). Conversion of the labeled PCB to watersoluble metabolites was effected by the fungus (Eaton, 1985), while partitioning characteristics of the Aroclor did not change on sterile incubation. GC analysis of the dichloromethane extracts showed that virtually all the congener peaks were lost or greatly diminished by the action of P. chrysosporium. Recent observations by D. H. Pieper (unpublished observations) showed a 2 to 6% mineralization of 4-chloro[14C]biphenyl over 4 weeks by cultures of P. chrysosporium starved of either nitrogen or carbon. A crude ligninase preparation exhibited no activity against 4-chloro- or 4,4'-dichlorobiphenyls. The cyanobacterium Oscillatoria sp. JCM para-hydroxylated biphenyl when illuminated in a carbon dioxide-enriched medium that allowed photoautotrophic growth (Cerniglia et al., 1980). XII. Conclusions

The widespread, recalcitrant, and bioaccumulating nature of PCBs has presented a major environmental problem. Bacteria are available, however, that grow aerobically on the parent hydrocarbon and cometabolize a range of PCBs to chlorobenzoates. Research to optimize the

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breakdown of PCBs has been directed firstly at the isolation of species especially competent in the removal of a spectrum of Aroclor components, and secondly, at the bringing together of pathway segments with a view to constructing an organism that can use PCBs as sole carbon source. Several naturally occurring strains grow on 4-chlorobiphenyl, but only Shields et al. have demonstrated mineralization of this compound by a natural isolate; the others simply metabolize the unsubstituted ring and cause accumulation of 4-chlorobenzoate. A number of approaches have been described to obtain growth on other chlorobiphenyls. The combination of biphenyl and chlorobenzoate degraders by nutrient agar mating or in a multiple chemostat system has yielded strains able to mineralize 3-chlorobiphenyl. The growth range of the recombinant produced is related to the versatility of the parents, but may not be a simple composite of the two; for example, only some of a set of chlorobenzoate-degradinggenes may appear in the hybrid. A more predictable method of strain construction would be to insert the cloned biphenyl/PCB-degradative genes into a chlorobenzoate-degrading recipient. These genes have been found to be both plasmid- and chromosome-encoded, and homology has been established for biphenyl gene clusters identified in organisms from diverse geographical locations. The observation of metabolites such as 2,5,2',5'-tetrachlorobiphenyl-3,4-dihydrodiolor chloroacetophenones in strain H850 suggests that the degradation of biphenyl derivatives involves reactions other than those promoted by the four main enzymes which lead to benzoate. Selected strains have been applied with some success in soil bioremediation attempts, but the most efficient degradation of PCBs employs anaerobic pretreatment, when, for example, river sediment microorganisms use highly chlorinated congeners, which often resist attack by oxygen, as terminal electron acceptors. Electron acceptors are usually limiting in anaerobic environments, so that a selection pressure is exerted by PCB contamination in river sediments. The microbial population adapts to the Aroclor present and to achieve optimal dehalogenation of say, Aroclor 1260, we should look at sites contaminated by this mixture for suitable experimental inocula. The contribution of white rot fungi to PCB degradation has not been fully explored, but the results of mineralization of a variety of xenobiotics (2,4,6-trinitrotoluene, pentachlorophenol, polycyclic aromatic hydrocarbons) suggest that the nonspecific ligninolytic system could be harnessed in the elimination of PCBs as well. Such activity is driven by the breakdown of readily available lignocellulose materials such as wood chips and may prove more economical than the applica-

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tion of biphenyl to allow cometabolism of PCBs by aerobic bacteria. Mondello’s report of constitutive cometabolism of a range of congeners by E. coli bearing the bphABCD genes of strain LB400 has been suggested as a way of avoiding the need for biphenyl induction, but how well the recombinant strains would compete with indigenous microflora has yet to be established. Competition with existing microorganisms for space and nutrients is of course a limiting factor for all new inoculants, even for soil biphenyl degraders provided with biphenyl. ACKNOWLEDGMENTS

I am most grateful for the helpful suggestions of Dr. Dennis D. Focht (UCR) and the financial assistance of Occidental Petroleum (Grand Island, NY) and the U.S. Environmental Protection Agency. REFERENCES Adriaens, P., and Focht, D. D. (1990). Continuous coculture degradation of selected polychlorinated biphenyl congener by Acinetobacter spp. in an aerobic reactor system. Environ. Sci. Technol. 24, 1042-1049. Adriaens, P., Kohler, H.-P. E., Kohler-Straub, D., and Focht, D. D. (1989). Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4’-dichlorobiphenyl. Appl. Environ. Microbiol. 55, 887-892. Ahmad, D., Masse, R., and Sylvestre, M. (1990).Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorinated biphenyl-degrading genes in other bacteria. Gene 86, 53-61. Aust, S. D. (1990). Degradation of environmental pollutants by Phanerochaete chrysosporium. Microb. Ecol. 20, 197-209. Aust, S. D., and Bumpus, J. A. (1987). “Biodegradation of Halogenated Hydrocarbons,” EPA Environ. Res. Brief 600-M-87-012. U.S. Environ. Prot. Agency, Cincinnati, Ohio. Axcell, B. C., and Geary, P. J. (1975). Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. Biochem. J. 146, 173-183. Ballschmiter, K.,and Zell, M. (1980). Analysis of polychlorinated biphenyls by capillary gas chromatography. Fresnius Z. Anal. Chem. 302, 20-31. Barton, M. R., and Crawford, R. L. (1988). Novel biotransformations of 4-chlorobiphenyl by a Pseudomonas sp. Appl. Environ. Microbiol. 54, 594-595. Bayly, R. C., and Dagley, S. (1969). Oxoenoic acids as metabolites in the bacterial degradation of catechols. Biochem. J. 111, 303-307. Beall, M. L. (1976). Persistence of aerially applied hexachlorobenzene on grass and soil. J. Environ. Qual. 5, 367-369. Bedard, D. L., and Haberl, M. L. (1990). Influence of chlorine substitution pattern on the degradation of polychlorinated biphenyls by eight bacterial strains. Microb. Ecol. 20, 87-102.

Bedard, D. L., Unterman, R., Bopp, L. H., Brennan, M. J., Haberl, M. L., and Johnson, C. (1986). Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls. Appl. Environ. Microbiol. 51, 761-768.

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Bedard, D. L., Haberl, M. L., May, R. J., and Brennan, M. J. (1987). Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850. Appl. Environ. Microbiol. 53, 1103-1112. Bewley, R. J. F. (1986). A microbiological strategy for the decontamination of polluted land. In “Contaminated Soil” (1. W. Assink and W. j. van der Brink, eds.), pp. 759768. Nijhoff, Dordrecht, Netherlands. Bewley, R. J. F., and Thiele, P. (1988). Decontamination of a coal gasification site through application of vanguard organisms. In “Contaminated Soil ‘88” (K. Wolf, W. J. van den Brink, and F. J. Colon, eds.), pp. 739-743. Kluwer, Dordrecht, Netherlands, Bopp, L. H. (1986). Degradation of highly chlorinated PCBs by Pseudomonas sp. LB400. J. Ind. Microbiol. 1, 23-29. Boyd, S. A., Shelton, D. R., Berry, D., and Tiedje, J. M. (1983).Anaerobic biodegradation of phenolic compounds in digested sludge. Appl. Environ. Microbiol. 46, 50-54. Carnahan, J. C., Peng, H., and Wagner, R. E. Brown, j. F., Bedard, D. L., Brennan, M. I., (1987a). Polychlorinated biphenyl dechlorination in aquatic sediments. Science 236, 709-712. Brown, J. F., Wagner, R. E., Feng, H., Bedard, D. L., Brennan, M. J., Carnahan, j. C., and May, R. J. (1987b).Environmental dechlorination of PCBs. Environ. Toxicol. Chem. 6, 579-593. Brunner, W., Sutherland, F. H., and Focht, D. D. (1985). Enhanced biodegradation of polychlorinated biphenyls in soil by analog enrichment and bacterial inoculation. J. Environ. Qual. 14, 324-328. Bumpus, J. A., and Aust, S. D. (1986). Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chrysosporium: involvement of the lignin degrading system. BioEssays 6, 166-170. Bumpus, J. A,, and Aust, S. D. (1987). Biodegradation of chlorinated organic compounds by Phanerochaete chrysosporium, a wood-rotting fungus. In “Solving Hazardous Waste Problems: Learning from Dioxins” (1. H. Exner, ed.), ACS Symp. Ser., No. 338, pp. 340-349. Am. Chem. SOC.,Washington, D.C. Bunce, N. j., Kumar, Y., and Brownlee, B. G. (1978). An assessment of the impact of solar degradation of polychlorinated biphenyls in the aquatic environment. Chemosphere 7 , 155-164. Burton, N. F., Day, M. J., and Bull, A. T. (1982).Distribution of bacterial plasmids in clean and polluted sites in a South Wales river. Appl. Environ. Microbiol. 44, 1026-1029. Cane, P. A,, and Williams, P. A. (1986). A restriction map of naphthalene catabolic plasmid pWW60-1 and the location of some of its catabolic genes. J. Gen. Microbiol. 132, 2919-2929. Carey, A. E., and Harvey, G. R. (1978). Metabolism of polychlorinated biphenyls by marine bacteria. Bull. Environ. Contam. Toxicol. 20, 527-534. Carney, B. F., and Leary, j. V. (1989). Novel alterations in plasmid DNA associated with aromatic hydrocarbon utilization by Pseudomonas putida R5-3. Appl. Environ. Microbiol. 55, 1523-1530. Catelani, D.,and Colombi, A. (1974). Metabolism of biphenyl. Structure and physacid, the metaicochemical properties of 2-hydroxy-6-0~0-6-phenylhexa-2,4-dienoic cleavage product from 2,3- dihydroxybiphenyl by Pseudomonas putida. Biochem. J. 143, 431-434. Catelani, D., Mosselmans, G., Niehaus, j., Sorlini, C., and Treccani, V. (1970). Microbial degradation of aromatic hydrocarbons used as reactor coolants. Experientia 26,922923. Catelani, D., Colombi, A,, Sorlini, C., and Treccani, V. (1973). Metabolism of biphenyl. 2-

DEGRADATION OF BIPHENYL AND ITS DERIVATIVES

159

hydroxy-6-oxo-phenylhexa-2,4-dienoate: the meta-cleavage product from 2,3-dihydroxybiphenyl by Pseudomonas putida. Biochem. J. 134, 1063-1066. Cerniglia, C. E. (1984). Microbial transformation of aromatic hydrocarbons. In “Petroleum Microbiology” (R. M. Atlas, ed.), pp. 99-128. Macmillan, New York. Cerniglia, C. E., van Baalen, C., and Gibson, D. T. (1980). Oxidation of biphenyl by the cyanobacterium OsciIIatoria sp. strain JCM. Arch. Microbiol. 125, 203-207. Chatterjee, D. K., and Chakrabarty, A. M. (1981). Plasmids in the biodegradation of PCBs and chlorobenzoates. In “Microbial Degradation of Xenobiotics and Recalcitrant Compounds” (T. Leisinger, R. Hutter, A. M. Cook, and J. Nuesch, eds.), pp. 213-219. Academic Press, London. Chatterjee, D. K., and Chakrabarty, A. M. (1983).Genetic homology between independently isolated chlorobenzene-degradative plasmids. J. Bacteriol. 153, 532-534. Chatterjee, D. K., and Chakrabarty, A. M. (1984). Restriction mapping of chlorobenzoate degradative plasmid and molecular cloning of the degradative genes. Gene 27, 173181.

de Frank, J. J., and Robbins, D. W. (1977). p-Cymene pathway in Pseudomonas putida: ring-cleavage of 2,3-dihydroxy-p-cumate and subsequent reactions. J, Bacteriol. 129, 1365-1374. Dmochewitz, S., and Ballschmiter, K. (1988). Microbial transformation of technical mixtures of polychlorinated biphenyls (PCB) by the fungus Aspergillus niger. Chemosphere 17, 111-121. Dodge, R. H., Cerniglia, C. E., and Gibson, D. T. (1979). Fungal metabolism of biphenyl. Biochem. J. 178,223-230. Eaton, D. C. (1985). Mineralization of polychlorinated biphenyls by Phanerochaete chrysosporium: a ligninolytic fungus. Enzyme Microb. Technol. 7 , 194-196. Engesser, K.-H., Fietz, W., Fischer, P., Schulte, P., and Knackmuss, H.-J. (1990). Dioxygenolytic cleavage of aryl ether bonds: 1,2-dihydro-l ,2-dihydroxy-4-carboxybenzophenone as evidence for initial 1,2-dioxygenation in 3- and 4-carboxy biphenyl ether degradation. FEMS Microbiol. Lett. 69, 317-322. Ensley, B. D., Gibson, D. T., and Laborde, A. L. (1982). Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB9816. J. Bacteriol. 149, 948-954. Fairbanks, B. C., O’Connor, A., and Smith, S. E. (1987). Mineralization and volatilization of polychlorinated biphenyls in sludge-amended soils. J. Environ. Qual. 16,18-25. Fathepure, B. Z., Tiedje, J. M., and Boyd, S. A. (1987). Reductive dechlorination of 4chlororesorcinol by anaerobic microorganisms. Environ. Toxicol. Chem. 6 , 929-934. Fedorak, P. M., and Westlake, D. W. S. (1983). Selective degradation of biphenyl and methylbiphenyls in crude oil by two strains of marine bacteria. Can. I. Microbiol. 29, 497-503. Franklin, F. C. H., Bagdasarian, M., Bagdasarian, M. M., and Timmis, K. N. (1981). Molecular and functional analysis of the TOL plasmid genes for the entire regulated aromatic ring-cleavage pathway. Proc. Natl. Acad. Sci. U S A . 78, 7458-7462. Furukawa, K. (1982). Microbial degradation of polychlorinated biphenyls. In “Biodegradation and Detoxification of Environmental Pollutants” (A. M. Chakrabarty, ed.), pp. 33-57. CRC Press, Boca Raton, Florida. Furukawa, K., and Chakrabarty, A. M. (1982). Involvement of plasmids in total degradation of chlorinated biphenyls. Appl. Environ. Microbiol. 44,619-626. Furukawa, K., and Miyazaki, T. (1986). Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes. J. Bacteriol. 166, 392-398.

160

FRANK K. HIGSON

Furukawa, K., and Suzuki, H. (1988). Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds. Appl. Microbiol. Biotechnol. 29, 363-369. Furukawa, K., Matsumara, F., and Tonomura, K. (1978a). Alcaligenes and Acinetobacter strains capable of degrading polychlorinated biphenyls. Agric. Bi d . Chem. 42,543548. Furukawa, K., Tonomura, K., and Kamibayashi, A. (1978b).Effect of chlorine substitution on the biodegradability of polychlorinated biphenyls. Appl. Environ. Microbiol. 35, 223-227. Furukawa, K., Tomizuka, N., and Kamibayashi, A. (1979). Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls. Appl. Environ. Microbiol. 38, 301-310. Furukawa, K., Tomizuka, N. and Kamibayashi, A. (1983). Metabolic breakdown of kaneclors (polychlorobiphenyls) and their products by Acinetobacter sp. Appl. Environ. Microbiol. 46, 140-145. Furukawa, K., Hayase, N., Taira, K., and Tomizuka, N. (1989). Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon. I. Bacteriol. 171, 5467-5472. Ghosal, D.,You, 1.-S., Chatterlee, D. K., and Chakrabarty, A. M. (1985). Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4. Proc. Natl. Acad. Sci. U.S.A. 82, 1638-1642. Gibson, D. T., Roberts, R. L., Wells, M. C., and Kobal, V. M. (1973). Oxidation of biphenyl by a Beijerinckia sp. Biochem. Biophys. Res. Commun. 50, 211-219. Gliick, M., and Lingens, F. (1988). Heteroxanthine demethylase, a new enzyme in the degradation of caffeine by Pseudomonas putido. Appl. Microbiol. Biotechnol. 28, 59-62. Gray, P. H. H., and Thornton, H. G. (1928). Soil bacteria that decompose certain aromatic compounds. Zentralbl. Bakteriol., Parasitenkd. Infektionskr., Abt. 2 73, 74-96. Griffin, R. A., and Chow, S. F. J. (1981). Movement of PCBs and other persistent compounds through soil. Water Sci. Technol. 13, 1153-1163. Hada, H. S., and Sizemore, R. K. (1981). Incidence of plasmids in marine Vibrio spp. isolated from an oil field in the northwestern Gulf of Mexico. Appl. Environ. Microbiol. 44, 199-202. Halpaap, K.,Homing, M. G., and Homing, E. C. (1978). Metabolism of biphenyl in the rat. J. Chromatogr. 166, 479-490. Hayase, N., Taira, K., and Furukawa, K. (1990). Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis and expression in soil bacteria. I. Bacteriol. 172, 1160-1164. Heinaru, A. L.. Dugglesby, C. J., and Broda, P. (1978). Molecular relationships of degradative plasmids determined by in situ hybridization of their endonuclease-generated fragments. Mol. Gen. Genet. 160, 347-351. Hernandez, B. H., Higson, F. K., and Focht, D. D. (1990). Studies on the bacterial degradation of chlorobenzoic acids. Abstr. Am. SOC.Microbiol. Hickey, W. J. (1990). Enhanced mineralization of Aroclor 1242 in soil by inoculation with chlorobenzoate- and chlorobiphenyl-degradingbacteria. Ph.D. Thesis, Univ. of California, Riverside. Hickey, W. J,, and Focht, D. D. (1990). Degradation of mono-, di- and trichlorobenzoic acids by Pseudomonas aeruginosa JB2. Appl. Environ. Microbiol. 56, 3842-3850. Higson, F. K., and Focht, D. D. (1989a).The construction of ortho-halobiphenyl-degrading bacteria by a multiple chemostat method. Abstr. Annu. Meet. Am. SOC.Agron.

DEGRADATION OF BIPHENYL AND ITS DERIVATIVES

161

Higson, F. K., and Focht, D. D. (1989b). Bacterial metabolism of hydroxylated biphenyls. Appl. Environ. Microbiol. 55, 946-952. Higson, F. K., and Focht, D. D. (1990). The bacterial degradation of chlorinated acetophenones. Appl. Environ. Microbiol. 56, 3678-3685. Higson, F. K., Gibson, D. T., Nadim, L. M., and Singh, S. C. (1987). Oxidation of selected PCB congeners by Pseudomonas putida LB400 and Alcaligenes eutrophus H850. In “Research and Development Program for the Destruction of PCBs,” 6th Prog. Rep., pp. 5-8. General Electric Co., Schenectady, New York. Hiramoto, M., Ohtake, H., and Toda, K. (1989). A kinetic study on total degradation of 4chlorobiphenyl by a two-step culture of Arthrobacter and Pseudomonas strains. J. Ferment. Bioeng. 68, 68-70. Horvath, R. S. (1972).Microbial cometabolism and the degradation of organic compounds in nature. Bacteriol. Rev. 36, 146-155. Huang, C.-M. (1988). Strain construction strategies for chlorinated aromatic hydrocarbonutilizers by multiple chemostat. Ph.D. Thesis, Univ. of California, Riverside. Hutzinger, O., Blumich, M. J., von der Berg, M., and Olie, K. (1985). Sources and fate of PCDDs and PCDFs: a n overview. Chemosphere 14,581-600. Ishigooka, H., Yoshida, Y., Omori, T.. and Minoda, Y. (1986). Enzymatic dioxygenation of biphenyl 2,3-diol and 3-isopropylcatechol. Agric. Biol. Chem. 50, 1045-1046. Jezequel, S. G., Kaye, B., and Higgins, I. J. (1984). 0-dealkylation-a newly-discovered class of reactions catalyzed by the soluble monooxygenase of the methanotroph Methylosinus trichosporium OB3B. Biotechnol. Lett. 6 , 567-570. Kamp, P. F., and Chakrabarty, A. M. (1979). Plasmids specifying p-chlorobiphenyl degradation in enteric bacteria. In Plasmids of Medical, Environmental and Commercial Importance” (K. N. Timmis and A. Puhler, eds.), pp. 275-285. Elsevier, Amsterdam. Khan, A., and Walia, S. (19891. Cloning of bacterial genes specifying degradation of 4chlorobiphenyl from Pseudomonas putida OU83. Appl. Environ. Microbiol. 55,798805. Khan, A., Tewari, R., and Walia, S. (1988). Molecular cloning of 3-phenylcatechol dioxygenase involved in the catabolic pathway of chlorinated biphenyl from Pseudomonas putida and its expression in Escherichia coli. Appl. Environ. Microbiol. 54, 2664-2671. Kim, H. Y., Kim, T. S., and Kim, B. H. (1990). Degradation of organic sulfur compounds and the reduction of dibenzothiophene to biphenyl and hydrogen sulfide. Biotechno]. Lett. 12, 761-764. Kimbrough, R. D. (1987). Human health effects of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). Annu. Rev. Pharmacol. Toxicol. 27, 87-111. Kochetkov, V. V., Stasovoitov, I. I., Boronin, A. M., and Skryabin, G. K. (1982). Pseudomonas putida plasmid pBS241: plasmid-mediated biphenyl degradation. Dokl. Akad. Nouk SSSR 226, 241-243. Kohler, H.-P., Kohler-Straub, D., and Focht, D. D. (1988a). Cometabolism of polychlorinated biphenyls: enhanced transformation of Aroclor 1254 by growing bacterial cells. Appl. Environ. Microbiol. 54, 1940-1945. Kohler, H.-P. E., Kohler-Straub, D., and Focht, D. D. (1988b). Degradation of 2-hydroxybiphenyl and 22’-dihydroxybiphenyl by Pseudomonas sp. HBP1. Appl. Environ. Microbiol. 54, 2683-2688. Kong, H.-L., and Sayler, G. S. (1983). Degradation and total mineralization of monohalogenated biphenyls in natural sediment and mixed bacterial culture. Appl. Environ. Microbiol. 46, 666-672. Krockel, L., and Focht, D. D. (1987). Construction of chlorobenzene-utilizing recombi-

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nants by progenitive manifestation of a rare event. Appl. Environ. Microbiol. 53, 2470-2475. Levy, S. B., and Miller, R. V. (1989). “Gene Transfer in the Environment.” McGraw-Hill, New York. Lunt, D., and Evans, W. C. (1970).The microbial metabolism of biphenyl. Biochem. J. 118, 54P-55P. Masse, R., Messier, F., Peloquin. L., Ayotte, C., and Sylvestre, M. (1984). Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls. Appl. Environ. Microbiol. 47, 947-951. Masse, R., Badr, M., Ayotte, C., and Sylvestre, M. (1985). Gas chromatographic-mass spectrometric characterization of bacterial metabolites of &nitrobiphenyl formed in gram-negative strain B-206. Toxicol. Environ. Chem. 10,225-246. McCann, J., Choi, E., Yamasak, E., and Amer, B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72, 5135-5139. Meigs, R. A. (1987). The constitutive 7-ethoxycoumarin 0-deethylase activity of human placental microsomes-relationship to aromatase. Biochem. Biophys. Res. Commun. 145, 1012-1018. Mokross, H., Schmidt, E., and Reineke, W. (1990). Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads. FEMS Microbiol. Lett. 71, 179-186. Mondello, F. M. (1989). Cloning and expression in Escherichia coli of Pseudomonas sp. LB400 genes encoding polychlorinated biphenyl degradation. J. Bacteriol. 171, 1725-1732. Muller, R., and Lingens, F. (1980). Enzymatische Bildung und Isolierung von 2-Hydroxymuconsaeure, ein Metabolit im bakteriellen Abbau des Herbizids Chloridazon. 2. Naturforsch., C 35C, 346-347. Nadim, L. M., Schocken, M. J., Higson, F. K., Gibson, D. T., Bedard, D. L., Bopp, L. H., and Mondello, F. J. (1988). Bacterial oxidation of polychlorinated biphenyls. Proc. U.S. EPA Annu. Res. Symp. Land Disposal, Remedial Action, Incineration Treat. Hazard. Waste, 13th, Cincinnati, Ohio. Ogunseitan, 0. A . , Tedford, E. T., Pacia, D., Sirotkin, S. M., and Sayler, G. S. (1987). Distribution of plasmids in groundwater bacteria. J. Ind. Microbiol. 1, 311-317. Ohisa, N., and Yamaguchi, M. (19791. Cfostridium species and gamma-BHC degradation in paddy soil. Soil Biol. Biochem. 11, 645-649. Omori, T., Sugimura, K., Ishigooka, H., and Minoda, Y. j1986a). Purification and some acid hydrolyzing enzyme properties of a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic from Pseudomonas cruciviae S93B1 involved in the degradation of biphenyl. Agric. Biol. Chem. 50, 931-937. Omori, T., Ishigooka, H., and Minoda, Y. (1986bj. Purification and some properties of 2hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) reducing enzyme from Pseudomonas cruciviae S93B1 involved in the degradation of biphenyl. Agric. Biol. Chem. 50, 1513-1518. Omori, T., Ishigooka, H., and Minoda, Y . (1988). A new metabolic pathway for meta ringfission compounds of biphenyl. Agric. Biol. Chem. 52, 503-509. Opperhuizen, A,, Gobas, F. A. P. C., van der Steen, J. M. D., and Hutzinger, 0. (1988). Aqueous solubility of polychlorinated biphenyls related to molecular structure. Environ. Sci. Technol. 22, 638-646. Parkinson, A,, Safe, S. H., Robertson, L. W., Thomas, P. E., Ryan, D. E., Reik, L. M., and Levin, W. (1983). Immunochemical quantitation of cytochrome P450 isozymes and epoxide hydrolase in liver microsomes from polychlorinated or polybrominated

DEGRADATION OF BIPHENYL AND ITS DERIVATIVES

163

biphenyl-treated rats: a study of structure-activity relationships. J. Biol. Chem. 258, 5967-5976.

Patel, T. R., and Gibson, D. T. (1976). Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological properties. J. Bacteriol. 128,842-850. Pettigrew, C. A., Breen, A., Corcoran, C., and Sayler, G. S. (1990). Chlorinated biphenyl mineralization by individual populations and consortia of freshwater bacteria. Appl. Environ. Microbiol. 56, 2036-2045. Pfeifer, F., Schacht, S., Klein, J., and Triiper, H. G. (1989). Degradation of diphenyl ether by Pseudomonas cepacia. Arch. Microbiol. 152, 515-519. Quensen, J. F., 111, Boyd, S. A., and Tiedje, J. M. (1990). Appl. Environ. Microbiol. 56, 2360-2369.

Rhee, G.-Y., Bush, B., Brown, M. P., Kane, M., and Shane, L. (1989). Anaerobic biodegradation of polychlorinated biphenyls in Hudson River sediments and dredged sediments in clay encapsulation. Water Res. 23, 957-964. Rosenberg, E., Perry, A., Gibson, D. T., and Gutnick, D. L. (1979). Emulsifier of Arthrobacter RAG-1: specificity of hydrocarbon substrate. Appl. Environ. Microbiol. 37, 409-413,

Sayler, G. S., Hooper, S. W., Layton, A. C., and King, J. M. H. (1990).Catabolic plasmids of environmental and ecological significance. Microb. Ecol. 19, 1-20, Schwartz, R. D. (1981). A novel reaction: meta-Hydroxylation of biphenyl by an actinomycete. Enzyme Microb. Technol. 3, 158-159. Shields, M. S., Hooper, S. W., and Sayler, G. S. (1985). Plasmid-mediated mineralization of 4-chlorobiplienyl. J. Bacteriol. 163, 882-889. Smith, R. V., and Rosazza, J. P. (1974). Microbial models of mammalian metabolism. Aromatic hydrocarbons. Arch. Biochem. Biophys. 161, 551-558. Smith, R. V., Davis, P. J., Clark, A. M., and Glover-Milton, S. (1980). Hydroxylations of biphenyl by fungi. 1. Appl. Bacteriol 49, 65-73. Smith, R. V., Davis, P. J., Clark, A. M., and Prasatik, S. K. (1981).Mechanism of hydroxylation of biphenyl by Cunninghamella elegans. Biochem. J. 196, 369-371. Suflita, J. M., Horowitz, A., Shelton, D. R., and Tiedje, J. M. (1982). Dehalogenation. A novel pathway for the anaerobic biodegradation of haloaromatic compounds. Science 218, 1115-1117. Sylvestre, M., Masse, R., Messier, F., Fauteux, J., Bisaillon, J.-G., and Beaudet, R. (1982). Bacterial nitration of 4-chlorobiphenyl. Appl. Environ. Microbiol. 44,871-877. Takase, I., Omori, T.. and Minoda, Y. (1986). Microbial degradation products from biphenyl-related compounds. Agric. Biol. Chem. 50, 681-686. Tittmann, U., and Lingens, F. (1980). Degradation of biphenyl by Arthrobacter simplex strain BPA. FEMS Microbiol. Lett. 8, 255-258. Tomasek, P. H., and Crawford, R. L. (1986). Initial reactions of xanthone biodegradation by an Arthrobacter sp. 1. Bacteriol. 167, 818-827. Tsuda, M., and Ilino, T. (1987). Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWW0. Mol. Gen. Genet. 210, 270-276. Unterman, R., Bedard, D. L., Brennan, M. J., Bopp, L. H., Mondello, F. J., Brooks, R. E., Mobley, D. P., McDermott, J. B., Schwartz, C. C., and Dietrich, D. K. (1988). Biological approaches for polychlorinated biphenyl degradation. In “Environmental Biotechnology-Reducing Risk from Environmental Chemicals Through Biotechnology“ (G. S. Omenn, ed.). Plenum, New York. Viney, I., and Bewley, R. J. F. (1990). Preliminary studies on the development of a microbiological treatment for polychlorinated biphenyls. Arch. Environ. Contam. Toxicol. 19, 789-796.

164

FRANK K. HIGSON

Waid, J. S., ed. (1986). “PCBs and the Environment,” 3 vols. CRC Press, Boca Raton, Florida. Walia, S., Khan, A., and Rosenthal, N. (1990). Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes i n toxic organic-contaminated soil environments. Appl. Environ. Microbiol. 56, 254-259. Weisshaar, M. P., Franklin, F. C. H., and Reineke, W. (1987). Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13. J. Bacteriol. 169, 394-402. Wittich, R.-M., Schmidt, S., and Fortnagel, P. (1990). Bacterial degradation of 3- and 4carboxybiphenyl ether by Pseudornonas sp. NSSZ. FEMS Microbiol. Lett. 67, 157160. Yates, J. R., and Mondello, F. J. (1989). Sequence similarities in the genes encoding polychlorinated biphenyl degradation by Pseudomonas strain LB400 and Alcaligenes eutrophus H850. J. Bacteriol. 171, 1733-1735. Yeh, W.-K., Gibson, D. T., and Liu, T.-N. (1977). Toluene dioxygenase: a multicomponent enzyme system. Biochern. Biophys. Res. Commun. 78,401-410.

The Sensitivity of Biocatalysts to Hydrodynamic Shear Stress ALESPROKOP* AND RAKESHK. B A J P A I ~ * Department of Chemical Engineering Washington University St. Louis, Missouri 63130 +

Department of Chemical Engineering Engineering Building University of Missouri Columbia, Missouri 6521 1

I. Introduction 11. Cell Architecture and Its Relationship to Hydrodynamic Shear Stress 111. Fluid Mechanics A. Stirred Tank Reactors B. Other Reactor Types IV. Methods of Assessing Shear Sensitivity V. Sensitivity of Biocatalysts to Hydrodynamic Stress A. Enzymes B. Prokaryotes C. Lower Eukaryotes D. Mammalian Cells E. Insect Cells F. Plant Cells G. Nematodes H. Comparative Study VI. Summary and Outlook A. Physical Effects B. Biological Effects VII. Nomenclature References

The purpose of this article is to review the current status of shear sensitivity of microorganisms and other biocatalysts. The structural features of biocatalysts serve as a basis for rational explanation of effects of shear damage. The fluid mechanics in a real reactor provides a basis for shear field characterization. Experimental assessment of shear sensitivity is then covered, followed by a detailed discussion of different types of organisms and biocatalysts as affected by shear. Physical and biological mechanisms of shear damage and ways of overcoming the adverse effects of shear are then summarized. 165 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 37 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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I. Introduction One of the key parameters in the development of processes utilizing biocatalysts is their sensitivity to hydrodynamic shear stress. Bioreactors can generate strong forces in the region of the impeller, along the walls, and at air-liquid interfaces. These forces are strong enough to inhibit or permanently destroy the activity of biological catalysts. Enzymes, fungi, and plant, mammalian, and insect cells have all been reported to be sensitive to forces generated in bioreactors and during the bioprocessing. This review article examines what is known about the sensitivity of enzymes and organisms to shear stress, the magnitude of the forces experienced by biocatalysts in various reactor types, and the implications for the design and operation of biochemical processes. A comparison of the behavior of the biocatalysts between themselves might be useful and can provide a basis for a rational approach. Considering the importance of this topic, there have been relatively few studies on the shear sensitivity of biocatalysts, and there are no review articles presently in the literature. This may be due in part to the fac that microorganisms, which have been employed in the biotechnology industry for several millenia, are relatively insensitive to mixing forces. Plant, insect, and mammalian cell cultures, which have come into the focus only in the past 20 years, however, are believed to be very sensitive to these forces. There are two important aspects to this problem. The first is cell structure and physiology, which dictates how a cell will respond to hydrodynamic stress. The second is the type of forces exerted on a cell and the magnitude and duration of these forces. li. Cell Architecture and Its Relationship to Hydrodynamic Shear Stress

Some insight into how an organism will respond to hydrodynamic shear stress can be obtained by examining four key aspects of cellular anatomy: (I)the cell membrane and/or cell wall which surrounds the cell and acts as the first line of defense against shearing forces; (2) the cell cytoskeleton and the highly compartmentalized and viscous cytoplasm which, in addition to the outer cell boundary, absorbs some of the energy generated by turbulence; (3) the cell size, which determines to what extent the kinetic energy present in turbulent flow will be adsorbed by the cell; and finally, (4) the presence of a mechanism to receive and amplify fluid mechanical stimuli. The widely varying degrees of sensitivity among different organisms can be traced to fundamental differences in these criteria. Figure 1 provides schematics of the

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SHEAR SENSITIVITY OF BIOCATALYSTS

Gram (-)

,

Gram (+)

chromosome (DNA)

plasma membrane

I

Approximately 1 -5 pm

4

FIG. 1. Architecture of a typical prokaryote.

typical architecture of bacteria; Fig. 2 (Finean, 1978) that of mammalian and plant cells. All cells are surrounded by a lipid bilayer membrane, embedded with enzymes and structural proteins, which mediates communication between the cell and the environment. For bacteria, yeasts, and plant cells, however, an additional barrier between the cell and the environment exists: the cell wall, which can be thought of as a complex organelle carrying out information processing, metabolism, and immunological duties. The cell walls of bacteria, yeasts, and plant cells, although differing considerably in composition, are built on the same structural principles. There is a relatively thick amorphous polysaccha-

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ALES PROKOP AND RAKESH K. BAlPAI

Animal

Plant

Gel! wall

Nuclear membrane with pores

plasma membrane

K

Approximately 10 p m

>I

FIG. 2. Architecture of a typical eukaryote (cytoskeleton not shown). (After Finean, 1978.)

ride gel in which are embedded long fibers, composed of protein, carbohydrate, or both, which reinforce the gel (Rogers, 1968). The net effect is a rigid barrier between the cell and the environment. Cells which lack a cell wall will generally be more susceptible to hydrodynamic stress-induced damage to the cell ultrastructure. The turbulent eddies perform work on the cell wall or cell membrane and expend their energy through the process of viscous dissipation. If the external cell boundary is deformable, a portion of the work done on this boundary will be transmitted to the interior of the cell (Fischer, 1980). The cell envelope may experience microscopic deformation, in which a significant portion of the wall or membrane is affected. In some cases, deformation may occur only at the molecular level. For example,

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stress-bearing components of the cell envelope may experience considerable deformation without the structure, as a whole, experiencing any microscopically visible effect. The cytoplasm of most cells is a dense, highly viscous gel (Fulton, 1982). Several methods have been used to measure the viscosity of the cytoplasm. Viscosities on the order of several thousand poise have been measured by various methods (Valberg and Albertini, 1985). Current models of the cytoplasm characterize it as a viscoelastic polymer network (Sato et a]., 1984; Buxbaum et a]., 1987) with both fluid-like and solid-like properties (Sato et a]., 1987). Some data on viscosities of cytoplasm and cellular components are presented in Bereiter-Hahn (1987).

The mechanical properties of the cytoplasm are largely determined by the cytoskeleton (Sato et a]., 1984; Buxbaum et a]., 1987). The cytoskeleton is composed of several proteins which are capable of polymerizing into long fibrous networks. The primary components are three interacting filament systems: the microtubules (microfilaments), actin filaments, and the intermediate filaments. This milticomponent system mediates movements of the cell (Dunn and Heath, 1976) and of intracellular organelles (Williamson, 1986) and plays an important role in determining cell shape (Watt, 1986; Lloyd, 1982). The cytoskeleton is intimately associated with the cell membrane through specific protein interactions. When the plasma membrane of certain cell types is acted on by mechanical stimuli, the cytoskeleton undergoes a rapid and dramatic reorganization of its components (Franke et al., 1984). The mechanical properties of eukaryotic cells are reviewed in Bereiter-Hahn (1987) and Hiramoto (1987) and those of representative mammalian cell types are discussed in Prokop (1991). Some organisms possess receptors, either in the cell wall or cell membrane, which are sensitive to mechanical stress. The presence of such receptors implies that these cells have evolved specific regulatory responses to mechanical stimuli. Evidence for such receptors has been found in yeast (Gustin et a]., 1986), higher plants (Falke et a]., 1986), and mammalian cells (Guharay and Sachs, 1984,1985; Stockbridge and French, 1988), suggesting that they may be ubiquitous. Stretch-sensitive receptors are actually believed to be ion channels which pass specific ions when mechanically stimulated (Guharay and Sachs, 1984). Localized membrane tension causes a subpopulation of ion channels to open. The signal is rapidly transmitted throughout the entire cell by ion channel networking via cytoskeletal elements (Guharay and Sachs, 1984). The flood of ions into or out of the cell is believed to be one of the first events in a signal amplification cascade.

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Studies are now being conducted on plant cells which are known to be sensitive to mechanical forces such as friction, flexure, and contact with neighboring cells, to show that they possess a similar system (Falke et a]., 1986). Ill. Fluid Mechanics

A. STIRRED TANKREACTORS The forces acting on an organism suspended in a fluid are the result of the dissipation of kinetic energy, present in fluid in motion, to the external surface of the organism. The magnitude of these forces is a function of the fluid viscosity and the velocity gradient in the boundary layer surrounding a cell: T

= py =

-p(dv/dy)

(1)

where 7 is the shear stress, p is the fluid viscosity, and y is the rate of shear strain. The rate of shear strain (dvldy) is the velocity gradient normal to the surface of the organism. Chemical engineers usually distinguish between two types of flow: laminar, in which the fluid flows in smooth layers or laminae, and turbulent, in which there is a vigorous interchange of small packets of fluid between adjacent layers of the fluid. The type of fluid flow field present in a given system is characterized by the dimensionless Reynolds number: Re

= pfvtlp

(2)

where pf, v, and p are the fluid density, velocity, and viscosity, respectively, and t is the characteristic length of the system. In an agitated reactor, typically, the characteristic length would be the impeller diameter. Most bioreactors are operated in the fully turbulent regime which is characterized by a Reynolds number of 1000 or greater (Nagata, 1975). In laminar flow the shear stress can be easily calculated from the fluid viscosity and the velocity profile of the system [Eq.(I)].When an organism is suspended in the bulk flow, one must consider the cell (particle) Reynolds number to determine the stress acting on the cell (Bird et a]., 1960): Re, = pd,/u = pfydglp (3) If the cell Reynolds number is <1, then viscous forces dominate and the shear stress acting on the cell can be calculated by the following expression (Stokes flow):

SHEAR SENSITIVITY OF BIOCATALYSTS

Re, < 1

T = 3py/2

171 (4)

When the cell Reynolds number is >1, inertial forces dominate and a fluid drag coefficient must be considered. The drag coefficient is USUalIy obtained from empirically derived correlations. The stress is then calculated from the following formula (Bird et al., 1960): r

=

CDpfv2/2

1

< Re, < 100

(5)

where C, = 18/(Rep)3’5. 1. Turbulent Regime The situation is far more complex in the turbulent flow regime. Here, the instantaneous velocity vector may be written as the sum of the timeaveraged velocity and a randomly fluctuating time-dependent velocity (illustrated in Fig. 3). The intensity of turbulence in a given system is directly related to the magnitude of the fluctuating velocity vector. The shear stresses generated in a turbulent flow field are thus of a different nature from those present in laminar flow due to this fluctuating component of the velocity vector. A cell suspended in a laminar flow field will experience mean shear stress that is independent of time, while a cell suspended in a turbulent flow field can experience bursts of shear stresses, as well as dynamically oscillating forces (Batchelor, 1967). A second feature of turbulence which is relevant to this discussion is the scale at which energy dissipation occurs. In a stirred tank bioreactor, kinetic energy is imparted to the fluid by the rotating impeller blade. In a bubble column, the rising gas bubbles transfer kinetic energy to the liquid. Initially, this energy is transported by the eddies, which

FIG. 3. Instantaneous velocity vector as broken into time-averaged (v)and fluctuating velocity (v’) components.

172

ALES PROKOP AND RAKESH K. BAJPAI

are characterized by a length scale approximately equal to the diameter of the impeller in the case of a stirred tank reactor, or the diameter of a rising gas bubble in the case of a bubble column. The kinetic energy present in these eddies is rapidly passed down to smaller and smaller eddies in an energy cascade. Most of the energy that is introduced in a bioreactor by agitation is transferred to the fluid internal energy through viscous dissipation of small eddies. These eddies are characterized by a length scale called the Kolmogoroff microscale (Tennekes and Lumley, 1972). The kinetic energy of turbulence is transferred from microscale eddies to the organisms suspended in the turbulent flow field, to the extent determined by the relative sizes of the eddy and the cells. When the eddy size is significantly larger than the characteristic length of an organism, an insignificant amount of kinetic energy is dissipated on the cell surface and the cell may be thought of as floating in a pool of quiescent fluid bounded by the dimensions of the eddy (Moo-Young and Blanch, 1981; Prokop and Rosenberg, 1989). Microscale eddies, which are approximately the same size as the characteristic dimension of the suspended organism, will perform mechanical work on the cell (Moo-Young and Blanch, 1981).Microscale eddies that are significantly smaller than a suspended cell may perform work on the cell if the energy content of these eddies is large enough. The Kolmogoroff microscale in stirred tank reactors (Nagata, 1975; Okamoto et al., 1981; Cutter, 1966) is on the order of 25 to 200 pm in laboratory-scale stirred tank reactors. The microscale is even larger in bubble column reactors (Schiigerl, 1982). Since plant and mammalian cells have diameters in this range, they experience a large fraction of the kinetic energy present in the reactor in the form of shear work done on the cell surface. Microcarrier beads used to grow mammalian cells have dimensions in the range of 150-250 p m . The cells anchored on these beads are not free to rotate, as are the suspended cells in suspension. Hence, in this case, the characteristic size of microcarriers is of importance, not that of the individual cells. Prokaryotes and some lower eukaryotes are an order of magnitude smaller than the Kolmogoroff microscale present in most bioreactors, and thus are not affected by the majority of the turbulent energy spectrum. When microorganisms form larger structures or aggregates (such as filaments and pellets), however, these organisms will be subjected to the turbulent energy spectrum present in microscale eddies. Under these conditions, microorganisms do display sensitivity to hydrodynamic stress. Another complication results from a nonhomogeneity of the flow field in reactors. In a stirred tank reactor there are regions of high shear

SHEAR SENSITIVITY OF BIOCATALYSTS

173

stress and regions of low shear stress as shown in Fig. 4. The highest shear stresses are generated in the boundary layers surrounding the impellers (van't Riet and Smith, 1975). The maximum time-averaged stresses occur in the trailing vortices in the wake of the blades generated as the impeller slashes through the liquid (van't Riet and Smith, 1975). These stresses are directly proportional to the impeller Reynolds number. The mean shear stress in the bulk fluid drops off dramatically as a cell travels farther away from the impeller region (Nagata, 1975). A third consideration in agitated reactor vessels is that suspended cells may experience cell-cell collisions and/or cell-impeller collisions. Damage will be a function of collision frequency and the amount of energy transmitted to the cells during the collision event (Cherry and Papoutsakis, 1986). The flow field will be modified by approaching collision partners such that suspended cells will be subjected to transiently increased shear stress levels. If the kinetic energy of the collision partners is sufficiently large, a severe collision event may occur. Ways of evaluating collision severity are discussed later. To understand the phenomena involved in hydrodynamic damage better, a more fundamental description of flow can be adopted using results from the fluid-mechanical approach via basic equations of continuity, motion, energy balance, etc. The stress is defined as force per

mpeller region

bulk fluid region

FIG. 4. Three-region model of a stirred tank reactor. Each region is characterized by different turbulent intensities and mechanisms.

174

ALES PROKOP AND RAKESH K. BAJPAI

unit area. In a viscous fluid, the stresses on an element of fluid (or on a solid particle, such as cell suspended in it) are of two types (for simplicity, only one plane is depicted in Fig. 5) (Sabersky and Acosta, 1964; Kay and Nedderman, 1985):normal stress {pxx),acting on a plane perpendicular to the direction in which it acts, and tangential shear stress, whose direction is parallel to the surface on which they act (e.g., 7xr T ~ J .The normal stress has typically two contributions: the first from isotropic fluid pressure (local, time-averaged pressure), the second from normal viscous stresses ( T ~ ~p x) x: = T~~ - p. Note that pressure p is regarded as being positive for compression whereas the normal stress components pxx, etc. are defined as being positive for tension. To illustrate the situation once more in terms of forces: term total forces

(1) =

(2)

normal forces -P + Txx

(3)

+ tangential forces +

(Txy, Txx,

(6)

etc.1

All the viscous stresses are conveniently put together into one stress

+

FIG. 5. Stress action on yz-plane.

SHEAR SENSITIVITY OF BIOCATALYSTS

175

tensor. The stress tensor thus has a normal [diagonal) and shear stress components. As described earlier, both normal and tangential stresses can be expressed, for incompressible fluids, as functions of velocity gradient (Sabersky and Acosta, 1964): T~~

c~

p(dvi/dxi)

7xy 'x

(dilatation = 0)

p(dvi/dyi + dv,/dx,)

(7) (8)

where vi, vi (and vk) are the components of the velocity vector and represent velocities in the xi, xjand xk direction. It will be appropriate to examine the physical implications of forces a fluid element (particle) will experience. Term (1)of Eq. (6) causes a translation, while term (3) is responsible for rotation. Terms (2) and (3) together can cause distortion (deformation). A mere translation does not, however, result in a stress. Term (3) itself is the true shearing term. An illustration of these terms is attempted in Fig. 6 (Sabersky and Acosta, 1964). Another way of representing particle-fluid eddy interactions is shown in Figure 1 7 of Prokop and Rosenberg (1989). A generalized momentum conservation equation can be written as follows (Kay and Nedderman, 19851:

..- - - -....?

Translocation

Angular distortion (stretching and compression)

't?. Volume

Rotation

distortion

FIG. 6. Basic components of the motion of a fluid element. (After Sabersky and Acosta, 1964.)

176

ALES PROKOP AND RAKESH K. BAJPAI

p

Dv

=

-V p

-

[V.71

+ pg

(9)

where DIDt is the total derivative and V (del) is the grad-operator. For time-averaged (steady] conditions in incompressible fluids, the term on the left-hand side of Eq. (91 is zero. The first and second terms on the right-hand side represent pressure and viscous stress contributions. Such an equation, although valid for both laminar and turbulent motion, cannot be used directly to solve problems in turbulent flow where the instantaneous velocities (such as vi) can be decomposed into a time-averaged (macroscopic mean) value (overbar denotes time-averaging) and fluctuation v' around that value (v = 0 + v') (Fig. 3). Resulting modification represents, in fact, the introduction of additional stress components into the equation of motion (Kay and Nedderman, 1985). Thus Eq. (9) becomes

*

(10) where dt) is the turbulent momentum tensor, whose components are commonly known as the Reynolds stresses. These are defined as

In an energy cascade smaller eddies, arising due to a turbulent mixing, finally transfer kinetic energy into the inertial energy of the fluid by the molecular transfer process which is termed viscosity. Accordingly, the Reynolds stresses may be expressed as follows:

where is eddy or turbulent (dynamic) viscosity. It is not a constant and depends on both flow pattern and fluid properties. The practical interpretation of Eq. (10) is then as follows. The timeaveraged shear stress thus has two components, only one of them associated with turbulent phenomena:

In Eq. (13), d") stands for momentum transfer by microscopic mixing (viscous dissipation) and df)is due to turbulent inertial [macroscopic) mixing. It can be shown (Prokop and Rosenberg, 1989; Reynolds, 1971) that the ratio of the turbulent to viscous contributions of the stress can be equated to the Reynolds number characterizing the mean flow prop-

SHEAR SENSITIVITY OF BIOCATALYSTS

177

erties. One may expect that turbulent stresses will be dominant in a flow with high Reynolds numbers, except very near the vessel wall. Laminar flow (and associated stresses) is only a less significant component of the total picture under turbulent conditions. In this context, laminar (viscous) flow is an abnormal state which only prevails at low values of the Reynolds number when the viscous forces are sufficiently large. Under turbulent conditions,

~ not a constant, Eq. (14) merely says that the proportionality Since l ( is in this equation breaks down at turbulent flow and the viscosity effect is somewhat lost. As the formulation of energy or force balance [above) with time-dependent variables does not provide exact solutions to a real situation, the only practical approach involves dimensional analysis guided by certain physical simplifications. The eddy viscosity method already represents such an approach. Other approaches include the mixing length method of Pradtl or the isotropic turbulence theory of Kolmogoroff. These have been used to approximate the Reynolds stresses in Eq. (10). The criticism of these approaches was presented by Leslie (1973). These models have been sometimes applied indiscriminately. For example, Prandtl’s method has been discussed in relation to the viscous dissipation term of Eq. (lo) (Sabersky and Acosta, 1964), as opposite to Reynolds stress (Taylor et al., 1980). The Kolmogoroff theory has been typically applied to evaluate inertial Reynolds stresses, although the theory is strictly applicable for eddies in the equilibrium energy-dissipation range.

2. Stresses

on Cells in Turbulent Regime

Having considered limitations of the fluid mechanical approach and, particularly, realizing that the total stress has several important components, we can now proceed toward some practical evaluations of these forces. However, it should be kept in mind that they represent only estimates. To estimate the forces acting on a cell in a bioreactor, one can categorize these forces in the form of time-independent and time-dependent shear stresses as above. The more stable time-averaged component [time-independent] will be addressed first because it is easier to measure. a . Time-Independent Forces. In a stirred tank reactor, cells will experience high steady shear stresses in the boundary layers surrounding

178

ALES PROKOP AND RAKESH K. BAJPAI

the impellers and much lower levels of stress in the bulk fluid phase. The organism will experience a maximum time-independent shear rate at the impeller tip as given by a semiempirical correlation (Wichterle et al., 1984): y,

=

N[1

+ 5.3p)l’P [Re’)l/(P+’)

115)

where N is impeller speed and (Re’)is a generalized Reynolds number. As the latter also contains N, the shear rate in Eq. (15) is proportional to N to the power p [ = 3/(n 1)1, where p varies from 1.5 to 2.5 as the flow index n varies from 1to 0.2. This dependence is in contrast to the estimate of mean shear rate as given by Calderbank and Moo-Young (1959) and others (Metzner and Otto, 1957; Metzner and Taylor, 1960):

+

Ymean =

Idv

(16)

where the average shear rate is proportional to N. The constant k is a function of the viscosity of the medium and the geometry of the impeller (Atkinson and Mavituna, 1983). The measured shear rates described by Eq. (15) are up to 100 times larger than those given by Eq. (16). New measurements on the front face of the Rushton turbine blade performed by Robertson and Ulbrecht (1987) agree well with the boundary layer theory (Schlichting, 1973). The suggested formula for maximum shear rate is y,

= 3.3N(Re)lI2

(17)

where Re = N D 2 / v and v is the zero-shear kinematic viscosity. The correlation has been confirmed from Re 100 to 29,000 using O.l-lo/o polyox solutions to simulate process conditions. Van’t Riet and Smith (1973) on the other hand got a formula similar to Eq. (16): Ymax ==

90N

(18)

Croughan et al. (1987) have suggested that the maximum shear stress on the microcarrier suspended in a turbulent field can be estimated by the formula derived for the creeping flow (laminar regime!): rmax ==

3p.Y

(19)

where Y is the one-dimensional gradient in time-averaged velocity [cf. Eq. (4)]. Y can be assessed from the following, based on previously reported measurements (Nagata, 1975; Oldshue, 1983):

Y = 0.4?rNdi [for flat blade impeller) (20) While overlooking these differences, there is a problem in the fact that

SHEAR SENSITIVITY OF BIOCATALYSTS

179

the shear rate in turbulent flows cannot be simply converted into stress by multiplying by viscosity [see nonconstancy of /.+ in Eq. (14)]. Alternatively, the stress at the surface of an impeller blade for a given distance x from the tip of the blade can be estimated from the boundary layer theory (Schlichting, 1973): T~ =

0 . 0 2 9 4 p ( 2 ~ d ~(N2)~~d ~ N x l v ) - ~ / ~

(21)

According to this, the shear stress should be proportional to Since cells will be excluded from the region immediately adjacent to the surface, due to steric hindrances and lift forces, the exponent of N will be somewhat less. Equation (17) would thus give the best estimates since it is based on real data. b. Time-Dependent Forces. To estimate the time-dependent stresses acting on a suspended flow, one must calculate the energy dissipation rate per unit of mass. This can be estimated from the power number and the reactor geometry (Nagata, 1975): E

= NpN3d:/V

(22)

The power number N, can be obtained from empirical curves (e.g., in Harnby et al., 1985). When the cell Reynolds number is small (
(23)

7(;) a p ( ~ / v ) * / ~

(24)

v;

a:

and and is independent of cell (particle) size d,. For practical evaluation of viscous stresses, the following equation can be used: dt) = 0.73p(~/v)*’~

(25)

Viscous-subrange turbulent stresses will occur primarily in the bulk fluid. The fluctuating velocity component of turbulence will also vary with the vessel location. For large particle sizes, the inertial subrange holds and, as derived on the basis of dimensional analysis (Matsuo and Unno, 1981), v;

CL:

(cdP)1/3

(26)

Time-dependent inertiahubrange turbulent stresses (in this case denoted as Kolmogoroff stresses) predominate in the impeller region and can be estimated by (Thomas, 1964; Toni and Bagster, 1978)

180

ALES PROKOP AND RAKESH K. BAJPAI

The Kolmogoroff stresses will only operate on suspended organisms if the microscale is of the same order of magnitude as the characteristic dimension of the organism. The Kolmogoroff microscale is given by (Tennekes and Lumley, 1972)

e,

=

(28)

(v3/€)1/4

Assuming turbulence-related cell damage to be limited only to situations where cellular dimensions equal or exceed the Kolmogoroff length, Croughan et al. (1988) have proposed and verified the following expression for eddy-related cell damage of microcarrier-grown cells: Dcldt

=

Dcldt

= p, -

p,

K,(E)~’~C

e < e, e 2 e,

(29) (30)

This treatment assumes that cells grow at a specific rate which is independent of turbulent structure around them, but the cells are also damaged by turbulence showing a first-order dependence on the “concentration of eddies” in the system. Finally, turbulent Reynolds shear stresses can be estimated from the laser-Doppler anemometry (Yianneskis et al., 1987). Yianneskis et al. measured both turbulent normal and shear stresses for a vessel fitted with a Rushton turbine. The Reynolds stresses are extremely position and direction/plane dependent, with the maximum value given by rR =

0.056p(~rND)~ = 0.17p(~Nd~)~

(31)

where (rNdi) is an impeller tip speed. The proportionality between the Reynolds shear stress and the square of the impeller tip speed conforms well with the early concepts of Rushton and Oldshue (1953) on Reynolds stress in stirred vessels. Such estimates are considerably larger than the stress estimates from Kolmogoroff’s theory in the viscous subrange (Oh et al., 1989). These in fact correspond to the dependencies to be expected for the inertial subrange [see Eqs. (22) and (27)]. Based on an analogy with disintegration of flocs in a turbulent field, one can assume that the physical mechanism of cell damage under the turbulent regime may include bulgy deformations, followed by a loss of cell membrane integrity (perforation) (Thomas, 1964). However, no experimental data are available to demonstrate such a cell damage. It is well documented that the dissipation rate is maximal near the impeller (Cutter, 1966; Placek and Tavlarides, 1985). The latter authors recommended using dp in place of the volume in Eq. (22). The maximum energy dissipation rate is then

181

SHEAR SENSITIVITY OF BIOCATALYSTS

E'

=

N,N3df

(32)

and should be used preferably in Eqs. (23) through (28) (Cherry and Papoutsakis, 1988). Rosenberg (1987) presented a comparison of stress estimates for his Haake viscometer provided with a two-bladed paddle impeller, 4 cm in diameter. He used this viscometer with a plant cell culture and considered an average aggregate size of 190 pm in his calculations. Estimates of both time-averaged and time-dependent stresses, together with a Kolmogoroff microscale estimate for the time-dependent situation, are presented in Table I. The proportions of viscous shear in bulk flow and Kolmogoroff shear are apparent from the last two columns of this table, demonstrating a predominance of the latter forces. The corresponding numbers for the calculations related to this paper are also listed in Table I. From Table I, it can be concluded that plant cells suspended in the bulk fluid will experience a mean shear stress on the order of 0.0965 dyn/cm2, depending on the impeller speed. As a cell aggregate moves into the impeller region, it will be subjected to forces on the order of 1.8-898 dyn/cm2. The Kolmogoroff scale varies between 242 and 22 pm in size, mostly below the aggregate size. From other studies with carrot suspension cultures in a 1.5-liter bioreactor (Rosenberg, 1987),it appears that cell viability and the growth rate were depressed when the Kolmogoroff scale was less than 80 pm in size. 3. Collision-Related Damage of Cells

An additional consideration in agitated reactor vessels is that suspended cells may experience potentially damaging cell-cell collisions TABLE I ESTIMATESOF TIME-AVERAGED AND TIME-DEPENDENT SHEARSTRESSES AS A FUNCTION OF IMPELLER TIP SPEED^ ~

Tip speed (cm/sec) 13.4 26.8 53.6 107.2 214.5 314.0 419.0

Equation

~

Trnea,

Tmax

rk

(dyn/cmz)

1, (Pm)

S p

(dynlcm')

(dynlcmz)

(dynlcmz)

0.09 0.18 0.36 0.72 1.44 54.3 64.4

1.8 6.4 22.2 78.1 270.0 526.9 898.0

242 147 90 57 35 26 22

0.1 0.4 1 .o 2.5 6.5 11.5 16.2

20.7 40.2 77.5 142.5 271.3 396.2 497.0

(3)-(5)

(15)

(22) and (28)

(24)

(271

Calculations are for an average aggregate size of 190 pm, Rosenberg (1987).

182

ALES PROKOP AND RAKESH K. BAJPAI

and/or cell-impeller collisions (Cherry and Papoutsakis, 1986). In this context, a collision is defined as the close approach of two bodies such that the shear field around them is significantly modified. In a typical stirred tank reactor, the suspended cells may collide with other cells, or with the impeller, baffles, walls, and internal probes. If the forces which push two cells (or beads) toward one another, or if the forces which drive a cell and an impeller blade together, are greater than the force required to remove the fluid film between them, then actual physical collision will occur. Cell damage will be a function of collision frequency and the amount of energy transmitted to the cells during the collision event. Energy transfer will be dependent on the deformability of the cell wall or plasma membrane. When the Kolmogoroff microscale is approximately the same as the distance separating individual cells, beads, or cell aggregates, then the frequency of cell-cell collisions can be estimated by an equation derived by Cherry and Papoutsakis (1988, 1990), based on the work of Hinze (1971) (first term of the right hand-side of equation):

N, = (va2/d;)(nd;/6a)

(33)

These authors defined the turbulent collision severity CS, as the product of the collision frequency and the kinetic energy of the collisions per cell (particle) rather than per volume: CS, = (~v)3/*+p,ad~/72 (34) in which a! is the volume fraction of cell (beads) in the system and the characteristic velocity (Nagata, 1975) of eddies in isotropic turbulence has been used for the relative cell (bead) velocity v: (35) Croughan et al. (1988) have used a similar concept to investigate the detrimental effect of agitation and number density of beads on the growth rate of mammalian cells on microcarrier beads. According to their model, the rate of damage of cells by cell-cell collision can be written as v =

Dcidf

=

[v41’4

K2E3’4Cm

(36)

where C, is the concentration of microcarrier beads in the reactor. This expression corresponds to the quadratic dependence of collision frequency on C,. These authors have found an excellent agreement between the experimental observations using an inert microcarrier addition (Sephadex beads) and this theory. At the same time, an experimental evidence, not available earlier, has been provided for the impeller collision mechanism (Croughan et al., 1987).

SHEAR SENSITIVITY OF BIOCATALYSTS

183

Cells will collide with impeller blades by a process of interception if they pass within a certain distance of the blade. A collision window, demarcated by one cell diameter on either side of the stagnation point of fluid streamline, can be defined. The collision window is set equal to the volume fraction occupied by cells ( a ) rather than by the reactor volume (Cherry and Papoutsakis, 1986). Any cell entering the collision window will collide with the impeller. The frequency of this event can be approximated by the following expression (Rosenberg, 1987): Ni

= 18nbd;Na/8d:

(37)

If particles actually touch the impeller during the collision event, then the energy of the collision will be approximately equal to the transfer of kinetic energy. The severity of collision can again be defined as the product of the frequency of collisions and the kinetic energy of the collision event. Collision severity is thus proportional to the third power of impeller tip speed and the fourth power of impeller diameter: CS,

= 1.71ppnbN3adpd4

(38)

Note that the first-order dependence on cell (bead) diameter (d,) makes this expression entirely different from the similar one suggested by Cherry and Papoutsakis (1986), arriving at the fourth power dependence. Since the diameter difference of cells and microcarriers is about a factor of 10, there will be still a lo-fold difference in severity. Some design implications of this impeller collision expression are discussed in Croughan et al. (1988).Collisions of the cells (beads) against impeller or other reactor internals should have similar effects to cell-cell collisions. The primary difference is that the kinetic energy of the impellercell collision is much higher. When a film of viscous fluid is forced out from between a pair of approaching bodies (this discussion applies to both cell-cell and cellimpeller collisions), pressures are developed which resist the tendency of these bodies to come together. The force required to squeeze out the liquid film can be estimated by (Rosenberg, 1987)

P

= 12p~/h3

(39)

If the kinetic energy of collision is greater than the film pressure, the probability of two bodies actually making contact increases. The biological response of an organism to a collision event may differ significantly depending on whether it actually makes physical contact with a collision partner or not. One can estimate the viscous film drainage time using a theory developed for the coalescence of bubbles in a twophase flow (Das et aI., 1987). Direct physical contact between collision partners has several impor-

184

ALES PROKOP AND RAKESH K. BAJPAI

tant consequences for the way in which cells will respond to this type of stress. If physical contact occurs, then kinetic energy is transferred between the collision partners. The equations presented above assume 100% efficiency for the transfer of kinetic energy, that is, the collision is viewed as an inelastic event. The actual efficiency will depend on the viscoelastic properties of the collision partners. Some of the kinetic energy of a collision event is dissipated as a cell deforms and will thus experience less damaging force as compared to a rigid surface. Direct physical contact may also stimulate metabolic responses that would not otherwise be influenced by interaction with fluid elements alone. The rest of the kinetic energy should be dissipated by viscous forces. The stress exerted on the collision partners is thus significantly less than if physical contact occurs. The elasticity of an event has not yet been brought into a theoretical treatment of the collision damage. Cell-cell collision frequencies and severities calculated for cells grown in a stirred vessel are presented in Table I1 (Rosenberg, 1987). A mean cell size of 190 pm was assumed for all calculations (plant cell culture). This could be also applied to other cell aggregates or cells growing on microcarriers. The calculations are based on the assumption of physical contact between collision partners. The values listed can thus represent rough approximations of the maximum forces imparted to cells during collision events. Cell-cell collision severities are probably much less than the values presented since physical contact rarely occurs. As seen from Table 11, the kinetic energy of cell-impeller collisions is several orders of magnitude greater than the kinetic energy of cell-cell collisions due to the high velocity of the impeller relative to the motion of a cell. Consequently, the frequency of cell-impeller collisions is thus much greater than cell-cell collisions. The operational variables present in expressions for cell-cell and cell-impeller severities [Eqs. (34) and (38)l are numerous. Those which may distinguish these expressions between themselves are viscosity, agitator speed, and design, cell (bead) concentration, and cell (bead) diameter. Few limited observations are available for differential comparison because the above variables have not been varied experimentally over a broad range of conditions (Cherry and Papoutsakis, 1988). In addition to growth characterization, cellular responses are also needed. Without such data further advance in delineating different mechanisms (including also those of damage by turbulent eddies themselves] is not possible. So far, the relative contributions of hydrodynamic shear and collisions have not been quantified. Some limited physiological observations are available on how mam-

185

SHEAR SENSITIVITY OF BIOCATALYSTS TABLE I1 CELL-CELL AND CELL-IMPELLER COLLISION FREQUENCY AND SEVERITY AS A FUNCTION OF TIP SPEEDO

cs,

Nc (l/sec)

csTx 109 (g-cmz/sec3)

N, (l/sec)

(g-cmz/sec3)

13.4 26.8 53.6 107.2 214.5 314.2 419.0

0.9 1.5 2.5 4.0 6.4 8.5 10.1

0.2 1.1 4.8 18.9 80.1 198.0 315.0

8,510 17,000 34,000 68,000 136,100 200,000 266,000

0.9 7.4 59.4 475.5 3,803.9 11,956.3 28,340.0

Equation

(33)

(34)

(37)

(381

Tip speed (cm/sec)

Calculations are for an average aggregate size of 190 km, Rasenberg (19871.

malian cells attached to microcarriers are affected by mixing (Cherry and Papoutsakis, 1988).Authors tried to relate the breaking of bridges formed between populated beads to the above mechanism. Another complication may occur, related to a capability of some cell lines to repopulate beads devoid of cells, originating either from cell detachment or from any of the above types of mechanical damage. Croughan and Wang (1990) identified that some CHO cell lines are capable of secondary growth on naked beads, clearly reducing sensitivity of such cells to mixing.

B. OTHERREACTORTYPES In the following, a short discussion of the calculation of fluid mechanical stresses generated in other reactor types is presented. Airlift and bubble columns are noted for their gentle mixing regimes (Schiigerl, 1982). Regions of high shear stress, as they are in stirred tank reactors, are not present in these reactors due to the complex hydrodynamics of these multiphase reactors. Yet, only an approximate estimate can be made on the shear stresses present in a bubble column or airlift reactors. The shear stress at the walls of a bubble column can be estimated from boundary layer theory using Eq. (21). In the bulk fluid, suspended organisms will experience transiently high levels of shear stress in the wakes of rapidly rising gas bubbles. The velocity of a suspended organism (relative to the fluid) in these wakes can approach 100 cm/sec (Mercer, 1981). Jones et aI. (1990) measured mean liquid velocities in a split bubble column (airlift) using

186

ALES PROKOP AND RAKESH K. BAJPAI

laser-Doppler velocimetry. Their velocities ranged from 19 to 36 cm/sec. The presence of microcarriers (up to 15 glliter) within this reactor may promote an increase in the turbulent dissipation of energy. The effect of serum addition on hydrodynamics was not, however, conclusive. The shear stress can be calculated by assuming transient, local isotropic turbulence in the wake of a rising bubble and using the equations for Reynolds stresses [Toni and Bagster, 1978). The Kolmogoroff microscale generated in the rising bubble wakes is on the order of 100 to 500 pm, which is generally larger than the size of most plant and mammalian cells but could affect large cell aggregates and mammalian cells grown on microcarriers (Schiigerl, 1982). In any case, shear stresses encountered in airlift reactors should be smaller than those of the bubble columns, perhaps due to lower relative velocities between the liquid and bubbles (Shi et a ] . , 1990). Thus, the airlift bioreactors should be more appropriate then the bubble columns for the cultivation of sensitive cells (plant and mammalian). Another deleterious effect of sparging is associated with the surface energy of the air bubble. Cells will absorb to air-liquid interfaces, where they will be subjected to significant surface tension forces. The forces due to surface tension can presumably stretch the cellular lipid bilayer and result in cell damage. Aunins et al. (1986) suggested a formula for minimal and maximal local shear rate due to bubble aeration. The minimum local rate is Ymin =

(I/Z)~,NO~~~

(40)

The upper limit in the trailing vortices of rising bubbles is ymax= ('/z)(~~//Rb)~'*

(41)

where vt is bubble terminal velocity and R, is bubble radius. The authors estimated ymimand ymaXto be 18 and 2900 sec-l, respectively. For 100 cm/sec velocity in an airlift column, the local maximum shear stress can be up to 200 dyn/cm2, high enough to cause some cell damage. The role of surface shear in cell physiology has not yet been sufficiently elucidated. Besides the cell damage at the air-liquid interface, product or nutrient denaturation should be also considered. Extracellular protein products are likely to be affected at these interfaces. The alteration of tertiary protein structure, denoted as denaturation, may result from the distortion at the air-liquid interface (Donaldson et a]., 1980). It is assumed that proteins form a spread film with surface pressure causing unfolding. As the protein denaturation seems to be reduced in the

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presence of surface-active substances, this fact may be brought into consideration during the downstream processing. Immobilized reactor configurations are designed to protect cells from hydrodynamic stresses. In a hollow-fiber reactor, for example, cells will be subjected to laminar flow conditions, with stresses much less than 1 dyn/cm2. For a high-density immobilized cell reactor, another factor should be considered. As the microbial population fills up the available space (e.g., that of extracapillary space in the hollow-fiber reactor, void volume of a macrocarrier particle, or interstitial space in gel particles) an organism can exert a stress on its surroundings. The growing organisms can deform gels and rupture hollow-fiber membranes. At the same time, a considerable distortion of cell shape and cell size reduction may occur (Inloes et al., 1983; Stewart and Robertson, 1987). Pressure exerted on cells represents a form of stress similar to turbulent pressure fluctuations in a mixed vessel, although of a steady nature. A similar effect may be exercised during centrifugation. Hamamoto et al. (1989) observed an inhibition of proliferation of hybridomas exposed at 50 g for 30 minutes. Also, a similar situation may exist in a “maintenance bioreactor” or with encapsulated cells (mammalian) (Prokop and Rosenberg, 1989). So far, very few researchers have looked into the metabolic alterations due to a moderate pressure applied on cells. Very high pressures are known to inhibit both transcription and postranscriptional events in the protozoa Tetrahymena (Tahir et al., 1988). Ben-Ze’ev (1985) demonstrated that some cytoskeleton components undergo dramatic changes in a dense epithelial cell culture as compared to a nondense one. Napolitano et al. (1987) observed the appearance of a previously undetected polypeptide, perhaps a novel stress protein, under similar conditions. Extensive cell-cell contact and moderate pressures generated may constitute a stressful situation resulting in the increased production of a class of proteins, accompanied by a decrease of ordinary protein synthesis. Such a mechanism may represent a way how a high-density mammalian culture switches to a maintenance nonproliferating state capable of sustained viability and expression of a desired class of proteins. IV. Methods of Assessing Shear Sensitivity

There are two basic approaches to the study of shear sensitivity. The first one involves applying the shear stress in the bioreactor of interest. The advantage of this method is that long-term biological responses, such as the specific growth rate, production rate, and product yield,

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can be measured as a function of the hydrodynamic environment. The problem with this approach is that most bioreactors are operated in the turbulent flow regime. Because of the chaotic nature of turbulent flow, one can only obtain empirical information about the sensitivity of the organism that is specific to the reactor type and geometry. Only with great difficulty can one extrapolate results obtained in one reactor type to other systems. The complexity of the flow patterns in bioreactors and the difficulty of quantifying the magnitude of shear stress on these cells under agitation makes it difficult to ascertain in detail how flow affects cell growth and metabolism. Difficulties in characterizing the hydrodynamic field of bioreactors have been covered in Section 111. Another problem is a limitation in the range of stresses imposed on microorganisms in a standard bioreactor due to their mechanical design. Development of new mixing and aeration designs also requires testing of shear sensitivity of microorganisms under such conditions. As an example, an aeration jet (free jet) in a loop system may be used, providing a somewhat more defined shear field in the jet vicinity, although only of short exposure times (Bronnenmeier and Markl, 1982; Markl and Bronnenmeier, 1985). A second approach which is increasingly being used is the exposure of organisms in a well-defined flow field. Such a flow field can be generated in various types of viscometers normally used for determining the rheological properties of fluids (Fredrickson, 1964). The advantage of using a well-defined flow field is that the results can be used to predict the behavior of biocatalysts in more complex flows, such as those in bioreactors. The disadvantage of this method is that the longterm biological responses, such as changes in specific growth rate, cannot be measured since cells cannot be easily cultured in viscometers. Exceptionally, sterilizable viscometers are available, allowing for cell culturing or for longer exposure times (Schiirch et al., 1988). A variety of apparatus has been developed for the purpose of subjecting anchorage-dependent cells to shear stress. The essential component of these instruments is a flow chamber, composed of two parallel plates, that allows for well-characterized conditions over a long period of time. Cells are typically grown attached to one or both plates. Flow rates should be uniform across the cell layer and accurately verified (Grabowski et d.,1985). The flow chamber should also allow for colIection of data and morphological observation. The ability to maintain a high cell-to-medium ratio may be important, particularly when metabolites of interest are synthesized in small quantities. As in any cell culture system, environmental conditions such as nutrient levels, gas concentrations, pH, and temperature must be maintained. In addition to a

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steady shear stress, in both laminar and turbulent regimes, a pulsatile flow can be also introduced (Frangos et al., 1985). Such instrumentation can be in principle applied for anchorage-independent cells. However, an appropriate shear-free cell transfer via pumping is virtually impossible to achieve. Once an organism has been stressed, a biological response to that stress should be measured. Biochemical engineers have concentrated their effort on measuring the shear stress necessary to cause a significant reduction in specific growth rates and to cause the cell lysis. These parameters are important for determining the maximum permissible agitation conditions for a given bioreactor configuration. The cellular viability can be assessed in different ways. Cook and Mitchell (1989) reviewed assays for viability measurements in mammalian cell systems [most of them can be applied to other organisms too). Of these, permeability, functionality, and morphological assays are of interest. The dye exclusion test (Trypan blue], fluorescein diacetate test (FDA), tetrazolium dye reduction test, lactate dehydrogenase (LDH) release, and nucleotide release come into the first category, all representing a cellular membrane integrity. A fluorometric FDA test has been suggested (Taya et al., 1989). The LDH release test provides a very sensitive assay (Freshney, 1987) and can be used in situ without cell sampling. Nucleotide release has been applied by Tanaka and Ueda (1975) for mycelial fungi, although it can be used with other organisms. Functionality tests include incorporation of labeled thymidine or amino acid, representing macromolecular synthetic capacity. Morphological observations may include cell surface or cytoskeletal morphology facilitated via electron microscopy and specific antibody labeling. Rapid evaluation of cell status is now possible via the employment of a suitable labeling technique coupled with flow cytometry. A FDA/FACS (fluorescence activated cell sorter) test has been suggested (Aeschbacher et al., 19861.Monitoring of surface markers or products (e.g., IgG) is now commonplace (Al-Rubeai et a]., 1989; Rupp et al., 1989; Meilhoc et al., 1989). More detailed discussion on fluorescent labeling of cell surfaces or cytoplasm is in Muirhead and Horan (1984) and Edidin (1989). Other modern approaches include, for example, the use of electrical properties to evaluate the effect of stress on insect cells (Freitag et al., 19891. Cell biologists have collected a large amount of data on sublethal phenomena such as the modulation of enzyme activity, protein synthesis rates, cell morphology changes, and plasma integrity. Some examples will be presented while discussing specific organism groups. To cite just one method, fluorescence ratio imaging allows one to follow the fate of intracellular and extracellular calcium as related to stress

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(Tsien and Poenie, 1986). Not only will these types of data allow the selection of the proper bioreactor and optimum operating condition, but they may eventually lead to the engineering (genetic) of organisms for a bioreactor environment. There are, however, no accepted (standardized) methods for determining shear damage. There is also a room for interpretation of different methods as related to different shearing modes. V. Sensitivity of Biocatalysts to Hydrodynamic Stress

A. ENZYMES Proteins are subjected to shear stresses in bioreactors and in various downstream processing steps. It has been hypothesized that these shear forces may be responsible for protein denaturation (Charm and Wong, 1970; Tirrell and Middleman, 1975). Initial studies showed that globular enzymes were partially inactivated when subjected to shear stress in a Couette viscometer (Charm and Wong, 1970; Thomas et a]., 1979). The rate of enzyme inactivation was correlated with the dimensionless product of the shear rate and the exposure time. More recent studies, however, have shown that globular proteins are insensitive to shear stress levels as great as 300 dyn/cm2 for several hours [Thomas et a]., 1979). It is now believed that the protein denaturation observed in early experiments was caused by surface tension forces present at the air-liquid interface. Lee and Choo (1989) demonstrated that direct shear-induced mechanical damage of lipase does not occur. Also, a sulfhydryl group oxidation by air has been ruled out. The most likely explanation is shear-induced interfacial effects on proteins. The true mechanism is not clear, since several effects can be encountered: protein adsorption at the air-liquid interface, followed by denaturation, be it unfolding, coagulation, or breakdown into subunits. Their conclusions should, however, be interpreted with caution as soluble starch was used to adjust viscosity (shear stress). Other possible interpretations may result from observations of the loss of enzyme activity during ultrafiltration through membranes where proteins are exposed to shear forces while passing through membrane pores. However, a careful material balancing revealed that the apparent loss of enzyme activity can be ascribed to protein adsorption to membranes (Truskey et a]., 1987). Manton-Gaulin homogenizers, which are capable of generating shear stresses u p to 5 x lo5 dyn/cm2,do not inhibit enzyme activity (Brookman, 1974). Since protein molecules are several orders of magnitude smaller than the universal-equilibrium eddies, it is reasonable to as-

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sume that enzymes should be insensitive to the shear stresses generated in equipment used for agitation in bioprocessing.

B. PROKARYOTES The prokaryotic cell may be surrounded by as many as three surface layers which differ in structure and composition. The innermost layer is the lipid bilayer membrane. This layer is surrounded by the cell wall, which is composed primarily of a peptidoglycan polymer. Between these two layers is the periplasmic space. Many, but not all prokaryotes, have an additional slime layer composed of various polysaccharides and enzymes. The slime layer has an ill-defined width and can be many times thicker than the underlying cell wall (Hammond et al., 1984). Prokaryotes can assume a variety of shapes, such as spheres or rods. These organisms are generally very small, with the longest dimension being on the order of 0.5 to 5 pm. Because they are typically smaller than velocity eddies, no effect on them is usually observed in an agitated vessel. Actinomycetes, which form filaments, on the other hand, do show shear sensitivity and will be discussed with the appropriate eukaryotic category. High-pressure Manton-Gaulin homogenizers can generate much higher shear stresses and can disrupt microorganisms. A typical goal of disruption is the extraction of protein products (Brookman, 1974; Chisti and Moo-Young, 1986). There are several theories regarding the mechanism of cell lysis in this device. Rapid pressure drop across the valve, cavitation, and turbulence have all been hypothesized to be the primary cause of cell disruption (Brookman, 1975; Doulah et al., 1975; Keleman and Sharpe, 1979). Cell size, shape, and cell wall characteristics are all known to influence the susceptibility of microorganisms to disruption (Keleman and Sharpe, 1979). C. LOWEREUKARYOTES Fungi, microscopic algae, and protozoa can exist as either single cells or multicellular aggregates of cells. Single-celled organisms enjoy a variety of shapes and sizes: from spheres of 1 to 5 pm in diameter, to rods with a width of 0.5 to 2 pm and a length of 1 to 10 pm. Furthermore, these organisms for multicellular pellets or complex branched chains of cells depending on the culture conditions. The morphology of the cells influences the viscosity of the media, mass transfer characteristics, and the way in which the cells will respond to shear stress (Roels et al., 1974; Kim et a]., 1983). The external boundary is a cell

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wall composed of a polysaccharide gel with reinforcing glycoproteins and fibrous polysaccharide chains (Arnold, 1983). The cell wall is very rigid, which, in addition to small cell size, renders cells relatively insensitive to shear when compared to higher eukaryotes. Many studies have been conducted on the effects of impeller tip speed on productivity of filamentous fungi (Taguchi et a]., 1968; Ujcova et a]., 1980). Tanaka and Ueda (1975) found that cell damage, as measured by the release of nucleic acids, increased with increasing agitation rates (Fig. 7). The rate of cell damage was found to depend on the culture conditions and the reactor geometry, as well as on the morphological status of cells (Taguchi et al., 1968). Smith et al. (1990) have measured the effect of agitation on morphology and product formation by Penicillium chrysogenum at two different scales of operation and concluded that the effect of agitation was scale-dependent. Hyphal damage was not related to the impeller tip speed, nor to the rate of dissipation of turbulence. It was found to be related to the mean circulation time in the reactor, with lower circulation rates beneficial. It was hypothesized that the hyphae are damaged in a dispersion zone around the impeller and higher circulation frequency increases the damage. Reuss (1988) has also reported breakage frequency as

-

1

3 Tip speed

(m/s)

FIG. 7. Leakage rate versus tip speed. Data of Tanaka and Ueda (1975) as replotted by van Suijdam and Metz (1981). ( x ) V = 5 liters, di = 0.064 m;(e)V = 5 liters, di = 0.082 m; (0) V = 5 liters, d, = 0.1m; (0) V = 10 liters, di = 0.1 m.

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= PIdPt,. Wase et al. (1985) found that extracellular cellulase production rates in radial turbine-agitated reactors were reduced as the agitation rate was increased (Elliot, 1975). Even though the overall mass transfer coefficient increased with agitation rate (a high mass transfer is necessary because of a high cell density and oxygen uptake requirement), there was a decreased yield of cellulase and an increased amount of cell damage, as inferred from the release of protein into the culture medium. Comparison studies performed in an airlift reactor showed that increasing the overall mass transfer coefficient, by increasing the superficial gas velocity, led to cellulase yields 20% greater than those observed in the stirred tank reactor. Wase et al. (1985) concluded that the impeller was damaging the mycelium, causing an increased release of intracellular protein and a decreased product yield. The addition of protein adsorbents such as alumina removed a suspected metabolic inhibitor present intracellularly in damaged cells and thus partially relieved the negative mixing effect. No estimates of shear stresses in both reactor configurations have been offered. Bronnenmeier and Mark1 [ 1982) measured the hydrodynamic shear stress capacity of the green alga Chlamydomonas reinhardi. Experiments were conducted in two different types of shear field. In the first set, the organism was grown in a stirred tank reactor at an agitation rate that was assumed not to damage cells. The cells were then passed to a second holding tank by negative pressure, and then recycled back to the stirred tank reactor through a nozzle. The pressure exerted on the cells as they passed through the nozzle, as well as the frequency with which an individual cell passed through the nozzle, could be calculated. Damage caused by the hydrodynamic stress load in the jet increased linearly with the increase in pressure in the jet. A critical pressure of approximately 15 bar (1.5 dyn/cm2)was found for the wildtype cells and 2 bar for a mutant strain that did not have a cell wall. Similar results were obtained when cell lysis was estimated. The release of intracellular substances from the damaged cells continued for up to 6 hours after the application of the shear stress, implying that the damage to the cell membrane was repaired during this time. Cell viability was also measured, and it was found that a critical shear load of 2-3 bar was necessary to begin damaging the wild-type cells. In another set of experiments, the same cells were grown in a chemostat at different agitation rates. The specific growth rate remained constant between 300 and 2400 rpm. Above 2400 rpm, the specific growth rate fell rapidly. The cell wall-less mutant was found to be more

194

ALES PROKOP AND RAKESH K. BAJPAI

sensitive to mixing. Again, no values of stresses have been made available for this reactor type. In one of the following pioneering studies on the shear sensitivity of biocatalysts, Midler and Finn (1966) examined the effects of shear stress on the protozoa Tetrahymena. Tetrahymena is a pear-shaped ciliated protist with an average size of 50 by 30 pm. The organism is surrounded by a series of lipid bilayer membranes (Elliot, 1975). The fact that Tetrahymena lacks a tough cell wall and is comparable in size to the microscale eddies renders it particularly sensitive to shear stress. Midler and Finn (1966) subjected Tetrahymena to shear stress in a Couette viscometer and in a stirred tank reactor. The critical shear stress at which lysis occurred in the Couette viscometer was on the order of 240 dyn/cm2. The critical shear stress was defined as a minimal stress applied over a long period of time causing noticeable damage as evaluated via a chosen method. A shear stress of 10,000 dyn/cm2 for 1 minute resulted in 80% lysis. The extent of cell damage was directly proportional to the magnitude of the shear stress and the exposure time. Cell lysis increased with fluid viscosity at a fixed shear rate, indicating that the cells were sensitive to shear stress rather than shear rate. It is also interesting to note that Tetrahymena appeared to be much more sensitive to turbulent flow than to laminar flow. The average turbulent shear stress required to lyse over 50% of the population within 10 minutes was on the order of 100 dyn/cm2. Under laminar flow conditions, a shear stress of 3,000 dyn/cm2 was required to cause the same amount of damage. In a second set of experiments, Tetrahymena was subjected to hydrodynamic forces in a stirred tank reactor. Cell lysis was correlated with impeller diameter and impeller tip speed. Lysis increased dramatically at a critical tip speed of 170 cm/sec. Increasing the impeller diameter from 5 to 10 cm resulted in a significant increase in the extent of lysis. These data are in agreement with the collision theory of Cherry and Papoutsakis (1986). Cell lysis also increased exponentially with impeller tip speed, which implies that both shear stress and collisions may be damaging Tetrahymena. Experiments conducted in a French Press showed that the stress required to disrupt 50% of the cell population was 69,000 dyn/cm2for a filamentous Aspergillus fumigatus and 156,000 dyn/cm2 for Saccharomyces cerevisiae,which is much less than for bacteria (Kelemen and Sharpe, 1979). These organisms are approximately twice as large as the bacteria tested and therefore experience higher shear stresses and more frequent and stronger collision forces (cell-solid surface collisions) (Nagata, 1975; Cherry and Papoutsakis, 1986). Brookman (1974,

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1975), however, states that yeast cells are not sensitive to shear stress but rather to the magnitude and the rate of change of the pressure drop through the French Press. Stumpf et al. (1946) and Doulah (1977) investigated the lysis of Saccharomyces cerevisiae in a sonic disrupter. Ultrasonic cavitation generates an intensely turbulent flow field due to the implosion of microbubbles formed at nucleation spots in the fluid phase. Suspended cells are thus subjected to time-dependent stresses and cell-cell collision phenomena, but cell-solid surface collisions are virtually nonexistent. When the Kolrnogoroff microscale was reduced to approximately 5 pm, 90% of the yeast cell population was lysed within 10 min. These experiments illustrate that microorganisms that are usually thought of as shear-resistant exhibit shear-sensitive characteristics when shear stress is applied under the right conditions.

D. MAMMALIAN CELLS Mammalian cells, although far more difficult and expensive to work with than prokaryotes and lower eukaryotes, are enjoying a resurgence of interest because they possess the necessary intracellular machinery to fold and glycosylate protein products properly (Mizrahi, 1986). One of the major difficulties with large-scale mammalian cell culture is the fragility of these cells (Feder and Tolbert, 1983). Mammalian cells are on the order of 10 to 20 p m in diameter and are surrounded by a deformable cell membrane. The cell membrane encases the highly compartmentalized and viscous cytoplasm. Unlike prokaryotes and lower eukaryotes, mammalian cells do not possess a protective cell wall. 1. Anchorage-Dependent Cells

Anchorage-dependent mammalian cells are often cultured on microcarrier beads in stirred tank reactors (van Wenzel, 1967; Reuveny, 1983). The degree of agitation necessary to keep microcarriers suspended can damage the cells (Hirtenstein and Clark, 1980). Crouch et al. (1985) studied the shear stress necessary for anchorage-dependent mammalian cells to experience attachment and detachment to plastic or glass surfaces. The experiments were conducted in a small radial flow chamber where the velocity profile decreased linearly from the point of entry of the fluid to the periphery of the disk. By determining the radius at which cell attachment or detachment begins, the critical shear stress for these phenomena can be calculated. Shear stress levels on the order of 0.001 dyn/cm2 were required for cellular adhesion to either plastic

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or glass surfaces, while shear stress levels of 40 to 50 dyn/cm2 were necessary to cause detachment of the cells. It was also noted that the critical shear stress was strongly influenced by cell culture conditions such as pH. Croughan et al. (1985) studied the effects of shear stress on anchorage-dependent mammalian cells which were grown in small spinner flasks on Cytodex microcarrier beads having a diameter of 150 pm. The specific growth rate was correlated with the Kolmogoroff microscale. It was found that a minimum of 60 rpm was necessary for the suspension of Cytodex 1 microcarriers. The results of the experiment in which agitation rate was varied are shown in Fig. 8. Growth was modeled by the following set of equations: en(C/C,) = ( p - k)(t - to]

for t < t,

(431

en(C/Co] = -k(t - t,)

for t < t,

(44)

where p is the specific growth rate and k is the specific death rate. By assuming that cell death was due to shear stress effects only, the specifI"

l t \

2 2 0 rpm

101 0

50

100

150

Time (hrs )

FIG. 8 . Growth of human foreskin fibroblasts on Cytodex 1 microcarriers as dependent on agitation rate (Croughan et al., 1985).

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ic growth rate and specific death rate due to agitation were calculated from the data. In a similar experiment by Hirtenstein and Clark (1980), it was found that the optimum rpm for growth of fibroblast cells on microcarriers was 60 rpm and that growth fell off rapidly for agitation rates less than or greater than 60 rpm. Implicit in this type of mathematical modeling is the assumption that shear stress effects behave in a stochastic manner. If a cell encounters a fluid eddy of the certain size and energy content, the cell will die. Fluid eddies that are much larger or smaller than a suspended organism or which contain less energy than a critical minimum for cell death are essentially inert to the cells. The fact that these cells are immobilized on microcarrier beads has important implications for the types of forces that these cells will experience. Assume for a moment that the cell is not immobilized on a microcarrier. If the cell is relatively rigid and does not deform in a shear field, then it will behave as a rigid body. At low shear stress levels, and taking into account the highly viscous cytoplasm of mammalian cells, this is a reasonable assumption. In this case, a cell that is free to rotate and translate in a shear field will assume an orientation of minimum energy dissipation. The cell therefore minimizes the stress exerted on the plasma membrane. Immobilized cells, which lack translatability, experience significantly greater shear stresses and a higher frequency and severity of cell-cell and cell-impeller collisions. Croughan et al. (1985) believe that hydrodynamic shear stress is the primary mechanism of damage. They have correlated their data with the Kolmogoroff microscale. Fibroblasts were grown on Cytodex microcarriers with a diameter of 150 pm. When the microscale decreases from 130 to 100 pm, the maximum cell yield at the end of the process decreases dramatically (Fig. 9). One would expect that, if these same cells could be grown in suspension, a similar effect would be seen when the microscale is decreased to 25 to 50 pm. Earlier studies by Augenstein et al. (1971) examined the response of mammalian cells to turbulent stresses in a capillary tube. Mouse fibroblast cells were passed through the capillary tubes repeatedly, and cell viability, as measured by the vital strain erythrosin B, was measured. Viable cell count was found to decrease linearly with the number of passages through the capillary. The average length of time spent in the capillary per passage was on the order of 0.1 second and the mean turbulent shear stress at the capillary wall was on the order of 103 to lo4 dyn/cm2. The death rate kinetics were found to be first order. The transformed HeLa cell line was found to be less sensitive to hydrodynamic stress than the mouse fibroblast cell line. Since all of the experiments were conducted in turbulent flow it is difficult to con-

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-

s

u

r ” 40.-0

>

Shear stress (dyneskm 2,

0

-

A

AM

2.6 6.5 13.0 26.0 5L.O

I

1

I

2

4

8

<< I

24

Time ( h r s )

FIG. 9. Biomass yield of human foreskin fibroblasts on Cytodex 1 microcarriers as a function of the Kolmogoroff microscale [Croughan et ol., 1985).

clude whether hydrodynamic shear stresses, cell-cell collisions, or cell-wall interactions were dominant. Human embryonic kidney cells attached to the lower of two parallel plates in a confluent monolayer and subjected to shear stress levels between 6.5 and 54 dynicmZ for periods of up to 24 hours experienced significant changes in cell morphology and metabolism (Stathopoulos and Hellums, 1985). The average maximum cell diameter was found to increase linearly with increasing shear stress and increasing exposure time. At the highest shear stress level, cells elongated 3-fold over control cells after 24 hours of exposure. At low shear stress levels, the ability of cells to remain attached to the lower plate was unaffected, but as the shear stress was increased to 26 dyn/cm2,more than 20% of the cells became detached within 2 hours of exposure. Detachment (which the authors equated with viability) increased steadily with exposure time thereafter (Fig. 10). The release of an intracellular enzyme, urokinase, was also measured and found to increase with shear stress in the range of 2.6 to 6.5 dyn/cm2, but, interestingly, decreased to intermediate levels at greater shear stress levels (Fig. 11). The release of urokinase implies that shear stress alters membrane permeability. A great deal of data are available on endothelial cells in respect to shear stress. Although such studies have a medical significance, they may shed some light on the complexity of hydrodynamic effects on other cells. Endothelial cells serve not only as a barrier between the blood and extravascular space, but function as a source of molecules

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FIG. 10. Shear sensitivity of human embryonic kidney cells: viability as equated to detachment from surface (Stathopoulos and Hellums, 1985).

1.2

-r

0 X

0.8 -

a VI l E

c .x 2

3

0.4 -

-

c

0

.-VIc u

3

0

I

1

FIG. 11. Release of urokinase from human embryonic kidney cells attached to a substrate (Stathopoulos and Hellums, 1985).

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ALES PROKOP AND RAKESH K. BAIPAI

which affect the structural and functional integrity of circulation. Investigation into the biological properties of endothelial cells was facilitated by the development of techniques for their growth in culture (Jaffe et a]., 1973). However, it has become evident that the classical tissue culture methods which lack the dynamic flow characteristics of the in vivo environment are not suitable for studies concerning the functions of endothelial cells. Pulsating hemodynamic forces play an important role in endothelial cell function and morphology, including the modulation of endocytosis (Dewey, 1984), cytoskeletal structure (see later), cellular proliferation [Ando et al., 1987), histamine metabolism (Skarlatos and Hollins, 1987), arachidonic acid metabolism (McIntire et a]., 1987), and expression of proteins (e.g., of those of fibrinolytic activity (Diamond et a]., 1989) (Fig. 12). Human endothelial cells have been subjected to laminar and pulsatile flow between two parallel plates by Frangos et al. (1985). Primary human endothelial cells secrete prostacyclin, an arachidonate metabolite, which is a powerful inhibitor of platelet aggregation and a potent vasodilator. Pulsatile shear stresses, similar to those experienced in the human circulatory system and varying between 8 and 1 2 dyn/cm2,induced endothelial cells to secrete prostacyclin at levels 16fold greater than cells grown in stationary culture. Endothelial cells subjected to a steady shear stress of 10 dyn/cm2 secreted twice as much

0

10

20

30

Time ( hrs 1

FIG. 12. Cumulative secretion of tissue plasminogen activator (tPA) by human endothelial cells as affected by steady laminar shear (Diamond et al., 1989). NSR is the normalized (versus control) secretion rate. (0)Static culture [control]; shear stress: (0)4 dyn/cm2, (A) 1 5 dyn/cm*, (W) 2 5 dyn/cm*.

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prostacyclin as control cells grown in stationary culture. The release of prostacyclin-related compounds by fluid mechanical stresses is implicated in ultrastructural and morphological changes. At 10-20 dyn/cm2, for exposure times up to 2 hours there was no visible change in cell morphology. When these cells were subjected to 50 dyn/cmz,cell shape changes became apparent after 24 hours (Ives et al., 1986). Between 24 and 48 hours, the cells would elongate parallel to the direction of flow. Fluorescent antibodies to the microtubule component of the cytoskeleton revealed that cell morphology changes were accompanied by a redistribution of the microtubule network. Control cells exhibited a diffuse network of microtubules while cells exposed to 50 dyn/cm2 shear stress for 48 hours had microtubule networks aligned parallel to the direction of flow. Similar experiments, done by Franke et al. (1984), found cytoskeletal responses to shear stress at 2 dyn/cm2 after only 3 hours of exposure. Endothelial cells were sheared in a cone and plate viscometer and then stained with fluorescent antibodies to the actin, Sactinin, and myosin components of the cytoskeleton. Stress fibers, composed primarily of actin, appeared within 3 hours and were concentrated at the periphery of the cells. Since endothelial cells are subjected to pulsatile stresses in vivo, it is reasonable to assume that these results are indicative of a highly evolved response to fluid stresses. Grabowski et al. (1985) confirmed the increased prostacyclin production following a step increase in shear stress to 15 dyn/cm2. A more detailed mechanism of shear-induced prostacyclin production in endothelial cells has been proposed, based on specific inhibition studies (calcium mobilization, ion channels blocking, etc.) (Bhagyalakshmi and Frangos, 1989). Another study looked at the shear-induced cytoskeleton reorganization as related to cell adherence (Wechezak et al., 1989). Lansman et al. (1987) discovered the presence of stretch-activated calcium ion channels in the plasma membrane of endothelial cells. Membrane tensions of 1 dyn/cm2 were sufficient to open these channels. Lansman ef al. speculate that these ion channels may flood the cell with free calcium ions, which activates enzymes responsible for prostacyclin synthesis. Providing further evidence to support this model are the observations by Ando et al. (1988) that laminar shear stresses of 10 dyn/cm2 cause an almost instantaneous 3-fold rise in the intracellular free calcium level. Christensen (1987) observed stretchactivated calcium ion channels in epithelial cells, residing next to calcium- and voltage-activated potassium channels. He speculated on the subsequent activation of potassium channels due to the former process of membrane polarization. The flow-induced polarization has

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been also observed in endothelial cells (Olesen et aJ., 1988). The usual end product of a stimulus-response (see later) cascade is a protein synthesis (Prokop, 1991). The stimulation of endothelin mRNA and of peptide itself (vasoconstrictor peptide) has been reported by Yoshizumi et al. (1989), using a shear stress of 5 dyn/cmz in a viscometer. Komuro et aJ. (1990) in a unique observation documented a protooncogene (c-fos) and its mRNA synthesis in cardiocytes stretched on elastic silicone dishes. Tissue plasminogen activator messenger RNA levels were more than 10 times higher in endothelial cells subjected to shear stress for 24 hours compared to stationary controls (Diamond et a]., 1990). 2. Anchorage-Independent Cells

Hybridoma cells are used for the production of monoclonal antibodies (Randerson, 1985). These cells are created by fusing mammalian B cell lymphocytes with transformed mammalian myeloma cells (Yelton and Scharff, 1981). The resulting cell is similar to the mammalian cells discussed above, but there are some important differences. The cytoskeleton and plasma membrane of these cells are significantly different from those of nontransformed cell lines (Gowing et aJ., 1984). These differences undoubtedly influence the way these cells respond to hydrodynamic stress. One of the only studies on transformed cells found that they were more sensitive to fluid mechanical stresses than the anchorage-dependent (nontransformed) cell lines (Croughan et aJ ., 1985). Another report on the shear sensitivity of transformed cell lines, is that of Brooks (1984). Murine melanoma cells were subjected to shear stress in a cone and plate viscometer. Relatively low shear stresses of between 5.9 and 29.0 dyn/cm2 killed 20 to 70% of the population after approximately 6 hours of exposure. Hybridomas can be grown in laboratory-scale stirred tank reactors at low rpm. In one study, hybridoma cells were grown in 100-ml spinner flasks and a 500-ml stirred tank reactor employing two 3-bladed marine impellers (Dodge and Hu, 1986). An optimum specific growth rate was achieved at 60 rpm in a spinner flask, corresponding to a Reynolds stress of approximately 0.2 dyn/cm2 (our estimate). At 240 rpm, the specific growth rate was reduced and the final cell density was also lower. Above 240 rpm (Reynolds stress of 1.4 dyn/cm2)cell growth was inhibited. This report and several others (e.g., in Backer et aJ., 1988) clearly demonstrated that suspended mammalian cells can be successfully cultivated in stirred vessels provided the agitation rate (and type of agitator) is carefully selected. Backer et aJ. (1988) demonstrated that a tip speed up to 2.03 misecond (marine impeller) in a 150-liter tank was critical for obtaining healthy hybridoma cultures. It seems clear

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that, for individual applications, a selection for more resistant cells is usually attempted. For a laboratory culture, Lavery et al. (1985) observed that tip speeds above 0.9 m/second were detrimental to hybridomas. Abu-Reesh (1989) observed a significant adaptation to higher stresses in a modified spinner flask. A stepwise exposure to higher mixing rates resulted in a more robust hybridoma culture. His maximal tip speed used was about 0.7 m/second. No estimates of stresses were, however, made. Petersen et al. (1988) also observed similar adaptation effects. Estimates of stresses hybridomas experience in mixed bioreactors are very rare. Oh et al. (1989) thus came up with a 0.01-0.4 dyn/cm2 and 0.6-64 dyn/cm2 as average and maximal (near mixer) stresses, respectively, in surface-aerated 1.4 liter reactors fitted with propellers or Rushton turbines. This estimate (Kolmogoroff’sstress) was done under the assumption of the validity of Kolmogoroff’s theory of isotropic turbulence. The turbulent maximal Reynolds stresses varied between 77 and 1240 dyn/cm2. It should be also pointed out that hybridomas exposed to the maximal stresses reported were indistinguishable from controls (low mixing conditions) in terms of morphological characteristics as documented via scanning electron microscopy (as well as by the physiological characteristics such as growth, viability, antibody production, glucose consumption, and lactate production). On the other hand, Papoutsakis and Kunas (1989) estimated a critical (complete death) Kolmogoroff’s stress of 8 dyn/cm2 in their 2-liter (total volume) reactor fitted with a pitched 4-bladed impeller, based on cell count and viability (trypan blue) observations. There is clearly a need for further physical characterization of the mixing field in bioreactors to enable more meaningful estimates of stresses to be obtained. Some physiological responses of hybridomas stirred in small vessels have been followed by Al-Rubeai et al. (1990). DNA synthesis has been noted to be inhibited under conditions of increased mixing. On the other hand, metabolic activity (respiration via a formazan assay) increased in surface-aerated highly stirred cultures and in more sparged cultures. More defined studies have been performed via viscometers or in special devices (e.g., fitted with a capillary tube). C. G. Smith et al. (1987) were the first to report on the use of a Couette viscometer to study long-term effects of laminar flow (15 hours) on viability of the hybridomas as estimated by trypan blue exclusion and LDH release. A level of 1.6 dyn/cmz was determined to be critical. Petersen et al. (1988) employed a laminar device in the range of 0-50 dynfcmz for 10 minutes only. Abu-Reesh and Kargi (1989) used 50-100 dyn/cm2 for 0.5-3 hours under the laminar regime. In addition to the above viability crite-

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ria, they also employed a respiratory activity (formazan formation). Data from viscometers, although interesting, are not applicable to continuous shearing conditions under real situations. Similarly, wall shear stress applied in a loop formed with capillary tube can only provide rough estimates over short times because cells residence time is determined by residence time per passage and number of passages through the capillary. Thus, McQueen et al. (1987) and McQueen and Bailey (1989) employed turbulent as well as laminar flow regimes on their myelomas and hybridomas. Cell count and LDH release were used as viability criteria. An increase of medium viscosity is usually used to shift turbulent conditions to laminar ones in a viscometer and to achieve higher laminar stresses. Abu-Reesh and Kargi (1989) have used a dextran addition, among many others. McQueen and Bailey (1989) have observed no effect of dextran on hybridomas sheared in capillary tubes in both laminar and turbulent regimes. Papoutsakis and Kunas (1989) have observed a negative effect of dextran addition on the viability of hybridomas mixed in a turbulent regime in a 2-liter reactor. This is in contrast to the typical positive effects observed with anchorage-dependent mammalian and insect cells (see Sections V,D,1 and V,E). It is clear that the hydrodynamic conditions under different shearing conditions in viscometers or reactors are not adequately understood and that additional forces not yet identified have decisive effects. Serum at higher concentrations (5-10%) has been shown to have a positive influence on hybridoma cells with respect to mixing effects (Handa-Corrigan et al., 1989; Kunas and Papoutsakis, 1989). This effect is not due to a slightly higher viscosity but is primarily due to changes in cell physiological properties. Cells grown at low serum concentrations are mechanically weak (Abu-Reesh, 1989; Lee et al., 1989), perhaps due to modified cell membrane structure. Practically, in a production stage serum may be reduced to I%, following growth at higher serum concentrations. Bubbling of hybridoma cell suspensions may cause adverse shear effects. Negative effects of air sparging have been identified (e.g., in Oh et a]., 1989; Handa-Corrigan et al., 1989),and some researchers tried to avoid such effects by choosing appropriate mixers and by suppressing the bubble entrainment (Kunas and Papoutsakis, 1989). In an experimental setup, surface aeration is a solution (Oh et a]., 1989). A bubble column seems to be the best model reactor to study the bubble effects. It can be conveniently divided into three major zones as far as bubble influence: distributor zone, bubble riser zone, and bubble disengagement zone. Based on varying-column-length experiments, Handa-

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Corrigan et al. (1989) ascribed the damaging effect to the disengagement zone, where damage due to rapid oscillations caused by bursting bubbles and damage due to shearing in draining liquid films (or lamellae) in foams are two mechanisms proposed. Pluronic F68 addition has been shown to damp the damaging effects of bubbles. Bubble effects and polymers are discussed further in Section V, E. An integrated view of the effect of mixing and bubbling has been offered by Kunas and Papoutsakis (1990). It can be concluded that no damage is observed at low mixing rates in an agitated vessel provided with a surface aeration. At higher mixing rates, cell death results from interaction of hybridomas with turbulent liquid elements (eddies). At lower mixing rates, an adverse effect of large and slowly moving bubbles (perhaps due to their disengagement, i.e., bursting on the liquid top) is observed. Small and rapidly moving bubbles, originating from the vortex entrainment, do not necessarily cause cell damage. These observations may have a practical significance for suggestions of design conditions (direct sparging and mixing conditions) without cell damage. Other results can be cited in support of these conclusions (Gardner et a]., 1990). At a biological level, the ultimate change due to excessive mixing should be an expression of stress proteins. The only report3 data (Passini and Goochee, 1989) are negative. However, the authors used moderately high agitation rates in a 1-liter volume bioreactor provided either with surface aeration or with agitation via a scoping (nonvortexing) marine impeller. Special care was taken to avoid air entrainment from the gas phase above the hybridoma suspension. The pattern of intracellular protein synthesis as revealed in one- and two-dimensional polyacrylamide gel electrophoresis did not differ from that observed under static or low-mixing conditions. However, the upper mixing rates used by Passini and Goochee (1989) did not cause any detectable cell damage in terms of growth, viability, LDH release, and monoclonal antibody production. Experiments at higher agitation rates should be performed to answer this question. Papoutsakis et aI. (1991), in experiments designed to selectively inhibit specific components of the cytoskeleton proteins of hybridomas by means of drugs, demonstrated an importance of microfilaments (not microtubules) in the shear-induced injury. Cells were exposed in a viscometer at 50 dyn/cm2 for 10 minutes. While using cytochalasins, results indicate that contractile microfilaments (and other structures of polymerized actin) are important mediators of structural and mechanical integrity of cells. Blackshear (1972) conducted extensive studies on the behavior of

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human erythrocytes in a shear field. The erythrocyte can be visualized as a bag of viscous protein concentrate (hemoglobin) surrounded by a viscoelastic membrane (Fischer et a]., 1978). These cells contain no internal membraneous organelles, resulting in a cytoplasm that is much less structured and viscous than other typical mammalian cells. The cytoskeleton of the human erythrocyte plays an important role in the dissipation of shear stress. It is fairly simple compared to most eukaryotes, consisting of a fibrous intracellular network of microfilaments connected to a protein network anchoring it to the cell membrane (Branton et al., 1981). More is known about the molecular architecture of the red blood cell membrane than any other membrane system and attempts have been made to relate this information to how red blood cells respond to shear stress (Bennett, 1985). The shape of the erythrocyte membrane is believed to be controlled primarily by the highly charged integral membrane protein glycophorin A and elongated spectrin oligomers adjacent to the interior of the interior of the cell membrane (Elgsaeter et al., 1986). Erythrocytes are characterized by their remarkable deformability (Prokop, 1991; Sutera, 1978; Heath et al., 1982; Waugh and Hochmuth, 1987).At low shear stress levels (10-200 dyn/cm2),the erythrocyte will orient itself in the shear field such that a minimum amount of surface area is perpendicular to the flow field. The membrane will begin rotating about the relatively stationary cytosol inside. This effect is known as treadmilling (Fischer and Schmidt-Schonbeim, 1977; Sutera et al., 1983). As shear stress is increased from 200 to 1500 dyn/cm2,the cell membrane undergoes significant deformation. At 1500 dyn/cm2,highly elongated cells can be observed in the flow field (Sutera et al., 1975). Significant amounts of lysis occur at shear stresses greater than 2000 dyn/cm2 for exposure times of 5 to 10 minutes (Nevaril et al., 1968). Erythrocytes, due to the highly elastic membrane, are capable of experiencing stresses as high as 10,000 to 40,000 dyn/cm2 for very short periods of time and then of recovering their shape (Leverett et al., 1972). Cell biologists are now using sophisticated biophysical techniques to probe the relationship between the erythrocyte cell membrane and the cytoskeleton during the application of hydrodynamic stress (Chasis and Mohandas, 1986). Normal erythrocytes, as well as cells from individuals with hereditary defects in blood cell morphology and erythrocytes treated with specific compounds, have been examined during the application of shear stress. 2, 3-Diphosphoglycerate, which disrupts the association of spectrin with actin and spectrin with protein 4.1, causes erythrocyte membranes to have reduced deformability and re-

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duced stability in a continuously applied field of 575 dyn/cm2 (Chasis and Mohandas, 1986). Individuals with a rare blood disorder that specifically affects the binding of spectrin and protein 4.1 but not the spectrin-actin association have erythrocyte membranes with reduced stability but normal deformability. Chasis and Mohandas (1986) postulated that, in nondeformed cells, spectrin exists in a coiled conformation. The application of shear stress causes the cell to deform and the spectrin molecules to become uncoiled. At low levels of stress, energy is stored in spectrin oligomers giving the cell shape memory. As the magnitude of the stress increases, the spectrin-actin-band 4.1 interactions become dissociated, leading to compromised membrane integrity. This methodology has allowed the assignment of different functions, such as membrane deformability and stability, to specific membranecytoskeleton associations. These experiments illustrate the important role the cytoskeleton plays in the response to hydrodynamic stress. Unlike erythrocytes, leukocytes do possess internal organelles and more complex cytoskeletal apparatus. A few studies have been conducted with human leukocytes and they have shown that the percentage of lysis increases steadily above a “critical” shear stress of 150 dyn/cm2 for exposure times of 2 to 10 minutes (Dewitz et a]., 1977). A shear stress of 600 dyn/cm2 for 10 minutes caused 25% of cells to rupture. The enzymatic activity of the intracellular enzyme alkaline phosphatase, which is confined to the lysosomes, was greatly reduced at shear stresses greater than 15 dyn/cm2. The decrease in phosphatase activity occurs before a significant amount of the population is lysed. There are three explanations for this behavior. The lysis of part of the cell population may cause the release of factors which inhibit phosphatase activity. A second possibility is that shear stress of a sufficient magnitude activates intracellular control mechanisms which modulate phosphatase activity. The last possibility is that shear stress effects are transmitted into the interior of the cell, causing ultrastructural damage. Studies on cell suspensions containing human T and B cells and monocytes confirmed the above “critical” shear stress of 100-200 dyn/cm2 (10 minutes) Chittur et al., 1988). Authors observed that exposure of these cells to stress in a Couette apparatus resulted in a stress interference with the ability of phytohemagglutinin P (lectin) to induce the T cell activation process leading typically to proliferation. Such an observed decrease in the proliferation response may have important implications for clinical applications (blood contacting devices). Mazur and Williamson (1977) studied the deformability of guinea pig macrophages in a Couette viscometer. Cells were subjected to 1000 dyn/cm2 for 5 minutes and then treated with glutaraldehyde to freeze

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the plasma membrane. Macrophages were elongated to twice the length of control cells. Inhibitors of glycolysis were found not to significantly affect cell deformability under shear stress, although they do affect normal phagocytic processes which do require cell deformability. Microtubule-disrupting agents did not influence deformability but chemicals which interacted with microfilaments, actin, and myosin did affect deformability. From these experiments, it was concluded that deformability in the presence of shear stress was moderated by contractile protein elements of the cytoskeleton. Studies have also been done on the role of the cytoskeleton in the mechanical stress response of lymphocytes. Elson et al. (1983) have developed an instrument called a “cell poker” which applies a given force to a small part of the plasma membrane. By measuring the resistance to local indentation of the plasma membrane by the cell poker, one can estimate cell deformability parameters. Lymphocyte plasma membranes, and probably those of all higher eukaryotic cells, are populated with integral membrane protein receptors. These receptors are linked to the cytoskeleton through either covalent or hydrophobic interactions. The plasma membrane has a high degree of fluidity and membrane proteins possess a high degree of lateral mobility (Frye and Edidin, 1970). Reagents which cross-link these surface receptors into large, relatively immobile groups significantly decrease deformability. Reagent-induced cell stiffness can be reduced by cytoskeleton-disrupting agents (Pasternak and Elson, 1985). These experiments illustrate the intimate relationship between the plasma membrane and the cytoskeleton. Because of this relationship between the membrane and the underlying cytoskeleton, mechanical stimuli are rapidly transmitted to the cell interior, activating control mechanisms as well as causing physical damage. There is now evidence available that actin [not tubulin) may be involved in the ability of suspended cells to resist shear forces. Hybridomas treated with cytochalasins (depolymerization of actin) become more fragile (Petersen, 1989). E. INSECTCELLS Insect cells can be used for the production of viral insecticides and mammalian glycoproteins [Tramper and Vlak, 1986; Maiorella et al., 1988; Luckow, 1991; Luckow and Summers, 1988). These cells are approximately 20 pm in diameter and lack a cell wall. The sensitivity of Spodoptera frugiperda cells has been investigated in both a Couette viscometer and a bubble column (Tramper et al., 1986). The critical shear stress at which cell viability began to decrease occurred in the

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10-30 dyn/cm2 range for a continuous exposure time. In this case, exclusion of trypan blue was used as an indicator of cell viability. The decrease in cell viability is a linear function of both the magnitude of shear stress and the exposure time over the range of a few minutes to 2 hours (Fig. 13).Based on a literature survey, Wu et al. (1989) concluded on the same range of critical shear stress. Insect cells were also found to be sensitive to turbulence induced by rising bubbles in a bubble column or in airlift reactors (Tramper et a]., 1986, 1988). Significant shear stress levels can be generated in a bubble column at the gas distributor and at the top of the column as the bubbles burst through the liquid surface. Cell viability as a function of time in 200 cm3 bubble columns at different gas flow rates and with three different types of spargers was measured. The three different spargers produced bubbles of different sizes but this had a negligible effect on the cells. Increasing gas flow rates, however, led to significant decrease in cell viability as measured by trypan blue exclusion. A high pressure drop across the distributor has been identified as an important cause of shear damage at the bubble formation region near the gas distributor (Murhammer and Goochee, 1990). The increase in gas velocity would

0

Sheor stress (dy a 0 0

0

10

20

30

Exposure time

5 15 250

500

40

50

61

(minutes)

FIG. 13. Shear sensitivity of insect cells measured in a Couette viscometer: viability as trypan blue exclusion [Tramper et al., 1986).

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lead to an increase in turbulence in the sparger region and to more cell damage. The riser section does not seem to generate enough stresses. However, the disengagement (collapse) section has been shown to be critical for damage by Handa-Corrigan et al. (1989) with mammalian cells and the same should hold for insect cells. Related to the bubble formation/collapse mechanism described above is a concern on bubble incorporation via vortexing or cavitation. Typically, moderate agitation rates have negligible effects. When the agitation rate is increased, bubble introduction via vortexing or cavitation causes a rapid loss of insect cell viability (Murhammer and Goochee, 1990). The gas entrainment from the gas phase situated above the liquid is inevitable for higher agitation rates and can be suppressed by increasing reactor volume. The cavitation bubbles are formed in regions of low liquid pressure. It has been hypothesized that gas entrapped in small particles (cell debris) can serve as nuclei for their formation. The cavitation is characterized by extremely high specific energy levels in the fluid, leading to formation of turbulent eddies in the viscous dissipation region (Kolmogoroff). Medium supplemented with Pluronic F68 protected insect cells from detrimental effects of both vortexing and cavitation (Murhammer and Goochee, 1990). Murhammer and Goochee (1990) hypothesized that Pluronic polyols interact with cell membranes and protect cells. The generation of a layer of a Pluronic polyol on the cell surface, as well as on the surface of bubbles, would inhibit damaging interactions between cells and air-liquid interfaces. Some partial evidence for the above hypothesized mechanism is available for erythrocytes. C. M. Smith et al. (1987) demonstrated that treatment of sickled erythrocytes with Pluronic F68 reduces their rigidity. Murhammer (1989) also correlated the ability of polyol family agents to serve as a protective agent with hydrophilic-lipophilic balance, indicating that the hydrophobic portion of the molecule is important. This, perhaps, suggest a possibility of agent incorporation into cell membranes. The detrimental effect of agitation on insect cell growth has been reduced by addition of methylcellulose to the medium (Hink and Strauss, 1980). In this case, the mechanism should be different because the agent is a strong viscosity enhancer. Other suggested protection is via cell encapsulation (King et d.,1988), applicable to any cell type. The expression of recombinant proteins by cells infected with a genetically modified baculovirus has been also demonstrated in sparged bioreactors. Pluronic F68 did not improve the expression, perhaps because virally infected cells are more sensitive because their membranes may be stretched by internal virus accumulation (Murhammer, 1989).

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It can be concluded from the above that insect cells are more sensitive to mixing as compared to hybridoma cells. F. PLANTCELLS Plant cell bioreactors are being developed for the production of secondary metabolites that can be used as pharmaceuticals, flavors, and fragrances (Stafford et al., 1986). Plant cells have been shown to be sensitive to hydrodynamic shear stress (Shuler, 1981). For this reason, several different reactor designs have been proposed which address this problem. Plant cells are now grown in hollow-fiber reactors, airlift reactors, and stirred tank reactors using either marine or Rushton impellers at low rpm (Jose et al., 1983; Smart and Fowler, 1984; Drapeau et aI., 1986; Rajasekhar et al., 1971). The method and degree of agitation are known to influence both primary and secondary metabolism (Tal et al., 1983; Lindsay and Yeoman, 1983). Suspension-cultured plant cells possess a primary cell wall composed of five major polymer systems (McNeil et al., 1984). In whole plants, individual cells are often surrounded by an additional secondary wall. The current working model of the cell wall stipulates that cellulose microfibrils and cross-linked extensin molecules form two independent reinforcing matrices embedded in an amorphous pectin gel (Cooper et al., 19871. The cell wall is a dynamic structure in the sense that local gradients in protons, calcium ions, and water activity continually alter the viscoelastic properties of the wall (Cooper et al., 1987).

Single cells in a suspension culture can range from 20 to as much as 100 pm in diameter (Morris and Fowler, 1981). Suspended cells can

assume a wide variety of shapes, from spheres to ellipsoidal cells. In suspension culture, plant cells have a tendency to aggregate into clusters of 10 to 100 cells, depending on the culture conditions. This aggregation phenomenon is primarily due to incomplete cell separation after mitosis and electron micrographs show that the cell walls between adjacent cells in an aggregate are meshed together [Walker and Nevins, 1973). Individual cells as well as aggregates have a length scale similar to that of the Kolmogoroff microscale in stirred tank bioreactors and can thus be sensitive to mixing. The cytoplasm of plant cells, like that of most eukaryotic cells, is highly dense and structured. Plant cells also possess large vacuole compartments which are much less viscous than the cytoplasm (Matile, 1978). In some cases, the vacuole can occupy as much as 80% of the cell volume. The degree of vacuolization will thus affect the bulk viscosity

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of the cell interior, and may influence the propagation of hydrodynamic stresses into the cell. Early studies on the sensitivity of plant cells to mixing concentrated on the amount of agitation necessary to lyse plant cells in a stirred vessel. One of the first studies on optimal orbital shaking rates for suspension-cultured plant cells was reported by Street et al. (1971). They found that Acer pseudoplatanus exhibited optimal growth rates at 100 rpm and reduced growth rates at 80 and 120 rpm. Reduced growth rates at 80 rpm were attributed to retention of a volatile growth inhibitor in the head space. Increased cell lysis was noted, though not quantified, at 1 2 0 rpm. Later studies in agitated reactors showed that lysis occurs when turbine impellers in typical size laboratory bioreactors reach speeds in the range of 150 to 300 rpm (Fowler, 1982; Wagner and Vogelmann, 1977). Fowler (1982) has reported the isolation of a “shear-insensitive” Catharanthus roseus cell line which can withstand up to 300 rpm with a Rushton impeller without deleterious effects. Tanaka (1981) studied the effects of different agitation systems on the ability to grow Cudrania tricuspidata in shake flasks. Biomass yield after 15 days growth in batch culture decreased with increasing agitation rates. The maximum biomass yield was obtained with a tip speed of approximately 70 cm/sec. Changes in the aggregate size distribution from 15-day old cultures were noted. Tanaka therefore concluded that the decreased biomass yield was most probably the result of hydrodynamic stress. There have been several investigations to determine which bioreactor environment is most suitable for plant cell suspensions. Wagner and Vogelmann (1977) examined several different mechanical and air-driven agitation systems and concluded that bubble columns were optimal. Tanaka (1981) compared several different agitation systems, including shake flasks with and without baffles, Rushton impellers, paddle impellers, bubble columns, and airlift reactors. In terms of optimizing mass transfer rates and the hydrodynamic stress environment for plant cells grown at high cell densities (greater than 20 g/liter), Tanaka concluded that mechanical agitation with large paddle impellers at a low rpm was the best. The sensitivity of a given species may also vary considerably with factors such as the specific origin of the cell line (derived from leaf, root, stem, etc.), nutrient media, and growth conditions. Tal et al. (1983), for example, reported that Dioscorea deltoidia cells could not be grown with mechanical agitation (tip speed of approximately 50 cm/sec). Drapeau et al. (1986) reported, however, that Dioscorea deltoidia, grown in the same media, was not adversely affected by mechanical agitation at tip speeds of 80 cm/sec.

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Catharanthus roseus has received a great deal of attention due to the economically significant alkaloids it produces (Stafford et al., 1986; Curtin, 1983; Sahai and Knuth, 1985). It has been grown in reactors ranging from 1 to 200 liters in scale employing either mechanical or hydrostatic (bubble column) agitation methods. Wagner and Vogelmann (1977) observed extensive cell lysis at tip speeds as low as 35 cm/sec. Other investigators have observed optimal growth at tip speeds of 75 cm/sec. Fowler’s “shear-insensitive’’cell line withstands agitation rates up to 300 rpm (approximate tip speeds of 150-170 cm/sec) (Fowler, 1982). Typically, studies in plant tissue cultures concentrate on measuring three responses to hydrodynamic stress: cell lysis, changes in aggregate distribution, and changes in final biomass yield. Several criticisms can be made with respect to the quality of these data. The observations on cell lysis are qualitative rather than quantitative (Wagner and Vogelmann, 1977). No study reports a percent cell lysis, nor do any of the investigators state how cell lysis is being measured [Wagner and Vogelmann, 1977; Tanaka, 1981). Since plant cells form large aggregates, cell number per unit volume is not an easily accessible quantity. The data on aggregate distribution changes are scant, and there is no information on the statistical significance of these data (Tanaka, 1981). Finally, the reports on biomass yield vary greatly among different investigators, even though they may be working on the same cell line grown under similar conditions. More meaningful are data obtained under more defined conditions in a well-established shear field. Experiments conducted in a Couette viscometer under laminar flow conditions show that carrot cells exhibit a wide variety of biological responses to hydrodynamic shear stress depending on the magnitude of the applied force (Rosenberg, 1987). In the shear stress range of 5-1,000 dyn/cm2, carrot cells lose the ability to undergo further cell growth and division (Fig. 14). The rate of viability loss increases exponentially with the applied shear stress. Furthermore, stresses applied for as little as 15 seconds will affect the cells. Although carrot cells lose viability at relatively low shear stresses, they do not lose many other important cellular functions. Intracellular enzyme activity, as measured by the ability of plant cells to carry out electron transport activities, is not seriously affected by hydrodynamic shear stress until a force of 7,500 dyn/cm2 is applied. At lower shear stress levels, there is actually a transient stimulation of intracellular enzyme activity. This stimulation is believed to be the result of the competing influence of mechanical damage and the release of mass transfer limitations. At shear stresses above 30,000 dyn/cm2, intracellular enzyme activity is severely compromised. Membrane integrity,

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-

*

100-

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impeller Vessel Diom.(cm) Vol. ml

Symbol

60-

3.2

0

b

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150

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Kolmogoroff microscale ( p m ) FIG. 14. Regrowth of carrot cells exposed to laminar shear stress (Rosenberg, 1987).

as measured by the ability to concentrate the vital stain fluorescein diacetate is damaged by stresses in the 30,000-100,000 dynlcm2 range. Cell lysis caused by viscous shear stress does not occur to any significant degree until stresses in excess of 100,000 dyn/cm2are applied over a period of 1 hour (Fig. 15). In each case, the loss of the particular biological response could be characterized by an apparent first-order rate constant. A similar observation has been made for the death kinetics of mammalian cells (Augenstein et a]., 1971). Cell viability is clearly lost at shear stress levels two to three orders of magnitude below that required to do significant physical damage to the cells. This observation implies that plant cells must have a mechanism for sensing the applied stress and respond to it. One hypothesis is that plant cells possess receptors in either the cell membrane or the cell wall which are sensitive to mechanical stress (see, e.g., Lintilhac and Vesecky, 1984; Pickard, 1984). These receptors communicate the magnitude of the stress to control centers within the cell via second messengers. When shear stresses greater than 100 dyn/cm2 are applied to plant cells for 60 seconds or more, the stress receptors activate control pathways which selectively inhibit mitosis without affecting such functions as electron transport activity or plasma membrane integrity, Experiments in a turbulent flow field (Rosenberg, 1987) using a pad-

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-2

100

90

ul

d

80

'p, -

r

d

70

C 0

Z

60

50

I 0

100,000

10

20

,

I

I

30

40

50

Exposure time

j 60

(hours 1

FIG. 15 Lysis of carrot cells exposed to laminar shear stress (Rosenberg, 1987).

dle impeller, however, show that intracellular enzyme activity and cell lysis are seriously affected by turbulent stresses that are orders of magnitude below those necessary to generate the same degree of damage in laminar flow (Fig. 16). Plant cells may be particularly sensitive to some aspect of turbulence. In the laminar flow field in a viscometer, both cell-cell collisions and cell-wall interactions are negligible (Einav and Lee, 1973; Sutera, 1977). In turbulent flow, coHision phenomena and transient Kolmogoroff (Reynolds) stresses could predominate. An increased sensitivity to turbulent flow has been noted for endothelial cells and protozoa (Midler and Finn, 1966; Davies et a]., 1986a). Special attention has been recently paid to the employment of the hairy root culture system. This type of plant tissue (organ) culture has become of interest because of their indefinite and active proliferation in phytohormone-free medium and their sustained capacity to produce valuable products. Many plant species have been transformed through the infection of a soil bacterium, Agrobacterium rhizogenes (Mugnier, 1988). A stirred tank reactor is not suitable for hairy root cultures because root cells can be easily injured by the impeller or by stress in its vicinity. An airlift reactor worked well for horseradish hairy root cells (Taya et a]., 1989). Kondo et al. [1989) immobilized roots in a rotating basket reactor (stainless steel mesh) fitted with a turbine-blade mixer. An immobilization in a reticulate polyurethane foam is also possible in a rotating drum reactor. Because of higher densities achieved in such reactors, increased oxygen transfer has to be provided. The mechanism of physical damage in plant cultures is not well

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0 U

Tip speed (cm/secl

0

80

t I

0

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13.1 26.8 107.2

0

211.5

8

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40

80

120

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24

Residence time (minutes 1 FIG. 16. Lysis of carrot cells exposed to turbulent shear stress in a Haake viscometer provided with a paddle (Rosenberg, 1987).

understood. Although protected with cell wall, plant cells in a suspension culture have relatively weak and pliable cell walls, which deform or rupture readily (Hooker et al., 1989).An increase in cell size, associated with growth and expansion, may lead to a decrease in cell wall strength, causing an increase in susceptibility to shear until the wall components become stronger. The decrease in cell wall strength has been documented only indirectly by Hooker et al. (1989) through an elevated phenolics release in a Nicotiana tabacum species, perhaps due to increased permeabilization. Tanaka et ul. (1988) observed in Catharanthus roseus cell culture an increase in hemicellulose and cellulose components of the cell wall after extended cultivation under mild mixing conditions, implying a strong cell wall complex. The dynamics and physical adaptation to shear conditions should be further studied. Much less is known about the actual chemistry of shear damage (stimulus-response cascade). Knowledge on the events involved in any stimulus (stress] response is much more fragmentary as compared to that of mammalian cells (Ryan, 1988). Essentially, there is no published study dealing with biochemical details of hydrodynamic stress injury to plant cells in suspension. Some insight can be gained from closely

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related work on mechanical stress injury. Cytoplasmic calcium elevation and calmodulin-related enzymes have been documented for mechanically (rubbing) injured seedlings of soybean (Glycine max) (Jones and Mitchell, 1989). Such compounds belong to the category of second messengers involved in mediating responses. Another well-documented response to mechanical wounding is synthesis of numerous proteins responsible for cell wall synthesizing processes (glucan, callose, extensin, etc.). Such a response may substantially alter cell expansion and reduce wall extensibility (deformability). The production of “hydrodynamic stress proteins” in Nicotiana tabacum suspension cultures has been documented recently, although the delineation of exact cell physiological conditions is still a problem (A. Prokop and D. H.-T. Ho, 1990 unpublished observations).

G. NEMATODES Nematode worms can be used for the productian of insecticides. These worms are multicellular organisms with a length of 1 to 5 mm and a width of 0.5 mm. Such organisms are very large and much denser than water. Large-scale nematode culture is being considered as an alternative to chemically synthesized insecticides. One of the primary obstacles to large-scale commercial production of nematodes is that the agitation forces necessary to suspend the organism destroy the worms. Stresses on the order of 0.1 dyn/cm2 are necessary to suspend the organisms whereas Reynolds stresses on the order of 20 dyn/cm2cause physical damage (Pace et a ] . , 1986). An airlift reactor seems to be the most appropriate choice of reactor.

H. COMPARATIVE STUDY Extensive data on cell viability and shear stresses have been collected and somewhat categorized (Rosenberg, 1987). In many cases, the literature does not provide explicit information on exposure times and magnitudes of the applied force in the laminar as well as the turbulent flow regime. The exposure time is assumed to be equivalent to the residence time of the cells in the viscometer or bioreactor. In some cases, assumptions were made about such parameters as impeller dimensions and vessel geometry. Bacteria and lower eukaryotes are much less sensitive to hydrodynamic stress than higher eukaryotes due to their small size and the presence of a cell wall. When microorganisms form large aggregates through flocculation or hyphal growth, they experience a larger frac-

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tion of the fluid-phase kinetic energy, and exhibit shear-sensitive properties (Wase et a]., 1985). Similarly, when the Kolmogoroff microscale is reduced to a few microns, yeast cells exhibit cell lysis at much lower turbulent stresses (Doulah, 1977). Animal cells are very sensitive to the shear forces generated in bioreactors due to the relatively large size and the lack of a protective cell wall. The biological responses are concentrated in the region of smaller shear stresses. The studies reviewed here have concentrated on the shear forces required to cause cell lysis or reduce viability. In most cases, viability is implicitly equated with membrane integrity (Brooks, 1984; Tramper and Vlak, 1986) or the detachment of anchorage-dependent cells (Croughan et al., 1985; Stathopoulos and Hellums, 1985). This is not necessarily true in either case (Ciftci et al., 1981; Farmer et a].,1978; Ben-Ze’ev et aJ., 1980). The few examples where sublethal phenomena have been studied, such as changes in enzyme activity and reorganization of the cytoskeleton, imply that there are many biological responses to low levels of shear stress that remain to be characterized. Plant cells are even larger than animal cells. They form large aggregates, which means that they are subjected to a large fraction of the kinetic energy present in the fluid. As a first approximation, one would expect that plant cells would be less sensitive to hydrodynamic stress damage than anchorage-dependent mammalian cells grown on microcarrier beads. Microcarrier beads are roughly the same size as plant cell aggregates. As a result, microcarrier cultures can be grown under similar agitation conditions as plant cell cultures. Agitation by paddle impellers at tip speeds of approximately 25-50 cm/sec is typical for both plant and anchorage-dependent mammalian cell cultures. Plant cells thus appear to be as shear sensitive as mammalian cells which lack a protective cell wall. There is a growing body of evidence which suggests that animal cells display several different types of biological responses to hydrodynamic stress. The severity of the response is a function of the magnitude of applied force and exposure time. For example, at low levels of stress, enzyme activity (Grabowski et a]., 1985; Fragos et al., 1985; Stathopoulos and Hellums, 1985) and cytoskeletal organization (Franke et aJ., 1984; Ives et a]., 1986; Chasis and Mohandas, 1986) change in a highly regulated manner. At higher levels of stress, detachment from substrata (Croughan et al., 1985; Stathopoulos and Hellums, 1985), loss of membrane integrity (Brooks, 1984; Tramper and Vlak, 1986),and cell lysis occur. One may hypothesize that any organisms will also exhibit a set of

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graded responses to hydrodynamic stress. Biological responses to hydrodynamic stress will be a function of the magnitude of force and the duration of exposure to that force. Low levels of stress or short exposure times may cause subtle, transitory shifts in metabolism. Enzyme activity and gene expression may be influenced by relatively innocuous stress conditions. As the shear stress and/or exposure time increase, dramatic and irreversible changes in metabolism may occur. Loss of viability, aggregate disruption, and cell lysis may all occur as the severity of the hydrodynamic stress conditions increases. Finally, it can be predicted that, based on the animal cell shear sensitivity literature, a critical shear stress for a continuous exposure will exist for each biological response. Below that stress, there will be no adverse effects, regardless of the magnitude of the stress or the exposure time. Beyond the critical shear stress level, the biological response will be proportional to the magnitude of the stress and the exposure time, VI. Summary and Outlook

In the following some generalizations on the physical and biological mechanisms of hydrodynamic shear stress are attempted and indications are made as to the areas of inadequate treatment and knowledge which need further development. A. PHYSICALEFFECTS The theoretical treatment as depicted in Section I11 does not seem to be adequate, although the tools are available. Too many variables and nonavailability of suitable closing on balances represent a main drawback. The application of certain theoretical expressions should be carefully screened in terms of specific limitations and conditions. The concepts developed in fluid and material mechanics largely await further applications in the area of cell damage. Two basic mechanisms of cell damage are visualized: (I) stretch (compression) and breakup in the laminar field and (2) surface dislocation and perforation (loss of permeability barrier]; they await further experimental proofs. The questions of fundamental importance are how those two distinct regimes differ and what are the consequences for living organisms. There are enough experimental proofs to demonstrate their different modes of action for attaching (Davies et a]., 1984, 1986b; Levesque et a]., 1989) as well as nonattaching mammalian cells (Sallam and Hwang, 1984; Abu-Reesh, 1989).Data of Abu-Reesh (1989) on hybridomas are depicted in Fig. 17. Also, Rosenberg (1987) noted

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ALES PROKOP AND RAKESH K. BAJPAI

0

100

300 500 700 Shear stress ( dynes /cm2)

900

FIG. 17. Effect of flow regime on viability of the hybridomas (trypan blue exclusion test) for a 1-hour exposure time (Abu-Reesh and Kargi, 1989).

much higher sensitivity of plant cells in a culture to turbulent stress. Are these differences due to the fluctuating velocity component of the turbulent stress? In mammalian cells, some researchers noted a significant difference between steady and pulsating (low frequency) flow (Levesque et al., 1989); others did not (Yoshizumi et a]., 1989). Is the difference due to long relaxation times of biological material (treated here as viscoelastic bodies; see Prokop, 1991)?Perhaps the combination of a high turbulent frequency and a slow relaxation time results in the accumulation of energy in the form of shear strain. This effectively means that the cells do not have time to relax fully (recover to the original shape] from the previous encounter with a turbulent eddy before they hit the next turbulent eddy. Such accumulation of energy in the form of deformation may cause permanent damage to the stressbearing molecular network. In a laminar regime, whose frequency approaches zero, only when laminar shear stress is larger than the critical one does permanent damage due to stretchinglbreakup follow. This hypothesis would also explain why plant cells in a culture are particularly sensitive to certain turbulent fields. The plant cell cellulose-extensin network serves, perhaps, as a good sink of turbulent energy. The viscoelastic characterization of cells is somewhat fragmentary in terms of species coverage. In general, cells may behave either as elastic or rigid bodies depending on the degree and frequency of the physical disturbance. For short periods of time and moderate stresses cells can tolerate high forces. For higher stress (above critical) damage is a func-

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tion of stress level and exposure time. Such behavior is due to the dynamic properties of cells. Perhaps a breakdown into individual components, such as normal, shear, and viscous forces, would also help. Normal forces generated by pressure fluctuations in the turbulent field have been largely neglected in the analysis as contrasted to turbulent shear forces. Another intriguing question is how all these components interact. Can we simply use an additive rule, that is, do they function independently, and can we sum up individual contributions? Perhaps, turbulent stress can be added on the top of viscous forces, based on the concept of decomposition (Fig. 4). Typically, however, normal forces are neglected altogether. Carefully designed model experiments are thus required. The question will remain how these simplified situations reflect in a real case. A careful analysis of viscosity effects should also be added here. How does the viscosity affect transition from a laminar to a turbulent regime? Is there an upper limit of cell protection in a turbulent field via viscosity enhancers? The next question which should be addressed is a distinction between damage due to encounter with eddies themselves versus due to collision events. As mentioned previously, the numerous variables involved preclude a solid analysis at this moment. Another area requiring consideration is whether cells are present as individuals or in aggregates. Where is the physical effect of damage localized? Are only the surface cells hit or can the mechanical signal be propagated further down in the cell aggregate? Still another complication may be in the area of population sensitivity toward hydrodynamic stress. Are all cells equally responding or are some more sensitive or more robust? Some experimental evidence exists in support of heterogeneity of response because of a possibility of selection for a resistant population (Petersen et a]., 1988). Thus, global shear stress estimates should be substituted by differentiated damage procedures. A major engineering inadequacy is in the area of mapping the shear field throughout a reactor. Coupling of residence time characteristics with such shear field would allow the development of a more rational description of shear damage. Also, it would allow for scale-up on the basis of hydrodynamic similarity (Bliem, 1989). The best way of avoiding physical damage to biocatalysts is via a lowshear mixing (with predominant axial component), provided adequate oxygen transfer is available. At the same time, unsuspended solids (cells, carriers, etc.) should be eliminated to qvoid nutrient gradients. Some rational criteria applicable to any shear-sensitive biocatalyst have already been proposed (Prokop and Rosenberg, 1989). However, at pre-

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sent, an empirical route of trial and error is probably the safest. Obviously, one should operate in the subcritical regime. B. BIOLOGICALEFFECTS Based on previous sections, which presented some supporting evidence, biological effects can be categorized into subcritical and critical effects. Subcritical effects can be condensed into a hypothetical mechanistic scheme based on an analogy with other environmental stresses (Fig. 18).The first step involves signal reception and transduction including subtle molecular deformations of surface molecules (i.e., ion

Signal Reception/ Transduction

Molecular

Microscopic deformation

V Amplification

Expression

Biological response

Physical

Second messengers

I

eleton

mRNA synthesis I

IF~GL~~ ion

I

Respiration rate Growth rate Production rate Differentiation, etc

FIG. 18. Hypothetical scheme of the mechanism of the hydrodynamic shear stress signal-response cascade.

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SHEAR SENSITIVITY OF BIOCATALYSTS

channels), membrane movements and deformations, and changes in membrane fluidity, all mimicking the hormone-receptor response. The interaction between the local field and the dynamic response of cell surface components represents a major challenge in future. Resulting second messengers lead to a reaction cascade generating several other second messenger species, affecting specific mRNA and protein synthesis in a transient manner. The latter may lead on a longer time scale to numerous biological responses. Such a biochemical cascade may be partially bypassed by a physical signal propagation through a flexible cytoskeleton or cytoplasmic network directly to affect mRNA/protein synthesis (Packard, 1986). Both networks represent a physical connection from the plasma membrane [and cell wall) into the nucleus and complement the above chemical transducing system of the second messengers. The question remains open whether there are any specific hydrodynamic stress proteins (e.g., refoldases) involved in the cell recovery at higher stress exposures besides proteins already present in cells and accentuated as a result of stress (e.g., cytoskeleton proteins). The utilization of subcritical effects to our benefit, that is, an intentional use of controlled shear conditions to produce substances of interest is in the early stages of application. The critical responses involve adverse effects on cells. Table 111 summarizes a typical sequence of some selected criteria of cell physiology as observed with hybridomas and plant cells in culture. This table provides a guideline as to what functions can be expected to be affected as the level of shear field increases. More subtle effects are first observed (damage to surface molecules would be of the first priority], TABLE 111 APPROXIMATE SEQUENCE OF DAMAGE IN FUNCTIONS OBSERVED IN A SHEAR FIELD

Sequence of events Regrowth Respiration activity Membrane integrity Lysis Lactate dehydrogenase release

Hybridomas in viscometer and stirred culture (Abu-Reesh, 1989)

1 2 3 4

Plant cell culture in viscometer (Rosenberg, 1987) 1 2 3 4

-

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followed by more dramatic ones, and ending up with total cell destruction. There is a need to extend such a list and add some meaningful criteria for some specific conditions (e.g., substrate consumption, product formation) as well as to standardize criteria to achieve better comparison between different research groups. The establishment of generally accepted reference cell strains and lines as indices of shear effects would also be a great advantage (Sallam and Hwang, 1984). Finally, we discuss the biological ways of overcoming adverse effects of shear on biocatalysts, namely, cells. It is reasonable to assume that modulation of gene expression may be one of the most sensitive responses to hydrodynamic stress. At stresses well below those required to influence membrane integrity or growth rate the expression of specific genes may be enhanced or repressed. It is widely believed that heat-shock-effect proteins are to protect the cells from the damaging effect of stress (high temperature). If an analogy between these two stresses holds, one may speculate that either amplifying the above specific response (gene amplification) or increasing the sensitivity of the “receptor” response might be possible to increase the shear necessary to induce such biological responses as decreased metabolic or overall growth rate, loss of membrane integrity, or cell lysis. The end result is that cells would tolerate higher agitation rates, thus providing a mechanism for increasing nutrient supply and toxic wastes removal. Alternatively, the expression of unique hydrodynamic stress proteins may be detrimental to cellular metabolism. In this case, one would want to search for ways to turn off the signals which stimulate the expression of hydrodynamic stress genes. There are several potential routes whereby the signal could be short-circuited before reaching the hydrodynamic stress-sensitive promoter. Once again, the end product would be a cell that would experience higher mass transfer rates without generating harmful gene products. If a unique set of hydrodynamic stress proteins exists, then it may be possible to utilize the hydrodynamic stress promoter to control gene expression. In some instances, it may be possible to construct chimeric genes in which the hydrodynamic stress promoter is linked to the ratecontrolling enzyme, allowing for a switch-on of the particular product synthesis via an increase in the agitation rate. Historically, biochemical engineers have attempted to circumvent the problem of shear sensitivity by designing novel bioreactors. This strategy has met with limited success. Instead of designing bioreactors for shear-sensitive cells, it may be possible to design a cell specifically for the stressful environment of the bioreactor.

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VII. Nomenclature constant in Eq. (27); cell concentration initial cell concentration (seed] number density of beads drag coefficient cell-impeller collision severity turbulent (cell-eddy) collision severity impeller diameter cell (particle) size vessel diameter gravitationaUcentrifuga1 acceleration vector average acceleration vector specific death rate constant in Eq. (29) constant in Eq. (36) characteristic length Kolmogoroff length number of impeller blades impeller speed bubble concentration cell-cell collision frequency cell-impeller collision frequency power number

P Pxx

P Rb

Re *P

Re'

Q t V

V' V

t Vt Vf'

V X

Y

pressure normal stress force to squeeze out liquid film bubble radius impeller Reynolds number particle (cell) Reynolds number generalized Reynolds number volumetric flow rate time velocity; settling velocity; relative velocity fluctuating fluid component of velocity velocity vector average velocity vector bubble terminal velocity root mean square value of fluctuating velocity component of turbulence liquid volume coordinate; distance from impeller blade tip one-dimensional gradient in time-averaged velocity

Subscripts i, j, k

directional coordinates in threedimensional systems

x, y, z directional coordinates in threedimensional system

Overline overbar represents time-smoothed value

+

arrow represents vector

Greek letters Q

p y

S, E

E'

volume fraction of cells (particles) coefficient in Eq. (15) shear rate (strain) turbulent boundary layer thickness energy dissipation rate per unit of mass local dissipation rate

P PC

PT v Pf PP

T Tk dt)

fluid viscosity specific growth rate eddy viscosity kinematic viscosity fluid density cell (particle) density shear tensor Kolmogoroff stress turbulent stress tensor

226 dvj

dt) dij T~

ALES PROKOP AND RAKESH K. BAJPAI laminar stress tensor viscous turbulent stress inertial turbulent stress boundary layer shear stress

TR T , ~

T~,,. T~~

Reynolds stress normal stress component in x direction viscous shear stresses

REFERENCES Ahu-Reesh, I., and Kargi, F. (1989). J. Biotechnol. 9, 831. Abu-Reesh, I. (1989). Ph.D. Thesis, Washington Univ., St. Louis, Missouri. Aeschbacher, M., Reinhardt, C. A., and Zbinden, G. (1986). Cell Biol. Toxicol. 2, 247. Al-Rubeai, M. A., Rookes, S., and Emery, A. N. (1989). In ”Advances in Animal Cell Biology and Technology for Bioprocesses” (R. E. Spier, J. B. Griffiths, J. Stephenne, and P. J. Crooy, eds.), p. 241. Butterworth, London. Al-Rubeai, M., Oh, S. K. W., Musaheb, R., and Emery, A. N. (1990). Biotechnol. Lett. 12, 323.

Ando, J., Nomura, H., and Kamaya, A. (1987). Microvasc. Res. 33, 62. Ando, J., Komatsuda, T., and Kamiya, A. (1988). In Vitro Cell. Dev. Biol. 24, 871. Arnold, W. N., ed. (1983). “Yeast Cell Envelopes: Biochemistry, Biophysics, and Ultrastructure,” Vol. 1 . CRC Press, Boca Raton, Florida. Atkinson, B., and Mavituna, F., eds. (1983). “Biochemical Engineering and Biotechnology Handbook.” Nature Press, London. Augenstein, D. C., Sinskey, A. J., and Wang, D. I. C. (1971). Biotechnol. Bioeng. 8, 409. Aunins, J. G., Croughan, M. S., Wang, D. I. C., and Goldstein, J. M. (1986). Biotechnol. Bioeng. Symp. 17, 1. Backer, M. P., Metzger, L. S., Slater, P. L., Nevitt, K. L., and Boder, G. B. (1988). Biotechnol. Bioeng. 32, 993. Batchelor, G. K. (1967). “An Introduction to Fluid Mechanics.” Cambridge Univ. Press, London. Bennett, V. (1985). Annu. Rev. Biochem. 54, 273. Ben-Ze’ev, A. (1985). Exp. Cell Res. 157, 520. Ben-Ze’ev, A., Farmer, S., and Penman, S. (1980). Cell (Cambridge, Mass.) 21, 365. Bereiter-Hahn, J. (1987). In “Cytomechanics. The Mechanical Basis of Cell Form and Structure” [J. Bereiter-Hahn, 0. R. Anderson, and W.-E. Reif, eds.), p. 3. SpringerVerlag, Berlin. Bhagyalakshmi, A . , and Frangos, J. A . (1989). Biochem. Biophys. Res. Commun. 158, 31. Bird, R. B., Stewart, W. E., and Lightfoot, E. N. (1960). “Transport Phenomena.” Wiley, New York. Blackshear, P. L. (1972). In “Biomechanics: Foundations and Objectives” (Y. C. Fung, ed.), p. 501. Prentice-Hall, Englewood Cliffs, New Jersey. Bliem, R. (1989). Trends Biotechnol. 7, 197. Branton, D., Cohen, C. M., and Tyler, J. (1981). Cell [Cambridge, Mass.) 24, 24. Bronnenmeier, R., and Mark], H. (1982). Biotechnol. Bioeng. 24, 553. Brookman, J. S. G. (1974). Biotechnol. Bioeng. 16, 371. Brookman, J. S. G. (1975). Biotechnol. Bioeng. 17, 465. Brooks, D. E. (1984). Biorheology 21, 85. Buxhaum, R. E., Dennerill, T., Weiss, S., and Heidemann, S. R. (1987). Science 2 3 5 , 1 5 1 1 . Calderbank, P. H., and Moo-Young, M. B. (1959). Trans. Instn. Chem. Eng. 37, 28. Charm, S. E., and Wong, B. L. (1970). Biotechnol. Bioeng. 12, 1103. Chasis. J. A,, and Mohandas, N. (1986). J. Cell Biol. 103, 343.

SHEAR SENSITIVITY OF BIOCATALYSTS

227

Cherry, R., and Papoutsakis, T. (1986). Bioprocess Eng. 1, 29. Cherry, R. S., and Papoutsakis, E. T. (1988). Biotechnol. Bioeng. 32, 1001. Cherry, R. S., and Papoutsakis, E. T. (1990). In “Animal Cell Biotechnology” (R. E. Spier and J. B. Griffiths, eds.), Vol. 4, p. 71. Academic Press, San Diego, California. Chisti, Y., and Moo-Young, M. (1986). Enzyme Microb. Technol. 8, 194. Chittur, K. K., McIntire, L. V., and Rich, R. R. (1988). Biotechnol. Prog. 4, 89. Christensen, 0.(1987). Nature (London) 330, 66. Ciftci, T., Wang, S., and Constantinides, A. (1981). Biotechnol. Bioeng. 23, 1407. Cook, J. A., and Mitchell, J. B. (1989). Anal. Biochem. 179, 1. Cooper, J,, Chen, J., van Holst, G., and Varner, J. (1987). Trends Biochem. Sci 12, 24. Crouch, C. F., Fowler, H. W., and Spier, R. E. (1985). J. Chem. Technol. Biotechnol. 35B, 273. Croughan, M. S., and Wang, D. I. C. (1990). In “Animal Cell Bioreactors” (C. S. Ho and D. I. C. Wang, eds.], p. 213. Butterworth-Heinemann, Boston, Massachusetts. Croughan, M. S., Wang, D. I. C., and Hamel, J. F. (1985). AIChE Meet., Chicago, 111. Croughan, M. S., Hamel, J.-F., and Wang, D. I. C. (1987). Biotechnol. Bioeng. 29, 130.

Croughan, M. S., and Hamel, J.-F., and Wang, D. I. C. (1988). Biotechnol. Bioeng. 32, 97s. Curtin, M. (1983). BioiTechnology 1, 649. Cutter, L. A. (1966). AIChE J. 12, 35. Das, P., Kumar, R., and Ramkrishna, D. (1987). Chem. Eng. Sci. 42, 213. Davies, P. F., Dewey, C. F., Jr., Bussolari, S. R., Gordon, E. J., and Gimbrone, M. A,, Jr. (1984). J. Clin. Invest. 73, 1121. Davies, P. F., Gimbrone, M. A., Gordon, E. J., Reumuzzi, A,, and Dewey, C. F. (1986a). Biorheology 23, 195. Davies, P. F., Remuzzi, A., Gordon, E. J . , and Dewey, C. F., Jr. (1986b). Proc. Notl. Acad. Sci. U.S.A. 83, 2114. Dewey, C. F. (1984). J. Biorned. Eng. 106,31. Dewitz, T. S., Hung, T. C., Martin, R. R., and McIntire, L. V. (1977). J. Lab. Clin. Med. 90, 728. Diamond, S. L., Eskin, S. G., and McIntire, L. V. (1989). Science 243, 1483. Diamond, S. L., Sharefkin, J. B., Dieffenbach, C., Frasier-Scott, K., McIntire, L. V., and Eskin, S. G. (1990). J. Cell. Physiol. 143, 364. Dodge, T. C., and Hu, W.-S. (1986). Biotechnol. Lett. 8, 683. Donaldson, T. L., Boonstra, E. F., and Hammond, J. M. (1980). J. Colloid Interface Sci. 74, 441. Doulah, M. S. (1977). Biotechnol. Bioeng. 19, 649. Doulah, M. S., Hammond, T. H., and Brookman, J. S. (1975). Biotechnol. Bioeng. 17,845. Drapeau, D., Blanch, H. W., and Wilke, C. R. (1986). Biotechnoi. Bioeng. 28, 1555. Dunn, G. A,, and Heath, J. P. (1976). Exp. Cell Res. 101,1. Edidin, M. (1989). Methods Cell Biol. 29, 87. Einav, S.,and Lee, S. L. (1973). J. Multiphase Flow 1, 73. Elgsaeter, A,, Stokke, B. T., Mikkelsen, A., and Branton, D. (1986). Science 234, 1217. Elliot, A. M., ed. (1975). “The Biology of Tetrahymena,” p. 61. Dowden, Hutchinson & Ross, Stroudsburg, Pennsylvania. Elson, E. L., Daily, B. B., McConnaughey, W. B., Paternak, C., and Peterson, N. 0. (1983). In “Frontiers in Biochemical and Biophysical Studies of Proteins and Membranes” (T. Y. Lin, A. Sakakibara, A. Schechter, K. Yagi, H. Yagima, and K. T. Yasumobu, eds.), p. 399. Elsevier, New York.

228

ALES PROKOP AND RAKESH K. BAJPAI

Falke, L., Edwards, K. L., Misler, S., and Pickard, B. G. (1986). Plant Physiol. 80, Suppl. 9, Abstr. 40. Farmer, S., Ben-Ze’ev, A., Benecke, B., and Penman, S. (1978).Cell (Cambridge, Mass.) 15, 627.

Feder, J., and Tolbert, W. R. (1983). Sci Am. 249, 24. Finean, J. B. (1978). “Membranes and Their Cellular Function.” Halsted/Wiley, New York. Fischer, T. M. (1980). Biophys. J. 32, 863. Fischer, T.M., and Schmidt-Schonbein, H. (1977). Blood Cells 3, 351. Fischer, T. M., Stohr, M., and Schmidt-Schonbein, H. (1978). AIChE Symp. Ser. No. 182, p. 38. Fowler, M. W. (1982). Prog. Ind. Microbiol. 16, 207. Frangos, J. A., Eskin, S. G., McIntire, L. V., and Ives, C. L. (1985). Science 227, 1477. Franke, R. P., Grafe, M., Schnittler, H., Seiffge, D., and Mittermayer, C. (1984). Nature [London) 307, 648. Fredrickson, A. G. (1964). “Principles and Applications of Rheology.” Prentice-Hall, Englewood Cliffs, New Jersey. Freitag, R., Schiigerl, K., Arnold, W. M., and Zimmermann, U. (1989). J. Biotechnol. 11, 325.

Freshney, R. I. (1987). “Culture of Animal Cells. A Manual of Basic Technique,” 2nd Ed., p. 246. Alan R. Liss, New York. Frye, F., and Edidin, G. (1970). J. Cell Sci. 7, 319. Fulton, A. B. (1982). Cell (Cambridge, Mass.) 30, 345. Gardner, A. R., Gainer, J. L., and Kirwan, D. J. (1990). Biotechnol. Bioeng. 35, 940. Gowing, L. R., Tellam, R. L., and Banyard, M. R. C. (1984). J. Cell Sci. 69, 137. Grabowski, E. F., Jaffe, E. A., and Weksler, B. B. (1985). 1. Lab. Clin. Med. 105,36. Guharay, F., and Sachs, P. (1984). J. Physiol. (London) 352, 685. Guharay, F., and Sachs, P. (1985). J. Physiol. [London) 363, 119. Gustin, M. C., Martinac, B., Saimi, Y., Culbertson, M. R., and Kung, C. (1986). Science 233, 1195. Hamamoto, K., Ishimaru, K., and Tokashiki, M. (1989). J. Ferment. Bioeng. 67, 190. Hammond, S. M., Lambert, P. A., and Rycroft, A. N. (1984). “The Bacterial Cell Surface.” Kapitor Szalo, Washington, D.C. Handa-Corrigan, A., Emery, A. N., and Spier, R. E. (1989). Enzyme Microb. Technol. 11, 230.

Harnby, N., Edwards, M. F., and Nienow, A. W. (1985). “Mixing in the Process Industries.” Butterworth, London. Heath, B. P., Mohandas, N., Wyatt, J. L., and Shohet, S. B. (1982). Biochim. Biophys. Acta 691,211. Hink, W. F., and Strauss, E. M. (1980). In “Invertebrate Systems in Vitro” (E. Kurstak, K. Maramorosch, and A. Dubendorfer, eds.), p. 27. Elsevier, Amsterdam. Hinze, J. 0. (1971). Prog. Heat Mass Transfer 6,433. Hiramoto, Y . (1987). In “Cytomechanics. The Mechanical Basis of Cell Form and Structure’’ (7. Bereiter-Hahn, 0. R. Anderson, and W.-E. Reif, eds.), p. 31. Springer-Verlag, Berlin. Hirtenstein, M., and Clark, J. (1980). In “Tissue Culture in Medical Research” (R. Richard and K. Rajan, eds.), p. 97. Pergamon, Oxford. Hooker, B. S., Lee, J. M., and An, G. (1989). Enzyme Microb. Technol. 11,484. Inloes, D. S., Smith, W. J., Taylor, D. P., Cohen, S. N., Michaels, A. S., and Robertson, C. R. (1983). Biotechnol. Bioeng. 25, 2653. Ives, C. L., Eskin, S. G., and McIntire, L. V. (1986). In Vitro Cell Dev. Biol 22, 500.

SHEAR SENSITIVITY OF BIOCATALYSTS

229

Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973). J. Clin. Invest. 52, 2745. Jones, G. T., Erickson, L. E., and Glasgow, L. A. (1990). Ann. N.Y. Acad. Sci. 589, 431. Jones, R. S., and Mitchell, C. A. (1989). Physiol. Plant. 76, 598. Jose, W. H., Pedersen, H., and Chin, C. K. (1983). Ann. N.Y. Acad. Sci. 413, 409. Kay, J. M., and Nedderman, R. M. (1985). “Fluid Mechanics and Transfer Processes.” Cambridge Univ. Press, Cambridge, England. Kelemen, M. V., and Sharpe, J. E. (1979). J. Cell Sci. 35,431. Kim, 7. H., Lebeault, J. M., and Reuss, M. (1983). Eur. J. Appl. Microbiol. Biotechnol. 18, 11.

King, G. A,, Daugulis, A. J., Faulkner, P., Bayly, D., and Goosen, M. F. A. (1988). Biotechnol. Lett. 10, 683. Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y., Hoh, E., Takaku, F., and Yazaki, Y. (1990). J. Biol. Chem. 265, 3595. Kondo, O., Honda, H., Taya, M., and Kobayashi, T. (1989). Eur. J. Appl. Microbiol. Biotechnol. 32, 291. Kunas, K. T., and Papoutsakis, E. T. (1989). Biotechnol. Lett. 11, 525. Kunas, K. T., and Papoutsakis, E. T. (1990). Biotechnol. Bioeng. 36,476. Lansman, J. B., Hallam, T. J., and Rink, T. J. (1987). Nature (London) 325, 811. Lavery, M., Kearns, M. J., Price, D.G., Emery, A. N., Jefferis, R., and Nienow, A. W. (1985). Dev. Biol. Stand. 60,199. Lee, G. M., Savinell, J. M., and Palsson, B. 0. (1989). Hybridoma 8, 639. Lee, Y.-K., and Choo, C.-L. (1989). Biotechnol. Bioeng. 33, 183. Leslie, D.C. (1973). “Developments in the Theory of Turbulence,” p. 10.Oxford University Press (Clarendon), London. Leverett, L. B., Hellums, J. D., Alfrey, C. P., and Lynch, E. C. (1972). Biophys. J. 12,257. Levesque. M. J., Sprague, E. A., Schwartz, C. J., and Nerem, R. M. (1989). Biotechnol. Prog. 5, 1. Lindsay, K., and Yeoman, M. M. (1983). 1. Exp. Bot. 34, 1055. Lintilhac, P. M., and Vesecky, T. B. (1984). Nature (London) 307, 363. Lloyd, C. W. (19821. “The Cytoskeleton in Plant Growth and Development.” Academic Press, New York. Luckow, V. A. (1991). In “Recombinant DNA Technology and Applications” (A. Prokop, R. K. Bajpai, and C. S. Ho, eds.), p. 97. McGraw-Hill, New York. Luckow, V. A., and Summers, M. D. (1988). Bio/Technology 6,47. Markl, H. H., and Bronnenmeier, R. (1985). In “Biotechnology” (H. J. Rehm and G. Reed, eds.), Vol. 2, p. 369. VCH, Weinheim. Maiorella, B., Inlow, D., Shauger, A., and Harano, D. (1988). BioiTechnology 6, 1406. Matile, P. (1978). Annu. Rev. Plant Physiol. 29, 193. Matsuo, T., and Unno, H. (1981). J. Environ. Eng. Div. [Am. SOC.Civ. Eng) 107,527. Mazur, M. T., and Williamson, J. R. (1977). J. Cell Biol. 75, 185. McIntire, L. V., Frangos, J. A., Rhee, B. G., Eskin, S. G., and Hall, E. R. (1987). Ann. N.Y. Acad. Sci. 516, 513. McNeil, M., Darville, A. G., Frye, S . C., and Albersheim, P. (1984). Annu. Rev. Bjochem. 53, 625. McQueen, A., and Bailey, J. E. (1989). Biotechnol. Lett. 11, 531. McQueen, A., Meilhoc, E., and Bailey, J. E. (1987). Biotechnol. Lett. 9, 831. Meilhoc, E., Wittrup, K. D., and Bailey, J. E. (1989). J. Immunol. Methods 121,167. Mercer, D. (1981). Biotechnol. Bioeng. 23, 2421. Metzner, A. B., and Otto, R. E. (1957). AIChE J. 3, 3.

230

ALES PROKOP AND RAKESH K. BAlPAI

Metzner, A. B., and Taylor, J. S. (1960). AIChE 1.6, 109. Midler, M., and Finn, K. (1966). Biotechnol. Bioeng. 8, 71. Mizrahi, A. (1986). BiolTechnology 4, 123. Moo-Young, M., and Blanch, H. W. (1981). Adv. Biochem. Eng.lBiotechnol.19, 1. Morris, P., and Fowler, M. W. (1981). Plant Cell Organ Cult. 1, 15. Mugnier, J. (1988). Plant Cell Rep. 7, 9. Muirhead, K. A., and Horan, P. K. (1984). Adv. Cell Cult. 3, 57. Murhammer, D. W. (1989). Ph.D. Thesis, Univ. of Houston, Houston, Texas. Murhammer, D. W., and Coochee, C. F. (1990). Biotechnol. Prog. 6, 142. Nagata, S. (1975). “Mixing: Principles and Applications.” Wiley, New York. Napolitano, E. W., Pachter, J. S., and Liem, R. K. M. (1987). J. Biol. Chem. 262, 1493. Nevaril, C. G., Lynch, E. C., Alfrey, C. P., and Hellums, J. D. (1968). J. Lab. Clin. Med. 71, 784. Oh, S. K. W., Nienow, A. W., Al-Rubeai, M., and Emery, A. N. (1989).J. Biotechnol. 12,45. Okamoto, Y., Nishikawa, M., and Hashimoto, K. (1981). Int. Chem. Eng. 21, 88. Oldshue, J. Y. (1983). “Fluid Mixing Technology.” McGraw-Hill, New York. Olesen, S.-P., Clapham, D. E., and Davies, P. F. (1988). Nature (London) 331, 168. Pace, G. W., Grote, W., Pitt, D. E., and Pitt, J. M. (1986). Aust. Pat. wP86/01074. Packard, B. (1986). Trends Biochem. Sci. 11, 154. Papoutsakis, E. T., and Kunas, K. T. (1989). In ”Advances in Animal Cell Biology and Technology for Bioprocesses” (R. E. Spier, J. B. Griffiths,, J. Stephenne. and P. J. Crooy, eds.), p. 203. Butterworth, London. Papoutsakis, E. T., Peterson, J. F., and McIntire, L. V. (1991). Proc. Meet. Eur. SOC.Anim. Cell Technol. (ESACTJ, l o t h meeting. Passini, C. A,, and Goochee. C. F. (1989). Biotechnol. Prog. 5, 175. Pasternak, C., and Elson, E. L. (1985). J. Cell Biol. 100, 860. Petersen, J. F. (1989). Ph.D. Thesis, Rice University, Houston, Texas. Petersen, J. F., McIntire, L. V., and Papoutsakis. E. T. (1988). J. Biotechnol. 7,229. Pickard, B. G. (1984). Plant Cell Environ. 7, 171. Placek, J., and Tavlarides, L. L. (1985). AIChE J. 31, 1113. Prokop, A. (1991). In “Animal Cell Bioreactors” (C. S. Ho and D. 1. C. Wang, eds.), p. 21. Butterworth, Stoneham, Massachusetts. Prokop, A., and Rosenberg, M. Z. (1989). Biochem. Eng./Biotechnol. 39, 29. Rajasekhar, E. W., Edwards, M., Wilson, S. B., and Street, H. E. (1971).J. Exp. Bot. 22, 107. Randerson, D. H. (1985). J. Biotechnol. 2, 241. Reuss, M. (1988). Chem. Eng. Technol. 11, 178. Reuveny, S. (1983). Adv. Biotechnol. Processes 2, 1. Reynolds, A. J. (1971). “Thermofluid Dynamics.” Wiley, London. Robertson, B., and Ulbrecht, J. J. (1987). In “Biotechnology Processes. Scale-Up and Mixing” (C. S. Ho and I. Y. Oldshue, eds.), p. 31. Am. Inst. Chem. Eng., New York. Roels, J. A., van Den Berg, J., and Voncken, R. M. (1974). Biotechnol. Bioeng. 16, 181. Rogers, H. J. (1968). “Cell Walls and Membranes.” Spon, London. Rosenberg, M. Z . (1987). Ph.D. Thesis, Washington Univ., St. Louis, Missouri. Rupp, G. R., Tate, E., and Peterson, L. (1989). In “Advances in Animal Cell Biology and Technology for Bioprocesses” (R. E. Spier, J. B. Griffiths, J. Stephenne, and P. J. Crooy, eds.), p. 129. Butterworth, London. Rushton, J. H., and Oldshue, J. Y. (1953). Chem. Eng. Prog. 49, 161, 267. Ryan, C. A. (1988).Biochemistry 27, 8879. Sabersky, R. M., and Acosta, A. J. (1964). “Fluid Flow. A First Course in Fluid Mechanics.” Macmillan, New York.

SHEAR SENSITIVITY OF BIOCATALYSTS

231

Sahai, O., and Knuth, M. (1985). Biotechnol. Prog. 1, 1. Sallam, A. M., and Hwang, N. H. C. (1984). Biorheology 21, 783. Sato, M., Wong, T. Z., Brown, D. T., and Allen, R. D. (1984). Cell Motil. 4, 7. Sato, M., Schwarz, W. H., and Pollard, T. D. (1987). Nature [London] 325, 828. Schlichting, H. (1973). “Boundary Layer Theory,” 7th Ed. McGraw-Hill, New York. Schiigerl, K. (1982). Biochem. Eng.iBiotechno1. 22, 93. Schurch, U., Kramer, H., Einsele, A., Widmer, F., and Eppenberger, H. M. (1988). 1. Biotechnol. 7, 179. Shi, L. K., Riba, J. P., and Angelino, H. (1990). Chem. Eng. Commun. 89, 25. Shuler, M. L. (1981). Ann. N.Y. Acad. Sci. 369, 65. Skarlatos, S . I., and Hollins, T. M. (1987). Arteriosclerosis (DaIlasJ 64, 55. Smart, N. J., and Fowler, N. W. (1984). J. Exp. Bot. 35, 531. Smith, C. G., Greenfield, P. F., and Randerson, D. M. (1987). In “Modern Approaches to Animal Cell Technology” (R. E. Spier and J. B. Griffiths, eds.), p. 316. Butterworth, Stoneham, Massachusetts. Smith, C. M., Hebbel, R. P., Tukey, D. P., Clawson, C. C., White, J. G., and Vercellotti, G. M. (1987). Blood 69, 1631. Smith, J. J., Lilly, M. D., and Fox, R. I. (1990). Biotechnol. Bioeng. 35, 1101. Stafford, A., Morris, P. A., and Fowler, M. W. (1986). Enzyme Microb. Technol. 8, 578. Stathopoulos, N. A., and Hellums, J. D. (1985). Biotechnol. Bioeng. 27, 1021. Stewart, P. S., and Robertson, C. R. (1987). AiChE Summer Nat. Meet., Minneapolis, Minn. Stockbridge, L. L., and French, A. S. (1988). Biophys. J. 54, 187. Street, H. E., King, P. J., and Mansfield, K. (1971). Colloq. Int. C.N.R.S. No. 193, p. 17. Stumpf, P. K., Green, D. E., and Smith, F. W. (1946). J. Bacteriol. 51, 487. Sutera, S. P. (1977). Circ. Res. 41, 1. Sutera, S. P. (1978). J. Biomech. Eng. 100, 139. Sutera, S. P., Mehrjardi, M., and Mohandas, N. (1975). Blood Cells I, 369. Sutera, S. P., Tran-Son-Tay, R., Boylan, C. W., and Williamson, J. R. (1983). Blood Cells 9, 485.

Taguchi, H., Yoshida, T., Tomita, Y., and Teramoto, S. (1968).J. Ferment. Technol. 46,814. Tahir, S. K., Symington, A. L., and Zimmerman, A. M. (1988).Cell Biol. Int. Rep. 12,1005. Tal, B., Rokem, J. B., and Goldberg, I. (1983). Plant Cell Rep. 2, 219. Tanaka, H. (1981). Biotechnol. Bioeng. 23, 1203. Tanaka, H., Semba, H., Jitsufuchi, T., and Harada, H. (1988). Biotechnol. Lett. 10, 485. Tanaka, S., and Ueda, K. (1975). J. Ferment. Technol. 53, 27. Taya, M., Yoyama, A., Kondo, O., Kobayashi, T., and Matsui, C. (1989).J. Chern. Eng. Jpn. 22, 89.

Taylor, C., Hughes, T. G., and Morgan, K. (1980). In “Recent Advances in Numerical Methods in Fluids” (C. Taylor and K. Morgan eds.), p. 311. Pineridge Press, Swansea, England. Tennekes, H., and Lumley, J. L. (1972). “A First Course in Turbulence.” MIT Press, Cambridge, Massachusetts. Thomas, C. R., Nienow, A. W., and Dunnill, P. (1979). Biotechnol. Bioeng. 21, 263. Thomas, D. G. (1964). AIChE J. 10, 517. Tirrell. M., and Middleman, S. (1975). Biotechnol. Bioeng. 17, 299. Toni, D. T., and Bagster, D. F. (1978). Trans. Inst. Chem. Eng. 56, 1. Tramper, J., and Vlak, J. M. (1986). Ann. N.Y. Acad. Sci. 469, 279. Tramper, J., Williams, D. J., Joustra, D., and Vlak, J. M. (1986).Enzyme Microb. Technol. 8 , 33.

232

ALES PROKOP AND RAKESH K. BAJPAI

Tramper, J., Smit, D., Straatman, J., and Vlak, J. M. (1988). Bioprocess Eng. 3, 37. Truskey, G.A,, Gabler, R., DiLeo, A., and Manter, T. (1987). J. Parenter. Sci. Technol. 41, 180.

Tsien, R. Y., and Poenie, M. (1986). Trends Biochem. Sci. 11,450. Ujcova, E., Fencl, Z., Musilkovi, M., and Seichert, L. (1980). Biotechnol. Bioeng. 2 2 , 237. Valberg, P. A., and Albertini, D. F. (1985). J. Cell Biol. 101, 130. van Suijdam, J. C., and Metz, B. (1981). Biotechnol. Bioeng. 23, 111. van’t Riet, K., and Smith, J. M. (1973). Chem. Eng. Sci. 28, 1031. van’t Riet, K., and Smith, J. M. (1975). Chem. Eng. Sci. 30, 1093. van Wezel, A. L. (1967). Nature (London) 216, 64. Wagner, F., and Vogelmann, H. (1977). In “Plant Tissue Culture and Its Biotechnological Applications” (W. Barz, E. Reinhard, and M. H. Zenk, eds.), p. 245. Springer-Verlag, Ber I in. Walker, S. J., and Nevins, D. J. (1973). Am. 1. Bot. 60, 255. Wase, D. A. J., McManamey, W. J., Raymahasay, S., and Vaid, A. K. (1985). Biotechnol. Bioeng. 27, 1166. Watt, F. M. (1986). Trends Biochem. Sci. 11, 482. Waugh, R. E., and Hochmuth, R. M. (1987). In “Cytomechanics. The Mechanical Basis of Cell Form and Structure” (7. Bereiter-Hahn, 0. R. Anderson, and W. -E. Reif, eds.), p. 249. Springer-Verlag, Berlin. Wechezak, A. R., Wight, T. N., Viggers, R. F., and Sauvage, L. R. (1989). J. Cell. Physiol. 139, 136. Wichterle, K., Kadlec, M., Zak, S., and Mitschka, P. (1984). Chem. Eng. Commun. 26, 25. Williamson, R. E. (1986). Plant Physiol. 82, 631. Wu, J., King, G., Daugulis, A. J., Faulkner, P., Bone, D. H., and Goosen, M. F. A. (1989).Eur. J. Appl. Microbiol. Biotechnol. 32, 249. Yelton, D.E., and Scharff, M. D. (1981). Annu. Rev. Biochem. 50, 657. Yianneskis, M., Popiolek, Z., and Whitelaw, J. H. (1987). J. Fluid Mech. 175,537. Yoshizumi, M., Kurihara, H., Sugiuama, T., Takaku, F., Yanagisawa, M., Masaki, T., and Yazaki, Y. (1989). Biochem. Blophys. Res. Commun. 162,859.

Biopotentialities of the Basidiomacromycetes SOMASUNDARAM RAJARATHNAM, MYSORE NANJARAJURS SHASHIREKA, AND ZAKIABANO Fruit and Vegetable Technology Central Food Technological Research Institute Mysore 570 013, India

I. Introduction 11. Biology and Cultivated Species

A. Morphology and Life Cycle B. Species of Commercial Importance and Their Geographic Distribution C. World Production 111. Chemistry and Biomedicinal Values of Fruiting Bodies A. Chemical Composition B. Nutritional Value C. Biological/Medicinal Properties IV. Potential Lignocellulosic Substrates for Bioconversion A. Lignocehlosic Wastes B. Fermentation-Solid and Submerged States V. Biotransformation of Lignocehlosic Wastes A. Preparation of Substrates B. Cultural Conditions C. Biomass Conversion Efficiencies VI. Changes i n the Growth Substrates during Degradation A. Chemical B. Biochemical VII. Applications and Implications of Spent Substrate A. As an Upgraded Ruminant Feed B. Biogas Production C. CardboardiPaper Manufacture and Basidiomacromycetes as Pulping Agents D. Deodorization of Waste Gases E. Source of Saccharification Enzymes F. Recycling for Mushroom Culturing G. Production of Native Silica H. As Soil Ameliorant I. Single-Cell Protein Production J. Antiviral Activity of Water-Solubilized Lignin VIII. Applications of Functions of Fruiting Bodies/Mycehm A. Oxidation of Environmental Pollutants B. Peroxidase Production C. Indicators of Heavy Metal Accumulation D. Decolorization of Spent Kraft Liquor and Molasses Pigment E. Pyranose Oxidase Production F. Polymer Production G . Tenderization of Mushroom Stipes H. Retting of Flax Fibers 233 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 3 7 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

234

SOMASUNDARAM RAJARATHNAM ET AL. I. Hair Growth Stimulant J. Low-Alcohol Wines K. Use as Fungal Elicitors L. Use as Test Organisms M. Metabolic Substances N. Biodegradation of Degradable Plastic Polyethylene IX. Conclusions References

I. Introduction

Basidiomacromycetes represent a circumscribed group of higher fungi of the class Basidiomycetes, wherein the basidia bearing the exogenously produced basidiospores represent the fertile layer (hymenium), which in turn is lined up, encroached, and covered by tufts of tissues to form distinct objects of the Earth’s beauty, namely the basidiocarps. As many as 2000 species are widely used for human consumption (Chang and Miles, 1982), but only a few of them are used commercially; some are inedible and a few are toxic. In most cases, the basidiocarps are highly conspicuous. They are distributed all over the world, over a range of temperate to tropical climates; some are endemic to certain areas, whereas others are cosmopolitan (of course, during a particular season of a year). Their life cycle is elaborate and quite complicated, with a vast potential for diversification of the genome, which aids in generating the variants, resulting from their high chances for variability, adaptability, selectivity, and survivability. These fungi, during their growth (spawn run) and fructification (phase of formation of basidiocarps), have been demonstrated to display a wide range of chemical and biochemical, biodegradative, and biosynthetic reactions, a composite of anabolic and catabolic processes. They have a unique capacity to grow on a wide spectrum of lignocellulosic wastes, some peculiar to brown-rot species and others to the white-rot type, all, however, being cellulolytic. They exert a positive influence on the gradual clearance of accumulating inedible plant materials left unutilized on the earth. The edible species represent classic examples, which, during their growth, biodegrade the substrate to varied degrees, leaving behind some of the degraded substrate with increased solubility, with the rest being lost as CO, and H,O during fungal metabolism. The production of CO, and H,O can be as high as 70% of the dry organic matter of the substrate (Zadrazil, 1978a) and thus, these fungi play an invaluable role in the carbon cycle on the surface of our planet. Their efficiency in converting substrate to protein is far superior to that of several plant sources and even animal sources of protein (Table I). Their valuable capability to convert plant wastes

TABLE I PRODUCTION OF CRUDEPROTEIN AND GROSSENERGY BY CROPSAND ANIMALSCOMPARED WITH

Species Grass [perennial) Cabbage Peas Potatoes (main crop] Wheat [winter) Maize Rice Cattle, beef (sucker) Cattle [dairy) Pigs [bacon) Hen

Pleurotus sajor-caju Pleurotus flabellatus Agaricus bisporus Volvariella volvacea (on straw) Volvariella volvacea (on cotton waste)

Yield component THd TH Seed Tuber Seed Seed Seed Carcass Milk Carcass Eggs (edible portion) TH TH TH TH TH

“From Rajarathnam and Zakia Ban0 (1990) “Fresh weight. cDM, Dry matter. dTH, Total harvested.

THE

EDIBLEBASIDIOMACROMYCETES~

Yield DM (kgW

Crude protein (%)

Gross energy (MJW

Crude protein (kgW

Gross energy (MJW

10.0 11.0 86.0 21.0 86.0 86.0 86.0

12,000 6,000 2,159 5,800 3,779 3,995 4,876 360 3,386 875 624

17.5 13.6 26.1 9.0 12.4 9.8 7.7 14.8 3.5 12.0 11.9

18.5 17.5 18.9 17.6 18.4 19.0 18.0 10.9 2.6 16.5 6.6

2,100 316 566 52.5 469 392 375 53 118.5 105 74

222,000 105,000 40,805 102,080 69,534 75,905 87,768 3,924 8,770 14,438 4,118

248,888 133,333 189,000 10.864

10 10 10 10

24,888 13,333 18,900 1.086

22.5 21.6 26.3 22.5

12.5 11.3 13.7 12.7

5,599 2,880 4,970 244

312,344 151,196 259,308 13.792

22,222

10

2,222

22.5

12.7

500

28,219

Yieldb (ks)

DMc

60,000 54,545 2,511 27,621 4,394 4,654 5,670

(%1

236

SOMASUNDARAM RAJARATHNAM ET AL.

into food, the quality and biological properties of the formed food, and the wide range of biochemical properties displayed by the mycelium and the basidiocarp are discussed here. Also, a bird’s-eye view of their biology and the range of potential substrates that can be utilized as a result of the exploitation of these fungi for the advantage of mankind is presented. II. Biology and Cultivated Species

A. MORPHOLOGY AND LIFE CYCLE

The basidiomacromycetes represent the highest evolved group of the fungi. These fungi have two distinct phases in their life cycle: the vegetative phase, represented by the mycelium, and the reproductive phase, represented by the fruiting bodies (basidiocarps). In general, the former is long-lived, hidden in the growth substrate, and not highly conspicuous. The latter present great diversity in shape, size, color, texture, structure, and flavor. A typical basidiocarp has a cap and a stalk (stipe), resulting from a thick intertwining of the mycelium. The stipe may have a ring at its distal end called the annulus, as present in Agaricus, or a basal cup termed the volva, as in the case of Volvariella, or both of these structures may be absent, as in Pleurotus. Both the annulus and the volva are characteristically found in species of Amanita, the well-known poisonous mushrooms. Smith (1978) has described the morphological features and structural details of these fruiting fungi. The stages proceeding from spore to spore, the “life cycle,” are elaborate in most of the species, involving diverse nuclear segregation and aggregation, with the objective of favoring outbreeding, resulting in the generation of innumerable variants and offering greater chances for natural selection and successful survival. Raper (1978) has described nine steps constituting a typical life cycle (Fig. 1):(A) Germination of a basidiospore initiates development of (B) a haploid homokaryotic mycelium capable of indefinite, independent propagation. The mycelium may or may not go through an asexual cycle via the production of oidia or chlamydospores. Mating between two compatible homokaryotic mycelia through hyphal fusion (C) (plasmogamy) establishes the fertile mycelium, the dikaryon (D), that represents the heterokaryotic phase with conjugate divisions. The dikaryon is capable of independent and indefinite propagation and may or may not go through an asexual cycle via the production of oidia or chlamydospores. If asexual spores are produced and are uninucleate, homokaryotic mycelia of the parental

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

237

E

C

I FIG. 1. Diagrammatic representation of typical life cycle of a basidiomacromycete. A, Basidiospore germination; B, haploid homokaryotic mycelium; C, plasmogamy; D, heterokaryon; E, fruiting body; F, basidia formation; G, karyogamy; H, meiosis and formation of basidiospores; I, basidiospores.

types are regenerated; if the asexual spores are binucleate, the dikaryon is regenerated. Under suitable environmental conditions, the dikaryon produces (E) the fruiting body as an outgrowth of specialized tissue. (F) The spore-bearing tissue of the fruit body develops as a columnar layer of club-shaped, binucleate cells termed the basidia. (G) Fusion of the paired nuclei of the two parent mating types, karyogamy, establishes the diploid nucleus in a single-cell stage. (H) Meiosis, which follows immediately, involves recombination and segregation of the genetic material (of the mated parents), ultimately resulting in the production of four haploid nuclei, each of which on movement to the tip of a stalk-

238

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like structure, the sterigma, on the basidium, results in the formation of a basidiospore. Typically, four uninucleate spores are formed on each basidium. (I) The spores are discharged and undergo mitotic divisions that lead to basidiospore germination, the point at which the life cycle is reinitiated. A great amount of research interest is vested in the study of the patterns of sexuality of the basidiomacromycetes. This study is of paramount importance while exploiting the edible species for the production of flavored fruiting bodies for human consumption. The sexuality can be as simple as primary homothallism, as in Volvariella volvacea; secondarily homothallic, as in Agaricus bisporus; heterothallic and bipolar, as in Agaricus bitorquis, Pholiota nameko, and Auricularia auricula; or heterothallic and tetrapolar as in Auricularia polytricha, Lentinus edodes, Flammulina velutipes, Pleurotus ostreatus, Pleurotus flabellatus, and Coprinus fimetarius (Raper, 1978). Ultrastructural changes involved during the morphogenesis of fruiting bodies in species of Agaricus are described by Angeli-Papa and Eyme (1978). B. SPECIESOF COMMERCIAL IMPORTANCEAND THEIR GEOGRAPHIC DISTRIBUTION Basidiomacromycetes are found in most parts of the world and temperature has been the main factor controlling their distribution. It is well known that species of Agaricus, Lentinus, and Flammulina are temperate and Volvariella species are tropical; populations of Pleurotus are characteristically subtropical (Chang and Hayes, 1978). However, strains and species of the these genera have been found in other zones. The reason for this ultimately rests on outbreeding, generation of variants and their acclimatization through varied ecological adaptations to differences in climatic conditions. The distribution of some of the prominent and prevalent taxa is briefly considered here. 1. Agaricus

Agaricus is the oldest of the basidiomacromycetes cultured for human consumption. It is variously termed the Button or white or European mushroom. Agaricus bisporus is temperate, occurring in many parts of Europe, the United Kingdom, and the United States. Agaricus bitorquis is a strain that was introduced to suit growth conditions in subtropical climates. These species are humicolous, found characteristically growing on dead and decayed debris of plant wastes and soil.

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239

2. Lentinus

Lentinus edodes, commonly termed the Japanese mushroom or shiitake, is the second most prominently cultivated fungus in the world. In nature, Lentinus edodes has been reported to grow on broad-leaved trees, mainly on the members of Fagaceae, and is found in Japan, Taiwan, China, Indochina, Borneo, Wilhelm, and New Guinea (Tokimoto and Komatsu, 1978). 3. Volvariella

Volvariella volvacea, popularly known as the Chinese or paddy straw mushroom, like Agaricus bisporus and Lentinus edodes has been regarded as a high-price delicacy for many centuries in China and other Asian countries. This fungus characteristically grows on rice straw and is cultured in many parts of the world, including Korea, Japan, the Phillipines, Indonesia, Singapore, Malaysia, Thailand, India, Madagaskar, Nigeria, and in a few parts of the United States and Europe (Chang, 19 7 8a). 4. Flammulina

Flammulina velutipes, because of its property of fructification during winter, is commonly known as the winter mushroom. It occurs all over the world, in places such as China, Siberia, Asia Minor, Europe, Africa, North America, Australia, and Japan. It is one of the wooddestroying fungi found growing on the trunks or stumps of aspens, willows, elms, and other broad-leaved trees from the end of autumn to early spring. Although this species has been gathered in the wild and used as food in Japan for many centuries, it is now cultured on artificial media there, and such techniques are now becoming popular in Taiwan and parts of Europe (Tonomura, 1978; Wood and Smith, 1989). 5 . Pleurotus

Species of Pleurotus are popularly called Oyster mushrooms because of the characteristic nature of their fruiting bodies, with an eccentric stalk attached to the pileus, which opens up like an oyster shell during morphogenesis. The species enjoy a worldwide distribution in nature. Pleurotus ostreatus, a wood-destroying, saprophytic fungus, sometimes appearing as a parasite, is widespread in the temperate zone. Block et al. (1958) in the United States have reported on P. ostreatus. Pleurotus ostreatus var. Florida is a subtropical variety found in Florida. Pleurotus eryngii is a typical fungus of the flora of the subtropics and steppes. It is widespread in Southern Europe and areas of Central Asia

240

SOMASUNDARAM RAJARATHNAM ET AL.

and North Africa. Pleurotus sajor-caju has been isolated in the foothills of the Himalayas (India) and is found growing as a saprophyte on the stumps of Euphorbia reyeleana. Pleurotus flabellatus is a white, attractive oyster mushroom reported by Zakia Ban0 (1967) from India. Pleurotus abalone is another saprophytic fungus, cultivated in the Republic of China (Han et al., 1977). Zadrazil (1978a) and Rajarathnam and Zakia Ban0 (1987a) have detailed many more aspects of Pleurotus. 6. Pholiota

Pholiota nameko grows on dead trunks or stumps of deciduous trees, especially Fagus crenata, Fagus japonica, and Quercus magnolica. The fungus, thus far, has been collected only in Japan: however, its natural distribution is also recorded in Taiwan (Arita, 1978). 7. Auricularia

Auricularia has a worldwide distribution in temperate to tropical regions. Auricularia emeni is found in Africa; A. delicata and A. tenuis also grow only in the tropics. A. mesenterica, A. ornata, and A. polytricha occur in both tropical and subtropical regions. Auricularia auricula is a temperate species and is only occasionally found in the subtropics. This species commonly occurs on dead wood, especially on oak trunks (Cheng and Tu, 1978). 8. Tremella Tremella fuciformis, the white jelly fungus, also called “Silver ear” by the Chinese, has significant medicinal value in the curing of several kinds of diseases, including tuberculosis, hypertension, and the common cold. It is reported to improve vigor and to extend the human life span. Furthermore, it provides nutrients to the skin and can improve personal appearance. It is a subtropical fungus that appears during the rainy season growing on dead trunks or branches of hard woods. Tremella fuciformis is reported to be distributed in Argentina, Australia, Chile, China, India, Japan, Java, Mexico, and the United States (Chen and Hou, 1978). 9. Stropharia Stropharia rugoso-annulata is found in Berlin, Poland, Czechoslovakia, Hungary, and the Soviet Union. It can be grown on straw with simple, inexpensive procedures (Szudyga, 1978). 10. Tricholoma Tricholoma matsutake grows chiefly in Japan, on coniferous woods, and has been considered an autumn delicacy since ancient times. This

BIOPOTENTIALITIESOF BASIDIOMACROMYCETES

241

fungus develops fruiting bodies and forms mycorrhiza in the roots of Pinus and Tsuga. This fungus is also found in North America, Canada, Algeria, and Czechoslovakia (Tominaga, 1978). 11. Coprinus

Coprinus comatus, commonly termed “shaggy mane” is cultured for its fleshy fruiting bodies. A temperature range of 35-40°C is preferred by the fungus. Many species of Coprinus occur as weed molds during the cultivation of Pleurotus (Kurtzman, 1978a). C. WORLDPRODUCTION The various edible fruiting bodies known for their characteristic flavor and biting texture, in addition to their biological properties (to be described in a later section), command good prices in the world market. They have been suggested as a supplementary food source for the growing populations in the developing countries (Hayes, 1975). In looking at world production and percentage contribution by each type of mushroom (Table 11), it is evident that Agaricus, Lentinus, Volvariella, and Pleurotus are the dominant taxa cultured for human consumption (Brien, 1989). The United States leads the world, having produced 285,000 tons (23.2%) of the total Agaricus marketed in 1986 (Chang, TABLE I1 WORLD PRODUCTION OF EDIBLE BASIDIOMACROMYCETES AND SUBSTRATES UTILIZEDQ ~~

~

Production ( x 1000 tons)

As % of world production

1227

56.2

Composted straw

314

14.4

Volvariella volvacea

178

8.2

Pleurotus spp. Auricularia sp.

169 119

5.5

Flammulina velutipes Tremella fuciformis Pholiota nameko

100 40

4.6

25

1.1

Wood logs or sterilized sawdustlbran Rice straw or composted cotton waste Range of plant wastes Wood logs, sterilized sawdustlbran Sterilized sawdustlbran Wood logs Wood logs or sterilized sawdustlbran

10 2182

0.5 100.0

Mushroom Agaricus bisporusl A. bitorquis Lentinus edodes

Other species Total

aFrom Zakia Bano and Rajarathnam (19881.

7.7

1.8

Substrate

242

SOMASUNDARAM RAJARATHNAM ET AL.

1987). With regard to the increase of mushroom production from 1980 to 1985, Agaricus, Lentinus, and Pleurotus top the list, with major increases noted in Ireland, Mainland China, Holland, Spain, and Great Britain. Delcaire (1978) has reported in detail the economics of cultivated mushrooms. Brien (1989) has reviewed mushroom marketing worldwide. About 5 million kilograms of species of Pleurotus have been produced in Europe since 1973 (Heltay, 1978), indicating industrial use. Species of Agaricus, Pleurotus, Lentinus, Auricularia, Flammulina, and Tremella are cultivated on a commercial scale in Thailand (Natalaya, 1978). 111. Chemistry and Biomedicinal Values of Fruiting Bodies

A. CHEMICAL COMPOSITION

The data published in food composition tables are often presented in different units of measurement based on the highly variable fresh or dry weight, making the comparison of such data difficult. In order to ensure a universal comparison of values among different species, it would be appropriate to express all of the analytical data on a moisture-free basis. Significant composition changes occur with a strain as a result of differences in age or the particular stage of development, lapse of time after harvest, study of different portions of a single fruiting body, the inaccuracies inherent in the methods of analysis, and the relative precision of the analyst (Crisan and Sands, 1978). The chemical nature of the substrate influences the chemical composition of fruiting bodies (Zadrazil, 1980a; Rajarathnam et al., 1986). Protein and amino acid contents in cultivated mushrooms are approximately two times higher than those in wild ones (Gotoh, et al., 1985). In general, fruiting bodies, on a dry weight basis, contain about 39-88% carbohydrates, 17-46% proteins, 2-9% fat, with the rest as ash, constituting the minerals (Table 111). Crisan and Sands (1978), Chang (1978a), and Zakia Ban0 and Rajarathnam (1982b, 1988) have reviewed the chemistry and food value of fruiting bodies. Mannitol and trehalose represent the bulk of the free sugars (Rajarathnarn, 1981), while most of the carbohydrates are in the polymeric form, with starch as such being absent, presenting, therefore, a food item suitable for diabetics. Protein is the constituent next in concentration after carbohydrates. Because of the presence of nonprotein nitrogen, represented by chitin, and further, because of the decreased digestibility of crude mushroom protein, the use of a conversion factor equal to 70% N x 6.25, i.e., N x 4.38, has been suggested. The result is a closer approximation of mushroom protein content. This

243

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES TABLE 111

APPROXIMATE COMPOSITIONO OF FRUITING BODIESOF BASIDIOMACROMYCETES~

Species Agaricus bisporus Auricularia auriculajudae Auricularia polytricha Boletus edulis Cantharellus cibarius Collybia albuminosa Coprinus cornafus Flammulina velutipes Lentinus edodes Lycoperdon lilacinurn Pholiota nameko Pleurotus florida Pleurotus eous Pleurotus Jimpedus Termitomyces microcarpus Tricholoma spp. Volvariella esculenta

Crude protein (N x 4.38) 26.3 8.1 7.7 29.7 21.5 26.6 25.4 17.6 17.5 46.0 20.8 18.9 17.5 38.7 27.4 16.7

34.4

Fat 1.8 1.5 0.8 3.1 5.0 4.0 3.3 1.9 8.0 7.5 4.2 1.7 1.0

9.4 4.3 3.1 20.6

Carbohydrate

Fiber

Ash

Energy value (kcals)

59.9 81.0 87.6 59.7 64.9 67.5

10.4 6.9 14.5 8.0 11.2 8.1 7.3 3.7 8.0 12.3 6.3 11.5 12.0 27.6 2.2 12.9 11.2

12.0 9.4 3.9 7.5 8.6 7.0 12.5 7.4 7.0 7.7 8.3 9.3 9.1 5.3 14.1 8.3 13.3

328 356 347 362 353 365 366 378 387 358 372 265 261 313 364 342 396

58.8

73.1 67.5 38.8 66.7 58.0 59.2 46.6 54.2 71.9 31.7

OOn dry weight basis. bAfter Crisan and Sands (1978) and Zakia Bano and Rajarathnam (1988)

factor has been adopted for mushrooms in several food composition tables (FAO, 1970; FAO/WHO, 1972; Leung et al., 1972). Zakia Ban0 et al. (1978) and Rajarathnam et al. (1986) have found that Pleurotus flabellatus grown on rice straw supplemented with groundnut cake, sesame cake, cottonseed powder, and yeast cake after the spawn run resulted in an increased protein content of the fruiting bodies compared to those of the unsupplemented control. There was a twofold increase in the protein content of the fruiting bodies grown on cottonseed-supplemented straw substrate. Similar results have also been observed with Pleurotus sajor-caju (Zakia Ban0 and Rajarathnam, 1982a). Zadrazil (1980a) observed a similar increase in the protein content of the fruiting bodies of P. SQjOr-CajLI,when the growth substrate was supplemented with alfalfa and soybean meal. Albumins and globulins predominate in the protein fraction of Boletus edulis and Cantharellus cibarius (Dzamic and Miljkovic, 1984). It has been claimed that nearly one-fifth of the total nitrogen is in the form of free amino acids, predominantly alanine, glutamic acid, and glutamine (Sato et al., 1985). Of the total amino acid content, 25-40% comprises essential amino acids

244

SOMASUNDARAM RAJARATHNAM ET AL.

(Maggioni and Renosto, 1970; Le Roux and Danglot, 1972). The amino acid content-bound and free-has been reported by a number of workers (Doyle and Levenberg, 1974; Sugahara et al., 1975; Abe et al., 1982; T. Fujita, 1990). Mushroom protein, unlike animal protein, is incomplete, lacking some of the essential sulfur-containing and aromatic amino acids (Crisan and Sands, 1978). The fat content includes representatives of all classes of lipid compounds, including free fatty acids; mono-, di-, and triglycerides; sterols; sterol esters; and phospholipids. A large variety of free and combined fatty acids occur, with especially high concentrations of palmatic, stearic, oleic, and linoleic acids (Hughes, 1962; Holtz and Schister, 1971). Linoleic acid is especially abundant (Khanna and Garcha, 1983; Hadar and Cohen-Arazi, 1986), comprising approximately 70% of the fatty acid content of the neutral lipid fraction and 90% of the polar lipid fraction (Holtz and Schister, 1971). The fruiting bodies generally contain about 10% ash, on a dry weight basis, which comprises mainly potassium, phosphorus, and magnesium; the presence of ferrous iron has also been established (Memuna and Chakrabarti, 1982; Ikari et al., 1985). Organic acids such as succinic, malic, fumaric, citric, a-ketoglutaric, oxalic, lactic, acetic, and formic acids are contained in the fruiting bodies as taste components [Yoshida et al., 1983; J. Fujita et al., 1990). Thiamine, niacin, riboflavin, and folic acid represent the major vitamins contributed by the mushrooms (Crisan and Sands, 1978; Zakia Ban0 and Rajarathnam, 1988). About 70% of the total sterols has been reported to be ergosterol. Invariably, ribonucleic acid is found to be the predominant nucleic acid [Khanna and Garcha, 1986). Compared to the nucleic acid content of algae (Viikari and Linko, 1977), yeast, and other microbes (Kihlberg, 1972),the nucleic acid content of Pleurotus mushrooms is quite low (Zakia Ban0 and Rajarathnam, 1988): mushroom nucleic acid content is about one-third to one-fourth that of other microbes. On the basis of PAG recommendations and the findings of the First International Conference on single-cell proteins held at the Massachusetts Institute of Technology in 1967 (Scrimshaw, 1975), consumption of Pleurotus-type mushrooms should not cause gout or gouty arthritis, which results from consumption of food microbes high in nucleic acid content. Flavor is the most important inducement for past and present widespread consumption of wild and commercially grown edible mushrooms. Maga (1976) has reviewed the subject of volatile fractions of mushroom flavor. As many as 150 volatile compounds have been identified in various mushroom species (Pyysalo, 1978). A series of eight-

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

245

carbon (C,) compounds are believed to be the most important volatile flavor compounds. It has been demonstrated that some of the C, and C,, compounds can be enzymatically formed from linoleic and linolenic acids, both of which are usually predominant in fruiting bodies (Tress1 et al., 1982). The preponderance of 1-Octen-3-01in fresh fruiting bodies of Cantharellus cibarius, Coprinus atramentarius, and Leucocoprinus elaeidis has been investigated. 1-Octen-3-one has the highest aroma value in fresh fruiting bodies of Psalliota bispora (Pyysalo, 1978). Threshold values of flavors and their characteristics are described in Table IV. A difference in the relative quantities of the volatile components of Pleurotus flabellatus fruiting bodies effected through the supplementation of the rice straw substrate with cottonseed powder has been investigated (Rajarathnam et al., 1990). Of the several classes of flavor components identified in the volatile extracts of Agrocybe aegerita, 1-octen-3-01 is found to be the main component (Takama et al., 1978). The aromatic substances formed by the basidiomacromycetes have been discussed by others (Drawert et a1., 1983; Komai et al., 1986; Iwade, 1987). Thus, the edible basidiomacromycetes contain a range and variety of chemical constituents. The presence of these chemical constituents in the fruiting bodies despite their absence, as such, in the growth substrates results from the multifarious biosynthetic capacities of the basidiomacromycetes. The relatively long duration of the vegetative TABLE IV

THRESHOLD VALUES AND ODORCHARACTERIZATION OF THE MAIN VOLATILES IN FRESH FRUITINGBODIES"

Compound

Threshold value (PPml

Characterization ~

1-Octen-3-01 1-Octen-%one

0.010

trans-2-Octen-1-01 trans-2-Octenol 3-Octanol 3-Octanone Octanol 1-Octen-3-yl acetate 1-Octen-3-yl propionate Nonanol

0.040

OAdapted from Pyysalo (1978).

0.004

0.003 0.018 0.050 0.480 0.090

0.022 0.090

~~

~

~

Mushroom-like Like boiled mushroom, metallic at higher concentrations A little medicine-like, oily sweet Sweet, phenolic Like cod-liver oil Sweet, fruity, musty Detergent or soapy Mushroom-like, soapy Rich odor, sweet, fruity, mushroom-like Detergent or soapy, sweet

246

SOMASUNDARAM RAJARATHNAM ET AL.

phase as represented by the mycelium and the shorter phase of fructification (lasting a few hours to 2-3 days), taking into account the biomass output per unit time, lead to the conclusion that the metabolic turnover is significantly higher during fructification. The fruiting bodies are directly palatable, appreciated for their biting texture and pleasant flavor-all of which are again a result of their biosynthetic potentialities. Their carbohydrate composition, free and bound amino acid pools, relative preponderance of unsaturated fatty acids, and low nucleic acid content obviously place these fungi in the uppermost rank of unconventional sources of single-cell proteins (Zakia Ban0 and Rajarathnam, 1988). B. NUTRITIONALVALUE

The information available on the nutritive value of the edible fruiting fungi is quite meager. Investigations carried out by Quackenbush et al. (1935), Lintzel (1943), and Fitzpatrick et al. (1946) contain information confined to Agaricus bisporus and Agaricus campestris. Very little information on the feeding of edible species of mushrooms to animals is available. Samajpati (1979) observed an increase in body weight of experimental mice supplied with a diet containing mycelia of Pleurotus sajor-caju and Pleurotus ostreatus during feeding trial experiments. No morphological or histopathological abnormalities were found in any of the experimental mice at the end of the experimental period. Other animal feeding studies were carried out for Pleurotus sajor-caju where 84.1% digestibility, 89.4% biological value, and 75.1% net protein utilization were reported (Thayumanavan and Manickam, 1980).In the absence of animal feeding studies, several other methods have been used for determining or predicting the nutritional value of foods based on their essential amino acid content (EAA) (FAO, 1970; FAO/WHO, 1973). Chemical score or amino acid score (FAO, 1968),essential amino acid index (FAOIWHO, 1965; Oser, 1959), and nutritional index (NI) (Crisan and Sands, 1978) have also been used as the criteria for evaluating the nutritional status of edible mushrooms (Table V). According to Crisan and Sands (1978), the most nutritive mushrooms, which have a high EAA index and amino acid score, are almost equal in nutritional value to meats and milk, whereas the mushrooms showing the least nutritive value, rank considerably lower but are comparable to some vegetables such as carrots and tomatoes. When the NI of the mushrooms is taken into consideration, they rank above all vegetables and legumes except soybeans. Hence, it is very difficult to make a general statement about the nutritive value of mushrooms. Some spe-

247

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES TABLE V

ESTIMATED NUTRITIVE VALUEOF EDIBLEBASIDIOMACROMYCETES~

Species

Essential amino acid index

Biological value

Nutritional index

Amino acid scoreb

Agaricus bisporus Boletus edulis Cantharellus cibarius Lentinus edodes Pholiota mutabilis Pleurotus eous Pleurotus florida Pleurotus flabellatus Pleurotus sajor-caju Pleurotus ostreatus Russula vesca Termitomyces microcoirpus Volvariella displasia

55.8 76.6 86.2 55.8 85.5 95.7 84.5 82.7 70.9 64.8 88.9 74.7 87.9

49.1 71.8 82.3 49.1 81.5 92.7 80.4 78.4 59.2 58.9 85.2 69.7 84.1

17.0 9.3 3.0 9.8

36 37 68 40 67 59 67 47 67 40 70 45 71

NAG 16.7 15.9 17.8 14.4 13.6 6.0 20.5 25.1

OAfter Crisan and Sands (1978). busing egg as reference protein. cNA. Not available.

cies of Pleurotus eous provide nutritive value comparable to that of meats and milk, but Pleurotus ostreatus, due to low protein content and deficiency in some essential amino acids, has a low nutritive value (Zakia Ban0 and Rajarathnam, 1982b).Origins and utilizations of several newhnconventional protein sources, including mushrooms, are listed in Table VI. In view of the high cost and low efficiency of some forms of meat production, future diets are likely to emphasize the vegetarian, thus giving mushrooms a special status as a useful food in the future. In view of the potential of mushroom production in low-income countries, increased world production is inevitable. In order to sustain the progress of mushroom cultivation, the consumption of fresh mushrooms should be encouraged, its value as a food should be emphasized, new outlets should be sought, production costs should be lowered, and a greater degree of international collaboration, in both research and marketing, should be encouraged (Hayes, 1975). C. BIOLOGICAL/MEDICINAL PROPERTIES

Edible mushrooms are known to display several interesting biological properties. They have a notable place in folklore throughout the

248

SOMASUNDARAM RAJARATHNAM ET AL TABLE VI ORIGINSAND UTILIZATION OF SOME NEWSOURCES OF PROTEINS~ Protein source

Comments ~~

Spirulinab

Chlorellab

Fish protein concentrate

Whey proteins Proteins from the wastes of paper industry Single-cell proteins Synthetic amino acids Mushrooms

A microscopic alga produced in Mexico and eaten by certain ethnic groups: also harvested naturally and eaten in Central Africa This alga is produced in Taiwan and consumed by humans in the United States in the form of dietetic preparations Mainly manufactured in Sweden, which supplies to developing countries through world organizations, e.g., WHO Principally used for animal nutrition: about one-third enters into the composition of biscuits and baby food Bisulfite liquors are the raw materials used mainly by the sauce-making industry in the United States Grown on ethanolic substrates obtained from maize: they enter into the composition of sauce Mainly as lysine: consumed in Japan, particularly in some school canteens Cultured for edible fruit bodies of pleasant flavor: economic means of help of disposing of natural lignocellulosic wastes; the fruit bodies are directly acceptable/ palatable

oFrom Zakia Bano and Rajarathnam (1988).

bBoth of these algae are mainly (-80%) used in aquaculture for growing fish.

world, and the traditions of many cultures, past and present, describe medical effects of variously employed fungi (Lucas, 1959). Medical effects of mushrooms, although unexploited, are not unexplored (Shivrina, 1965). Benedict and Brady (1972) have indicated that polyacetylenes appear frequently among compounds characteristically occurring in Aleurodiscus, Clitocybe, Daedalea, Marasmius, Merulius, Pleurotus, Polyporus, Poria, Psathyrella, and Tricholoma. Bohus et al. (1961) found a high prevalence of antibacterial activity in higher fungi tested against penicillin-resistant bacteria. Pleurotin, a polycyclic compound isolated from Pleurotus griseus, is reported to display antibiotic properties (Grandjean and Huls, 1974). Examples of mushrooms with antifungal activity include Lentinus edodes (Herrman, 19621, Coprinus comatus (Bohus et a]., 1961), and Oudemansiella mucida (Musilek et al., 1969). 6-Deoxyilludin M, a new antitumor antibiotic obtained from the fermentation cultures of Pleurotus japonicus, is markedly active against murine leukemia (Hara et al., 1987). Gregory et al. (1966) screened more than 7000 cultures of basidio-

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

249

mycetes for antitumor activity in rodent tumor systems. A slow-growing, spontaneous mammary tumor in Ps strain mice was inhibited by the culture filtrate of Hohenbuehelia geogenius (Riondel et al., 1981). Antitumor polysaccharides have been reported from CorioJus consors, Coriolus versiocolor, Corticium centrifugum, Crepidotus sp., Flammulina velutipes, Ganoderma applantatum, Phellinus Jinteus, Pholiota nameko, Pleurotus ostreatus, Poria cocos, Schizophyllum commune, and Tricholoma aggregatum (Ikekawa et al., 1982). In fact, U.S. patents have been issued to Komatsu et aJ. (1973) and Ohtsuka et aJ. (1977) for their processes for production of antitumor polysaccharides from members of the basidiomycetes. Lentinan, extracted from Lentinus edodes, shows significant inhibition of the growth of subcutaneously implanted Sarcoma 180 in laboratory animals (Kirin Brewery Co. Ltd., 1980). Schizophyllan and its ultrasonicate derivative Neoschizophyllan, obtained from Schizophyllum commune, are effective against a range of tumors such as M-32, Ehrlich carcinoma, and Yoshida sarcoma, but are inactive against ascitic tumors (Komatsu et al., 1969); neoschizophyllan is absorbed into blood and tissues more rapidly than schizophyllan (Kikumoto et al., 1978). An acidic polysaccharide fraction obtained from Pleurotus ostreatus displays antitumor activity (Yoshioka et al., 1975). Most of these antitumor polysaccharides are glucans composed of glucose molecules with p-~-(1,3) and P-D-(1,6) linkages. The subject of carcinostatic activity of the mushrooms is reviewed by Mouri (1976), Cochran (1978), Mori et al. (1987), and Jong and Donovick (1989). Kajuno et al. (1991) have investigated the structural features of alkali-soluble polysaccharides from the fruiting bodies of Lyophyllum ulmarium; the two glucans showed the pdominant linkages with varying degrees of branching. The biological activities of polysaccharides from Auricularia auricula and TremeJla fusiformis on the biosynthesis of lymphocytic DNA/RNA, as well as their antiulcer effects, have been reported (Xia et aJ., 1987; Xua et al., 1987). Species of Boletus, Clavaria, Lactarius, Russula, Pholiota, and FJammuJina have been demonstrated to possess lectin properties causing hemagglutination of erythrocytes (Chung et al., 1987). Two crystalline forms of lectins from Flamrnulina velutipes have been reported (Hirano et al., 1987). Lentinus edodes and Grifola frondosa decreased hypertension and plasma cholesterol in rats (Kabir et aJ., 1987). Kaneda and co-workers (1964; Kaneda and Tokuda, 1966) observed a lowering of plasma cholesterol from 24 to 45% diet fed to rats was supplemented with 5% ground, dried fruit bodies of Lentinus edodes. A hypocholesterolemic property of mushrooms is reported by others (Arakawa et al., 1977; B. Huang, 1982; Tam et al., 1986). An

250

SOMASUNDARAM RAJARATHNAM ET AL.

antileukosis substance has been isolated from Coprinus radiatus (Anisova et al., 1987). Dihydrofolate synthetase activity, involved in the synthesis of folic acid, has been reported in species of Pleurotus, Lentinus, Flammulina, and Tricholoma (Iwai et al., 1977). Species of Pleurotus, because of their high content of dietary fiber, find a place in diet therapies for hyperlipemia, diabetes, etc. (Kurosawa et al., 1982). Various endogenous cytokinins isolated from the fruiting bodies of Pleurotus sajorcaju are thought to contribute to the long storage life of the fruiting bodies (Dua and Jandaik, 1979). Pleurotus ostreatus and other related species can destroy nematodes through the release of toxins (Thorn and Barron, 1984). An oligonucleotidase from Flammulina velutipes (Kurosawa et al., 1984) degrades oligonucleotides exonucleolytically from the 3’hydroxy end (Kurosawa et al., 1990).A selective inhibitor of myxovirus multiplication, AC,P, was isolated from the aqueous extract of Lentinus edodes fruiting bodies by acetone precipitation (Yamamura and Cochran, 1974). Chihara (1978) has described some more antitumor and immunological properties of polysaccharides from fungal origin. Antitumor glucan polysaccharides are also reported from Coriolus versicolor (Atomachi, 1988) and Polyporus umbellatus (It0 and Hitaka, 1981). GMP production from the mycelium of Lentinus edodes in liquid cultures has been studied (Shangguan, 1988). After Pleurotus ostreatus is cultured on saw dust the spent substrate can be utilized for hydroxylation of lithocholic acid into uredoxycholic acid or selective reduction of 4-androstene-3,17-dione into testosterone (Terada et a]., 1985). Antineoplastic constituents are reported from the cultured mycelia of Laccaria laccata and Ganoderma lucidum (Kang et al., 1981; Kim et a ] . , 1984). The extracellular production of folic acid was enhanced greatly when Coprinus lagopus was grown in the dark in the presence of a reducing agent (ascorbic acid) (Ghosh and Sengupta, 1981). The effects of the immunomodulator PSK on growth of human prostate adenocarcinoma in immunodeficient mice has been reported (Mickey et al., 1989).

IV. Potential Lignocellulosic Substrates for Bioconversion A. LIGNOCELLULOSIC WASTES A wide range and variety of inedible plant wastes, agricultural and industrial, are useful in studies of biotransformation. These wastes are

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

251

chemically composed mainly of cellulose, hemicellulose, and lignin, and thus they are aptly designated as “lignocellulosics.” Bassham (1975) has estimated the net productivity of dry biomass due to photosynthesis by plants on the Earth to be 155.2 billion tons per year. About two-thirds of the biomass production occurs on land, and about one-third occurs in the oceans. Most terrestrial plant material occurs in forests (65%), with a bit more than 15% generated in grasslands and cultivated lands. Mc Hale (1970) has calculated that about three-quarters of the approximate 24 million tons of biomass generated on the cultivated lands and grasslands is waste or residue. About 1.25% of the total land biomass is projected to be eventually used for human food, with about 9% lost during the processing operations and the rest accounting for the magnitude of availability of lignocellulosic wastes (Rajarathnam and Zakia Bano, 1989). About 40% of the various cultivated crops consists of marginal foods and feeds (Zetelaki-Hovrath, 1984). A large proportion of these materials are lignocellulosic wastes produced mostly in the agriculture and food industries. It has been estimated that 1400 million tons of straw and corn stalks are produced annually worldwide. The potential use of the huge quantity of such wastes as a renewable carbon source is of great importance. Type and availability of natural lignocellulosic wastes in any particular geographic region depend on factors such as climate and environment, use and disuse, culture of people, and type and nature of the regional technology. Thus, while rice straw is more prevalent in the Far East and southern Asia, wheat straw and maize by-products are far more abundant in Europe and North America. Wheat straw, soybean stover, and corn stover are each produced in excess of 100 million tons per year, while sorghum stover, oat straw, and corn cobs are produced in amounts ranging from 15 to 56 million tons per year in the United States (Dunlap, 1979). As much as 364 million tons of dry plant waste material per annum is reported to be available in the United States (Young et al., 1986). Approximately 200 million tons of cellulosic agricultural wastes are produced each year in the United States. Much of these unused resources is disposed of by burning, a method which has been increasingly the subject of criticism because of the resultant air pollution. Also, in the United Kingdom considerable amounts of wheat straw are disposed of by burning (Wood, 1986). On the other hand, in many of the southeast Asian countries, large fractions of the available straws enter into uneconomical uses such as thatching for roofs, mulching, and even cattle feed. It has also been estimated that around half the

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total production of plant residues from agricultural and industrial processes remains unused (straw, leaves, wood, bark, etc.), but much of this material, if not burnt, is shredded and/or composted for landfill or improvement of soil types (Zadrazil and Grabbe, 1983). While such residues accumulate and methods for their disposal remain a problem, biological processes using microorganisms that economically convert lignocelluloses into useful products continue to be sought (Wood, 1986). 1. Chemical Nature

Lignocellulosic wastes are composed primarily of cellulose, hemicullulose, and lignin. Their nitrogen content is very low. Depending on the source of material, age, or stage of growth, these components vary in relative quantities. Lignocellulose compositions of a number of plant wastes have been reviewed elsewhere (Bassham, 1975; Chang et al., 1981; Janshekar and Fiechter, 1983; Rajarathnam and Zakia Bano, 1989). In general, cereal straws contain less lignin than do the several types of wood. a . Cellulose. Cellulose is the predominant polysaccharide in plant waste, constituting between 30 and 60% of the total dry weight. It is a major component of the cell wall and a linear polymer of D-glucose units linked by @-(I,4)-glucosidic bonds with a high molecular weight of approximately 0.5 million. b. Hemicellulose. Hemicellulose(s), accounting for up to 40% of plant materials, constitutes the next most abundant fraction of plant materials. Hemicelluloses are composed of various hexoses, pentoses, uronic acids, and other minor sugars. In principle, they are the short, branched-chain heteropolysaccharides of mixed hexosans and pentosans that are easily hydrolyzed. D-Xylose and L-arabinose are the major constituents of pentosans, while D-xylose, D-mannose, and Dgalactose constitute the hexosans. c . Lignin. A considerable part of the photosynthetic activity in plants is devoted to the conversion of atmospheric CO, to lignin. Lignin constitutes about 40% of the solar energy conserved in plants, playing a highly significant role in the carbon cycle. It is a noncarbohydrate heteropolymer of phenylpropane units consisting of vanillin yielding moieties with a characteristic absorption at 280 nm. Lignin differs from all other polymers in that its seemingly random threedimensional structure does not have repetitive linkages between the monomeric building blocks. A second unusual feature is that despite its potentially high energy content (calorific value 7.1 kcal gl), compared to that of coal and oil, it is not used as a sole energy source by any

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

253

known organism. Third, the organisms and the mechanisms by which the lignin macromolecule is degraded are exceptional (Leisola and Garcia, 1989). In plant tissue, lignin functions as a preservative and as a cement between the individual fibers. The lignin matrix joins the cellulosic fibrils together with hemicellulose to form a protective sheath against microorganisms. Lignocellulosic materials comprise about 95% of the Earth’s landbased biomass, about 25% of which is lignin (Janshekar and Fiechter, 1983). There is no exact estimate of the worldwide occurrence of lignincontaining materials. According to various sources, the annual production of lignin wastes in the United States should be between 900 and 3000 million tons. In 1975, 300 million tons of forest products were consumed in the United States, which corresponds to the production of 70 million tons of lignin. United States kraft mills produce about 2 1 million tons per year of kraft lignin; annual production worldwide should be about three times that amount. Lignin is composed of highly branched polymeric molecules, consisting of phenylpropane-based monomeric units linked together by different types of bonds, including alkyl-aryl, alkyl-alkyl, and arylaryl ether bonds. The relative proportion of the three cinnamyl alcohol precursors incorporated into lignin varies not only with the plant species, but also within the plant cell wall. Ecological factors, such as age of the wood, climate of the environment, plant sustenance, and amount of sunlight, also affect the chemical structure of lignin. The hydrolysis of native lignocellulosics is slow. The heterogeneous enzymatic degradation of lignocellulosics is primarily governed by their structural features, since (1)cellulose present in the biomass possesses a highly resistant crystalline structure, (2) lignin surrounding the cellulose forms a physical barrier, and (3) the sites available for enzymatic attack are limited. Any means that will increase the amorphous content of cellulose will enhance the hydrolysis rate (Cowling, 1975; Tsao, 1978; Fan et al., 1980a,b). The presence of lignin forms a physical barrier against enzymatic attack; therefore, treatments causing disruption of the lignin seal will increase the accessibility of cellulose to the enzyme molecule and eventually its hydrolysis rate. The limitation of available sites for enzymatic attack stems from the fact that the average size of the capillaries in the biomass is too small to allow the entry of large enzyme molecules; thus, enzymatic attack is confined to the external surface (Fan et al., 1982). Pretreatment, therefore, is an essential prerequisite to enhance the susceptibility of lignocellulosics to enzyme action. An ideal pretreatment would accomplish reduction in lignin content, concomitant with

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SOMASUNDARAM RAJARATHNAM ET AL.

reduction in crystallinity and increase in surface area. The variety of pretreatments can be classified into physical, chemical, and biological, depending on their mode of action. Lignin is not an organic material that can be easily degraded in the laboratory; usually, one has to employ a strong acid, accompanied by high temperature. Pretreatments to disrupt the lignin barrier-with chemicals such as NaOH, or with solvents, or by physical treatments such as fine grinding-contribute to the enhancement of the enzymatic hydrolysis. However, the physical methods require a large amount of energy, while the chemical methods lead to environmental pollution. Although a wide range of microorganisms are capable of degrading cellulose, far fewer produce enzymes that can degrade the natural lignocellulosic wastes, because lignin limits access to cellulose. In fact, the only proven microbial means of converting unmodified lignocellulosics is found in the production of enzymes by various types of mushrooms (Zetelaki-Hovrath, 1984; Schurz, 1978).This leads us to the consideration of global availability of lignocellulosic wastes. 2. Global Availability

a. Cereal Straws. Annually, about 2946 million tons of cereal straws (Table VII) are produced in the world (FAO, 1989). Significant quantities of the cereal straws are disposed of by burning in many parts of the world (Penn, 1976; Hayes and Lim, 1979; Wood, 1986). Clearly, this is a terrific loss of energy conserved through the process of photosynthesis by the green plants-the phenomenon that ultimately contributes to most of the energy generated on the Earth. Enactment of rigorous air pollution laws that restrict burning of straws and the increasing cost of animal feeds have revived interest in the study of lowquality crop residues as a ruminant feed (Crompton and Maynard, 1988). Subjecting these straws to biodegradation and bioconversion by the edible basidiomacromycetes would contribute to the production of directly palatable food and the degraded substrates would merit consideration for a number of diversified applications (See Section VII). The possible biotransformation of straw into the edible fruiting bodies and the spent substrate in various parts of the world, calculated on the basis of bioconversion efficiencies of the basidiomacromycetes, is projected in Table VIII. b. Pulse Plant Residues. A number of pulse plants are cultivated in various parts of the world and for every ton of the pulse that is harvested, two to three times of the inedible plant residue is available. In general, these plant residues contain more nitrogen than those of cereal straws. Further, their lignocellulose composition is more suitable for

255

BIOPOTENTIALITIESOF BASIDIOMACROMYCETES TABLE VII PRODUCTION OF PLANT WASTES IN THE WORLD IN

19890

Plant wastes ( x 1000 tons) Continent and country World Africa Egypt Kenya North (Central) America Canada United States of America Mexico South America Argentina Brazil Asia Afganistan China India Iran Pakistan Europe Australia

Cereal strawsb 2,946,040 165,272 17,988 7,250 500,102 58,713 437,713 45,710 153,661 43,547 81,842 1,135,508 8,734 534,439 236,980 20,987 30,901 550,518 35,507

Pulse plant wastesc 166,178 9,069 1,775 NAf 49,520 2,095 43,889 2,692 37,376 10,296 24,057 51,767 NA 22,826 16,070 725 985 10,055 a37

Oil crop wastesd 141,674 11,223 1,848 65 21,938 60 19,476 331 9,766 4,845 5,094 61,743 177 29,808 14,211 1,523 8,638 8,883 1,706

Plantation crop wastese 548,026 34,530 4,638 2,287 84,261 NA 15,476 18,692 147,058 6,806 115,813 174,923 57 28,655 88,632 1,044 16,862 983 12,462

oDerived from data by FA0 (1989). bStraws of wheat, rice, maize, barley, rye, oats, sorghum, and millets. CWastes of beans, broad beans, peas, chick-peas, lentils, and soybean. dPlant wastes of groundnut, sunflower, sesame, and cottonseed. .Plant wastes of coffee, sugarcane, and cotton. fNA, Not available

the economic bioconversion to produce edible fruiting bodies. However, the one problem that may be anticipated while using the pulse plant residues as culturing substrates is the necessity to size and trim the residues to maintain the bed texture to facilitate optimum mycelial ramification and gaseous exchange. Annually, about 1.7 million tons of this residue are available worldwide (FAO, 1989). c. Oil Seed Crop Plant Residues. Edible oil is one of the basic components of food consumed by people. After the oil seeds are harvested, significant quantities of the plant residues remain unutilized. For instance, in the case of sunflower, the plant residue does not have any value other than serving as fuel. Annually, about 1.4 million tons of oil crop residues are available over the world for the study of bioconversion.

TABLE VIII POSSIBLEPRODUCTION OF Pleurotus FRUITING BODIES FROM CEREAL STRAWSAND Continent or country

Population (X

lOOO]

25% of total cereal strawb used ( x 1000 tons)

Fruiting bodies producedc ( x 1000 tons, dry)

1,278,697 41,318 4,497 1,812 125,025 14,678 109,428 11,427 38,415 10,886 20,460 283,877 2,183 133,610 59,245 5,247 7,725 137,629 8,877

127,869 4,131 449 181 12,502 1,467 10,942 1,142 3,841 1,088 2,046 28,387 218 13,361 5,924 524 772 13,762 887

PER

CAPITAAVAILABILITY PER DAY^

Availability (g, drylpersonlday]

Fertilizer producedd ( x 1000 tons)

~~

World Africa Egypt Kenya North America Canada United States Mexico South America Argentina Brazil Asia Afghanistan China India Iran Pakistan Europe Australia

5,114,788 609,922 51,553 23,077 417,276 25,932 246,069 84,884 285,024 31,536 144,428 2,994,005 14,893 i,ioo,gga 819,482 53,123 115,042 496,812 16,353

68.4 18.5 238.6 21.5 82.0

155.0 121.8 36.9 36.9 94.5 38.8 25.9 40.1 33.2 19.8 27.0 18.4 75.8 148.6

338,854 10,949 1,191 480 33,131 3,889 28,998 3,028 10,179 2,884 5,421 75,227 578 35,406 15,699 2,047 2,047 36,471 2,352

GFrom Zakia Ban0 and Rajarathnam (1988). bValues for straw were obtained from the grain yields given in the FA0 Production Year Book 1989, using conversion factors 1.8 for wheat, 1.0 for rice, 2.4 for maize, and 1.7 for other cereals. GValues can be arrived at for Agaricus and Volvariella using the yield conversion factors of 7 [Schisler and Patton, 1978) and 1.5 (Chang, 1982), respectively, instead of 10 as used for Pleurotus in the table. Calculated based on the fact that starting with 1 ton rice straw, 265 kg spent straw (as fertilizer) is obtained after the harvest of fruiting bodies.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

257

d. Plantation Crop Residues. Coffee pulp is a major by-product of the coffee industry, representing about 28.7% of the coffee bean on a dry weight basis during the wet coffee processing method (Bressani, 1979). Through research on the use of Pleurotus for bioconversion of coffee pulp, done at the National Institute of Biotic Resources by Martinez and co-workers, it has been possible to set up a mushroom-producing plant on a semi-industrial scale in Mexico, designed to work on 1 ton of coffee pulp every day, to effect a total daily production of 110-130 kg of fresh mushrooms (Guzman and Martinez, 1986). The fibrous residue remaining after extracting juice from the sugarcane stalk is referred to as bagasse. In sugar mills, most bagasse is used as fuel; energy-efficient units can have 2 tons (dry weight) of surplus bagasse for every ton of fresh sugar cane processed (Paturau, 1982). The lignocellulosic residue left after sucrose conversion into ethanol by the EX-FERM is referred to as EX-FERMented sugarcane chips (Rolz et al., 1987), which contain 44.8, 37.7, and 13.5%, respectively, cellulose, hemicellulose, and lignin (bagasse samples contained 42.1, 37.0, and 14.6% cellulose, hemicellulose, and lignin, respectively). In Israel, 1.5 tons of straw are produced per acre of cotton cultivated (Platt et al., 1981). Its high lignin content (-25%) has limited the value of cotton straw as a direct ruminant feed (Gohl, 1981). Cotton boll locules, plentiful in cotton-growing areas, have been shown to be a promising substrate for bioconversion by the mushroom (Khan and Ali, 1981).

e. Industrial Residues. Citronella (Cymbopogon winterianus) bagasse and lemon grass (Cymbopogon citratus) are the lignocellulosic residues of steam distillation of freshly harvested lemon grass and citronella leaves to recover their essential oils (Rolz et al., 1986). The essential oil content is low-0.5 to 1.3% by weight of fresh grass-and its recovery is not complete. After steam distillation, the bagasse is partially dried in the field and a fraction is burnt to generate steam for the stripping; the rest is left in the fields, where natural biodegradation takes place. Its use as a ruminant feed is limited, due to animal rejection because of residual aroma and flavor. According to information on essential oil production (Robbins, 1981), there is an estimated worldwide availability of about 200,000 tons of dry bagasse per year that could be used as a source of lignocellulosic wastes. Shive is a bulky, woody by-product of flax (Linum usitatissimum L.) that is left after scutching and has little value (Sharma, 1987). For every ton of fiber produced, 2.5 tons of shive will be left after scutching. The high lignin content prevents ruminants from utilizing the cellulose (Jung and Fahey, 1983). Orange peel and distillery grape stalks are two

258

SOMASUNDARAM RAJARATHNAM ET AL.

of the wastes produced in large quantities in Italy (Nicolini et al., 19871, and they cause disposal and environmental problems. In 1985, the orange juice industries have processed 600,000 tons of citrus fruits, with a residual waste that constitutes 60% of the weight of the treated fruits. This waste contains a considerable amount of residual sucrose and macromolecular carbohydrates, but has a low protein content; therefore, it has a good digestibility level, but is of low nutritional value. Distillery grape stalks have a low carbohydrate content as well as a low protein content, but have a high level of lignocellulosic material. These characteristics limit their use as animal feed and they can thus be potential substrates for raising mushrooms. A compost of lime sludge has been found useful for mushroom production; this involves treatment of waste water from mandarin orange canneries, and thus its useful disposal (Mourin et al., 1981). Paper mill sludge and apple pomace (from the fruit processors) are inexpensive and plentifully available and can also serve as substrates for growing mushrooms (J. C. Mueller et al., 1984). Paper mill sludges (thermomechanical as well as h a f t sludges) provide some advantages: their moisture content of 65-80% happens to be optimum for mushroom growth, and their pH of 8-11, though alkaline, seems to provide enough buffering capacity to neutralize the effects of CO, and the release of organic acids during spawn run. Apple pomace, on the other hand, is found to be an effective additive to sludge substrates, as it provides mainly carbohydrates, proteins, and vitamins. A combination of sludge and pomace in equal proportions along with 2-3% soya is found to be very productive for mushroom culturing. The residual pulp waste from shochu production, containing about 32% carbohydrates and 29% proteins (on a dry weight basis], was found to be suitable for raising Flarnmulina velutipes (Ogawa and Toyama, 1982). Industrial wastes such as corrugated paper, saccharification waste, distillery slop, peat, and grass clippings have been evaluated to assess their suitability as substrates for the industrial production of Pleurotus ostreatus (Terashita and Kono; 19841. Use of tobacco waste (midrib) (Tolentino, 1981) and soft/hardwoods for culturing of Volvariella volvacea and Lentinus edodes (Dar et al., 19881, respectively, has been reported. Use of sulfite pulp waste for raising Flarnrnulina velutipes (Inaba et al., 1984) and bagasse for Volvariella cultivation (Zakhary et al., 1984) has also been reported. f. Forest Plant Residues. The number of forest trees, such as willow, poplar (Pilat, 1935; Ferri, 1972; Pirazzi et al., 1978), and alder (J. C. Mueller et al., 1984), have been successfully used for mushroom cultivation. Alder compost (previously used for Agaricus production)

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

259

in combination with pulp mill sludges can be recycled for culturing Pleurotus sajor-caju; the spent alder compost alone cannot support the mushroom yield. Since alder (which grows rapidly in British Colombia) is considered a weed throughout most of the forest industry, its utilization along with cellulose pulp mill sludges (available without any cost) as a substrate base for raising two distinct varieties of mushrooms appears to offer an interesting and economical prospect for a local mushroom industry, particularly in the vicinity of British Colombia C. Mueller, et al., (1984). g. Other Residues. The use of substrates like, corn stover, stems of the castor-oil plant, and leaves of mulberry for Pleurotus sajor-caju (Madan et al., 1987; Chahal, 1989), sawdust for Lentinus (Miyao, 1989), milled dry cassava roots for Sporotrichum puIveruientum (Smith et aI., 1986), and cottonseed husk and corn stalk for Pleurotus (Qin et al., 1989) growth, biodegradation, and bioconversion has also been described. Thus, the magnitude of available lignocellulosic wastes is quite high (TableVII). Their constant accumulation over the years would result in large quantities of unwanted wastes unless a significant and economic use is defined. Their availability is varied, depending on geographic location and season. It is also correct to surmise that their exploitation for valuable and economic use is underdeveloped and obviously merits study to create better utilization. Utilizing them in the culturing of and biodegradation by the basidiomacromycetes is one of the best channels of utilizing these lignocellulosic wastes for conversion into a useful form of food. In fact, harvesting of fruiting bodies is the easiest possible method of separating edible biomass from the substrate in a solid-state fermentation (Zadrazil and Grabbe, 1983). It is estimated that each year 1 acre of forest land is covered by nearly 2 tons of debris. Without these fungi-the decomposers-to do the clean-up job, our trees would be buried in no time by their cast-off leaves and branches (Booth and Harold, 1982).

u.

B. FERMENTATION-SOLID

AND

SUBMERGED STATES

Lignocellulosic materials are insoluble, and hence biodegradation of such materials would also be in the solid state, wherein the substrate is moistened, often with a thin layer of water on the surface of the particles, but without enough water present to make a fluid mixture. Weight ratios of water to substrate in solid-state fermentation (SSF) of lignocellulose are usually between 1 : 1 and 1 : 10. SSF offers many advantages over submerged fermentation for biodegradation: (1) smaller fer-

260

SOMASUNDARAM RAJARATHNAM ET AL.

mentor volumes (because there is no excess water), (2) lower sterilization energy costs, (3) easier aeration, (4) reduced or eliminated capital and operating cost for stirring, (5) lower cost for product recovery and drying, (6) a more natural environment for lignin degrading fungi, and (7) a less favorable environment for many bacteria. 1. Fungi for Biodegradation

Successful degradation of lignocellulosics is dependent on ligninolytic activity and thus, white-rot basidiomycetes have been given more attention; several reports of the brown-rot fungi are also available. A range of lignocellulosic wastes have been screened against a number of basidiomycetes (Table IX), but despite a considerable amount of work, it is not yet possible to offer lists of recommended species to delignify particular substrates (Reid, 1989). 2. Factors Influencing Degradation

Varying degrees of substrate degradation have been observed by several workers with different or identical species, grown on different or similar substrates. These differences in the relative abilities of the speciedstrains of Pleurotus have to be ultimately attributed to the interaction between the genome (heritable genetic material) and the environmental factors-physical, chemical, and biological. A study of these factors is essential in order to define the conditions required for optimum degradation of a particular substrate with any particular speciedstrain. The achievement of maximum degradation of the substrate should eventually increase the biomass conversion efficiency and incidentally, also, increase substrate solubilization. 3. Monoculturing: Probabilities and Improbalities

Ensuring monoculture of a basidiomycete on a lignocellulosic substrate (under conditions of nonsterilization) is one of the major difficulties posed during SSF. A delignifying fungus that would invade nonsterile substrates, outcompete the indigenous microflora, and then exclude other microorganisms would make the SSF process simpler and cheaper (Reid, 1989). Lignocellulosic materials generally carry a heavy inoculum of microorganisms that germinate and grow rapidly when the substrate is moistened (Gyurk6, 1977; Zadrazil and Peerally, 1986; Schuchardt and Zadrazil, 1986; Reid, 1989). These indigenous microflora usually inhibit colonization of the substrate by white-rot fungi (Schuchardt and Zadrazil, 1986). Under laboratory conditions, to trace all changes brought about by

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

261

the basidiomycete species under study, the growth substrate is sterilized by steam under pressure (Rajarathnam, 1981; Reid, 1985, 1989; Rajarathnam et al., 1991; Agosin et al., 1989). Due to conditions of restricted oxygen supply, though the spectrum of changes brought about in the growth substrate (compared to in vivo degradation under nonsterile conditions) may be similar, quantitatively significant differences do arise in the relative amounts of different constituents of lignocellulosics that are biodegraded. This results from the fact that fructification and a higher degree of biotransformation occur under growth conditions in vivo with free availability of oxygen for the growing mycelium and the fruiting primordia. Subjecting substrates to aerobic composting (as for Agaricus bisporus) (Wood, 1984) or anaerobic fermentation (as for Pleurotus) (Zadrazil and Peerally, 1986) and maintaining high CO, and low 0, concentrations (Schuchardt and Zadrazil, 1986) have aided in limiting growth of indigenous microbes, favoring the growth of mushroom mycelium. 4. Substrate Particle Size and Water Content Substrate particle size and water content coherently influence substrate degradation. While adding defined amounts of water per unit weight of substrate, the course of SSF is influenced by a number of interrelated factors. As the water content increases, the gas phase is reduced and gas exchange is thus increasingly impeded; finally, conditions become anaerobic in the substrate suspension. On the other hand, at low water content, the fungal growth conditions are also suboptimal because water retention is high and the degree of substrate swelling is low. The influence of water-to-air ratios in the substrate during solidstate culturing of Pleurotus serotinus, Pleurotus ostreatus, and Pleuro tus sajor-caju has been studied by Zadrazil and Brunnert (1981). Particle sizes ranging from 1 to 8 mm had no significant effect on SSF of wheat straw by Phanerochaete chrysosporium or Dichomitus squalens with passive air diffusion (Zadrazil and Brunner, 1982). Hammermilled aspen wood was delignified by Phlebia tremellosa as quickly as finer particles, but significantly faster than shavings or chips, all with active aeration (Reid, 1989). Degradation of wafers of birch and spruce by Phanerochaete chrysosporium and Panus conchatus was inversely proportional to their thickness in the range 1-10 mm (Yu and Eriksson, 1985).The optimum water content for delignification of aspen wood by PhIebia tremellosa in air was 2 g water/g wood (Reid, 1985). The fungi could generally digest the straw over a range of water: straw ratio from 1: 1 to 10 : 1 (Zadrazil and Brunnert, 1981, 1982).

TABLE IX

RESEARCHREPORTSON SOLID-STATE FERMENTATION OF LIGNOCELLULOSIC MATERIALS BY BASIDIOMACROMYCETES~ Angiosperms Dicot Species Agrocybe aegerita

N

m

Armiliariella mellea Coliybia fusipes, C. radicata Coprinus cinereus Coprinus comatus Coprinus fimetarius Coriolus hirsutus

N

Coriolus versicolor

Monocot straw and bagasse Zadrazil (1977, 1980b, 1985b); Nicolini et al. (1987); Rolz et al. (1986, 1987) -

Herbaceous Nicolini et al. (1987); Zadrazil (mob) Nicolini et al. (1987)

Zadrazil (1985b)

-

Barrows et al. (1979) Zadrazil (1985b) Rolz et al. (1986, 1987) Zadrazil (1985b); Zadrazil and Brunnert (1981), Kewalramani et al. (1988); Levonen-Munoz et al. (1983a)

-

Zadrazil (1985b); Rolz et al. (1986, 1987); Milstein et al. (1986); Levonen-Munoz et al. (1983b); Arora and Sandhu (1986); Viesturs et ai. (1985); Mueller and Trosch (1986); Hatakka (1983)

Woody Zadrazil (1980b)

-

Babitskaya and Shcherba (1987)

Rolz et al. (1988)

Setliff and Eudy (1980); Afans’eva (1984); Akamatsu et al. (1984); Solov’ev et al. (1982) Golovlev et al. (1983); Setliff and Eudy (1980); Reid and Seifert (1982); Otjen et al. (1987); Akamatsu et al. (1984); Solov’ev et al. (1982); Blanchette et al. (1985, 1987); Kirk (1973); Kirk and Moore (1972); Reade and Mc Queen (1983)

Gymnosperms -

Cyathus africanus, C. berkeleyanus, C. bulleri Cyathus canna Cyathus earlei, C. helenae, julietae, C. limbatus, C. pallidus, C. pygrnaeus Cyathus stercoreus

Dichornitus squalens

N

Flarnmulina velutipes

Wicklow et al. (1984) Wicklow et al. (1984) Wicklow et al. (1984)

Wicklow et al. (1984) Rolz et al. (1988) -

Wicklow et al. (1984)

Agosin and Odier (1985); Wicklow et al. (1980, 1984); Agosin eta]. (1985b, 1986) Zadrazil (1985b); Rolz et al. (1986, 1987); Agosin and Odier (1985); Levonen-Munoz et al. (1983b); Agosin et al. (1986); Zadrazil and Brunnert (1982) Zadrazil (1985b); Rolz et al. (1986, 1987);Mueller and Trosch (1986) Zadrazil (1985b)

Rolz et al. (1988)

Fornes fomentarius Fomes lividus

Zadrazil (1985b) Ibrahim and Pearce (1980)

Ganoderma applanaturn

Zadrazil (1985b); Zadrazil and Brunnert (1981); Rolz et al. (1986, 1987); Levonen-Munoz et 01. (1983b); Mueller and Trosch (1986)

Ibrahim and Pearce (1980) Rolz et al. (1988)

0)

W

Fornes annosusHeterobasidion annosum

Wicklow et al. (1984) Wicklow et al. (1984)

Setliff and Eudy (1980); Otjen et al. (1987); Blanchette et al. (1987); Reade and Mc Queen (1983); Myers et al. (1988) -

Otjen et al. (1987)

Golovlev et al. (1983); Otjen et al. (1987); Blanchette et al. (1985) Hartley et al. (1974)

Otjen et al. (1987)

Solov’ev et al. (1982); Blanchette et al. (1985); Kirk (1973); Kirk and Moore (1972); Reade and Mc Queen (1983) [continued)

TABLE IX (Continued) Angiosperms

Species

N

Monocot straw and bagasse

Grifola frondosa

Zadrazil (1985b)

Hymenochaete corrugata, H. tabacina Irpex lacteus

Zadrazil (1985b)

m

ip

Lentinus edodes Lentinus lepideus Lentinus tigrinus Lentinus betulina

Merulius lacrymans =Seropula lacrymans Merulius trernellosus = Phlebia trernellosa

Zadrazil (1985b); Levonen-Munoz et a]. (1983a,b); LevonenMunoz and Bone (1985) Zadrazil (1985b); Mueller and Trosch (1986) -

Zadrazil (1985b) Zadrazil (1985b); Zadrazil and Brunnert (1981) Zadrazil (1985b)

Dicot Herbaceous

Woody

Gymnosperms

-

Setliff and Eudy (1980); Reid and Seifert (1982); Kirk m d Moore (1972) -

-

Setliff and Eudy (1980)

Reid and Seifert (1982) Golovlev et al. (1983) Afans’eva (1984); Akamatsu et al. (1984) Setliff and Eudy (1980); Reid and Seifert (1982); Blanchette et al. (1987); Blanchette and Reid (1986); Reid (1985); Mes-Hartree et af. (1987) Hartree et al. (1987)

Ander and

Phanerochaete affinis Phanerochaete chrysosporium

Phanerochaete flavido-alba, P. hydnoides Phanerochaete sordida Phellinus igniarius Phellinus megaloporus Phellinus pini

Phlebia brevispora Phlebia livida Pholiota adiposa Pholiota lubrica ssp. squariosa Pholiota mutabilis Pleurotus cornucopiae Pleurotus corticatus, P. cystidiosus, P. dryninus, P. elangatipes

-

Zadrazil (1985b); Rolz et al. (1986, 1987); Agosin and Odier (1985); Levonen-Munoz et al. (1983a,b); Agosin et al. (1985b, 1986); Zadrazil and Brunnert (1982); Ibrahim and Pearce (1980); Levonen-Munoz and Bone (1985); Contrereas et al. (1985); Al-Ani and Smith (1988); Golovlev et al. (1983)

Ibrahim and Pearce (1980); Rolz et al. (1988); Myers et a ] . (1988); Johnsrud and Eriksson (1985); Mudgett and Paradis (1985); Reid (1983b)

Otjen et a ] . (1987) Golovlev et al. (1983); Eriksson et al. (1976); Setliff and Eudy (1980); Reid and Seifert (1982); Otjen eta]., (1987); Akamatsu et al. (1984); Reade and Mc Queen (1983) Otjen et al. (1987)

-

Ibrahim and Pearce (1980)

-

Zadrazil (1985b) Zadrazil (1985b) -

Zadrazil (1977, 1985b); Tsang et aJ. (1987) Zadrazil (1985b)

Otjen et al. (1987)

-

Hatakka (1983) Zadrazil (1985b) Zadrazil (1985b); Ibrahim and Pearce (1980)

Eriksson (1977) Otjen et a ] . (1987) Otjen et a ] . (1987); Johnsrud and Eriksson (1985); Ander and Eriksson (1977)

Hartley et al. (1974) Otjen et a]. (1987); Blanchette et al. (1987) Otjen et a!. (1987) Setliff and Eudy (1980)

-

Otjen et al. (1987)

Otjen et al. (1987) -

Zadrazil (1980b); Sharma (1987)

Otjen et al. (1987) Zadrazil (1980b)

Otjen et al. (1987)

-

(continued)

TABLE IX (Continued) Angiosperms

Species Pleurotus eryngii Pieurotus flabellatus Pleurotus ostreatus var. Florida

N Ln

m

Pleurotus liguatiiis, P. mutilis Pieurotus ostreatus

Pleurotus sajor-caju

Monocot straw and bagasse Zadrazil (1985b); Kamra and Zadrazil (1986) Zadrazil (1985b); Rolz et al. (1986, 1987) Zadrazil (1977, 1980b, 1985b); Mueller and Trosch (1986)

Zadrazil (1985b) Zadrazil (1985b); Zadrazil and Brunnert (1981); Nicolini et al. (1986); Agosin and Odier (1985); Kaneshiro (1976); Levonen-Munoz et al. (1983b); Muller and Trosch (1986); Hatakka (1983); Ibrahim and Pearce (1980); Tsang et al. (1987); Zadrazil and Peerally (1986); Ginterova and Lazarova (1987); Lindenfelser et al. (1979); Beg et al. (1986); Bisaria et al. (1984); Ortega Cerrillia et a ] . (1986) Zadrazil (1985b); Zadrazil and Brunnert (1981); Kewalramani

Dicot Herbaceous

Woody

Gymnosperms

Rotz et al. (1988) Zadrazil (1980b); Rolz et al. (1988); Sharma (1987); Platt et 01. (1981) -

Zadrazil (1980b)

Nicolini et al. (1987); Ibrahim and Pearce (1980); Sharma (1987)

Reid and Seifert (1982); Kirk and Moore (1972); Hiroi (1981); Kovics-Ligetfalusi (1977)

Bisaria et al. (1983)

-

Hiroi (1981); Ander and Eriksson (1977)

Pleurotus salmoneostramineus Pleurotus sapidus Pleurotus serotinus Pleurotus sp. Pleurotus spodoleucus, P. ulmarius Polyporus arcularius, P. brumalis, P. galactinus, P. varius Polyporus berkeleyi

Poria latemarginata Poria medulla-panis Pycnoporus cinnabarinus

Pycnoporus sanguineus

et al. (1988); Tsang et al. (1987); Kamra and Zadrazil (1986); Bisaria et al. (1983, 1984); Zafar et al. (1981) Zadrazil (1985b) Zadrazil (1985b); Tsang et al. (1987) Zadrazil (1985b); Zadrazil and Brunnert (1981) Zadrazil (1985b); Hatakka (1983) Zadrazil (1985b) Zadrazil (1985b)

Rolz et al. (1988) -

Agosin and Odier (1985); Hatakka (1983); Agosin et al. (1985b)

Stereum sanguinolentum Stropharia rugoso-annulata

Zadrazil (1985b); Rolz et al. (1986, 1987) Zadrazil (1985b) Zadrazil (1977, 1980b, 1985b); Rolz et al. (1987); Kamra and Zadrazil (1985)

Trametes cingulata Volvariella volvacea

Zadrazil (1985b)

After Reid (1989)

-

Rolz et al. (1988) -

Zadrazil (1980b); Rolz et al. (1988)

Setliff and Eudy (1980); Kirk (1973); Kirk and Moore (1972) Setliff and Eudy (1980) Blanchette et al. (1987) Setliff and Eudy (1980); Reid and Seifert (1982); Akamatsu et a]. (1984) Setliff and Eudy (1980) Zadrazil (1980b)

Setliff and Eudy (1980) -

-

Ander and Erikkson (1977)

268

SOMASUNDARAM RAJARATHNAM ET AL

5. pH Most white-rot fungi grow best at a slightly acid pH between 4 and 5. Many of these fungi acidify lignocellulosic substrates as they grow in them (Zadrazil, 1977; Daugulis and Bone, 1977; Zadrazil and Brunnert, 1981, 1982; Reade and Mc Queen, 1983; Agosin and Odier, 1985; Reid, 1985). 6. SpecieslStrain under Study The extent and rate of degradation of a lignocellulosic waste vary from species to species and among strains of the same species (Zadrazil and Brunnert, 1979; Kamra and Zadrazil, 1986; Sharma, 1987). Culturing Pleurotus ostreatus along with the bacterium Erwinia carotovora enhanced the degradation and also the in vitro digestibility of wheat straw (Streeter et al., 1982). 7. Nature of the Substrate Variations in substrate type greatly influence the rate of degradation (Zadrazil, 1978b). Wheat straw, straw reeds, sunflower straw, beechwood sawdust, and rape straw were studied. The good decomposition rate of straw could most probably be attributed to the relatively narrow range of the C:N ratio, 100 to 50 : 1 (while beechwood sawdust has a C : N ratio of 500 : I). Further, the chemical nature of the substrate in terms of sugars, phenols, amino acids, cellulose, and lignin greatly influences substrate degradation. Thus, coffee pulp first subjected to fermentation to reduce the phenolic content could only then promote the growth of Pleurotus ostreatus (Martinez et a]., 1984). 8. Inoculum Conidia; mycelium grown in liquid medium or stripped from the surface of agar plates or agar plugs; spawn grown on cereal grains; and lignocellulose waste precolonized by the fungus have been used successfully to inoculate growth in SSF; however, the efficacy of each inoculant on the biodegradation has not been critically investigated. Mycelial inoculum of Phanerochaete chrysosporium gave faster lignin degradation in bagasse than conidial inoculum (Contrereas et al., 1985). Liquid-grown mycelium was as effective as precolonized wood for inoculation of aspen wood with Phlebia tremellosa (Reid, 1989).In liquid culture containing oak wood extracts, the adaptation of the mycelium of Lentinus edodes to the toxins of the wood allowed the successful colonization of the wood (Leatham and Griffin, 1984). Further, the rate of inoculation also influences the rate of substrate degradation (Mudgett and Paradis, 1985; Reid, 1989).

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

269

9. Incubation Time and Temperature

In general, basidiomycete species cause an initial drop in the free soluble components of the substrate, before they attack lignin or cellulose (Zadrazil, 1977; Zadrazil and Brunnert, 1981, 1982; Kamra and Zadrazil, 1985, 1986; Agosin and Odier, 1985; Kewalramani et al., 1988). During the increase in incubation time, the existing fungal cells start secreting the hydrolytic and oxidative enzymes, which tend to cause further solubility of the substrate. Maximum degradation typically requires 3-8 weeks in hardwoods (Reade and Mc Queen, 1983; Reid, 1985) and 3-4 weeks in straw (Zadrazil and Brunnert, 1982). Kirk and Moore (1972) were the first to report that lignin removal was more selective during the early stages of lignin degradation than later. Subsequent studies of the time course of biological delignification have confirmed this pattern (Agosin and Odier, 1985; Lindenfelser et al., 1979; Zadrazil and Brunnert, 1982; Reade and Mc Queen, 1983; Reid, 1985). Zadrazil (1976b) has observed that the extent of degradation of wheat straw by Pleurotus cornucopiae and P. ostreatus var. Florida was maximum at the end of 120 days as measured by the loss of organic matter, substrate solubility, and increase in reducing sugars. Most white-rot fungi are mesophils, with temperature optima between 20 and 30°C. Pleurotus serotinus displayed reduced wheat straw degradation at 30"C, whereas the temperature had no influence on Pleurotus ostreatus and Pleurotus sajor-caju. 10. Oxygen Availability

Mycelial growth may not be effected at CO, levels even as high as 22% (Zadrazil and Schliemann, 1977), whereas morphogenesis and

fructification proceed only with pronounced oxygen availability. Oxygen content lower than 20% in the gaseous phase adversely affected the loss of organic matter, lignin degradation, and change in straw digestibility with both Pleurotus sajor-caju and Pleurotus eryngii (Kamra and Zadrazil, 1986). Replacing air with an atmosphere of 0, stimulates lignin degradation by many white-rot fungi growing on straw (Hatakka, 1983; Levonen-Munoz and Bone, 1985; Kamra and Zadrazil, 1985, 1986) or wood (Reid and Seifert, 1980; Reid and Seifert, 1982; Mudgett and Paradis, 1985; Yu and Eriksson, 1985). Increasing 0, pressure beyond 1 atm did not further stimulate lignin degradation by Phanerochaete chrysosporium (Reid and Seifert, 1980) and 0, pressures of 2 atm (Mudgett and Paradis, 1985) or 3 atm (Reid and Seifert, 1980) were inhibitory. Phlebia tremellosa degraded aspen wood lignin well at 0, partial pressures as low as 0.07 atm (Reid, 1985). Straw lignin degradation by Stropharia rugosa-annulata or Pleurotus species was only par-

270

S O M A S U N D A M RAJARATHNAM ET AL.

tially inhibited when the 0, level was reduced to 0.05 atm (Kamra and Zadrazil, 1985,1986). In general, oxygen enrichment of the atmosphere stimulates the degradation of lignocellulosics (Levonen-Munoz and Bone, 1985; Kamra and Zadrazil, 1985, 1986; Yu and Eriksson, 1985; Reid and Seifert, 1980). Liquid culture fermentation studies have been mainly concerned with the formation and production of enzymes by the basidiomycetes. This is dealt with in a later section. V. Biotransformation of Lignocellulosic Wastes

A. PREPARATION OF SUBSTRATES A range of pretreatments has been investigated to support the growth of the Basidiomacromycetes. The type, nature, and duration of the treatment are dependent on the growth substrate in question and the species that is to be grown. Sterilization ensures aseptic culturing on a substrate that is absolutely free from contamination. After the wet substrate is filled into containers, it is subjected to sterilization in a special steaming room and autoclaved at 90-100"C for 90 minutes or at 121OC for about 1hour under pressure and cooled to ambient temperature before spawning. Although this method can ensure very efficient growth of the fungal monoculture, it is quite uneconomical on a commercial scale. Sterile substrates are used for culturing at the Max Planck Institute in Hamburg (Lange and Hora, 1963), Pleurotus abalone in the Republic of China, and Pleurotus ostreatus in Japan (Zadrazil and Kurtzman, 1982). A sterilized substrate of sawdust and rice bran is used for culturing Auricularia and Flarnrnulina (Ho and Han, 1978). Pasteurization was introduced for large scale cultivation of Pleurotus by Zadrazil and Schneidereit (1972) and is routinely followed for the culturing of Agaricus (Wood and Smith, 1989). In this technique, the substrate is subjected to 60-100°C for a few hours and then cooled before spawning. The aim of pasteurization is to kill the myceloid and thin-walled microbes in the substrate. Zadrazil (1973) has suggested continuous production of substrate. This technique is very well illustrated during the culturing of the button mushroom. Here, a lignocellulose base such as wheat, rice, or jungle straw undergoes wetting with water and supplementation with nitrogen sources such as urea, ammonium sulfate, and oilseed cakes, followed by heaping, leads to an increase in core temperature involving the growth and succession of microbes. During this process, the water-soluble portion of the growth

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

271

substrate is reduced, and a lignin-humus complex that is selectively utilized by the growing mushroom is built up. The microbial cell wall polysaccharides formed and left behind during composting and after microbial death, respectively, serve to support the growth of Agaricus (Fermor and Wood, 1982). The possibility of using peat as a substrate for mushroom cultivation has been reported (Grabbe, 1980). Stanek and Rysava (1971) have described the fermentation under control conditions with the introduction of bacterial Streptornyces thermovulgaris and Pseudomonas species. In Mexico, coffee pulp is heaped on open grounds, which results in the reduction of caffeine content and allows growth of the mushroom mycelium (Martinez et al., 1985). Further, in such instances wherein a lignocellulose such as rice straw is directly used for culturing, as, for example, for the growth of Pleurotus species, the straw is pretreated with antifungal agents to free the substrate from contamination (Rajarathnam and Zakia Bano, 1988). ScIerotium rolfsii is a serious substrate-borne contaminant that reduces mushroom yield by 80-96%. Treating straw with carboxin, hot water (Rajarathnam et al., 1979b), methyl bromide (Rajarathnam et al., 1977), ethyl formate (Zakia Ban0 et a]., 1981), or liquid ammonia (Rajar athnam et al., 1983) effectively controls contamination. Treating straw with an aqueous suspension of benomyl controls the growth of Penicillium digitatum (Zakia Ban0 et al., 1975) and also encourages and supports thick growth of Pleurotus mycelia. Ecological factors play a vital role in influencing the control of weed molds in the growth substrates during the culturing of fruiting fungi (Sinden, 1971; Rajarathnam and Zakia Bano, 1988). The necessities and implications of pretreatments of wheat straw to serve as an ideal substrate for growing Pleurotus are discussed by Lanzi (1991). Thus, the purpose of pretreatments is to effect fast and thick growth of the cultured fruiting fungi, selectively overcoming the possible occurrence of unwanted fungi that compete for the mushroom nutrition. The treatments are meant to extract and remove the water-soluble components (which invite substrate contamination) and to cause the cellulose fibrils to swell, allowing easy accessibility for enzymatic degradation by the fruiting fungi. B. CULTURALCONDITIONS

Ever since the identification of edible fruiting bodies of Basidiomacromycetes for consumption as food items, valued for their flavor and texture, there have been concerted efforts to exploit these fungi by way of artificial culturing. In many parts of the eastern and western

272

SOMASUNDARAM RAJARATHNAM ET AL.

hemispheres, research for culturing on commercial scales has gained impetus since the middle of the present century (Ho and Han, 1978). Much research data has accumulated on the culturing of various edible species. In order to unravel the properties and potentialities displayed by these fungi, it is necessary to consider, briefly, the aspects of culturing, including the factors affecting bioconversion efficiency. Reviews and text books have appeared describing the conditions required to cultivate and harvest the edible species (Chang and Hayes, 1978; Chang and Quimio, 1982; Rajarathnam and Zakia Bano, 1987a,b; Wood and Smith, 1988). In general, the cultivation of these edible fungi can be classified into five categories (Fig. 2):

A. Cultivation in wood; examples: Lentinus edodes, Pholiota nameko, Pleurotus ostreatus, Auricularia polytricha; B. Bottle cultivation; examples: Flammulina velutipes, Pleurotus ostreatus; C. Vat cultivation; examples: Pholiota nameko, Pleurotus ostreatus; D. Bed cultivation; examples: Agaricus bisporus, Volvariella volvacea; E. Polyethylene bag cultivation; examples: species of Pleurotus, Auricularia, and Coprinus (Shio et al., 1974; Ho and Han, 1978). The most prominent genera of basidiomacromycetes of worldwide commercial importance are compared in Table X. A number of species are exploited for commercial production of edible fruiting bodies (Fig. 3). A large number of factors have been found to be responsible for controlling growth, duration of cropping, and productivity. 1. Physical

a. Temperature and Relative Humidity. Temperature is a vital factor in regulating the growth and yield of any species. In fact, the geographic distribution of various species is based mainly on the temperature. For any species to grow, a certain temperature range must be maintained. In general, the required temperature range for mycelial ramification is wide and that for fructification is narrow. The required temperatures for different species can be determined by considering the temperature optima of the various enzymatic systems operating during two phases of the life cycle: spawn run and fructification. On the basis of their temperature requirements for fruiting, the cultured species can be classified into temperate (up to 20°C; e.g., Agaricus), subtropical (20-30°C; e.g., Pleurotus), and tropical (above 30°C; e.g., Volvariella) species. The role of relative humidity (RH) is mainly to prevent the desicca-

273

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

Substrate

Spawning

Culturing

Fruiting body

..... ....... ....... ....... ...... ..... ......

A

B

FIG. 2. Culturing methods of edible basidiomacromycetes. (A) Cultivation in wood, (B) bottle cultivation, (C] vat cultivation, (D) bed cultivation, (E) polyethylene bag cultivation.

TABLE X COMPARISON OF DIFFERENT EDIBLEBASIDIOMACROMYCETES~ Aspects

Agaricus

Volvariella

Pleurotus

Lentinus

Nature of substrate

Needs composting

Grows directly on rice straw

Colonizes rapidly on unfermented natural lignocellulosic wastes

Temperature requirement Total time taken to complete the production of yield Yield (g fresh/kg dry substrate Protein conversion efficiency from the substrate (protein yield glkg dry substrate) Loss during substrate preparation Cost of production (U.S. dollars per kg fresh) Uses of spent substrate

<20"C 60-90 days

32-36°C 20-30 days

15-30°C 25-35 days

Wood logs or sterilized sawdust/ bran 12-20°C -105 days

700

300-400

1000-1150

-250

18.41

11.40

21.73

-

ZO%,during composting 3.0

-

-

2.0

1.0

As casing material, as manure

As garden manure

As upgraded form of ruminant feed; can be recycled for Agaricus cultivation, for biogas, as garden manure, for paperlcardboard manufacture

0

From Rajarathnam and Zakia Ban0 (1988).

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

275

FIG. 3. Species exploited for commercial cultivation. (a) Agaricus bisporus on compost, (b) Volvariella volvacea on cotton waste, (c) Lentinus edodes on sawdust, (d) Lentinus edodes on wooden logs, (el Pleurotus flabellatus on rice straw, [f) Pleurotus flabellatus on rice straw (urban model), (g) Pleurotus flabellatus on rice straw (rural model), (h) Stropharia rugoso-annulata on wheat straw with casing, (i) Stropharia rugoso-annulata on wheat straw without casing, (j) Flammulina velutipes on sawdust-

276

SOMASUNDARAM RAJARATHNAM ET AL.

FIG. 3. D-F rice bran substrate, (k)Pholiota aegerita on sawdust-rice bran substrate, (1) Pholiota narneko o n sawdust-rice bran substrate, (m) Dictyophora duplicata on sawdust-bagasse and bamboo trash, (n) Coprinus cornatus on wheat straw. (Courtesy of Vedder, Chang, Nutalaya, Balazs and Kovacsne, Arita, Lin et al., and Lanzi.)

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

277

FIG. 3. G-H

tion of the substrate and rowing fruiting bodies. Although various ranges of humidities have been proposed to effect optimum yields (Block et a]., 1958; Zakia Bano, 1967; Cailleux et a]., 1976; Jandaik and Kapoor, 1976; Zadrazil, 1976a; Han et a]., 1977; Singh and Rajarathnam, 1977; Gramss, 1977; Rajarathnam and Zakia Bano, 1987b), a minimum of 80-90% RH serves to favor heavy fructification. In fact, it is the combination of temperature and RH optima that determines the growth and yield of the various species. When species of Pleurotus are cultivated outdoors, during winter in particular, when ambient temperature falls below lS"C, the fruiting bodies that are pro-

2 78

SOMASIJNDAKAM KAJAKATHNAM ET AL.

FIG. 3 . I-K

duced look thin and desiccated, even when enough water is made available in the substrate. This is possibly due to physiological dryness; the mycelial cells are apparently unable to utilize the substrate water, due to low ambient temperature. Likewise, at temperatures of 28-31°C encountered during summer, though mycelial growth is age enhanced normal fructification is affected, evidently due to low RH (Rajarathnam and Zakia Bano, 1987b). The yield of Pleurotus florida was influenced when the spawn run substrate was chilled (Leong, 1982).

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

FIG.3. L N

2 79

280

SOMASUNDARAM RAJARATHNAM ET AL.

b. Water Content and Particle Size of the Substrate. These are two interrelated factors that affect mycelial growth and, hence, the yield. Cultivation of Pleurotus on whole straw beds, did not yield well, because of the many air spaces that led to active desiccation (Rajarathnam and Zakia Bano, 1987b). Chopped rice straw pieces (2-3 cm long) with 75% water content have been found ideal for cultivation. Moisture content above 80% allows bacterial growth accompanied by fungal contaminations such as Coprinus and Pluteus. c. Light. Generally speaking, light is not a critical factor affecting growth and yield, and age-old methods recall the cultivation of mushrooms in caves and tunnels. Normally, these fungi yield well under shade or diffused light; with exposure to strong daylight, darkening of fruiting bodies occurs, possibly due to the oxidation of phenols, thus reducing the marketing quality. d. pH. An initial substrate pH of 6.5-7.0 is preferred to effect optimum mycelial growth; with the progression in mycelial growth, the substrate pH falls (-4.0) due to the secretion of oxalic acid (Garibova et al., 1982),which might serve to overcome the natural contaminants to a certain extent. Soaking rice straw in water for more than 20 hours before spawning results in an increase in substrate pH (-9.0), combined with an increased bacterial population, followed by the growth of weed molds (Rajarathnam and Zakia Bano, 1987b). e. Oxygen and Carbon Dioxide. The two phases of the life cycle are differently affected by 0, and CO,. In general, the requirement for 0, is greater during fructification in consonance with the fast rate of metabolic activity, involving the construction of fruiting bodies. Zadrazil and Schliemann (1977) reported the effect of carbon dioxide on several species of Pleurotus; many species grow faster when the atmospheric CO, level is high-even as high as 22%. This is a very remarkable feature of Pleurotus, and it stands in sharp contrast to Agaricus (Tschierpe, 1959), which is well known to be oxygen sensitive. Ginterova (1973) has also confirmed that the oxygen requirement of Pleurotus is lower than that of many other fungi. Pleurotus flabellatus and Pleurotus sajor-caju were found to display a differential response when grown in polyethylene bags of varying thicknesses and aeration, under identical conditions (Rajarathnam and Zakia Bano, 1987b). Under conditions of inadequate aeration and enrichment with CO, in the atmosphere, fruiting is not normal, thus affecting the yield and quality of fruiting bodies. Kurtzman (1978b) has devised a vertical tray system, covered on either side with wire mesh, for growing Pleurotus that allows adequate aeration and facilitates handling and harvesting.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

281

2. Chemical

a. Sugars and Phenols. Substrates rich in phenols adversely affect growth, while the presence of sugar-rich substrates after spawning invites contamination. Hot water treatment of the substrate results in the removal of these substances, thus improving the fungal growth. b. Cellulose, Hemicellulose, and Lignin. The relative amounts and chemical composition of these three polymers have a bearing on the growth and fructification. While composting leads to the formation of a lignin-humus complex that is attacked by Agaricus, substrates rich mainly in cellulose with decreased lignin contents support Volvariella, and Pleurotus can even utilize unfermented natural lignocellulosic wastes (Rajarathnam and Zakia Bano, 1991). c. Extra Nutrient Supplementation. Supplementation of the growth substrates with extra nutrients has invariably improved yields (Randle, 1983; Perry, 1987);however, it is essential to know the time of supplementation, as well as the nature and concentration of the supplement. In general, supplementing the growth substrate with nitrogen sources while spawning leads to substrate contamination, whereas supplementation after spawn run results in a significant increase in yield; an increase in protein content and changes in flavor and texture have also been observed (Rajarathnam et al., 1986). Organic nitrogen supplements such as yeast cake, cottonseed meal, and soybean meal increase the yields; addition of inorganic nitrogen sources-urea, in particularduring cultivation of Pleurotus adversely affected mycelial growth, possibly due to ammonification (Zadrazil, 1980a; Rajarathnam et al., 1986). The effect of delayed-release nutrients in augmenting the yields of Agaricus has been reviewed by Randle (1983). The addition of delayedrelease nutrients to the compost increased the yields of Agaricus (Schisler and Patton, 1978). Tokimoto and Kawai (1975) have examined the nutritional factors influencing fructification in Lentinus edodes. 3. Biological a. Contaminants and Diseases. Trichoderma, Plicaria, Papulaspora, Conidiobolus, Paecilomyces, and Peziza are sometimes fungal contaminants in compost beds of Agaricus (Gandy, 1974; Fletcher, 1977; Fergus, 1978). Similarly, Trichoderma, Monilia, Fusarium, Penicillium, Mucor, Sclerotium, Coprinus, Pluteus, Papulaspora occur in straw beds of Pleurotus. Parasitic diseases of the fruiting bodies (Gandy, 1985; Atkey, 1985) significantly affect the yield. It is only recently that

282

SOMASUNDARAM RAJARATHNAM ET AL.

bacterial diseases of Pleurotus due to Pseudomonas have been reported (Ferri, 1984). b. Pests. Slugs, flies, mycophagus flies, sowbugs, larvae, worms, and maggots are known to cause problems on cultures of mushrooms (Olah et al., 1979). White (1985) has dealt with the ways in which the major mushroom pests infest and colonize a mushroom and how they, and the pesticides used to control them, can affect cropping and subsequent yield. Thus, a large number of factors influence the growth and fructification of these fungi. Their capacity to grow on a range of treated or untreated lignocellulosic wastes is dependent on their relative growth rates, which serve to increase or reduce their rate of colonization in the substrate relative to the occurrence of other microbial populations. It is a concept of microbial ecology and fungal physiology-a thorough understanding of which is helpful in designing economic bioconversions and the conversion of the substrates to fruiting bodies.

C. BIOMASSCONVERSIONEFFICIENCIES Conversion of lignocellulosic wastes into edible fruiting bodies is the most vital aspect of any commercial endeavor. It is usually expressed as biological efficiency (BE), which is defined as the dry weight of fruiting bodies produced per dry substrate. This scale of expression allows a comparison of the results of various authors, overcoming the inaccuracies borne out due to the variations in the substrate moisture content and/or moisture content of the fruiting bodies under question. Biological efficiency varies from species to species, within the same species on different substrates, and, further, within the same species on the same substrate under different cultural conditions. The performance of these fungi on growth substrates is a manifestation of several of their characteristics [Table XI). Zadrazil (1976a) has reported a BE of 10% for Pleurotus ostreatus var. Florida on wheat straw. Pleurotus sajor-caju when cultured on wet, chopped, unfermented rice straw in perforated polyethylene containers on commercial scales had a recorded BE of 11.10% (Zakia Ban0 and Rajarathnam, 1982a). The authors could draw a significant correlation with the loss of dry weight of the substrate and production of biomass during different seasons of the year. Pleurotus ostreatus on a substrate of poplar and willow stumps effected a bioconversion of 9 to 20 kg per quintal of wood (Anselmi and Deandrea, 1978). A BE of 11.34% for Pleurotus ostreatus cultured on fresh coffee pulp has been reported (Martinez et al., 1985). During a 40-day incubation

TABLE XI

EDIBLEFRUITING FUNGICULTURED O N CEREAL STRAWSUBSTRATE, GROWTH CHARACTERISTICS, AND DIGESTIBILITY OF SUBSTRATEO.~ ~~

Species Agaricus arvensis Agaricus bisporus Agaricus bitorquis Agrocybe aegerita Auricularia judea Coprinus comatus Coprinus fimetarius Flammulina velutipes Kuhneromyces mutabilis Lentinus edodes Lepista nuda Mocrolepiota procera Macrolepiota rhacodes PIeurotus abalone Pleurotus cornucopiae Pleurotus eryngii Pleurotus ostreatus var. Florida Pleurotus flabellatus Pleurotus ostreatus Pleurotus sapidus Pleurotus sajo-caju Pholiota nameko Stropharia rugoso-annulata Volvariella volvacea

Growth on sterile straw substrate

Saprophytic colonization ability

1 1

1 1

4 4 4 5 5 5 5 5 5 4 5 5 5

3 3 3 3 3 3 3 3 2 3 3

1 3 2 2 2

3 3 2

1

Rate of decomposition

Fructification

Yield

Straws

Lignin

Increase Of straw digestibility

10 10 10 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11

12-13 12 13 13 12-13 12

20 20 20 20 20 20

12 12 13 12 12 12 13 13 13 13 13

14b 16a 16a 15b 14b 14a 14a 14a 14b 14-15b 14b 14b 14b 15-16a 16a 16a 16a 15a

17 18 18 17 17 18 17 18 18

20 20 21

6 5 6 6

7 7 7 8 8 7-8 7-8 7-8 7-8 7-8 7 7 7 9 9 8 9 9

19 19 19 19 19

21 21 21 21 21

6 6 6 5 6 6

9 9 9 8 8 8

11 11 10 10 11 10

12 13 13 13 12 13

16a 16a 15-16a 14-15b 14-15a 14-15a

19 19 19 17 19 17

21 21 21 20 21 20

6

Degradation

18

After Zadrazil (1965a). 1, Slow; 2, good: 3 , very good: 4 , low; 5, good; 6, very good: 7, low: 8, medium; 9, high; 10,for good yield, extra supplementation of the substrate necessary: 11,for good yield, extra supplementation of the substrate not necessary; 12, fructification below 16°C: 13, fructification above 18°C; 1 4 , yield of fruiting bodies low; 15, yield of fruiting bodies good; 16, yield of fruiting bodies very good; 17, no or low; 1 8 , good, 19; very good; 20, no or low: 21, good or very good. c a, Commercially cultivated: b, at experimental stage. 0

b

284

SOMASUNDARAM RAJARATHNAM ET AL.

period, P. sajor-caju, on rice straw under nonsterile conditions, effected a BE of about 10% (Bisaria and Madan, 1984). Over a 45-day period, P. ostreatus effected 10% bioconversion of peat-moss-based substrate (Manu-Tawaiah and Martin, 1986). The ability of Pleurotus sajor-caju to grow, degrade, and fructify on Saccharum munja, a profusely growing weed, has been reported (Gujral et al., 1987). One kilogram of dry cotton straw yielded about 700 g of fresh fruiting bodies after the first flush; there was a loss of 50% of the dry matter content of cotton straw due to fungal growth (Platt et al., 1982). Chopped rice straw with the addition of rice bran (10-15%) effected better biomass yield by P. ostreatus than pine sawdust or rice hull substrates (Takahashi, 1976). Pleurotus flabellatus, during culture on citronella bagasse (CB);wheat straw (WS);coffee pulp (CP);and the combinations CB + CP, CB + WS, and CP + WS, effected bioconversions of 13, 64, 41, 52, 83, and 96%, respectively (De Leon et al., 1983). Coprinus comatus on soya-enriched waste cellulosic residues {clarified sludge) from the bleached kraft pulp mill yielded about 25% of the wet substrate weight; the average weight of the fruiting bodies was two to three times higher on the soya-kraft sludge substrate than on conventional manure compost (Mueller et al., 1985). A substrate of pure oak sawdust, supplemented with 10% rice bran and filled into polypropylene bags, yielded 150 g of fresh Lentinus edodes per kilogram (Campbell and Slee, 1987). The BE of Lentinus edodes on supplemented sawdust is up to 145% in 6 months compared to 35% over a 6year period with natural log cultivation (Leatham, 1982; Royse et al., 1985). Thus, less time is required, greater BE is attainable, and fresh market consistency is maintainable on supplemented sawdust substra., compared to crop production on natural logs (Royse et al., 1985; Diehle and Royse, 1986; Miller and Jong, 1987). Bech (1979) has observed a yield of 700 kg (fresh) per ton of dry substrate with Agaricus bisporus while preparing a productive commercial compost. The BE of Volvariella varies from 25 to 65% (Chang, 1978b); the average efficiency is about 15% for straw substrate under natural conditions and 33% for cotton waste compost under controlled conditions. The term BE here refers to the yield of the fruiting bodies in proportion to the dry weight of compost at spawning, as described by Tschierpe and Hartman (1977). Volvariella volvacea, during its growth on rice straw, cotton waste (CW),sugarcane rubbish (SCR), SCR + wheat bran, and CW + rice straw, effected mushroom yields of 21.6,45.2, 12.4,21.2, and 27.0%, respectively of dry weight of compost (Hu et al., 1974). Quimio and Abilay (1983) have observed the abundant formation of fruiting bodies of Collybia reinakeana on rice straw-corn meal substrate only when cased with garden soil; they did not mention quan-

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

285

titative bioconversion of the substrate. A substrate of sawdust (of suitable broad-leaved trees] with 20-25% rice bran loaded into a plastic bag measuring 50 x 10 cm could yield 350 g of dried fruiting bodies of Tremella fuciformis; neither the biological efficiency nor the weight of substrate per bag was mentioned (N. L. Huang, 1982). On a substrate of sawdust, bagasse, and bamboo trash, the fresh yield of Dictyophora duplicata was found to be as high as 148% (Lin et al., 1982). A yield of 20% for Pleurotus ostreatus using Cassia substrate has been reported (Mueller, 1987). Hashioka and Arita (1978), while culturing Pholiota adiposa, Pleurotus cornucopiae, Lyophyllum decastes, Oudemansiella canarii, Panellus serotinus, and Coprinus micaceus, have observed the indispensability of steaming (1.6 kg for 60 minutes) the straw, which was found to produce an average yield of 1 to 1.5 and 0.6 to 0.7, respectively, for bottle and box cultures. These yields represent the ratio of fresh weight of fruiting bodies to dry weight of rice straw. Ratio yields of 3.5, 2.7, and 1.6 (based on culture bed weight) were produced by Flammulina velutipes, Ganoderma lucidum, and Polyporus species, respectively, on a substrate of fermented sugar cane bagasse (Nanci Togyo Co., 1979). A review with 50 references on the biomass of saprophytic basidiomycetes in litter and soil, and their effect on cycling of nutrients, other than carbon, especially in temperate woodlands is presented by Frankland (1982). More data on the BE are presented in Table XII. Thus, it can be seen that the biomass output per unit time is maximum with species of Pleurotus and lowest with species of Auricularia. These differences in the output yields are ultimately reflected by the growth substrate, the treatment received by the growth substrate, and, above all, the genetic constitution of the species in question, which determines its growth rate and capacity to degrade the substrate nutrients and to compete with other microbes on the growth substrate. More knowledge based on physiological studies of these species, related to the type, nature, and duration of substrate treatment and ultimately linked with the commercial feasibility, should aid in adding more and more species to the list of those already available for commercial exploitation. Transformation of plant energy into proteins by Pleurotus in quite remarkable when compared with sources of animal protein (Fig. 4). VI. Changes in the Growth Substrates during Degradation A. CHEMICAL

Fruiting fungi vary greatly in their relative abilities to colonize a lignocellulosic substrate, the type of substrate they can colonize, their

286

SOMASUNDARAM RAJARATHNAM ET AL.

Plant energy

Chicken

Pleurotus

MilWegg

Pork

0.113

0.111

0.048

0.031

Beef

u 1 0.030

FIG. 4. Energy transformation of plant wastes into proteins

rate of growth and degradation of the substrate, and ultimately the capacities to fructify and bioconvert the inedible waste into edible biomass (Table XII). The growth of a species on a substrate produces, in the course of time (incubation period), various changes in the different constituents of the lignocellulosic substrates (both qualitative and quantitative). Various criteria employed by several authors to estimate the varied degrees of substrate biodegradation and biotransformation are discussed here. 1. Loss of Organic Matter

Loss of organic matter (LOM) is the simplest criterion adopted to evaluate, in a crude manner, the extent of degradation of a growth substrate. It takes into account, that, as a result of growth of the fungus and decomposition of the substrate, certain amounts of CO, and H,O are lost during the metabolic activity of the cultured fungus. Studying the degradation of various substrates by Pleurotus ostreatus var. Florida and Pleurotus cornucopiae, Zadrazil (1976b) found that rape and sunflower substrates showed higher LOM, whereas relatively low decomposition rates were found for beech sawdust and rice husks. The differences in degrees of decomposition of the substrates are related to the duration of decomposition and the species under study, whether they produced the fruiting bodies or not (Zadrazil, 1977).

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

287

The LOM while culturing these fungi for their fruiting bodies is relatively high because of the exposure of the growth substrate to adequate levels of aeration to promote fructification and hence bioconversion of the growth substrate. This loss, in general, is greater during the short phase of fructification compared to the long phase of spawn run (Rajarathnam et al., 1979a; Wood and Fermor, 1981), evidently indicating the high rate of fungal catabolic activity that is associated with the anabolic activity of building up fruiting bodies. A correlation could be established between the yield and the LOM when PIeurotus sajor-caju was cultured on rice straw during all three growing seasons of the year (Zakia Ban0 and Rajarathnam, 1982a). Extra nitrogen supplementation of the growth substrate during fructification leads to increased LOM, in consonance with higher yields harvested (Zadrazil and Brunnert, 1980). The addition of extra nitrogen favors the degradation of cellulose, and active degradation of cellulose eventually results in enhanced yields. 2. Biomass Yield

The inoculated substrate under sterile conditions is colonized by the mushroom mycelium. The formation of fruiting bodies is not very common, possibly due to the inadequate availability of oxygen. Zadrazil (1978b) and Nicolini et al. (1987) have observed the formation of fruiting bodies on a number of substrates. It is our observation that species such as Pleurotus flabellatus form fruiting initials in the headspace of Erlenmeyer flasks, above the straw substrate, but can grow to completion only when the cotton plugs are removed (effecting exposure to the atmosphere). The subject of biomass yield assumes greater importance during the in vivo degradation of lignocellulosic wastes by the fruiting fungi as discussed. It is also true that the extent of degradation of the growth substrate is greater under conditions of fructification, which in turn is greatly dependent on atmospheric oxygen. Wood (1979) employed measurement of laccase activity to estimate the biomass of Agaricus mycelium in compost. It was shown that laccase activity is directly proportional to the mycelial growth in axenic compost cultures. 3. Substrate Solubility

Substrate solubility involves measurements of the release of watersoluble substances including sugars (Zadrazil, 1978a). Pleurotus cornucopiae and P. ostreatus var. Florida effected a decrease in water-soluble

TABLE XI1 GROWTHAND FRUCTIFICATION OF EDIBLEBASIDIOMACROMYCETES~

Species N

m

Production time

Substrate

Yield (kg fresh per 100 g dry substrate)

From nonaxenic culture

methods

m

1. Without/little pretreatment

Lentinus edodes

2.

Wood logs

Auricularia sp.

Wood logs

Tremella fuciformis

Wood logs

Kuehneromyces mutobilis

Wood logs

Pleurotus sajor-cajub

Unfermented rice straw

Some pretreatment Volvariella volvacea Pleurotus sp.

Rice straw, cotton waste Pasteurized/fermented cereal straws

3-6 years

spring/autumn 2-5 years spring/autumn 3-6 years 7 monthsiyear 2-10 years 3 harvests/year 35 days

-

7-10 weeks 5-6 weeks 8-12 weeks

40 -12

-20 27-30

110

6-10 40 100

Stropharia rugoso-annulata Coprinus fimetarius Long camposting process Agaricus bisporus

3.

Coprinus comatus Lepista nuda

From axenic culture methods Flammulina velutipes

Lentinus edodes

N

co W

Auricularia species

0

b

After Smith et 01. (1987). From Zakia Ban0 and Rajarathnam (1982b).

Cereal straw Straw bales (soaked in CaNO,)

10-40 weeks 4-5 weeks

-20 -60

Composted cereal straw/animal manure mixtures Composted straw Compost and straw mixture (1:l)

14-16 weeks

65-80

14-16 weeks 18-20 weeks

40-50 15-25

Sterilized sawdust and rice bran mixture (polypropylene bottles) Sterilized sawdust and rice bran mixtures (polyethylene bags) Sterilized sawdust and rice bran mixture (polyethylene bags)

12-20 weeks

70-100

4-6 weeks

60-100

8-10 weeks

70-75

290

SOMASUNDARAM RAJARATHNAM ET AL

substances and reducing sugars of wheat straw substrate during the initial period of incubation. After 20 days, there was a progressive increase in these water-soluble substances; after 120 days, there was an eight- and a fivefold increase in reducing sugars (at 25"C), respectively, for these two species of Pleurotus. These fungi start growing immediately after inoculation, utilizing, first, the available free sugars from the substrate. They then shift to the use of carbon from polysaccharides. Agrocybe aegerita had the least ability to degrade the substrates and to liberate water-soluble substances (Zadrazil, 1978a). The amount of water-soluble substances released from cotton straw increased from 19.5 to 57% during the first 2 1 days of growth of Pleurotus ostreatus var. Florida (Platt et al., 19821. 4. Hexosamine

Hexosamine content is defined as the estimated amount of glucosamine in the degraded substrate and is a measure of the synthesis of fungal biomass in solid-state degradation, because the cell walls are constituted mainly of chitin, which is composed of glucosamine. Rajarathnam (1981) has observed a progressive increase in hexosamine content of straw degraded aseptically by Pleurotus flabellatus up to a period of 75 days of incubation. There was an increase in the hexosamine content from spawning until the end of spawn run and a significant decrease during fructification, resulting from its involve: ment in the growth of fruiting primordia to form mature fruiting bodies. Plassard et al. (1982a,b) have also reported on the estimation of fungal biomass by measurement of fungal chitin. 5. Heat of Combustion

Zadrazil (1977) has studied the changes in heat of combustion in the substrate during solid-state fermentation by species of Pleurotus. Heat of combustion decreased only slightly during fermentation, perhaps due to differences in the rates of degradation of cellulose and lignin. Lignin has a higher heat of combustion (-5200 cal g-1) than cellulose (-4030 cal g- l). The loss of energy caused by fungal metabolism correlated with the extent of decomposition of straw. During the decomposition of beech and spruce wood by Pleurotus and Lentinus, combustion heat was practically unchanged even at a high degree of fungal decomposition; for both fungi it fluctuated around the values measured in nondegraded wood (Dobry et a ] . , 1986). Energy losses and bioenergetic

291

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

efficiencies of various strains of Pleurotus have been evaluated (Table XIII). 6 . pH

In general, the pH of the substrate decreases during its degradation. Thus, the starting pH of 6.5 to 7.0 of wheat/rice straw drops to a final pH of 5.1 (Pleurotus ostreatus var. Florida) (Zadrazil, 1978a) or even as low as 4.2 (P. flabellatus) (Rajarathnam, 1981). Secretion of oxalic acid by Pleurotus and Agaricus (Garibova et al., 1982) adds to the decrease in substrate pH. Daugulis and Bone (1977) did not observe any drop in pH during culturing of Pleurotus sapidus on maple and cedar barks, which was indicative of lack of growth. This result is consistent with other studies (Umezurike, 1970; Carroad and Wilke, 1977; Moo-young et al., 1977) of cellulolytic fungi growing on carbohydrate substrates, in which the pH drops significantly during growth of the organism and then rises after the cessation of active growth. 7. Sugars, Amino Acids, and Phenols

A typical system of solid-state substrate degradation is effected by Pleurotus flabellatus (Rajarathnam, 1981; Rajarathnam et al., 1987); an increase in the incubation period (under sterile conditions) resulted in the release of sugars and amino acids, whereas phenol levels showed a decline. In a study of the bioconversion of rice straw into fruiting bodies by Pleurotus flabellatus on a mass scale (under nonsterile conTABLE XI11 ENERGYLOSSES(EL) AND BIOLOGICAL ENERGETIC EFFICIENCY (BEE) OF VARIOUSPleurotus STRAINS= Spent substrate energy

Fruiting body energy

EL

EL

BEE

Strain

(kJl

(kJ)

(kJ)

(%I

(%1

014

242 265 235 255 319

59.6 58.2 60.5 50.7 34.0

181 160 187 177 139

37.4 33.1 38.8 38.6 28.9

24.8 26.7 24.4 21.4 19.7

030 096 133 159 a

After Ginterova and Lazarova (1989). Inoculated substrate energy

=

483 kJ,

292

SOMASUNDARAM RAJARATHNAM ET AL.

ditions), there was a constant increase in the free sugar content from spawning until the end of cropping; amino acids increased until the end of spawn run (the onset of formation of fruiting primordia). During fructification their content decreased, evidently due to their utilization in the construction of fruiting bodies. The phenol levels showed a progressive decrease from the spawning until the end of cropping (due to the secretion of oxidizing enzymes by the fungus) (Rajarathnam, 1981). 8. Nitrogen

During the course of degradation of wheat straw under sterile conditions, by monocultured species of Pleurotus and Stropharia, the amount of nitrogen (N) in the substrate increased relatively, due to CO, loss. The greatest difference in the relative N accumulation was detected after 2 1 days with Pleurotus cornucopiae and Stropharia rugosoannulata; the increase in N, however, did not correlate with the in vitro digestibility (Zadrazil, 1977). An increase in the N content of wheat straw, orange peel, and grape stalks was also observed during the growth of Pleurotus ostreatus, but a tendency for reduction of N content was observed under the conditions of fructification (e.g., on wheat straw and orange peel + grape stalks) (Nicolini et al., 1987). A significant decrease in N content was observed during the fructification phase while culturing Pleurotus flabellatus on rice straw under nonsterile conditions to effect maximum bioconversion (Rajarathnam et al., 1979b).As supporting experimental evidence for this observation, extra supplementation of a suitable N source to the straw substrate during the fructification resulted in an enhanced mushroom yield and protein content of the fruiting bodies (Rajarathnam et al., 1986). 9. Cellulose, Hemicellulose, and Lignin Species of fruiting fungi resort to the degradation of cellulo-hemicellulosic polymers soon after the depletion of available free sugars in the growth substrate. Species of white-rot fungi display a pronounced ligninolytic activity and the degradation of lignin favors increased degradation of holocellulose from lignin-rich growth substrates. The decrease in relative amounts of these components is smaller under sterile conditions due to a restricted supply of oxygen. Decreased amounts of these components in wheat straw, rice straw, orange peel, grape stalks, and sugarcane bagasse have been reported (Riaz et al., 1977; Zadrazil, 1978a; Platt et al., 1981; rung and Fahey, 1983; Rolz et al., 1987). The decrease in cellulo-hemicellulosic content of the growth substrate in vivo may be more pronounced and varied, depending on the species

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

293

and yield produced. The decrease in the cellulose content was greater during fructification of Pleurotus ostreatus on rice straw and poplar sawdust (Hong, 1978) and Pleurotus flabellatus on rice straw (Rajarathnam, 1981). This is a result of the high rate of metabolic activity of the mushroom during fructification. The decrease in lignin content was prominent during the long phase of spawn run wile culturing Pleurotus flabellatus on rice straw. Similar observations have been reported by Geritts (1969) while growing Agaricus bisporus on compost. Sermanni et al. (1979) also noticed a progressive decrease in cellulose content from spawning until the end of cropping of Pleurotus ostreatus. Zadrazil (1978a) found that the absolute quantities of cellulose, hemicellulose, and lignin complexes (calculated on a dry weight basis) remain unchanged during growth and fructification of Pleurotus on wheat straw. Pleurotus sajor-caju, during its growth on Saccharum munja with and without rice straw, effected more cellulose decomposition in the former than in rice straw. Different strains of the same species degraded varying amounts cellulose, and the decrease was in consonance with the yields produced (Gujral et al., 1987). Phanerochaete chrysosporium degraded cellulose faster with organic N sources, whereas glucose transiently stimulated degradation of lignin (Reid, 1983). Otjen et al. (1987) have assessed the ability of 30 white-rot basidiomycetes to selectively degrade lignin. Data on the degradation of cellulose and lignin by white-rot fungi are reviewed by Eriksson, 1981. A mixture of ground straw and sulfite waste liquor inoculated with Trametes versicolor, after an 8-week incubation at 28"C, decreased lignin and cellulose by 20 and 30%, respectively (Trojanowski and Leonowicz, 1985). Kern (1983) has also reported on the degradation of lignosulfonates by white-rot species. Vine sprout wastes are efficiently degraded by Coriolus hirsutus, Coriolus versicolor, and Phanerochaete chrysosporium (about 30-40% lignin) (Kvestiadze et al., 1988). Heterobasidium annosum, Pleurotus ostreatus, and Stereum purpureum caused the degradation of hardwood h a f t lignin as shown by spectrophotometric studies at 280 nm; Stereum purpureum demonstrated the highest ligninolytic activity (Stevanovic-Janezic et al., 1988). During the culture of Volvariella volvacea on a cotton waste compost, cellulose and hemicellulose were utilized during mycelial growth but not during fructification; lignin remained more or less constant throughout the culture period (Kwan and Chang, 1981). Hong (1978) observed a progressive decrease in lignin content of both rice straw and poplar sawdust during growth and fructification of Pleurotus ostreatus. Decreases have been reported in lignin content of flax shieve (Sharma, 1987) during growth of several strains of Pleurotus

294

SOMASUNDARAM RAIARATHNAM ET AL.

and of wheat straw (Zadrazil, 1976a,b) during growth of Pleurotus. All these reports indicate that the decrease in lignin content is greatest during spawn run. Possibly, the fungus forms a lignin-humus-protein complex that is utilized rapidly by the growing mycelium (Gerrits, 1969).Changes in lemon grass and citronella bagasse by white-rot fungi and in pine sawdust by brown-rot fungi have been studied (Tables XIV and XV). The ability of 45 strains of basidiomycetes to degrade wheat straw and beechwood has been reported; Pleurotus eryngii caused a TABLE XIV

BIODEGRADATION OF LEMON GRASSAND CITRONELLA BAGASSEB Y WHITE-ROT FUNGIO Lemon grass Soluble Lignin lossb solids (9)

Species Bondarzewia berkeleyi Coriolus versicolor Pleurotus flabellatus Sporotrich um pulverulentum Coprinus fimetarius Pycnoporus sanguineus Phanerochaete chrysosporium Flammulina velutipes Ganoderma applanatum Ischnoderma recinosum Dichomitus squalens Agrocybe aegerita Controlc

13.6 12.0 16.2 15.2 10.2 14.4 15.5 13.2 15.5 13.8 16.8 18.8 16.2

Bondarzewia berkeleyi Coriolus versicolor Pleurotus flabellatus Sporotrichum pulverulentum Coprinus fimetarius Pycnoporus sanguineus Phanerochaete chrysosporium Flammulina velutipes Ganoderma applanatum Ischnoderma recinosum Dichornitus squalens Agrocybe aegerita Controlc

13.4 13.3 15.1 13.2 17.7 14.8 12.2 14.1 14.8 13.5 12.4 12.5 23.3

Cellulose lossb

Hemicellulose lossb

40.7 29.0 22.6 35.6 17.8 9.7 1.7 6.7 21.4 0.0 5.4 0.0

37.0 27.8 32.4 31.8 27.3 24.7 15.7 16.7 38.6 14.1 8.3 7.9

64.2 55.4 42.6 56.2 33.9 31.3 40.9 19.3 18.2 30.5 20.3 19.4

-

-

IVDMD 34.7 30.0 28.7 26.1 23.3 18.7 16.5 16.5 15.7 13.9 13.4 12.9 12.7

Citronella bagasse

~

~

~

37.2 45.5 30.0 44.8 25.4 27.3 32.0 19.8 22.0 27.5 5.0 19.1

~

~

_

_

29.8 31.5 36.6 31.1 1.5 13.9 15.7 2.1 0.0 7.7 1.8 1.5

42.6 32.3 45.0 35.2 0.0 20.2 18.1 22.7 12.7 26.5 26.7 15.5

-

__

43.3 39.7 36.6 34.1 33.7 28.3 26.3 21.7 18.8 16.9 14.8 13.0 19.3

_ ~

.After Rolz et al (1986) bPercentage of original sample Control values for uninoculated substrate which received same sterilization process, incubation, and aeration

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

295

TABLE XV CHANGES IN PINE SAWDUST DURING SSF B Y THREEBROWN-ROT FUNGP

Strain Uninoculated Gloeophyllum trabeum

Wolfiporia

COCOS

Lentinus iepideus

Incubation time (weeks) 0" 1

4 8 1 2 4 8 1 n

L

4 8

Lossb

pH

Weight

Cellulose

Hemicellulose

Lignin

5.0 3.6 3.5 3.4 3.3 2.9 3.6 3.3 3.2 4.0 3.9 3.6 3.5

0 11.9 15.2 23.3 39.4 9.2 11.8 13.6 17.8 9.7 12.8 13.6 21.9

0 (45.4)

0 (28.7) 19.2 27.9 40.4 58.9 13.3 21.3 22.0 34.7 17.0 19.9 21.6 34.3

0 (25.7) 3.3 4.3 6.6 8.7 4.1

10.8 11.7 20.5 42.8 9.0 9.2 11.1 14.9 6.2 12.6 12.8 19.7

aAfter Agosin et al. (1989). bhsults are expressed as percentage of original component. .Values in parentheses indicate the content of each structural component in sound wood

preferential degradation of lignin during the transformation of straw (Valmaseda et al., 1990). 10. Ash and Minerals Total ash was found to show a relative increase from the time of spawning until the end of cropping because of the constant utilization of organic matter. However, the total amount of ash could be assumed to remain constant throughout the life cycle, realizing that a fraction of it also enters into the developing fruiting bodies. Hence, in order to know the exact magnitude of a constituent from a unit weight of substrate, we suggest calculation of the degradation analytical values on a constant ash basis, with all values expressed as percentages of the dry matter during spawning, as described by Geritts (19691while culturing Agaricus bisporus. Kurtzman (1976) has employed SiO, content of the substrate for evaluating N balance in straw substrate, during the growth and fructification of Pleurotus ostreatus. A general decrease in the P, K, Ca, Mg, and Fe contents of rice straw, wheat straw, and poplar sawdust has been observed during the growth of Pleurotus ostreatus (Hong, 1978) and Pleurotus ostreatus var. Florida (Zadrazil, 1978a). These elements enter into the construction of the fruiting bodies and even par-

5.0 6.2 3.8 5.0 6.3 7.0 9.2

296

SOMASUNDARAM RAJARATHNAM ET AL.

ticipate in prosthetic groups during enzymatic reactions within the harvested fruiting bodies. 11. Acid and Alkali Extracts

Zadrazil (1978a) has employed spectroscopic analyses to trace the changes of the wheat straw substrate samples degraded separately by Pleurotus ostreatus var. Florida and Pleurotus eryngii at various growth stages. The individual fractions were separated and their solutions were read spectroscopically. With the progressive growth of both species, there was a clear leveling-off of the extinction curves at 280 nm, indicating that water-soluble phenolics were attacked by both species. A similar tendency was observed with wheat straw substrate in 0.1 N NaOH solution and in fulvic acid. A humic acid fraction peak remained very distinct at 280 nm until the end of cropping, in contrast to the NaOH and fulvic acid fraction peaks. The spectroscopic characteristics of the extracts are influenced by the extent of mycelial growth, changes in the substrate, and the reduction in the phenolics. 1 2 . Dry Matter Digestibility

Dry matter digestibility is a simple criterion for assessing the extent of substancednutrients available in a substrate for microbial growth. Rumen microflora act on these substrates, thereby making them digestible by ruminants. The terms in vitro dry matter digestibility (IVDMD) and in vitro dry matter enzymatic digestibility (IVDMED)are also used to refer this property. A detailed insight into this subject has been reported by Zadrazil (1979a), who observed that some white-rot fungi (including species of Pleurotus) can colonize wheat straw, liberate water-soluble substances from the substrate polymer complex during solid-state fermentation (Zadrazil, 1976c), and use lignin (Rypacek, 1966). During this process, the digestibility of degraded substrates increases for ruminants (Schanel et al., 1966; Pidgen and Heaney, 1969; Hartley et a]., 1974; Burrows et a]., 1979; Jilek et a]., 1979). To confirm the increased ruminant digestibility of a biodegraded lignocellulosic waste, it is necessary to feed ruminants for long periods (many months), which inevitably requires huge amounts of degraded (spent) substrate. Furthermore, the need to maintain a large number of replicates would also contribute to the consumption of spent substrate. To avoid this difficulty, and to predict digestibility using small amounts of spent substrates in a laboratory, stepwise determination of enzymatic digestibility has been advocated by various authors. Instead of rumen fluid, several authors have used fungal cellulases for the prediction of

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

297

in vitro digestibility (Adegbola and Paladines, 1977; Goto and Minson, 1977; Mc Leod and Minson, 1980; Roughan and Holland, 1982). The in vitro digestibility of wheat straw at pH 9.0 with a moisture content of 65% in solid-state fermentation by Coprinus sp. at 37°C for 2 1 days increased from 40 to 65.5%. However, this period did not conform to the time required for maximum delignification. Total delignification is not a proposed prerequisite for maximizing digestibility (Yadav, 1988). Most of the fruiting fungi significantly improved digestibility of biodegraded residues of Citronella bagasse and lemon grass (Rolz et al., 1986). Degradation of oat straw by Pleurotus ostreatus and Pleurotus cornucopiae did not increase digestibility, possibly due to slow growth rates of the fungus. Digestion of straw degraded by Pleurotus ostreatus and stropharia was not affected (Friedel et al., 1983). However, this is surprising in view of the increased solubility and decreased lignin content of the substrate. Sommer et al. (1978) made a similar observation-a decrease in digestibility of lignin and cellulose after culturing with and harvesting of Pleurotus ostreatus. It is only recently that Tsang et al. (1987) reported on the in vitro digestibility of spent wheat straw remaining after growth and harvest of various strains of Pleurotus. Using commercial cellulase and glucosidase (Roughan and Holland, 1982), the authors concluded that the simultaneous production of mushrooms and a highly digestible delignified residue from straw does not appear to be practical. The dry weight of the mushrooms produced was much lower than the weight loss of straw; there was no apparent correlation between mushroom yield and weight loss, or digestibility of spent straw. Zadrazil (1977) showed that digestibility of straw can be increased by a longer fermentation and by foregoing the harvest of fruiting bodies. B. BIOCHEMICAL

The chemical changes brought about by the basidiomycetes on a growth substrate (discussed above) are the ultimate result of the enzymatic potentialities of the fungus. 1. Degradatory Enzymes

The ability of a basidiomycete to grow on a wide spectrum of lignocellulosic materials reflects its ability to secrete a range of degradatory enzymes, both saccharifying (Hong, 1976; Trutneva, 1978) and oxidizing [Gavarilova and Grigor’eva, 1983; Vetter, 1983; Bollag and Leonowicz, 1984). While assaying the activity of these degradatory en-

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zymes during growth on natural lignocellulosic wastes, it is essential to observe (1) the method of enzyme preparation, (2) the method adopted for enzymatic assay, and (3) the unit of expression of enzymatic activity. To ensure universal comparisons among values of enzymatic activities reported for various species/strains, it is advantagous to follow one defined set of conditions for the functions cited above. One such method, followed for extraction of cellulase and laccase/protease from rice straw substrate during culturing with Pleurotus flabellatus, has been described (Rajarathnam, 1981). The efficiency of enzyme extractions in acetate buffer (50 mM in the pH range 4.0-6.0) indicated that the extractions were very efficient at pH 5.4 and all the enzymes (Cl, C,, FPD, and P-glucosidase) studied were stable at this pH. The acetone powder prepared from the rice straw substrate during growth of Pleurotus flabellatus upon extraction with 100 mM phosphate buffer (pH 7.1) at 5°C for 1 hour, yielded stable activities of laccase and protease (Rajarathnam et al., 1987a). a. Cellulases. Pleurotus flabellatus secretes various classes of cellulases, namely C,, C,, and P-glucosidases (necessary for complete saccharification of crystalline native cellulose, as proposed by Reese et al., 1950), while being cultured on wet chopped unfermented rice straw (Rajarathnam, 1981). The development of carboxymethylcellulase (CMCase) activity on waste paper substrate, related to the growth and fructification of Pleurotus sajor-caju, has been studied (Thayumanavan, 1982). Cellulase activity was high during the onset of fruiting body formation; it was maintained for some time, and then decreased as the yield decreased. Forty-seven cultures of Pleurotus were screened for their ability to secrete cellulase(s) (Ginterova et al., 1980). Cellulase activity was measured by liquefaction of gel from a modified cellulose (Zemek and Kunaik, 1978). The best producers of cellulase were obtained from cultures of Pleurotus cornucopiae and Pleurotus japonicus. The in vitro activities of the cellulases of these fungi are low, however, in comparison with that of Trichoderma, the international standard for cellulase production. Under in vivo cultural conditions, the enzymatic activities vary, depending on the rate of growth of the species in question, the substrate on which it is grown, and the stage of growth-being maximum during fructification and also at the end of cropping (Rajarathnam, 1981). A positive correlation was found to exist between the yield of Volvariella species and production of cellulase(s) by the fungus (Rangaswamy and Kandaswamy, 1976). In a study of the dynamics of exocellular hydrolases of Agaricales

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species (Flammulina velutipes, Pleurotus ostreatus, Armillaria mellea, and Lentinus tigrinus), there was a distinct relation between the accumulation of reducing sugars and cellulolytic activity in some cultures, which was also dependent on the age of the culture (Mel’nichuk and Danilyak, 1981). When cultured on a substrate of filter paper and cotton, the endoglucanase activity was highest on the loth 6th, and 3rd days of mycelial growth for Flammulina velutipes, Lentinus tigrinus, and Pleurotus ostreatus, respectively. The highest level of exoglucanase activity was observed in culture filtrates of Lentinus tigrinus, Pleurotus cornucopiae, Flammulina velutipes, and Hypholoma sublateritium (Meln’chuk and Danilyak, 1981). The activities of carboxymethylcellulase, avicelase, xylanase, polygalacturonase, p-D-ghcosidase, and pectin transeliminase of Lentinus edodes on a sawdust medium as a function of pH and temperature have been studied (Hong et a]., 1986). Production of cellulolytic enzymes from Armillaria mellea, Panus tigrinus (Danilyak et al., 1989); Chrysosporium lignorum (Canevascini and Gattlen, 1981; Phillip et al., 1984), Daedalea guersina (Cavazzoni and Manzoni, 1988), Irpex lacteus (Kanda et al., 1983; Kubo and Nishizawa, 1983; Cavazzoni and Manzoni, 1988), Polyporus hirsutus (Danilyak and Katsan, 1987; Danilyak et al., 1989),Polyporus versicolor (Danilyak et al., 1983, 1989; Semichaevskii et al., 1984, 1985, 19881, Polyporus species (Nigam and Prabhu, 1988), Phanerochaete chrysosporium (Jafelice et a]., 1988,1990), Sporotrichum celIuIophilum (Kim et al., 1985; Kinoshita et al., 1986; Tamada et al., 1986), Sporotrichum dimorphosphorum (Klesov el al., 1985, 1986a,b), Sporotrichum pulverulentum (Chkhartishvili and Kvachadze, 1985; Ishihara et al., 1985; Kvachadze et al., 1985; Urushadze et al., 1989), Sporotrichum thermophilum (Canevascini and Gattlen, 1981; Canevascini et al., 1983; Kashiwagi et al., 1985; Cossar and Canevascini, 1986; Bhat and Maheshwari, 1987), and Termitomyces clypeatus (Rouland et al., 1988; Sengupta and Sengupta, 1990) has also been reported. The cellobionolactone formed due to the phenolic compounds in the growth medium effected an increase in cellulase production by Tramates versicolor (Mueller et al., 1988). The carbohydrases of the mushroom spawn from Pleurotus flabellatus have been studied (Rajarathnam, 1981). The 3-month-old spawn (used for obtaining optimum yields of the mushroom on rice straw) did not show CMCase or hemicellulase activity, but did show fairly high amounts of P-glucosidase activity. It was also rich in soluble carbohydrates and reducing sugars. The absence of a polysaccharase system might be due to the presence of adequate amounts of soluble carbohy-

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drates and reducing sugars. Various enzymes noted at various stages of spawn growth of Pleurotus sajor-caju occurred in the order polygalacturonase transeliminase (PGTE) > polygalacturonase (PG) > cellulase (C,) > polymethygalacturonase (PMG) > pectin transeliminase (PTE) (Rawal et al., 1981). The order of enzymes detected in Calocybe indica during the spawn growth was PG > PMG > C, > PGTE > PTE (Doshi et al., 1987). A new strain of Sporotrichum ceIlulophyIlum (isolated from soil) produced as much cellulase activity at 50°C for 3 days as Trichoderma viride for 7-10 days at 30°C; characterization of multiple cellulases is reported by their enzymatic properties and immunological relationship (Kinoshita et al., 1991). The role of oxalic acid in the culture depolymerizing system of brown-rot fungi has been investigated; Gloeophyllum trabeum, which accumulates the lowest amount of oxalic acid during decay of pine holocellulose, showed the highest polysaccharide-depolymerizing activity (Espejo and Agosin, 1991). b. Hemicellulase(s). These include xylanase, arabinase, and mannase, all of which are involved in degradation of the corresponding polymers representing the hemicellulose fraction of the growth substrates. In a study of the development of hemicellulase activity, as measured on hemicellulose-B isolated from the rice straw substrate, Pleurotus flabellatus was found to display higher hemicellulase activities at the end of the spawn run and maximum activity was reached after cropping (Rajarathnam, 1981). This could be a result of corresponding amounts of hemicelluloses depleted from the straw substrate. A similar trend in the development of hemicellulases was observed when PIeurotus sajor-caju was cultured on rice straw (Madan and Bisaria, 1983). The secretion of xylanase by Pleurotus ostreatus (Hong, 1976) and Pleurotus ostreatus var. Florida on flax shive (Sharma, 1987) has also been observed. An enzyme preparation from Agaricus bisporus hydrolyzed rice straw hemicellulose to xylobiose, glucose, and arabinose, but not to xylose (Hashimoto and Takahashi, 1970). The production of hemicellulases/xylanases has also been observed by several authors (Adhikary et al., 1982; Margaritis et al., 1983; Durand et al., 1984; Mora et al., 1986; Wang and Wang, 1989). A major xylanase detected in cultures of Lentinus edodes grown on a commercial oat wood medium has been extracted, purified, and characterized with a view to the involvement of the xylanolytic enzymes of basidiomacromycetes in industrial bioconversion (for example, production of edible mushrooms), biopulping wood, retting flax fibers, upgrading or refining chemical or mechanical pulps, enhancing animal feed digestibility, converting lignocellulosic sugars into liquid feed stocks or fuels, and processing food (clarification of juices) (Mishra et a]., 1990).

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c. Proteases. The fruiting bodies, on a dry weight basis, contain about 20-35% protein, with a conversion factor of 4.38. This protein is built essentially from nitrogenous substances obtained from the substrates; only a fraction of the substrate is in the form of free amino acids and the rest is evidently complexed in the protein form. Obviously, the species in question would degrade the available substrate proteins to build up the fruiting bodies. Pleurotus flabellatus was found to secrete proteases when grown in vitro and in vivo on rice straw (Rajarathnam, 1981; Rajarathnam et al., 1987). However, the in vivo activity was greater, since this set of cultural conditions involved aerobic production of fruiting bodies, with an economic turnover of 650 g fresh mushrooms per kilogram of dry straw. Protease activity increased continuously during the spawn run, and the activity was enhanced during fructification. A decrease in protein content in the straw substrate correlated with an increase in protease activity (Rajarathnam et al., 1987). There was a similar increase in protease activity during differentiation of other fungi (Hoffmann and Huttermann, 1975; Schwalb, 1975). Evidently, the increase in protease activity enables the fungus to degrade more protein in the straw substrate and to divert the degradation products toward the formation of fruiting bodies. Flammulina velutipes cultivated in a synthetic liquid medium produced proteases with thrombolytic activity, milk coagulating activity, and caseinolytic activity. Peptone was more effective than urea in supporting protease production (Falina, 1980). The secretion and properties of proteases from Phanerochaete chrysosporium (Eriksson and Pettersson, 1982, 1988; Kaneda et al., 1985; Chao and Gruen, 1987; Elshafei and Penninckx, 1989; Dosoretz et al., 1990b); T. columbetta (Lamaison et al., 1980);Boletus, Clitocybe, Coprinus species (Denisova et al., 1989); Flammulina velutipes (Gavrilova et al., 1980); Flammulina velutipes, Auricularia auricula-judae, Lentinus edodes, and Sporotrichum dimorphosporum (Tapia et al., 1981);and Pleurotus species (Hong and Kim, 1981) have been investigated. d. Other Enzymes. Ffammulina velutipes is reported to secrete pectinase and amylase on wood fillings and wheat bran (Wang and Wang, 1989). Paneolus papillionaceus and Agaricus bisporus growing in starch and glucose media produced the highest levels of inulinase and invertase (Mukherjee and Sengupta, 1985). e. Phenoloxidases and Ligninolytic Enzymes. Certain mushroom species can secrete various types of oxidizing enzymes, such as phenoloxidases, peroxidases, and catalases. The lignocellulose-degrading activities exhibited by species of Auricularia, Coriolus, Flammunlina, Favolus, Lentin us, Lenzites, Pholio ta, PI eurot us, Stereum , Tramates,

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Tremella, and ?iyromyces have been described (Toyama and Ogawa, 1974), and compared with those of other white-rot fungi. The phenoloxidase activity of Pleurotus flabellatus is of a laccase type and is unable to oxidize tyrosine (Rajarathnam et al., 1987). The inability of laccase-deficient strains of Sporotrichum pulverulentum to degrade lignin (Ander and Eriksson, 1978) or polymeric dyes (Glenn and Gold, 1983), which are used as substrates for the fungal lignin degradation system, and the involvement of laccase in demethoxylation processes (Ander et al., 1983) suggest that laccase has a role in the degradation of lignin. Laccase activity in the rice straw substrate increased during the spawn run and reached a maximum at the beginning of fructification; this can be deduced from the decrease of corresponding amounts of lignin (estimated chemically) in the straw substrate (Rajarathnam et al., 1987). A similar increase in laccase activity during the spawn run of Agaricus bisporus in compost has been observed (Turner et al., 1975). Sermanni and Luna (1981) reported the comparative laccase activities of Agaricus bisporus and Pleurotus ostreatus. Activity was greater in the former than in the latter, probably due to the higher lignin content of the substrate. These authors have also described the properties of the laccases from these two types of mushrooms. Laccase was more prominent and peroxidase activity was not significant when Pleurotus ostreatus var. Florida was grown on cotton straw (Platt et a]., 1984). The activities reached a maximum only after 6-8 days of growth. Different strains of Pleurotus ostreatus showed various levels of laccase activity; this appears to be the reason for different degrees of lignin and cotton straw degradation. Laccase secretion by Pleurotus ostreatus on wood meal-rice bran increased during mycelial growth and then declined rapidly at the start of fruiting (Iwahara et al., 1981). Extracellular ligninases isolated from pure cultures of Pleurotus ostreatus displayed substrate specificity for guaiacol and hydroquinone and yielded a positive syringaldazine test (Hira et al., 1978). A mixed culture of Pleurotus ostreatus and Fusarium culmonarum produced the highest levels of cellulase and ligninolytic enzymes in a synthetic medium (Afans’eva and Kadyrov, 1980). The enzymatic activity of the white-rot fruiting fungi in lignin removal was stimulated by the addition of ferulic acid and 3-indolylacetic acid to the nutrient medium (Adamski and Zielinski, 1985). Pleurotus ostreatus grown on fir-wood meal produced the highest levels of polyphenoloxidases, peroxidases, and laccase (Feniksova et al., 1972). The dependence of production of humus-like substances on extracellular phenoloxidase activity has been reported (Haars et al., 1985). Several other authors have also observed the secretion of oxygenases and peroxidases by the white-rot

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species (Ceruti-Scurti et a]., 1981;Higuchi et a]., 1983;Glenn et al., 1983,1989;Leisola et al., 1985;Kuwahara and Asada, 1987;Morohoshi et al., 1989). Ligninase excretion by mutant strains of Phanerochaete chrysosporium hyperproducing cellulases (Galliano et al., 1988) and production and properties of ligninases from Coriolus, Merulius, and Polyporus species and Phanerochaete chrysosporium (Biswas-Hawkes et al., 1987;Haemmerli et a]., 1986;Sanglard et al., 1986)have been reported. Most of our knowledge on the ligninolytic enzymes is due to the findings on Phanerochaetae chrysosporium by Kirk and colleagues from the University of Wisconsin. An extracellular enzyme from Phaerochaete chrysosporium in the presence of added H,O, catalyzes oxidative C-C bond cleavage of lignin model systems as well as in spruce and birch lignins (Tien and Kirk, 1983).The relationship between growth, nutrient nitrogen assimilation, and the appearance of ligninolytic activity has been examined in stationary batch cultures of Phanerochaete chrysosporium, grown under conditions optimized for lignin metabolism. Appearance of ligninolytic activity occurs irrespective of the presence of lignin; lignin did not induce additional activity. Addition of NH,+ to cultures immediately prior to the appearance of the ligninolytic system delayed its appearance, suggesting that the NH,+ interfered with the synthesis of the enzyme system. It is suggested that all essential protein components of the ligninolytic enzyme system are synthesized as a part of a series of physiological (secondary metabolic) events that are initiated by the nutrient nitrogen starvation (Keyser et a]., 1978). Glenn et al. (1983)have also characterized the ability of hydrogenperoxide-dependent enzymes to oxidize veratryl alcohol to veratraldehyde, which can be detected in culture fluids, following fungal growth, under conditions of nitrogen or carbon limitation that allow expression of the ligninolytic system. A strain of Phanerochaete chrysosporium produces high levels of lignin peroxidase in a nitrogen-sufficient medium with glycerol as the major carbon source (Buswell et al., 1984). Fungal strains that grew poorly on glycerol produced the highest titers of lignin peroxidase and the enzymatic activity was maximal when the initial pH of the culture medium was set at 5.0 (Roch et al., 1989). Extracellular lignin degrading enzymes isolated from Phanerochaete chrysosporium are found to catalyze nonstereospecifically, several oxidations in the alkyl side chains of lignin-related compounds, oxidation of benzyl alcohols to aldehydes or ketones, intradiol cleavage in the phenylglycol structure, and hydroxylation of benzylic methylene groups (Tien and Kirk, 1984).The enzyme also catalyzes oxidative

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coupling of phenols, perhaps explaining the long-recognized association between phenol oxidation and lignin degradation. A proposed scheme for enzymatic degradation of lignin is illustrated in Fig. 5 (Leisola and Garcia, 1989). Extracellular enzymes associated with lignin degradation by Phanerochaete chrysosporium are lignin peroxidase (LIP), manganese peroxidase (MnP), and the H,O,-generating enzyme glyoxal oxidase (Kirk and Farrell, 1987). Lip oxidizes aromatic nuclei of lignin to cation radicals that react nonenzymatically, resulting in cleavages in both the aliphatic side chains and the aromatic nuclei. MnP, related to Lip, oxidizes Mn2 to Mn3+. Mn3 can oxidize phenolic units of lignin and has also been suggested to be involved in lignin fragmentation (Wariishi et d., 1989). In addition to these enzymes, an aromatic metabolite, 3,4-dimethoxybenyl alcohol (veratryl alcohol), seems to be a component of the ligninolytic system of Phanerochaete chrysosporium. Veratry1 alcohol appears to have multiple roles in lignin degradation. It is synthesized de novo by the fungus. It apparently induces expression of Lip (Faison and Kirk, 1985) and it protects Lip from inactivation by H,O, (Tonon and Odier, 1988). Further, veratryl alcohol has been shown to potentiate Lip oxidation of compounds that are not good Lip substrates (Harvey et al., 1986). Popp et al. (1990) have observed the formation of Mn3 in reaction mixtures containing Lip, MnZ , veratryl alcohol, malonate buffer, H,O,, and 0,. No Mn3+ was formed when veratryl alcohol or H,O, was omitted. Mn3 formation also showed an absolute requirement for oxygen. Evidence is provided that veratryl alcohol functions in part as an electron transfer agent or mediator of oxidations of non-LiP substrates. Lips are unusual oxidizing extracellular peroxidases produced by +

+

+

+

+

- lignin peroxidase active oxygen mediators

-

Mn-dependent peroxidase

C, , C2: C3 - fragments --+ C02 aromatic ring cleavage C02 quinones aromatic aldehydes and acids

aromatic alcohols 4 hydroquinones

I

dioxygenases ?

-

quinone oxidoreductases aromatic ring cleavage

co2

FIG. 5. Proposed scheme for lignin degradation. (Adapted from Leisola and Garcia, 1989.)

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most of the ligninolytic fungi that cause white rot of wood (Hammel et al., 1986; Buswell and Odier, 1987; Kirk and Farrell, 1987; Tien, 1987). In the presence of H,O,, they catalyze the one-electron oxidation of a wide variety of aromatics to yield, as initial products, aryl cation radicals that subsequently undergo substituent-dependent reaction of both radical and ionic nature. These enzymes bring about the oxidative cleavage of lignin model compounds (Buswell and Odier, 1987; Kirk and Farrell, 1987; Tien, 1987; Umezawa and Higuchi, 1987) and are though to be of key importance in lignin biodegradation. Lips are heme glycoproteins found in the extracellular broth of secondary metabolic cultures of Phanerochaete chrysosporium (Tien and Kirk, 1983; Gold et al., 1984), from which various isoenzyme forms have been purified and studied (Tien and Kirk, 1984; Gold et al., 1984; Paszczynski et al., 1986; Farrell et al., 1989). Lip has also been isolated from Phlebia radiata (Kantelinen et al., 1988) and Tramates versicolor (Jonsson et al., 1987). Details on Lip production, purification, and estimation are presented by Kirk et al. (1990). Lip has been compared with horseradish peroxidase (HRP) and laccase in the oxidation of methoxybenzenes as a homologous series of substrates (Kersten et al., 1990). These enzymes affect their substrates similarly, and whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized Lip is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes. Evidence that laccase, like Lip, plays a role in lignin degradation by fungi has also been presented (Kawai et al., 1988). Ligninase-mediated phenoxy radical formation and polymerization are shown to be unaffected by cellobiose: quinone oxidoreductase (Odier et al., 1988).A simple method has been described in which Lip and MnP isozymes are selectively produced in a 7-liter airlift bioreactor with Phanerochaete chrysosporium by varying Mn(I1) concentrations in the culture medium. Mn(I1) at low levels (0.3 ppm) produced high levels of Lip isozymes, and, at 400 ppm, MnP isozymes were preferentially produced (Bonnarme and Jeffries, 1990). Attempts are in progress to optimize the cultural conditions for maximal secretion of lignin peroxidase by Phanerochaetae chrysosporium (Kirk et al., 1986; Aleksandrova et al., 1989; Kirkpatrick and Palmer, 1989; Asther et al., 1990) and to produce lignin peroxidase in fermenters (Linko et al., 1986; Michel et al., 1990). Enzyme production is maximized by manipulation of the levels of veratryl alcohol and phospholipids in the culture medium (Linko et al., 1986; Asther et al., 1990). The effect of oxygen supply

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pattern on the onset and development of the lignolytic enzyme system of Phanerochaete chrysosporiurn in submerged culture has been studied (Dosoretz et al., 1990a). The method of oxygenation used in liquid culturing on N-limited media has a crucial influence both directly on the production of ligninase and Mn peroxidase and indirectly by regulating the levels of polysaccharides produced and the extent of the extracellular protease activity. Production of lignin peroxidase by Phanerochaete chrysosporium in liquid cultures in a N-limited glycerol medium supplied with solid manganese(1V)oxide increased to a high level (Kern, 1990). Lignin peroxidase, Mn peroxidase, and glyoxa1 oxidase activities of a mutant of Phanerochaete chrysosporium under nonlimiting (nutrient) conditions were 4- to 10-fold higher than those of the wild type under N-limiting conditions. The usefulness of this isolate for production of lignolytic enzymes in large amounts for biochemical study and the possibility of scale-up production for practical use have been discussed by Orth et al. (1991). Milgram (1985), writing on “ligninase: biotechnology’s new money spinner,” stated that a fungal enzyme that can decontaminate water and make plastic from straw could make biotechnologists rich-if only they knew how to mass-produce it. An interdisciplinary approach involving a combination of microbiologists, biochemists, and biotechnologists might accomplish this goal. 2. Properties of Degradatory Enzymes

When studying the secretion of degradatory enzymes by the fruiting fungi, it is useful to know their properties and the factors affecting their production and activity. This, in turn, helps to fix the cultural conditions to effect optimum generation of the degradatory enzymes and hence faster degradation of the growth substrates. When Pleurotus ostreatus was grown in a medium containing cotton, cellobiose, or filter paper, the endoglucanase and cellobiase produced had highest stability at pH 4 and a temperature 30-40°C. The phenoloxidizing ability of the enzyme preparations obtained from culture filtrates of four strains of Pleurotus ostreatus was studied by measuring the rate of 0, uptake using the Clark 0, electrode (Semichaevskii et a ] . ,1984). These enzyme preparations were highly stable and catalyzed the oxidation of a range of phenolic substrates such as p-cresol, phydroxybenzoic acid, hydroquinone, guiacol, pyrocatechol, and 3,4dihydroxybenzaldehyde, manifesting properties of both o-dihydroxyphenoloxidase and p-dihydroxyphenoloxidase.Of 15 species of Pleurotus investigated, 4 produced laccase but not tyrosinase (Tsuruta and

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Kawai, 1983). Syringic acid and vanillin induced the greatest increases in laccase and peroxidase activity of Pleurotus ostreatus (Kozlik, 1980). The induction of new forms of laccase by phenolic substrates such as ferulic acid has been observed (Leonowicz and Trojanowski, 1975; Molitoris, 1978). The addition of several components of lignin into the culture medium effected a considerable stimulation of intra- and extracellular laccase production by Pleurotus ostreatus. Production of protocatechuate, 3,4-dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase) from Pleurotus ostreatus and its properties have been studied by Wojfas-Wasilewska et al. (1983). Characteristics of Mn peroxidase purified from Lentinus edodes cultures grown on a commercial oak wood substrate have been described; N-terminal amino acid sequencing showed that the Mn peroxidase had higher sequence homology with Mn peroxidases than with the lignin peroxidases reported from Phanerochaete chrysosporium (Forrester et al., 1990). Properties of the degradatory enzymes of several species are summarized in Table XVI. 3. Role of Degradatory Enzymes during Spawn Run and Fructification Discovering and describing the changes in physiological and biochemical activities on the substrate during fruiting body formation, and distinguishing the differences typical of the various stages of development from spawning until the end of cropping of basidiomyceteous fruiting fungi, have been undertaken by several authors (Eger, 1970; Jablonsky and Schanel, 1979; Thayumanavan, 1979). Studies of the activities of laccase, peroxidase, and protease in various growth layers of a sterilized shredded corn cob substrate incubated with Pleurotus ostreatus, Pleurotus ostreatus var. Florida, Lentinus edodes, and Agrocybe aegerita have confirmed that the complexity of fructification involves a species specificity of the metabolic pathways in different strains of fungi. Thus, both Pleurotus species displayed high levels of laccase and protease activities, while the opposite trend was observed in the other two species. It has been shown that laccase activity appears in compost during colonization by the mycelium of Agaricus bisporus and that it declines rapidly during fruiting. Further work showed that cellulase(s) behaved in the opposite fashion-a large increase in activity taking place at the time of fruiting (Turner et al., 1975). Wood and Goodenough (1977) provided further evidence about the timing of the activity of these enzymes during fruiting by studying enzyme production by Agaricus bisporus with a consideration of whether the changes were associated with fruiting initiation or with later events during fruiting.

TABLE XVI PROPERTIES OF DEGRADATORY ENZYMES OF FRUITING k’UNGIa Species w

Enzyme

Agoricus bisporus

CMCase

Agaricus bisporus

Laccase

Dichomitus squalens

Endoglucanase

Irpex lacteus

Cellulase E,-A E2-B

Pleurotus flabellatus

CMCase Hemicellulase Laccase

0

a

Properties Induced by cellulose and cellobiose; repressed by glucose and cellobiose in the presence of cellulose; two forms were discerned: one adsorbed strongly to cellulose and the other was nonadsorbable Produced constitutively in defined or complex media; inhibited by sodium azide and potassium cyanide; on electrophoresis displayed 4 bands; enzyme contained 15% carbohydrate and 2 atoms of Cu per molecule Inactivation at 70°C for 30 min; optimum activity at pH 4.8, 5OoC Specific activities for CM-cellulose and avicel were 0.092 and 0.016, respectively 76.4 and 0.039, respectively Randomness of E,-A for the hydrolysis of cotton and viscose rayon was relatively high Optimum at 50°C and pH 4.8; inactivated at 90°C (10min) Optimum at 50°C and pH 5.4; inactivated at 90°C (10min) Optimum at 40°C and pH 7.0

Ref. Manning and Wood (1983)

Wood (1980)

Rouau and Foglietti (1985) Kubo and Nisizawa (1983)

Rajarathnam (1981)

Pleurotus ostreotus

Xylanase Laccase

Pleurotus ostreotus

Pleurotus sajor-coju

Sporotrichum thermophile

Cellulase Xylanase Protease Avicelase CMCase 0-Glucosidase Cellulases FPA

cx c,

w

a 0

Volvariella volvacea

pGlucosidase CMCase p-Glucosidase

"From Rajarathnarn and Zakia Ban0 (1991).

Optimum at 50°C; inactivated at 70°C (10 min); optimum pH 5.0; inhibitor, Mn; activators, K, Mg, Ca Optimum at 50°C; optimum pH 5.5; stable pH range 5-6; inhibitors, chlorogenic acid, esculantine; activator, ferulic acid Optimum at 30°C; activator, CaCO, (5%) Optimum at 25°C; activator, CaCO, (2%) Optimum at 25°C; rice straw medium with pH 5-6 optimum for secretion Optimum at 50°C; inactivated at 70°C (10 min); optimum pH 5.5; inhibitor, Mg2+;activator, Ca Optimum at 40°C and pH 4.5; inhibitor, Ag+ Optimum at 50°C and pH 6.0 Temperature maxima: 68°C 68°C 55°C 72OC Optimum at 5 7 T , pH 7; inactivated at 60°C (2 hours) Optimum at 65°C; pH 5.8; inactivated at 70°C ( 3 hours)

Hong (1976) Lee et al. (1985)

Danilyak (1981)

Hong and Kim (1981) Hong et al. (1984) Margaritis and Creese (1981)

Wang (1982) Wang (1985)

310

SOMASUNDARAM RAJARATHNAM ET AL.

During the spawn run, Pleurotus, like Agaricus, behaves like a whiterot fungus, with the ability to degrade lignin. During fructification, it behaves like a brown-rot fungus, with high levels of cellulase activity (Rajarathnam, 1981). Changes in amounts and properties of phenoloxidases have been correlated with formation of fungal fruiting bodies (Leonard, 1971; Cutterbruck, 1972). Similarly, variations in the activities of cellulases have also been shown to be associated with the fungal fruiting body (Thomas and Mullins, 1967). It still remains to be proven whether the changes observed in enzyme activity or properties are directly correlated with the differentiation process or are merely secondary events of the developmental sequence (Mandelstam, 1976). An increase in protease activity during fructification of Pleurotus flabellatus has been noted (Rajarathnam et al., 1987). Likewise, fruiting body development in Agaricus bisporus is accompanied by large increases in intracellular protease activity. It is likely that several enzymes are involved, since activity increased at each of the three pH values (3.6, 7.0, and 9.0) studied. Fungi can synthesize at least three classes of proteases, namely acidic, neutral, and alkaline (Matsubara and Feder, 1971). Intracellular proteases, assuming prominence during fructificaiton, are known in basidiomycetes and other microorganisms (Iten and Matile, 1970; Betz and Weiser, 1976; Mandelstam, 1976). It is thought that these proteases are involved in increasing the rates of protein turnover associated with development. The protein turnover supplies amino acids to allow synthesis of proteins not previously present during development. Chang and Chan (1973) have shown that new proteins are formed both in different tissues and at different times of development of fruiting bodies by Volvariella volvacea. Netzer (1979) reported that proteases are present in all developmental stages of Pleurotus ostreatus. The activity increases significantly during primordia formation. This leads to the assumption that proteases may be involved in the differentiation process itself. Iwahara et al. (1981) have also discussed the role of extracellular enzymes during the life cycle of Pleurotus ostreatus. The roles of these various enzymes in substrate degradation and morphogenesis of fruiting bodies are illustrated in Fig. 6). 4. White-Rot Reactions

During their growth on a lignocellulosic waste, fruiting fungi such as Pleurotus and Agaricus degrade lignin preferentially, and the degraded substrate becomes whiter in color due to the exposed cellulose. Such fungal degradation is called white-rot decay. This phenomenon has been studied in three categories: (1) ability of the fungal cultures to

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

c1-cellulase

I Altered form I Cx -cellulase

Hemicellulase(s) IXylanase

Oxidizing enzymes Laccase Ligninases Lignin Peroxidases

Protease(s)

311

Lipase(s) IEsterase(s)

color phenolic media, (2) secretion of phenoloxidases, and (3) capacity to degrade lignin by fungal monocultures. The first will be discussed here; the latter two have already been considered. Pleurotus flabellatus produced intense color reactions with most phenolic compounds, but not with tyrosine (Rajarathnam et al., 1987). These reactions are similar to those of Pleurotus ostreatus (Kirk and Kelman, 1965). The association of phenoloxidase production with lignin-degrading ability was emphasized by Bavendamm (1928), who used a simple detection test [the coloration of phenolic media by the fungus under study) to separate white-rot from brown-rot fungi. The correlation between the capacity of a fungus to degrade lignin as manifested by formation of white-rot decay and the secretion of extracellular phenoloxidases is well documented (Lyr, 1958; Kirk et al., 1976). Phanerochaete chrysosporium is able to decolorize a polymeric dye during secondary metabolism and this is strongly dependent on oxygen concentration; this ability is suppressed in cultures containing high levels of N (Gold et al., 1983). Platt et al. (1985) demonstrated the ability of Pleurotus to decolorize the polymeric dye polyblue (polyvinylamine sulfonate-anthraquinone). Pleurotus ostreatus var. Florida decolorized the dye in both solid and liquid media. Decolorizing ability developed in the absence of the dye, but only when the fungus had been previously cultivated on lignin-containing substrates. Thus, it ap-

312

SOMASUNDARAM RAJARATHNAM ET AL.

pears that decolorization is a useful indication of the lignin-degrading ability of an organism. However, it may only throw light on one of the many steps in the long and complex process of lignin degradation, since lignin itself does not induce the lignin-degrading system (Kirk, 1983). 5. Aspects on Lignocellulose Degradation

The mechanism of enzymatic cellulose and hemicellulose degradation is a vast subject. The requirement of various kinds of cellulolytic and hemicellulolytic enzymes for the degradation of corresponding polysaccharides, along with secretion of different classes of the polysaccharases by the fruiting fungi, supports the capacity of these fungi to degrade holocellulose. However, the ability of these fungi to delignify the substrate is a unique feature. Many reviews appear from time to time on this subject. A general picture of lignin degradation by whiterot fungi is depicted in Table XVII. White-rot fungi convert lignin to a large array of disparate degradation products (Chen and Chang, 1985). References dealing with the fruiting fungi are noted below. TABLE XVII CHANGES I N LIGNINS FROM DIFFERENTWOODS BY WHITE-ROTFUNGI^ Changeb Property

Increase

Carboxyl content Hydroxyl content Phenolic Aliphatic Carbonyl content Hydrogen/carbon Oxygenlcarbon Methoxyllcarbon Yield of methoxylated aromatic acids on oxidative degradation after methylation Yield of principal acidolysis products Yield of major products on nitrobenzene oxidation

+ +

+

Decrease

Method of analysisc C, UV, IR, PMR

+ + +

+ +

C, UV, PMR C, PMR UV, IR, PMR C C ,L C

+

C

+

C

OFrom Rajarathnam and Zakia Ban0 (1989): Komparison of degraded lignins with sound lignins. cC, Various chemical methods; UV, IR, PMR. ultraviolet, infrared, and proton magnetic resonance spectroscopy, respectively.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

313

While growing Pleurotus on wheat straw, Ginterova and Lazarova (1987) observed a decrease of 20% dry weight and a rapid decrease in hemicellulose and lignin during spawn run. During fructification, the substrate loss ranged up to 45% and cellulose consumption was higher than that of lignin. Compared to Agaricus bisporus (which degraded 712% lignin), the highest rate of lignin degradation (37-40%) was observed with Pleurotus ostreatus and Pleurotus ostreatus var. Florida when they were grown in a synthetic medium containing wheat straw as a carbon source (Friedel et al., 1983). Pleurotus is able to degrade lignin more rapidly than Sporotrichum and Lentinus (Goloviev et al., 1983). Two strains of oyster mushrooms (K-6 and 108) differed in the dynamics of lignin and cellulose utilization when grown on aspen, ash, alder, and oak trees. The xylosis index during growth of these strains on various woods ranged from 0.26 to 0.68 (Kawakami, 1979). Pleurotus ostreatus cultured on aspen wood degraded more cellulose than lignin: 44 and 10.5Y0, respectively, after 30 months, and 75 and 33% after 48 months (Bisko et a]., 1984). With a high N content (30 mM) in the medium, lignin degradation was not accomplished by Lentinus edodes and Pleurotus ostreatus; cultures of Phlebia brevispora and Pholiota mutabilis showed weak to moderate lignin-degrading activity, whereas low N content ( 2.6 mM) stimulated ligninolytic activity by Phlebia brevispora (Leatham and Kirk, 1983). Commanday and Macy (1985)raised Pleurotus ostreatus on solid media containing either growth-limiting (1 mM) or excess (10 mM) NH,Cl and found that the amount of 14C0, released from I4Clabeled corn stover lignin in a low-N medium was three times that in the N-rich medium. Supplementation of the low-N medium with glucose (0.3%) further enhanced ligninolytic activity (Commanday and Macy, 1985). At 20 mM N, lignin degradation was suppressed by 15% with Pleurotus ostreatus (Freer and Detroy, 1982). Lignosulfonic acid fractions isolated from sulfite waste liquor were degraded by liquid cultures of Pleurotus ostreatus using glucose as an additional carbon source. The rate of degradation was inversely proportional to the molecular weight of the fraction (Wojfas-Wasilewskaet al., 1980). Biodegradation of natural and synthetic (melanoidin) humic acids by Phanerochaete chrysosporium was demonstrated by decolorization in the batch culture; this occurred during secondary metabolism of the fungus under N-limiting conditions (Blondeau, 1989). Lignin degradation by Lentinus edodes and Grifola frondosa was faster in an atmosphere of oxygen than in air. Pleurotus degraded lignin at equal rates in oxygen and in air. The influence of oxygen on lignin degradation is shown in Table XVIII. These results reflect on the ecolog-

TABLE XVIII DEGRADATION OF ['C]LIGNINASPEN WOOD^

BY THE

BASIDIOMYCETES IN ATMOSPHERES OF AIR AND OXYGEN^

14C metabolized (%) Species

Atmosphere

Phanerochaete chrysosporium

Air 0,

Coriolus versicolor

Air

Gloeoporus djchrous

Air

Polyporus brumalis

Air

Merulius trernellosus

Air

Pycnoporus cinnabarinus

Air

Lentinus edodes

Air

Bondarzewia berkeleyi

Air

Pleurotus ostreatus

Air

Grifolo frondosa

Air

Control

Air

0, 0, 0 2

0 2

0 2

0, 0, 0 2

0 2

Weight loss (%I

To CO,

Totalc

12.3 41.4 13.2 30.0 8.5 12.9 11.1 22.4 9.4 19.4 9.5 19.2 9.0 19.7 4.9 7.2 10.6 11.6 5.2 5.4

10.8 35.2 14.6 35.5 9.7 18.1 16.6 33.0 14.0 22.3 13.6 22.6 9.7 18.0 9.0 13.8 11.7 11.6 9.2 10.6

23.6 54.5 31.9 58.7 29.8 49.9 43.5 63.0 36.4 43.9 29.2 39.6 21.3 35.9 25.8 29.8 27.6 26.8 19.8 24.1 1.8

0 2

.Incubated at 25°C for 8 weeks. bAfter Reid and Seifert (1982). c;Water-soluble radioactivity plus 1 4 ~ 0 , . dMeasured by H,SO, hydrolysis. %Sum of weight removed by water extraction and cellulase digestion. flncubated at 39°C for 3 weeks.

Lignin lossd (%)

Digestibility (water soluble]

Totale

13 40 24 46 22 24 19 33 30 40 18 37 18 41 25 27 17 17 8 15

7.3 12.8 8.5 15.5 9.5 10.5 7.3 8.8 9.8 12.6 7.2 8.0 8.2 10.2 8.0 13.8 5.8 6.1 6.0 5.2 5.2

16.5 18.6 26.3 32.8 28.0 28.2 26.2 22.1 37.4 41.0 36.0 38.6 36.9 47.9 31.7 34.0 19.9 17.1 33.2 31.7 14.6

-

-

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

315

ical considerations of these fungi (Reid and Seifert, 1982). In a study of biodegradation of wood components by Pleurotus ostreatus, lignin in poplar bed-logs was strongly decomposed, with a progressive decrease in methoxyl and ester groups. The syringyl moiety of lignin was decomposed more rapidly than the guaicyl moiety (Haider and Trojanowski, 1975). Syringyl-type substructures are more suscepible to oxidative cleavage of C-C bond than guaicyl propane substructures, as demonstrated in Phanerochaete chrysosporium by 13C-NMR (Tai et al., 1990). Two different pathways of lignin degradation may exist within the genus Pleurotus: the route in which splitting is the first process and the path whereby demethylation is followed by ring splitting (Platt et al., 1983). Spruce wood lignin degradation by white-rot fungi has been shown to involve oxidation with the formation of substantial amounts of carboxylic acids, both aromatic and aliphatic in nature; cleavage of aryl ethers; and oxidative modification of side-chain structures. Carboxylic acid may involve oxidative ring cleavage. These results are consistent with the involvement of oxygenases in the degradation of the lignin molecule (Chang et al., 1980). Cleavage mechanisms for side chains and aromatic rings have been elucidated (Umezawa, 1988). The addition of xylose to the growth medium facilitated depolymerization of lignin and prevented the polymerization of low-molecular-weight fractions of lignocarbohydrate complexes by Coriolus versicolor (Milstein et al., 1983). When a culture of Pleurotus ostreatus was grown on l3C-enriched DHP-lignin followed by 13C-NMR spectra of the degraded lignin, it was evident that both the aliphatic side chains and aromatic nuclei were degraded (Haider et a]., 1981). [14C]-Lignosulfonicacid released more 14C0, when incubated with Pleurotus ostreatus than did natural plant lignin. Preincubation of the first substrate with Pseudomonas putida followed by Pleurotus ostreatus effected a marked increase of 14C0, release (Agosin et al., 1985a). Pleurotus salignus and Pleurotus ostreatus displayed the highest rate of lignin degradation as measured by formation of 14C0, (when cultured in synthetic medium containing 14C-labeled lignin from wheat straw), as compared with Agaricus bisporus and Phyllotopsis nidulans (Nizkovakaya et al., 1981). The role of the biosynthesis of veratryl alcohol and also methanol formation in relation to lignin degradation in Phanerochaete chrysosporium has been discussed (Schimada et al., 1981; Leisola et al., 1984a; Ander and Eriksson, 1975). Kirk et al. (1985) have produced evidence for the noninvolvement of free hydroxyl radical in an important reaction of lignin degradation by Phanerochaete chrysosporium. Lignin degradation, and the mechanism thereof, and the factors

316

SOMASUNDARAM RAJAARATHNAM ET AL.

favoring and limiting it have been discussed by various authors (Reid and Seifert, 1980, 1982; Shimada, 1980, 1984; Ander et al., 1981; Barlev and Kirk, 1981; Buswell et al., 1981; Jeffries et al., 1981; Gold et al., 1982; Kutsuki and Gold, 1982; Umezawa et al., 1981,1982; Andreeva et al., 1983; Faison and Kirk, 1983; Kamaya and Higuchi, 1983; Kirk and Tien, 1983; Odier and Roch, 1983; Tai et al., 1983; Ulmer et al., 1983; Fenn and Kirk, 1984; Leisola et al., 198413; Ramasamy et al., 1985; Reid et al., 1985; Skryabin et al., 1985; Higuchi, 1986; Kadam and Drew, 1986; Eriksson, 1987; Kirk, 1988). This subject has also been reviewed by many workers: on Phanerochaete chrysosporium (Leisola, 1983; Kirk, 1988); on Tyromyces lacteus and Coriolus versicolor (Li and Zhu, 19871, and on several species of white-rot fungi (Rabinovich et al., 1985; Harvey et al., 1987; Higuchi, 1989). Free exocellular lignin peroxidase can also efficiently enhance mineralization of lignin by Phanerochaete chrysosporium (Kurek and Odier, 1990). The mineralization of 14C-ring-, 14C-side-chain-,and 14C-methoxy-labeled synthetic guaicyl ligning by Dichomitus squalens and the expression of extracellular Mn peroxidase were dependent on the presence of Mn(II), suggesting that Mn peroxidase is an important component of this organism’s lignin degrading system. The expression of laccase was independent of manganese. Lignin degradation by Dichomitus squalens proceeded in the cultures containing excess C and N [Perie and Gold, 1991).Crude Phanerochaete chrysosporium lignin peroxidase, in the presence of hydrogen peroxide and veratryl alcohol, catalyzed the partial fragmentation of p-14C-labeledhardwood lignin in Na acetate (pH 4.5): N, N-dimethyl formamide, 9:1. Gel permeation chromatography of the treated lignin demonstrated that fragments with molecular weights as low as -170 were products of this reaction (Hammel and Moen, 1991). Oxidative depolymerization was suggested as the first step in the degradation of DHP ( 14C-labeled synthetic lignin, dehydrogenative polymerizate) by Phlebia tremellosa (Reid, 1991a). Phlebia (Merulius) tremellosa (Reid, 1991b; Reid and Deschamps, 1991) and biodegradation products of lignin degradation (Reid, 1991c), as well as a clarification of the role of lignin peroxidase in vivo (Sarkanen et al., 19911, have been discussed recently. VII. Applications and Implications of Spent Substrate

A. AS

A N UPGRADEDRUMINANTFEED

The polysaccharides in lignified plant materials are abundant, potentially economical sources of sugars for ruminant feeds [Scott et al.,

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

317

1969). However, before these polysaccharides can be enzymatically hydrolyzed, they must be liberated from their association with lignin (Millett et al., 1976). Use of treatments such as steaming, ball milling, electron irradiation, and ammonia and sodium hydroxide (Heaney and Bender, 1970; Millett et al., 1970; Ololade et al., 1970) to increase the digestibility of wood and straw involve costs that have prevented their extensive use. Another alternative to accomplish the goal is to harness the lignin-degrading capacities of the white-rot fungi. Many of these fungi can degrade lignin markedly and increase the digestibility of lignocellulosic wastes (Kirk and Moore, 1972; Hartley et al., 1974; Reid and Seifert, 1982; Reade and Mc Queen, 1983) and straw (Lindenfelser et a)., 1979; Wicklow et al., 1980; Zadrazil, 1980a; Zadrazil and Brunnert, 1980, 1981, 1982; Hatakka, 1983) by enzymatic hydrolysis. A considerable modification of sawdusts of beach (Fagus sylvatica), oak (Quercus robur), poplar (Populus tremuloides), and barley (Hordeum distichnon) was effected by fungal treatment with Fomes lividus (Hartley et a]., 1974). Unfortunately, many white-rot fungi take several weeks to delignify their substrate to a useful extent and obligately consume some carbohydrate to support lignin degradation (Kirk et al., 1976; Eriksson et al., 1980). To make the biological delignification process economical, it is essential to maximize both the rate and the specificity of lignin breakdown by using an organism (wild-type or mutant) that requires little carbohydrate to support ligninolysis and by providing conditions that favor lignin degradation and discourage carbohydrate consumption. The efficiency of Merulius tremellosus to remove lignin selectively from both soft woods (Ander and Eriksson, 1977) and hardwoods (Reid and Seifert, 1982) and of Cyathus stercoreus to effect a substantial loss in lignin during fermentation of wheat straw (Wicklow et al., 1980) has been reported. The resultant biodegradation products were primarily high-molecular-weight lignin-carbohydrate complexes and carbon dioxide, with small amounts of aromatic and aliphatic acids (Abbott et al., 1983). Solid-state fermentation of aspen (Populus tremuloides) wood with Merulius tremellosus for 8 weeks removed 52% of lignin, but only 12% of the total wood weight, and increased cellulose digestibility to 53% from 18% (Reid, 1985). Of the 1 2 species of Cyathus screened to degrade “C-labeled lignin in kenaf (Hibiscus cannabinus), Cyathus pallidus, Cyathus africanus, and Cyathus berkeleyanus delignified kenaf most rapidly (Abbott and Wicklow, 1984). The sum of the amount of 1% released into solution plus that released into gas phase over a 32-day fermentation period was used to determine average daily rates of lignin biodegradation.

318

SOMASUNDARAM RAJARATHNAM ET AL.

Microscopy studies (Bauchop, 1979; Akin et al., 1983) have indicated that the fungi are better able than the bacteria to colonize the lignocellulosic components of plants and that they could effectively degrade and weaken lignified tissues (Akin and Rigsby, 1987). Structural changes and the resulting IVDMD were determined during the course of solid-state fermentation of wheat straw, using the lignindegrading white-rot fungi Sporotrichum pulverulentum, Pycnoporus cinnabarinus, and Cyathus stercoreus (Agosin et al., 1985b). The latter two fungi preferentially degraded hemicelluloses with a high improvement in IVDMD (maximum increase of 63 and 9470, respectively) with limited dry weight losses (12 and 18%) after 7 and 13 days, respectively. Lignin was preferentially degraded as compared to polysaccharides. The amount of lignin removed, as determined by Klason, correlated well with IVDMD improvement (r = -0.97), while aciddetergent-degraded lignin showed a lower correlation (r = -0.86). Cyathus stercoreus and Dichomitus squalens facilitated the highest improvement in IVDMD (68% against 38% for the sound straw) after 20 and 15 days of cultivation, respectively, on wheat straw. A study of the fate of [14C]lignin during fermentation using these two fungal strains showed that maximal levels of I4C-labeled water-soluble compounds are reached before peak levels of 14C0, evolution, suggesting that these compounds are intermediates in lignin degradation. The possible relationships between water-soluble lignins and improvement in IVDMD have been discussed (Agosin and Odier, 1985). The occurrence of lignin along with cellulose and hemicellulose decreases the feed value of cellulosic wastes (Faist et al., 1970; Baker, 1973; Han young and Callihan, 1974; Millett et al., 1976; Lindenfelser et QJ., 1979; Streeter and Horn, 1980; Jung and Fahey, 1983). Enactment of rigorous air pollution laws that restrict burning of straws and the increasing cost of animal feed have revived interest in the study of the use of low-cost residues as ruminant feed (Crompton and Maynard, 1988). In light of the impracticality of commercial exploitation of such treatments as with acid, alkali, sulfide, or chloride (Rexen, 1969 Kolfstein, 1978; Sundstol et al., 1978; Horton and Steacy, 1979) for delignification, because of their cost and environmental pollution, biological delignification through organisms of the classical type represented by the white-rot fungi could be a possible solution. The fungal mycelia produced during the course of substrate degradation could also provide valuable compounds such as amino acids, vitamins, and fats. Species of Pleurotus have a specific and decided advantage over other species in that (i) they can colonize and fruit economically on a wide range of unfermented lignocellulosic wastes and (ii) their strains can bio-

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

319

transform the substrates over a range of temperatures (15-30°C) and they can, therefore, grow well in most parts of the world (Rajarathnam and Zakia Bano, 1987b). Changes in the growth substrate are influenced by the form and availability of nutrients during fungal growth and the cultural conditions. The best edible fungi yield well and cause an increase in in vitro digestibility (Zadrazil, 1985a). Strophraria rugoso-annulata increased the digestibility of straw from 40 to 70%, and Pleurotus species to about 65%. The material remaining after mushroom harvest may be useful for animal feeding (Zadrazil, 1979a; Calzada et a]., 1987a). Pleurotus species, when grown under conditions that arrest mushroom formation, are known to increase ruminant digestibility (Zadrazil, 1977, 1980a, 1981, 1985b; Kamra and Zadrazil, 1986; Zadrazil and Brunnert, 1980) and cellulose digestibility (Hatakka, 1983; Miller and Jong, 1987). Suspectibility to enzymatic hydrolysis of the substrate is an easier and alternative way to measure digestion of the substrate. Although a correlation between in vivo and in vitro [enzymatic digestibilities) results has been demonstrated for forages (Dowman and Collins, 1982), the correlation has been low (Reade and Mc Queen, 1983) with biologically delignified lignocellulosic waste materials. The data for straw decayed by Stropharia rugosaanulata (Kamra and Zadrazil, 1985), Pleurotus eryngii, and Pleurotus sajor-caju (Zadrazil and Brunnert, 1980) show good correlations between enzymatic digestibility and in vitro ruminant digestibility, although the regression line is different for each fungus and the enzymatic digestibilities reported are unusually low. The highest value (-58%) of digestible dry matter from straw has been recorded with Lentinus edodes and Polyporus galactinus (Zadrazil, 1985b); aspen wood decayed with Polyporus berkelevi recorded digestibility of 61% (Kirk and Moore, 1972). Hardwoods naturally decayed by white-rot fungi, particularly Ganoderma applanata, in the Chilean rain forest have long been used as feed under the name “Palo Podrido,” and the selective delignification has been attributed to the environment under which the decay takes place (Zadrazil et al., 1982) and the low nitrogen content of the wood. Digestibilities that are lower than the values predicted from the equation derived by Reid (1985) ID, - DDM = K[L - Lo), where D, is residual dry weight, DDM is digestible dry matter, K is a parameter expressing the effectiveness of lignin in protecting carbohydrate from digestion, L is residual lignin, and Lo is the amount of lignin that is not involved in complexing polysaccharides], can be due to (1)the phenolic acids of many graminaceous materials esterified to the hemi-

320

SOMASUNDARAM RAJARATHNAM ET AL.

cellulose (Jung and Fahey, 1983) or (2) the production of metabolites by some white-rot fungi that are inhibitory to rumen bacteria and interfere with the digestion of the delignified polysaccharides [for example, Tramates gibbosa (Leatham, 1979) and Agrocybe aegerita (Zadrazil, 1985b)l. Animal feeding trials are expensive and require large amounts of treated material, and they are unsuitable for screening experiments. Uden (1984) has reviewed the methods for assessing the digestibility of smaller samples. Ruminant digestibility can be estimated by suspending the samples in a porous nylon bag inside the rumen of a fistulated animal or by incubating the sample with rumen fluid. In either case, digestibility is a measure of the solubilization of the substrate. These techniques measure the susceptibility of the material to digestion by rumen microorganisms, but they require access to a fistulated animal. Although most references deal with in vitro studies, Zafar et al. (1981) have reported an in vivo study. They observed, by feeding the wheat straw biodegraded by Pleurotus sajor-caju in vivo to ruminants and subsequent analysis of the undigestible matter, a 34% increase in the in vivo digestibility compared with that of the undegraded straw. Before the value of spent straw substrate as cattle feed can be assessed, it is necessary to ascertain the nontoxicity of the spent substrate. Although the cultured edible fungi are nontoxic, because of the differences in the cultural conditions on a commercial scale, wherein the substrate is neither pasteurized nor sterilized and the possibility of monoculturing is ruled out, toxic substances might be generated by the foreign organisms in the growth substrates. The study of Zakia Ban0 et al. (1986) of the spent straw, after cropping Pleurotus sajor-caju fruiting bodies, by thin-layer chromatography, did not reveal the presence of any of 1 2 mycotoxins. Feeding of the spent straw to albino rats at 4 , 1 2 , and 24% of their diet for 90 days did decrease body weight or weights of vital organs. Histopathological and hematological studies of vital organs did not differ from those of the untreated animals. Feeding trials with the residues after culturing Pleurotus species on wheat straw did not show any problems of palatability or toxicity in buffaloes or lambs (Bakshi et a]., 1985; Calzada et al., 1987b).The cellulose in straw treated with Pleurotus sajor-caju was significantly more digestible than that in undigested straw (Calzada et al., 1987b). No toxic effect or illness was observed among chicks or pigs fed with wheat straw that had been incubated with the oyster mushroom, Pleurotus ostreatus. Culture medium, containing rice hulls, corn grits, or brice bran, after harvest of the mushrooms with fortification with organic acids has been found to be a better animal feed, promoting a gain in body weight

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

321

and an improvement in the meat quality (Ikeda, 1988). Lignin-rich plant wastes biodegraded by mushrooms are found to enhance their feed values (Ikeda, 1990). B. BIOGAS PRODUCTION H. W. Mueller et al. (1984; Mueller and Trosch, 1986) have studied the possibilities of utilizing wheat straw biodegraded by the fruiting fungi for the production of biogas. Twenty-two basidiomycetes, including Pleurotus ostreatus, Pleurotus ostreatus var. Florida, and the mutants of Pleurotus ostreatus var. Florida-PFP 343 and PFP 551-isolated from the basidiospores of Pleurotus ostreatus var. Florida (Ericksson and Goodell, 1974), were employed to degrade wheat straw in sterile, solid-state cultures. Pleurotus ostreatus, Pleurotus ostreatus var. Florida, and Stropharia rugoso-annulata degraded the most lignin; Pleurotus-treated straw released more sugars. The biodegraded straw was subjected to fermentation by methanogenic bacteria to produce biogas. An efficient means of biogas production during mushroom growth on cellulosic materials has been reported by Japanese workers of the Osaka Gas Co. (1984). Periodically, during the harvesting of the mushrooms, the medium is replenished with cellulosic materials; as the sugar content of the medium increases and mushroom yield decreases, the spent medium is subjected to methane fermentation. Biological pretreatment by Pleurotus sajor-caju enhanced the biogas yield from rice straw by 54 and 52% with Cyamopsis tetragonoloba (Bisaria et al., 1983).Increased susceptibility due to degradation and increased N content (approximately three times that of undegraded substrate) as a consequence of fungal growth seem to be responsible for enhanced production of biogas. Subjecting the spent residues of Ricinus communis and Morus alba (after the cultivation of Pleurotus sajor-caju) to anaerobic digestion resulted in the production of a high percentage of methane (Satyawati et al., 1989). In another study, there was an increase in biogas production which varied from 21.5% in the case of spent bagasse to 38.8% in the case of spent rice straw; biodegradation was accomplished by Pleurotus sajor-caju (Bisaria et al., 1990).

c.

CARDBOARD/PAPER MANUFACTURE AND BASIDIOMACROMYCETES AS PULPING AGENTS

The active ligninolytic property of Pleurotus has been exploited for the production of cardboard or paper manufacture. In fact, a patent

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SOMASUNDARAM RAJARATHNAM ET AL.

(Eisenstan, 1982) has been developed in West Germany, describing a method of delignifying lignocellulosic materials with Pleurotus ostreatus, resulting in a product suitable for paper or board manufacture. The process is less espensive to operate than a chemical pulping process and avoids pollution problems. Its only drawback is that biological delignification for this purpose is quite slow; therefore, it would be worthwhile to develop speciedstrains that can degrade lignin at a faster rate. Pleurotus ostreatus was found to cause an increase in water retention value (WRV) (Friedel et al., 1983) with improved breaking length; Polyporus versicolor and phanerochaete chrysosporium were also effective. Schizophyllum commune and Phellinus pines, despite their marked WRV increases, did not improve paper strength. Fukuzumi et al. (1983) have also assessed the suitability of white-rot fungi for biological pulping. In Sweden, extensive work has been carried out on the possibility of delignifying wood by white-rot fungi to reduce the energy requirement for mechanical pulping and improve the strength properties of the resulting pulp (Eriksson et al., 1976; Samuelson et al., 1980; Eriksson and Vallander, 1982; Ander and Eriksson, 1975). These results have been reviewed by Eriksson and Kirk (1979). Paper chips incubated for 60 days with Tramates sanguinea and Coriolus hirsutus required less energy input for thermomechanical pulping than sound chips and gave pulps with good breaking and tear strength but yield was reduced (Eriksson et al., 1976). Aspen chips biodegraded with Dichomitus squalens (7 weeks) or Phanerochaete chrysosporium (4 weeks) caused significant increase in burst, tear, tensile, and zero-span tensile strength of mechanical pulps (Myers et al., 1988). Phanerochaete chrysosporium growth on and degradation of bagasse reduced the energy demand in mechanical pulping by 55% and gave a pulp of acceptable strength (Gutierrez et al., 1987). Eriksson and co-workers (Eriksson and Vallander, 1982; Eriksson and Kirk, 1983) in Sweden have produced mutants of white-rot fungi that are selective for lignin and hemicelluloses. After removal of even a small amount of lignin from wood or mechanical pulp, these fungi lower the amount of energy required for mechanical refining. Pretreatment of a coarse thermomechanical pulp from red alder with Phanerochaete chrysosporium substantially reduced the energy requirement for secondary refining. Strength properties were not diminished by the fungal treatment in the presence of added glucose (Bar-lev et a]., 1982). “Mycelial papers” containing wood fiber with 5 to 10% fungal mycelia, grown on wastes, were found to show acceptable paper characteristics (Johnson and Carloson, 1978). Sachs et al. (1989) have examined the

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323

cellular attack and damage of wood chips subjected to biodegradation by Phanerochaete chrysosporium using scanning electron microscopy. Of the several species tested for biological pulping, the sheet made from thermomechanical pulp treated with Phlebia gigantea for 1 week showed the best improvement in properties (Fukuzumi et al., 1983).

A brief overview of the status of research aimed at applying biotechnology in pulp and paper manufacture and a synopsis of the status of relevant fundamental research are provided by Kirk and Eriksson (1990). Research on biopulping, based on the ability of white-rot wood decay fungi to colonize wood rapidly and degrade lignin, has been pioneered at the Swedish pulp and paper Research Institute (STFI), where cellulase-less mutants of white-rot fungi have been developed and used in the partial delignification of various lignocellulosic materials (Eriksson, 1988). A biopulping consortium in Madison, Wisconsin, has been comprehensively investigating the concept of biopulping (Kirk and Chang, 1990; Myers et al., 1988). The consortium includes 17 pulp and related companies, the USDA Forest Service, Forest Products Laboratory, and the University of Wisconsin and is in cooperation with the University of Minnesota. Lignin removal using selective white-rot fungi in bioreactors with aspen, spruce, and pine chips ranged from 3 to 37% in 4-week fungal pretreatments. Refiner mechanical pulps prepared from the fungus-pretreated chips and from untreated control chips have been evaluated (Table XIX). Energy requirements for refining decreased by as much as 50% with fungus-pretreated chips. Burst, tear, and tensile strength of hand sheets from the biopulped aspen materials increased, with the burst index increasing a maximum of approximately threefold over controls. Brightness and light scattering decreased, but bleachability was not adversely affected and properties of the hand sheets overall were comparable to those of chemically treated mechanical pulps. Biopulping of sugarcane bagasse has been studied in a cooperative venture between the STFI and a Cuban laboratory. An energy saving of about 40% is achieved in a process using a cellulase-less mutant of a lignin-degrading fungus followed by cold soda/thermomechanical pulping (Johnsrud, 1987). Improvement in pulp properties following treatments with various cellulases and xylanases has been reported (Noe et al., 1986; Jurasek and Paice, 1988; Senior, 1988; Fuentes and Robert, 1988; Uchimoto, 1988). Whole, living fungi have been shown to be effective in bleaching pine h a f t pulp in the laboratory (Kirk and Yang, 1979). Promising results have been obtained in Canadian laboratory studies with hardwood h a f t pulps (Paice, 1989).

TABLE XIX

EFFECTOF BIOPULPINGON PROPERTIES OF PULPSAND PAPERV Chip treatmentb (freeness, CSF) (ml) ~

Opacity

Scattering coefficient (mzw

Fiber length index (mm)

Pulping energy (W hkg-I)

64.4 42.9

93.2 93.0

61.8 37.9

0.1005 0.1060

2700 1560

40.5

94.8

39.9

0.1201

1480

Burst index (kPam2ig)

Tear index (mNm2/g)

Tensile index (Nmig)

Density (kg/m3)

Brightness

0.66 2.11

2.75 6.13

28.1 51.4

393 425

2.04

4.64

52.5

402

(YO)

(%I

~

RMP (120) BRMP (110) Phlebia BRMP (100) Phanerochaete

aAfter Kirk and Eriksson (1990). bRMP, Refiner mechanical pulp; BRMP, refiner mechanical pulps from fungus-pretreated wood

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325

D. DEODORIZATION OF WASTEGASES An odorous waste gas can be deodorized by passing it through a layer of a compost-based medium containing rice hulls and spent mushroom culture medium obtained after culturing of Pholiota nameko. Removal of H,S, NH,, methyl mercaptan, dimethylsulfide, and dimethyldisulfide was -99, 99, 99, 83, and 72%, respectively (Hitachi and Kogyo, 1983).

E. SOURCE OF SACCHARIFICATION ENZYMES It is well known that species of fruiting fungi produce cellulolytic and hemicellulolytic enzymes (Rajarathnam and Zakia Bano, 1990). The fresh spent substrate subsequent to the harvest of mushroom fruiting bodies has been demonstrated to contain significant activities of these saccharifying enzymes (Rajarathnam et al., 1979a; Madan and Bisaria, 1983). A suitable extract of such a substrate displaying C,, C,, FPD, and P-D-glucosidase in simple buffer solutions, which are least expensive, and further incubation with such cellulosic wastes such as waste paper and cotton waste should aid in further saccharification of these wastes. The sugar content in wheat straw degraded by Pleurotus species for about 14 days doubled compared to that in undegraded straw (Zadrazil and Grabbe, 1983). Subjecting the straw to biodegradation after alkali treatment enhanced the effectiveness of saccharification; however, the fungus alone can serve the function of NaOH (without environmental pollution) but does so slowly (Hatakka, 1983). Biological modification of wheat straw with Pleurotus ostreatus followed by treatment with cellulase allowed a 72% conversion of cellulose to glucose, while utilization of chemical treatments prior to cellulase digestion resulted in 73-80% conversion (Detroy et aI., 1980). Saccharification of woods after enzymatic hydrolysis by Pleurotus ostreatus was enhanced from 1 2 to 63% (Hiroi, 1981). Of 39 strains of fungi selected for the degradation of lignocellulosics, a mixed culture of Pleurotus ostreatus and Fusarium culmonarum produced the highest amounts of cellulolytic and ligninolytic enzymes, which can find application for saccharification of lignocellulosics (Afans’eva and Kadyrov, 1980). Biological treatment of pine and poplar woods with the basidiomycetes causes saccharification of the substrate, which might serve to upgrade feed values for ruminants (Ando et a]., 1987). The significance of the generation of maximum activities of exo- and intracellular p glucosidases, coinciding with the initiation of the deceleration phase of

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growth of Phanerochaete chrysosporium), in addition to the production of lignin peroxidase, has been reported (Jafelice et al., 1990). Termitomyces clypeatus (Sengupta and Sengupta, 1990) and Coriolus hirsutus (Danilyak and Katsan, 1987) are known to generate P-glucosidase. The saccharification of cellulose was investigated in a membrane reactor using cellulase from Sporotrichum cellulophilum. About 90% of the sugar produced was glucose and enzyme inactivation was observed during a 6-day experiment (Woranisrakul et al., 1988). A heterokaryotic mutant of Polyporus ostreiformis is reported to generate high yields of exo-1,4-P-~-glucosidase(2.54 against 0.42 units ml-1 for the parental strain) (Pal et al., 1986). The action of the culture filtrate of Sporotrichum dimorphosphosporum on xylan, mannan, and carboxymethyl cellulose was found to be inhibited in the presence of 1 mM mercuric chloride (Mora et al., 1986). The enzymatic extracts of Termitomyces clypeatus grown on cellulosic agricultural wastes at 28°C for 14 days effected 71% saccharification of bagasse (Sengupta et al., 1984).

F. RECYCLINGFOR MUSHROOM CULTURING

A spent substrate consisting of used tea leaves and waste paper, after the harvesting of Pleurotus ostreatus, provides the required nutritional conditions for the growth of Agaricus bisporus (Harsh and Bisht, 1981). The work involved the use of gypsum that provided a more granular structure with increased water-holding capacity (greatly improved mycelial growth with strand formation could be achieved by the use of tea leaves together with waste paper and mycelia of Pleurotus ostreatus). Used tea leaves are also reported to support the growth of many other basidiomycetes (Harsh and Bisht, 1984). The spent substrate supplies the decomposition products of cellulose, hemicellulose, and lignin (Sermanni et al., 1979), which are provided in traditional mushroom cultivation by the compost and the end products of microbial degradation (Hayes, 1978). Several studies by Zadrazil (1976a,b, 1978a) have shown that during culturing of Pleurotus species, cellulose-lignin complexes are decomposed by the fungal mycelium. During this process, free CO, is given off; the C:N ratio falls; and proportions of amino acids, proteins, vitamins, and minerals are altered. It is also known that proteins, organic N, a lowered C:N ratio, Ca, Mg, K, and vitamins are necessary to support the growth of Agaricus bisporus (Hayes, 1972). Thus, utilization of used tea leaves and waste paper can result in production of two mushroom crops in succession, first, a crop of Pleurotus ostreatus and, second, a crop of Agaricus bisporus. With this process,

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biodegradation and bioconversion of the waste materials are also accomplished. The approximate economics of this process have been worked out and reported (Harsh and Bisht, 1984). Growth of Pleurotus ostreatus on a mixture of straw and cores of maize cobs left much unused organic matter, increased the protein content of the substrate, and formed humus, which leads to formation of a N-rich lignin-humus complex (Sermanni eta]., 1979), the material used by Agaricus bisporus (Wood and Fermor, 1981).The possibility of using such a spent compost, after cropping Pleurotus ostreatus, with suitable supplementation of horse or chicken manure and inorganic salts to obtain the correct C:N ratio and an optimum concentration of soluble salts for the growth of Agaricus bisporus, has been investigated (Sermanni et al., 1979).

G. PRODUCTION OF NATIVESILICA The capacity of white-rot fungi to degrade rice straw in order to obtain increased ash and silica (for use in the manufacture of photovoltaic cells) has been reported (Rohtagi et a]., 1986). The silica present in rice straw and husk is highly reactive, amorphous, and has a high surface area like silica gel. Use of heat or pyrrolysis destroys the native amorphous structure and high surface area of the deposited silica. Incineration renders the native silica more coarse, crystalline, and dark in color so that it needs to be ground, requiring energy, to be suitable for making cement. Cyathus proved to be more efficient for biodegradation than Pleurotus. This would be an effective biological method for obtaining silica in its original deposited form without recourse to the heating that is associated with the ashing process. H. As SOILAMERLIORANT The white-rot fungi, due to their activity on lignin, cause the spent substrate to contain constituents such as humic/fulvic acid fractions that can add to soil fertility. Grabbe (1983)reported that spent substrate can be used as a soil-improving agent when supplemented with lowsalt-containing organic materials such as peat and bark. Mixtures of spent substrate with these materials in a 1:1 ratio supplies humic substances suitable for use as a substrate for tea nurseries. In fact, the spent straw resulting from culturing Volvariella has proved to be effective in raising profitable yields of such crops as tomato and radish (Chang, 1982). Use of spent substrate after mushroom culturing is more effective than fresh horse manure for composting and subsequent use in vegeta-

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SOMASUNDARAM RAJARATHNAM ET AL.

ble growing (Gapinski, 1982). The physical and chemical characteristics of spent mushroom compost and its use in soilless growth media for the production of vegetable transplants have been reported (Isabel, 1984). A patent has been issued on the use of organic acids from species such as Phanerochaete chrysosporium, for increasing plant productivity (Hugh, 1988). I. SINGLE-CELL PROTEINPRODUCTION Protein sources, essential in the nourishment of humans and animals, are in very short supply in many parts of the world (FAO, 1978). It is estimated that the world’s protein deficiency amounts to about 2.27 billion kg annually (Ahlgren, 1975). Because of the tremendous demand for conventional sources of protein, more attention is being devoted to unconventional sources of protein to help alleviate this critical shortage. Straw, feedlot manure, municipal refuse, agricultural discards, and food-processing wastes/waters exemplify a few of the materials that can support the production of single-cell proteins (SCP), the term coined by Humphrey (1974). Preparation of SCP on food-processing effluents not only generates a product having a significant monitoring value but also simultaneously reduces the biological oxygen demand (BOD) load of the waste stream. In a study assessing the ability of various fungi including Agaricus bisporus, Calvatia gigantae, and Pleurotus ostreatus to grow on selected brewery wastes, mushroom cultures were found to give better yields than yeasts and the highest net gain in dry weight (27.2 g/liter) was obtained with Calvatia gigantae. The highest levels of BOD removal from three types of brewery waste were achieved by Calvatia gigantae (56.2, 56.1, and 25.9%, respectively, for the grain press, trub press, and fermentation sludge liquors (Shannon and Stevenson, 1975a). With nitrogen [(NH,),SO,] supplementation, a maximum protein content of 44.5% was recorded for Calvatia gigantae (Shannon and Stevenson, 1975b). Increased saccharification and solubilization of lignocellulosic substrates, such as rice straw, as a result of culturing of Pleurotus can be used for the production of single-cell proteins (Rajarathnam et a]., 1979a).

J. ANTIVIRAL ACTIVITY OF WATER-SOLUBILIZED LIGNIN A highly condensed and polycarboxylated lignin (Suzuki et al., 1990) that is solubilized by Lentinus edodes, when cultured on a solid medium of sugarcane bagasse and defatted rice bran (Sugano et al., 1982),inhibits infection by tobacco mosaic virus by blocking the initial

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329

stage of the replicative cycle (Maeda et a]., 1983).In addition, the material inhibits the infectivity of human immunodeficiency virus (HIV) and cytopathic effects on the virus-infected cells in vitre (Tochikura et al., 1988; Suzuki et a]., 1989). Oral administration of this lignin, watersolubilized by the basidiomycetes, has been reported to be effective in AIDS therapy (Shirohata et al., 1989). Intrapertoneal administration of this substance modulates suppression of the proliferation of rat ascite hepatoma AH 414 and its hepatocarcinogenesis (Sugano et al., 1985). When administered orally, it promotes seroconversion from HBe antigen to anti-HBe antibody in chronic hepatitis B patients without any side effects (Harada and Kanetaka, 1987). VIII. Applications of Functions of Fruiting Bodies/Mycelium

A. OXIDATION OF ENVIRONMENTAL POLLUTANTS Many toxic or carcinogenic organohalides and other organics persist in the environment and tend to accumulate in the body fat of animals occupying higher trophic levels (Moore and Walker, 1964; Clark and Lamont, 1976). One reason for the environmental persistence of these compounds is that microorganisms are unable to degrade them or do so very slowly [Alexander, 1981). Studies with I4C-labeled compounds demonstrated that the tested organopollutants, namely, DDT, HCB, TCB, lindane, and benzopyrene, were oxidized to I4CO, by nitrogendeficient, ligninolytic cultures of Phanerochaete chrysosporium (Kirk, 1975). Leatham et al. (1982) have demonstrated that Phanerochaete chrysosporium contains a constitutive enzyme system capable of cleaving the aromatic ring of 36 aromatic compounds, including 2-chloroisovanillic acid. It is also able to dechlorinate alkyl chlorides (such as lindane, a nonaromatic compound that is chlorinated on every carbon atom, to CO,). It is proposed that biotreatment systems inoculated with Phanerochaete chrysosporium and fortified with a suitable carbohydrate source, under nitrogen-limiting conditions, may provide an effective and economical means for the biological detoxification and disposal of hazardous chemical wastes (Bumpus et al., 1989). Obviously, this will be of use in aerobic treatment of contaminated waste effluents, sludges, sediments, and landfills using procedure including activated sludge processes, aerated lagoons, aerobic digestion, trickling filters, rotary biological contractors, and aerobic composts (Peyton, 1984). In fact, about 1600 species of basidiomycetes have been associated with recycling the carbon bound in lignin (Gilberton, 1980) and this property is envisioned as a prospect in biodegrading organic, aromatic, environmental pollutants.

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SOMASUNDARAM RAJARATHNAM ET AL.

White-rot fungi, as ligninolytic agents, have been proposed to be useful in hazardous waste biotreatment programs (Kirk and Chang, 1981; Kirk and Farrell, 1987). The occurrence of such compounds as phenolics, anisoles, aryl-o-diethers, and biphenyls, common in both lignin and organic pollutants, have led many researchers to surmise that any organism capable of mineralizing lignin must have highly nonspecific oxidizing systems that could be applicable to other aromatic pollutants as well. Lyr (1963) has indicated the unusual xenobiotic capability of Trametes versicolor, which was resistant to pentachlorophenol, and degraded this fungicide through unspecified dechlorination reactions. Phanerochaete chrysosporium was found to decolorize paper mill effluent, a complex mixture of chlorinated lignin fragments, phenols, anisoles, and other low-molecular-weight components causing water pollution; degrade its residual kraft lignings, and remove its low-molecular-weight chloroorganics. These results were later applied to the development of a successful bench-scale method for effluent treatment by Phanerochaete chrysosporium (Kirk and Chang, 1981; Huynh, 1985). Eaton (1985) reported that this fungus also mineralized a significant fraction of a **C-labeled polychlorinated biphenyl (PCB) mixture (Aroclor 1254) that was added to stationary, nutrient-limited cultures. Phanerochaete chrysosporiurn is also known to mineralize chlorinated anilines and oxidize pentachlorophenol (Arjmand and Sandermann, 1985) and phenanthrene (Mileski et al., 1988) to CO,. Benzo[a]pyrene mineralization is enhanced in high-biomass agitated cultures (Bumpus et al., 1989). Phanerochaete is also known to oxidize polycyclic aromatic hydrocarbons (Hammel, 1986; Bumpus, 1989) and polychlorinated phenols (Haemmerli et al., 1986; Mileski et al., 1988) to quinones. The fungus also displays the ability to oxidize the heterocyclic aromatic thianthrene (Hammel and Tardone, 1988). Even though it is not yet apparent what the role of extracellular ligninolytic enzymes may be in the oxidation of organopollutants, the only eukaryotes so far known to cleave fused-ring aromatic hydrocarbons are the white-rot fungi (Gibson and Subramanian, 1984; Schreiner et al., 1988). Phanerochaete chrysosporium causes extensive biodegradation of 2,4,6-trinitrotoluene (Fernando et a]., 1990). This fungus is also known to biodegrade dyes such as orange 11, tropaeolin 0, congo red, and azure B (Cripps et al., 1990),as well as crystal violet (Bumpus and Brock, 1988). Cellulose and starch in the growth medium are observed to aid in the active mineralization of DDT (Fernando et al., 1989). The possible role of Phanerochaete chrysosporiurn in detoxifying soil and water due to its ability to biodegrade 2,4,5-trichlorophenoxyaceticacid has been dis-

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331

cussed (Ryan and Bumpus, 1989). Bumpus and Aust (1987a) have observed that the ability of Phanerochaete chrysosporium to mineralize chlorinated organic compounds is favored in nutrient N-deficient cultures. The capacity of Phanerochaete chrysosporium to decompose environmental pollutants has been reviewed (Bumpus and Aust, 1987b). The wood-decaying capabilities of Gloeophyllum sepiarium, Gloeophyllum trabeum, Lentinus lepidus, Phanerochaete gigantea, Phlebia brevispora, Polyporus versicolor, Poria carbonica, Poria placenta, Poria radiculosa, and Poria xantha have been reported by Eslyn (1986). Phanerochaete chrysosporium rapidly oxidized benzo[a]pyrene to CO, and several organic-soluble and water-soluble compounds in agitated pellet cultures during secondary metabolism (Sanglard et al., 1986). The use of coimmobilized Phanerochaete chrysosporium to degrade pentachlorophenol and its potential utility using simulated contaminated soil extract and sand have been investigated by Lin et QI. (1991). B. PEROXIDASE PRODUCTION

Coprinus macrorhizus has been employed to produce peroxidase in liquid cultures using a medium that contained iron salts to enhance enzyme yield (Noda and Matsui, 1987).

C. INDICATORS OF HEAVY METALACCUMULATION Mushrooms collected in the vicinity of a chloroalkali factory contained 10 times more mercury than those grown in other areas. Use of the commonly occurring Lycoperdon gemmatum, Mycena pura, and members of Collybiaceae as indicators in the study of mercury pollution has been advocated (Rauter, 1975). Accumulation of heavy metals such as lead, cadmium, mercury, copper, and zinc is observed in mushrooms grown on sewage sludge (Grabbe and Domsch, 1976). Likewise, Agaricus arvensis and Agaricus bitorquis growing on the roadside have been found to contain higher levels of cadmium and zinc compared to mushrooms grown artificially (Woggon and Bickerich, 1978). Tyler (1980) has reported mineral analyses of 130 species of fruiting basidiomycetes. This study indicates that bioconcentration is a common occurrence in basidiomycetes; however, this can be predicted only to a limited extent from the taxonomic status of the species or from the properties of the substrate or soil. Undesirable heavy metal contaminants like lead, cadmium, and selenium in the fruiting bodies have been observed by several workers (Higgs et a]., 1972; Collet, 1977; Enke

332

SOMASUNDARAM RAJARATHNAM ET AL.

et al., 1977; Thomas et a]., 1972). Brunnert and Zadrazil (1980, 1981, 1983) have studied the uptake of cadmium and mercury by Pleurotus species, under controlled culture conditions, in comparison with uptake by Agaricus, Flammulina, and Agrocybe. Pleurotus flabellatus and Pleurotus sajor-caju were found to be the best heavy metal collectors. Pleurotus ostreatus had a tendency to bioaccumulate cadmium from the culture medium in the mushroom fruiting bodies; the increase in concentration was linear (Yasui et a]., 1988).

D. DECOLORIZATION OF SPENTKRAFT LIQUOR AND MOLASSES PIGMENT Bleached kraft mills usually discharge large volumes of brown effluents as a result of the different operations used in processing wood and pulp. To minimize pollution, these effluents are treated in most mills by biological oxidation in aerated lagoons and/or activated sludge systems before being released into streams. Such aerobic treatments have the ability to reduce the BOD and COD of the effluents, but they fail to reduce color efficiently. The brown color of the effluents originates mainly from lignin degradation products, most of them chlorinated. It is well known that species of Pleurotus, as representative examples of white-rot fungi, are able to degrade and metabolize not only native lignin, but also kraft lignin and lignosulfonates (Rajarathnam and Zakia Bano, 1989). Pleurotus ostreatus and Polyporus versicolor removed 40% of the color after 8 days (Livernoche et al., 1983). Phanerochaete chrysosporium achieved over 60% decolorization after 4 days of cultivation, but thereafter, the color increased slightly. Work is in progress at North Carolina State University in collaboration with the U.S. Environmental Protection Agency and the Forest Products Laboratory, on the MyCoR method, which uses Phanerochaete chrysosporium and white-rot species in rotating biological contractors to decolorize, dechlorinate, and detoxify chlorolignins and low-molecular-weight chlorinated aromatics (Pellinen, 1988). Recent findings indicate that combinations of MyCoR and bacterial treatments can reduce AOX (organically bound halide) from 6.1 to 1-2 kg/ADT pulp, with 5O-7OYO color removal. A modification of the MyCoR method, Mycopor, is being studied in Austria (Kirk and Chang, 1990; Farcher, 1985). Phanerochaete chrysosporium is immobilized on foam squares, from which a trickling filter is constructed. Decolorization of 80% in 24 hours was achieved for a first extraction stage effluent from a chlorine ieachery using sulfite pulp. AOX was reduced at about the same rate (Kirk and Eriksson, 1990).Incubation of hardwood kraft pulp

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

333

in agitated, aerated cultures of the white-rot fungus Trametes versicolor increased pulp brightness and decreased its residual lignin content. Immobilization of the fungus on polyurethane foam also allowed the repeated use of the same fungal biomass to bleach successive batches of pulp, either immediately or after storage at 4°C (Kirkpatrick et al., 1990). Of the 51 strains of ligninolytic basidiomycetes screened for their ability to decolorize phenolic industrial effluent, Lentinus edodes (4EC2019) strain proved the most efficient, removing 73% of color in 5 days without any extra carbon source (Esposito et al., 1991). Decolorization of kraft effluent by free and immobilized lignin peroxidases of Chrysonilia sitophila has been studied by Ferrer et al. (1991). Fawer et al. (1991) have described the immobilization of lignin peroxidase (partially purified) from Phanerochaete chrysosporium on glass beads and in activated agarose. Lankinen et al. (1991) have studied color removal by white-rot fungi grown on bleach plant effluents. Michel et al. (1991) have determined the roles of lignin peroxidases versus Mn peroxidases in the decolorization of pulp bleach plant effluent [BPE) by Phanerochaete chrysosporium. The results indicated that Mn peroxidases play an important role relative to lignin peroxidases in the decolorization of BPE. Decolorization of the pigment of molasses is a major problem, since molasses serves as one of the important raw materials for various fermentation industries, due to its low cost and availability. In a fermentation factory using molasses, waste water contains much dark brown pigment [molasses pigment, MP), which is a malanoidin and is not decolorized by the usual biological treatments such as the activated sludge process. Decolorization of this MP remains a problem to be solved (Amano et al., 1965; Aoshima et al., 1985). Aoshima et al. (1985) found that Pleurotus ostreatus decolorized the MP, as determined by the giant colony method. Testing this property in shake cultures, Pleurotus ostreatus displayed about 65% decolorization. Pleurotus ostreatus IF0 6519 has been selected among 24 white-rot basidiomycetes as suitable for decolorization of PVPP-treated molasses (Tamaki et al., 1986).

Fermentation wastes, especially those from alcoholic fermentation of molasses, are decolorized by incubation with Pleurotus or Auricularia (Hongo et al., 1974). Use of fruiting fungi for improving the biological treatment of phenol-containing waste water has been discussed (Kolankaya and Saglam, 1983; Timofeeva and Ustyuzhina, 1983; Timofeeva and Vatyuzhina, 1984). This is based on the exploitation of the property of basidiomycete cultures to produce phenol-oxidizing enzymes.

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SOMASUNDARAM RAJARATHNAM ET AL.

E. PYRANOSE OXIDASE PRODUCTION Pyranose oxidase (PROD) catalyzes the oxidation of n-glucose to Dglucosone with the reduction of molecular oxygen to hydrogen peroxide. The enzyme has been purified and characterized from Coriolus versicolor (Janssen and Ruelius, 1968; Ruelius et al., 1968; Machida and Nakanishi, 1984), Polyporus obstusus (Tamura and Arai, 39861, Phanerochaete chrysosporium (Eriksson et al., 1986), and Oudemansiella mucida (Volc et al., 1978). A new basidiomyceteous fungus (Strain 52) has been demonstrated to display a high activity of PROD (Izumi et al., 1990a). From a study of the enzyme properties (Machida and Nakanishi, 1984; Tamura and Arai, 1986; Izumi et a]., 1990b), it is recognized that PROD can be used for the measurement of blood glucose without the addition of mutarotase (Taguchi et al., 1985) and for assaying 1,5-anhydroglucitol, a marker for the diagnosis of diabetes (Nakamura et al., 1988). With regard to heat stability and increased tolerance to high NaCl concentrations the PROD isolated from basidiomycete Strain 52 is considered to be more advantageous in food industries for enzymatic assay of glucose than Penicillium (Nakamatsu et al., 1975). F. POLYMER PRODUCTION Five strains of Pleurotus have been investigated for potential use as high-viscosity polymer-producing organisms (Compere et al., 1980). The high viscosity biopolymers find applications in foods, enhanced oil recovery, cosmetics, and proprietary industrial preparations. Polymer production varied markedly from strain to strain; however, some of the strains of Pleurotus surveyed were capable of producing several grams of polymer per liter under the test conditions. This approaches the concentrations obtained with the industrial production strains of Sclerotiurn rolfsii, which produce a similar gum. Pleurotus strains required less N in the fermentation broth than did Sclerotium rolfsii. G.

~ N D E R I Z A T I O NOF

MUSHROOM STIPES

A protein extract of Pleurotus ostreatus consisting of chitinase, chitobiase, and glucanase (against alkali-insoluble glucan) was able to soften stipes of Pleurotus ostreatus, as well as those of Agaricus bisporus and Lentinus edodes (Schmitz and Eger, 1981).

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H. RETTING OF FLAXFIBERS Sharma (1987) has studied the use of polysaccharide-degrading enzymes for the removal of noncellulosic materials, such as hemicellulose, pectin, and lignin, present on the flax fibers, dew retted at 45°C. An enzyme extract of flax shieve colonized by a hybrid strain of Pleurotus ostreatus and Pleurotus ostreatus var. Florida was used. Xylanase, polygalacturonase, and laccase were found in high concentrations, along with minor amounts of pectinlyase and cellulase, in extracts of Pleurotus, as compared to other enzyme sources. All the enzyme-treated roves produced high-quality yarns compared to yarns spun from the untreated roves. Fluidity of all the yarns spun from enzyme-treated roves was low, suggesting that the enzymes did not affect the cellulose fibers. The importance of such an enzymatic treatment as an alternative to boiling with NaOH to save energy and to achieve the desired properties is discussed by Sharma (1987).

I. HAIRGROWTH STIMULANT An ointment liquefying at body temperature or a liquid emulsion has been formulated using a combination of waxes, fats, oils, and a citrus fruit juice supplemented with mushroom juice. The material remained stable for -3 months without stabilizers or antioxidants. The formulation is reported to stimulate hair growth (Rudder, 1984).

J. LOW-ALCOHOL WINES A low-alcohol wine is manufactured by fermentation of honey with yeast in the presence of culture broth from mushroom (Lentinus edodes) mycelia. The quality of the wine was superior to that produced by the conventional method (Yomeishu Seizo Co., 1984). K.

USE AS

FUNGAL ELICITORS

Production of acridone-epoxide, a phytoalexin, could be increased in tissue cultures of Ruta graveolens by the use of fungal elicitors of basidiomyceteous origin such as from Polyporus versicolor (Wolters and Eilert, 1982).

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L. USE AS

TEST

ORGANISMS

Because of the property of Lenzites palisoti and Ganoderma upplanatum to degrade heavily rubber wood, these fungi have been proposed to be used as test organisms for evaluating wood preservatives (Sujan et a]., 1980).

M. METABOLIC SUBSTANCES The various cultural conditions effecting the production of methylanthranilate, the characteristic flavor component of the Concord grape, have been described by Gross et al. (1990). Many species of Phellinus biosynthesize methyl esters of benzoic, salicylic, and furoic acids as natural products. Methylation of phenol by chloromethane in Phellinus pomaceus has been examined. Anisole was produced linearly with respect to time at a rate of 6.3 nmol per gram per hour over 16 hours when washed mycelia were incubated with phenol and labeled chloromethane (McNally and Harper, 1991). N. BIODEGRADATION OF DEGRADABLE PLASTIC

POLYETHYLENE

Recalcitrant plastics accumulate in the environment at a rate of 25 million tons per year. The fate of these organic compounds and the time required for mineralization to carbon dioxide is not yet fully understood. There is growing interest in enhancing the biodegradability of the plastics in landfills and compost. To the authors’ knowledge, this article is the first to report the pure culture study with Phanerochaete chrysosporium demonstrating that lignin-degrading microorganisms can actually degrade the oxidized polyethylene component of degradable plastics, as indicated by molecular weight reductions (Lee et al., 1991). IX. Conclusions

In conclusion, the basidiomacromycetes represent the most highly evolved group of fungi. There are two distinct phases in their life cycle, vegetative and fructification. These phases are mediated by a complex of physiological processes, defining primary and secondary metabolic events. The metabolic pattern of these fungi is a natural example of a highly evolved system for the dissipation of organic carbon and its return to inorganic carbon that effects the completion of the carbon cycle. Genetic incompatability factors (bipolarhetrapolar) allow these

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fungi great diversity in their ability to grow over a range of conditions: fermented or unfermented; pasteurized or unpasteurized; sterilized or unsterilized; on soft woods or hardwoods; on cereal straws, or oil crop residues, or plantation crop residues (the natural lignocellulosic wastes). Their growth is effected over a range of biomass turnovers, governed by physical and chemical factors. Factorial inheritance and multiple allelism are genetic assets in transmitting high degrees of variation in their biochemical properties (and hence their application) and these fungi contribute to the wide range of strains chosen for certain traits over numerous genetic variations. On the other hand, it is of utmost importance that biotechnologists ensure that the genetic stability and invariability of a straidspecies are fixed and identified for performance of a selected commercial task. Thus, inheritance and variation are equally important in this discussion of the basidiomacromycetes. It is critical to gain more insight into the patterns of genetic variation, because correction of these variations could, through the attainment of invariability, lead to better, sustained crop yields. The use of continued and repeated transfers and subcultures leads to the loss of culture potency. Genetic instability during the vegetative growth of Coprinus radiatus has been reported (Odile and Elisabeth, 1989). Breeding with monosporous cultures has aided in the circumvention and alleviation of such situations during continuous culturing of edible species, in an effort to bring about maximum bioconversion and resistance to growth competitors. Genetic breeding has indicated that bred dikaryons are superior to the component monokaryons/homokaryons, with regard to growth, patterns of growth, and yield (Schmidt and Kebernik, 1987). It is also equally important develop ideal means of preserving a selected culture for viability and potency. There is a paucity of information available on this subject and thus, this field is open to more research. Further, the type of medium used in the growth of a culture is of great importance in the achievement of culture stability. It has been noted that glucose in the growth medium often results in losses in culture stability, due to the ready supply of carbon. Maintenance of the culture on a complex carbon source, for example, malt extract (Chandrashekar et al., 1981) or even the substrate on which the fungus is found growing in nature, would overcome this problem to a great extent (Rajarathnam and Zakia Bano, 1987b). Basidiomacromycetes, as reviewed in the previous sections, display an array of physiological properties that have a range of useful applications. Edible species represent the best examples: they grow over a range of lignocellulosic substrates to yield fruiting bodies, and the de-

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SOMASUNDARAh4 RAJARATHNAM ET AL.

,--+--

Sunlight

I

I I

Organic plant matter

Food

Inediblewaste (cellulose, hemicellulose and lignin)

I

i I I-

Q

'7

I Fruiting bodies

-* -ICOz+ H@I

* Hairgrowth stimulant

+

Oxidizing enzymes

culture

celium

Ashuman Produc- Indica- Lowfood tion tors alwof perox- of Hg idases pdlu- wines tion

Oxidation of environmental zation of molasses pollutants spentKrah liquor

+

Degradative

Polymeric + .

enzymes

activity

pyranosoidase production

Retting flax fibers

Tender- pOk ization mer production of mushroom stipes

V

Upgraded ruminant feed

Biogas generation

Cardboard! Recyded Soil Production Deodorizaamelio- of SCP tion of Paper for wlmanufacture turing rant waste gases Agaricus

Production of native Si

FIG. 7. Role of basidiomacromycetes in the ecology of the carbon cycle and applications and implications of their culture. aAs high as 70% wheat straw substrate while culturing Pleurotus florida.

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graded substrates can in turn be used in a number of useful ways (Fig. 7). Biodegradation of natural lignocellulosic materials to CO,, H,O, and humic substances is one of the most natural processes (Kirk and Eriksson, 1990). The entire concept of bioconversion is based on the principle of biodegradation, which, in turn, depends on the strength of, and the ability to secrete, degradative enzymes (cellulases, hemicellulases, ligninolytics, proteolytics, etc). However, it is necessary to match the cultural conditions, and, more importantly, the stage of fungal growth and the physiological status of the fungus, to the desired application. In many instances, a preliminary genetic probe would be helpful in the selection of suitable variants and/or wild strains. Studies on genetic breeding and protoplast fusion would aid in the generation of new strains, their characterization, and the identification of their desirable traits and genetic stability. A monokaryotization method that allows the recovery of the two monokaryotic components (= neohaplonts) from a dikaryon has been described that is useful for genetic studies and breeding experiments on wood-rotting basidiomycetes (Leal-Lara and Eger-Hummel, 1982). Sufficient progress has been made in the identification and use of allozyme markers (Royse and May, 1982, 1989; Kerrigan and Ross, 1989) to characterize the types that would aid in the study of genetic breeding, and to routinely identify homo- and heterokaryons among regenerated single protoplasts (Sonnenberg et a]., 1988). The utility of DNA restriction fragment length polymorphisms in determining genetic relatedness among strains of Lentinus edodes and their potential applications in breeding and genetics have been elucidated by Rajiv (1991). Sufficient progress has also been made to release and regenerate protoplasts in several species (Moore, 1975; Ushiyama and Nakai, 1977; Abe et al., 1982; Akamatsu et ol., 1983; Yamada et al., 1983; Yanagi and Takebe, 1984; Yanagi et a]., 1985; Kiguchi and Yanagi, 1985). A peroxidase (Prx-) mutant was unable to degrade lignin, whereas a phenotypic revertant of Phanerochaete chrysosporium regained this capacity (Gold et al., 1980). It has been shown that dedikaryotization allows effective breeding of sporeless strains and protects them efficiently; this method has employed species of Pleurotus, Kuehneromyces, Flammulina, and Lentinus (Eger, 1978). Although DNA technology and biotransformation of the cultured fungal cells is a fascinating new research area, its use in commercial exploitation of a desired trait has been limited. These limited results should not discourage further research. Rather, efforts should be redoubled so that basic knowledge can be combined with new areas to achieve the goal of increased efficiency of applications. Studies are in

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progress to elucidate the molecular genetics of the enzymatic degradation of lignocellulosics by Phanerochaete chrysosporium (De Boer et al., 1988; Raeder et al., 1987; Sims et a]., 1988).Over the past few years, an unexpectedly large family of related genes has been described for lignin-degrading enzymes in Phanerochaete chrysosporium (Schalch et a]., 1989). In research, it is necessary to strike a balance between optimism and pessimism in the extrapolation of laboratory observations to industrial scales. It is time to transfer in vitro findings to in vivo targets and, in the process, to expose specific problems and consider them critically. To do so would be a step toward alleviating constraints and achieving the desired scale of commercialization. The search for new species of basidiomacromycetes and research on new and different ways to use these fungi should be a matter of priority. It is a task that requires much intelligence and perseverance. Increasing the list of their applications will depend on prolonged and continued research. It will take the ingenuity of mankind, coupled with nature’s mercy, to unravel the untapped potentialities of the basidiomacromycetes. REFERENCES Abbott, T. P., and Wicklow, D. T. (1984). Appl. Environ. Microbiol. 47, 585-587. Abbott, T. P., James, C., and Plattner, R. D. (1983). Am. Chem. Soc., Washington, D.C. pp. 267-284. Abe, H., Gotoh, S., and Aoyama, M. (1980). J. Jpn. SOC.Nutr. Food Sci. 33, 169-176. Abe, M., Umetsu, H., Nakai, T., and Sasage, D. (1982). Agric. Biol. Chem. 46,1955-1961. Adamski, Z.,and Zielinski, M. H. (1985). Chem. Technol. Chem. 7, 21-28. Adegbola, A. A., and Paladines, 0. (1977). J. Sci. Food Agric. 28, 775-785. Adhikary, D. K., George, U., and Ghose, T. K. (1982). Biotechnol. Lett. 4,197-202. Afans’eva, M. M. (1984). Mikol. Fitopatol. 18,210-215, Afans’eva, M. M., and Kadyrov, R. M. (1980). Mikol. Fitopatol. 14, 410-416. Agosin, E.,and Odier, E. (1985). Appl. Microbiol. Biotechnol. 21, 397-403. Agosin, E., Daudin, J. J., and Odier, E. (1985a).Appl. Microbiol. Biotechnol. 22, 132-138. Agosin, E., Monties, B., and Odier, E. (1985b). J. Sci. Food Agric. 36,925-935. Agosin, E., Tollier, M. T., Brillouet, J. M., Thivend, P., and Odier, E. (1986). J. Sci. Food Agric. 37, 97-106. Agosin, E., Jarpa, S., Rojas, E., and Espejo, E. (1989). Enzyme Microb. Technol. 11, 511517. Ahlgren, R. M. (1975). Proc. Indiana Waste Conf., 30th, Purdue Univ., Lafayette pp. 10361042. Akamatsu, K., Kamada, T., and Takemaru, T. (1983). Trans. Mycol. SOC.Jpn. 24, 173. Akamatsu, I., Yoshihara, K., Kamishima, H., and Fujii, T. (1984). Mokuzai Gakkaishi 30, 697-702. Akin, D. E., and Rigsby, L. L. (1987). Appl. Environ. Microbiol. 53, 1987-1995.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

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Akin, D. E., Gordon, G. L. R., and Hogan, J. P. (1983). Appl. Environ. Microbiol. 46, 738748. Al-Ani, F.,and Smith, J. E. (1988). J. Sci. Food Agric. 42, 19-28. Aleksandrova, G. P., Medvedeva, S. A., Bahkin, V. A., Solov’ev, V. A., Malysheva, 0. N., and Ivanova, S. 2. (1989). Khim. Drev. 6, 77-80. Alexander, M. (1981). Science 211, 132. Amano, H., Mizunuma, K., and Kanai, Y. (1965). Hakko Kyokaishi 23, 177. Ander, P.,and Eriksson, K. E. (1975). Sven. Papperstidn. 78, 641-642. Ander, P., and Eriksson, K. E. (1977). Physiol. Plant. 41, 239-248. Ander, P., and Eriksson, K. E. (1978). Prog. Ind. Microbiol. 14, 294. Ander, P., and Eriksson, K. E. (1985). Appl. Microbiol. Biotechnol. 2, 96-102. Ander, P., Eriksson, K. E., Maansson, P., and Pettersson, B. (1981). Ekman-Days 1981, Int. Symp. Wood Pulping Chem., SPCI, Stockholm 3, 71-74. Ander, P., Ericksson, K. E., and Yu, H. S. (1983). Arch. Microbiol. 136, 1. Ando, S.,Ito, K., Kakimoto, N., Kyofuji, K., and Hanai, S . (1987). Jpn. Pat. 87 181,794 A2; Jpn. Pat. 62181794; Jpn. Pat. Appl. 86 77,311; Jpn. Pat. Appl. 85 227,595. Andreeva, N. V., Murashkevich, N. V., and Stakheev, I. V. (1983). Vestsi Akad. Navuk BSSA, Ser. Biyal. Navuk 3, 56-59. Angeli-Papa, J., and Eyme, J. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 53-81. Academic Press, New York. Anisova, L. N., Bartoshevich, E., Efremenkova, 0. V., Krasilnokova, 0. L., Kudinova, M. K., Murenets, N. V., Klyuev, N. A., Chernyshev, A. I., and Shorshnev, S. V. (1987). Antibiot. Med. Biotechnol. 32, 735-738. Anselmi, N., and Deandrea, G. (1978). Mushroom Sci. 10,451-461. Aoshima, I., Tozawa, Y., Ohomoma, S., and Ueda, K. (1985). Agric. Biol. Chem. 49,20412045. Arakawa, N., Enomoto, K., Mukohyama, H., Nakajima, K., Tanahe, O., and Ingaki, C. (1977). Eiyo To Shakuryo 30, 29-33. Arita, I. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 475-496. Academic Press, New York. Arjmand, M., and Sandermann, H., Jr. (1985). J. Agric. Food Chem. 33, 1055-1060. Arora, D. S., and Sandhu, A. K. (1986). Acta Biotechnol. 6, 293-297. Asther, M., Capdevila, C., and Corrieu, G. (1990). Fr. Pat. 2,637,292 A l ; Fr. Pat. Appl. 8812887. Atkey, P. T. (1985). In “The Biology and Technology of the Cultivated Mushroom” (P. B. Flegg, D. M. Spencer, and D. A. Wood, eds.), pp. 241-260. Wiley, New York. Atomachi, R. (1988).Jpn. Pat. 88 37,087 B4; Jpn. Pat. 6337087; Jpn. Pat. Appl. 76 153,145. Babitskaya, V. G., and Shcherba, V. V. (1987). Mikrobiologiya 56,600-607. Baker, A. J. (1973). J. Anim. Sci. 36, 768. Bakshi, M. P. S., Gupta, V. K., and Langar, P. N. (1985). Agric. Wastes 13, 51-57. Bar-lev, S. S., and Kirk, T. K. (1981). Biochem. Biophys. Res. Commun. 99, 373-378. Bar-lev, S. S., Kirk, T. K., and Chang, H. M. (1982). Tappi 65, 111-113. Barrows, I., Seal, K. J., and Eggins, H. D. W. (1979). In “Straw Decay and Its Effect on Utilization and Disposal” (E. Grossbard, ed.), p. 147. Wiley (Interscience), New York. Bassham, J. A. (1975). Biotechnol. Bioeng. Symp. No. 5, 9. Bauchop, T. (1979). Appl. Environ. Microbiol. 38, 148-158. Bavendamm, W. (1928). Z. Pflanzen, Krankh. Pflanzenschutz 38, 257. Bech, K. (1979). Mushroom Sci. 10, 77-83. Beg, S.,Zafar, S. I., and Shah, F. H. (1986). Agric. Wastes 17,15-21. Benedict, R. G., and Brady, L. R. (1972). J. Pharm. Sci. 61, 1820-1822.

342

SOMASUNDARAM RAIARATHNAM ET AL.

Betz, H., and Weiser, U. (1976). Eur. J. Biochern. 62, 65. Bhat, K. M., and Maheshwari, R. (1987). Appl. Environ. Microbiol. 53, 2175-2182. Bisaria, R.,and Madan, M. (1984). Curr. Sci. 53, 322-324. Bisaria, R., Madan, H., and Mukhopadhyay, S. N. (1983). Biotechnol. Lett. 5, 811-812. Bisaria, V. S.,Saxena, S. K., Manihar, R. B., and Gopalakrishnan, K. S. (1984). Appl. Biotechnol. Biochern. 9, 341-342. Bisaria, R., Vasudevan, P., and Bisaria, V. S.(1990). Appl. Microbiol. Biotechnol. 33,607609. Bisko, N. A,, Bilai, V. T., and Churikova, E. K. (1984). Mikol. Fitopotol. 18, 435-439. Biswas-Hawkes, D., Dodson, A. P. J., Harvey, P. J., and Palmer, J. M. (1987). Colloq. INRA 40,125-130. Blanchette, R. A., and Reid, I. D. (1986). Appl. Environ. Microbiol. 52,239-245. Blanchette, R.A., Otjen, L., Effland, M. J., and Eslyn, W. E. (1985). Wood Sci. Technol. 19, 35-46. Blanchette, R. A . , Otjen, L., and Carlson, M. C. (1987). Phytopathology 77, 684-690. Block, S.S.,Tsao, G., and Han, L. H. (1958). J. Agric. Food Chem. 6,923. Blondeau, R. (1989). Appl. Environ. Microhiol. 55, 1282-1285. Bohus, G., Glaz, E., and Scheiber, E. (1961). Acta Biol. Acad. Sci. Hung. 12,1-12. Bollag, J. M., and Leonowicz, A. (1984). Appl. Environ. Microbiol. 48,849-854. Bonnarme, P., and Jeffries, T. W. (1990). J. Ferment. Bioeng. 70, 158-163. Booth, C., and Harold, H. B. (1982). “A Field Guide to Mushrooms and Their Relatives.” Van Nostrand-Reinhold, New York. Bressani, R. (1979). In “Pulpa de Cafe, Composicion, Technologia y Utilizacion” (J. E. Braham and R. Brassani, eds.). C. I. I. D., Bogota. Brien, A. 0. (1989). Mushroom J. No. 200, 253-263. Brunnert, H., and Zadrazil, F. (1980). Eur. J. Appl. Microbiol. Biotechnol. 10,145. Brunnert, H., and Zadrazil, F. (1981). Eur. J. Appl. Biotechnol. 12, 179. Brunnert, H., and Zadrazil, F. (1983). Eur. J. Appl. Microbiol. Biotechnol. 17, 358. Bumpus, J. A. (1989). Appl. Environ. Microbiol. 55, 154-158. Bumpus, J. A., and Aust, S. D. (1987a). ACS Symp. Ser. No. 338, 340-349. Bumpus, J. A., and Aust, S. D. (1987b). BioEssays 6, 166-170. Bumpus, J. A., and Brock, B. J. (1988). Appl. Environ. Microbiol. 54, 1143-1150. Bumpus, J. A.. Tin, M., Wright, D., and Aust, S. D. (1989). Science 228, 1434-1436. Burrows, I., Seal, K. J., and Eggins, H. 0. W. (1979). In “Straw Decay and Its Effect on Utilization and Disposal” (E. Grosshard, ed.), pp. 147-154. Wiley (Interscience), New York. Buswell, J. A,, and Odier, E. (1987). CRC Crit. Rev. Biotechnol. 6, 1-60. Buswell, J. A,, Ander, P., and Eriksson, K.E. (1981). Ekrnan-Days 1981, Int. Symp. Wood Pulping Chem., SPCI, Stockholm 3, 88-92. Buswell, J. A., Mollet, B., and Odier, E. (1984). FEMS Microhiol. Lett. 25, 295-299. Cailleux, R.,Diop, A., and Macaya-Lizano, A. (1976). Mushroom Sci. 9,595. Calzada, J.F.,De Leon, R., De Arriola, M. C., and Rolz, C. (1987a). Biol. Wastes 20, 217226. Calzada, J. F., Franco, L. F., De Arriola, M. C., Rolz, C., and Ortiz, M. A. (1987bl. Biol. Wastes 22, 303-309. Campbell, A. C., and Slee, R. W. (1987). Mushroom News Lett. Trop. 7, 127-134. Canevascini, G., and Gattlen, C. (1981). Biotechnol. Bioeng. 23, 1573-1590. Canevascini, G.,Fracheboud, D., and Meier, H. (1983). Can. J. Microbiol. 29,1071-1080. Carroad, P. A,, and Wilke, C. R. (1977). Appl. Environ. Microbiol. 33,836. Cavazzoni, V., and Manzoni, M. (1988). Ann. Microbiol. Enzyrnol. 38, 181-192.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

343

Ceruti-Scurti, J., Fiussello, N., Gullino, M. L., and Farina, F. (1981). Allionia 24, 61-70. Chahal, D. S. (1989). J. Ferment. Bioeng. 68, 334-338. Chandrashekar, T. R., Bano, Z . , and Rajarathnam, S. (1981).Trans. Br. Mycol. SOC.77,491. Chang, H., Chen, C., and Kirk, T. K. (1980). Lignin Biodegrad.: Microbiol., Chem., Potential Appl., Proc. Int. Semin., Madison, Wis., 1978 1, 215-230. Chang, M. M., Chou, T. Y . C., and Tsao, G. T. (1981). Adv. Biochem. Eng. 14, 93. Chang, S. T. (1978a). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 573-603. Academic Press, New York. Chang, S. T. (1978b). ASAIHL Lect., 1-38. Chang, S. T. (1982). In “Tropical Mushrooms-Biological Nature and Cultivation Methods” (S. T. Chang and T. H. Quimio, eds.), pp. 463-474. Chinese Univ. Press, Hong Kong. Chang, S. T. (1987). Mushroom J. Trop. 7, 117. Chang, S. T., and Chan, K. Y. (1973). Mycologia 65, 355-364. Chang, S. T., and Hayes, W. A. (1978). “The Biology and Cultivation of Edible Mushrooms.” Academic Press, New York. Chang, S. T., and Miles, P. G. (1982). In “Tropical Mushrooms-Biological Nature and Cultivation Methods” (S. T. Chang and T. H. Quimio, eds.), pp. 3-10. Chinese Univ. Press, Hong Kong. Chang, S. T., and Quimio, T. H., eds. (1982). “Tropical Mushrooms, Biological Nature and Cultivation Methods.” Chinese Univ. Press., Hong Kong. Chao, E. E., and Gruen, H. E. (1987). Can. J. Bot. 65, 518-525. Chen, C. L., and Chang, H. M. (1985). In “Biosynthesis and Biodegradation of Wood” (T. Higuchi, ed.), p. 535. Academic Press, San Diego, California. Chen, P. C., and Hou, H. H. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 629-643. Academic Press, New York. Cheng, S., and Tu, C. C. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.),pp. 605-625. Academic Press, New York. Chihara, G. (1978). Mushroom Sci. 10, 797-814. Chkhartishvili, D. K., and Kvachadze, L. L. (1985). Soobshch. Akad. Nauk Gruz. SSR 117, 409-41 2. Chung, K. H., Kim, M., and Chung, S. R. (1987). Yakhak Hoechi 31,213-218. Clark, D. R., Jr., and Lamont, T. G. (1976). Bull. Environ. Contam. Toxicol. 15, 1 . Cochran, K. W. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 169-187. Academic Press, New York. Collet, P. (1977). Dtsch. Lebensm. Rundsch. 73, 75. Commanday, F., and Macy, J. M. (1985). Arch. Microbiol. 142, 61-65. Compere, A. L., Griffith, W. L., and Greeve, S. V. (1980). Dev. Ind. Microbiol. 21, 180. Contrereas, 0.R., Roura, G., and Hernandez, D. (1985).Rev. Latinoam. Microbiol. 27,329334.

Cossar, D., and Canevascini, G. (1986). Appl. Microbiol. Biotechnol. 24, 306-310. Cowling, E. B. (1975). Biotechnol. Bioeng. Symp. No. 5, p. 163. Cripps, C., Bumpus, J. A., and Aust, S. D. (1990). Appl. Environ. Microbiol. 56, 11141118.

Crisan, E. V., and Sands, A. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 137-181. Academic Press, New York. Crompton, E. W., and Maynard, L. A. (1988). J. Nutr. 15, 383. Cutterbruck, A. (1972). J. Gen. Microbiol. 70, 423-435. Danilyak, N. I. (1981). Fermentn. Spirt. Promst. 4, 37. Danilyak, N. I., and Katsan, V. A. (1987). Mikrobiol. Zh. [Kiev] 49(4), 40-44.

344

SOMASUNDARAM RAJARATHNAM ET AL.

Danilyak, N. I., Mel’nichuk, G. G., and Yarovenko, V. L. (1983). Fermentn. Spirt. Promst. 4,33-37. Danilyak, N. I., Baglai, V. A,, and Yarovenko, V. L. (1989). Pishch. Promst. (Moscow) 3, 69-71. Dar, P. H., Clark, T. A,, and Chu-Chou, M. (1988). Process Biochem. 23, 156-160. Daugulis, A. J., and Bone, D. H. (1977). Eur. J. Appl. Microbiol. 4,159-166. De Boer, H. A., Zhang, Y. Z., Collins, C., and Adinarayana Reddy, C. (1988). Gene 69(2), 369. Delcaire, J. R. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 728-794. Academic Press, New York. De Leon, R., Morales, E., De Agreda. L., and Rolz, C. (1983). Mushroom News Lett. Trop. 4, 13-16. Denisova, N. P., Semenova, I. R., and Sukharevich, V. I. (1989).Mikol. Fitopatol. 23, 378381. Detroy, R. W., Lindengelser, L. A,, Julian, G., and Orton, W. L. (1980). Biotechnol. Bioeng. Symp. No. 10, 135-148. Diehle, D. A., and Royse, D. J. (1986). Mycologia 78, 929-933. Dobry, I . , Dziurzynski, A,, and Rypacek, V. (1986). Wood. Sci. Technol. 20, 137-144. Doshi, A., Munot, J. F., and Chakravarti, B. P. (1987). Mushroom J. Trop. 7, 83-85. Dosoretz, C. G., Chen, A., and Grethlein, H. E. (1990a). Appl. Microbiol. Biotechnol. 34, 131-137. Dosoretz, C. G., Chen, H. C., and Grethlein, H. E. (1990b). Appl. Environ. Microbiol. 56, 395-400. Dowman, M. G., and Collins, F. C. (1982). J. Sci. Food Agric. 33, 689-696. Doyle, R. R., and Levenberg, B. (1974). Phytochemistry 13, 2813-2814. Drawert, F., Berger, R. G., and Neuhaeuser, K. (1983). Eur. J. Appl. Microbiol. 18, 124. Dua, I. S., and Jandaik, C. L. (1979). Sci. Hortic. (Amsterdam) 10, 301. Dunlap, C. E. (1979). Final Rep. NSF Grant AER76-17912, Univ. of Missouri, Columbia. Durand, H., Soucaille, P., and Tiraby, G. (1984). Enzyme Microb. Technol. 6, 175-180. Dzamic, M., and Miljkovic, B. (1984). Arh. Poljopr. Nauke 45(160), 479-490. Eaton, D. C. (1985). Enzyme Microb. Technol. 7, 194-196. Eger, G. (1970). Arch. Microbiol. 74, 174. Eger, G. (1979). Mushroom Sci. 10, 415-420. Eisenstan, A. (1982). Eur. Pat. 60-467. Elshafei, A. M., and Penninckx, M. J. (1989). Egypt. J. Bot. 29/30(1/3), 273-278. Enke, M., Matschiner, H., and Achtzehn, M. K. (1977). Nahrung 21, 331. Eriksson, K. E. (1981). Ekman-Days 1981, Int. Symp. Wood Pulping Chem., SPCI, Stockholm 3, 60-65. Eriksson, K. E. (1987). Philos. Trans. R. SOC.London 321, 455-459. Eriksson, K. E. (1988). In “Biochemistry and Genetics of Cellulose Degradation” (J. P. Aubert, ed.), p. 285. Academic Press, New York. Eriksson, K. E., and Goodell, E. W. (1974). Can. I. Microbiol. 20, 371-378. Eriksson, K. E., and Kirk, T. K. (1979). In “Comprehensive Biotechnology” (M. Mooyoung, ed.), Vol. 4, pp. 271-294. Pergamon, Oxford. Eriksson, K. E., and Kirk, T. K. (1983). In “Comprehensive Biotechnology” (C. W. Ribson, ed.), Pergamon, Oxford. Eriksson, K. E., and Pettersson, B. (1982). Eur. I. Biochem. 124, 635-642. Eriksson, K. E., and Pettersson, B. (1988). Methods Enzymol. 160,500-508. Eriksson, K. E., and Vallander, L. (1982). Sven. Papperstidn. 85, R33-R38. Eriksson, K. E., Ander, P., Henningson, B., Nilsson, T., and Goodell, B. (1976). U.S. Pat. 3,962,033.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

345

Eriksson, K. E., Grunewald, A., and Vallander, L. (1980). Biotechnol. Bioeng. 22,363-376. Eriksson, K. E., Pettersson, B., Volc, J., and Musilek, V. (1986). Appl. Microbiol. Biotechnol. 23, 257. Eslyn, W. E. (1986). Holzforschung 40, 69-77. Espejo, E., and Agosin, E. (1991). Appl. Environ. Microbiol. 57(7),1980-1986. Esposito, E., Canhos, V. P., and Duran, N. (1991). Biotechnol. Lett. 13(8), 571-576. Faison, B. D., and Kirk, T. K. (1983). Appl. Environ. Microbiol. 46, 1140-1145. Faison, B. D., and Kirk, T. K. (1985). Appl. Environ. Microbiol. 49,299-304. Faist, W. C., Baker, A. J., and Tarkow, H. (1970). 1. Anim. Sci. 30, 832. Falina, N. N. (1980). Mikol. Fitopatol. 14, 40-42. Fan, L. T., Lee, Y. H., and Beardmore, D. H. (198Oa). Biotechnol. Bioeng. 22, 177. Fan, L. T., Lee, Y. H., and Beardmore, D. H. (1980b). Adv. Biochem. Eng. 14, 101. Fan, L. T., Lee, Y . H., and Gharpuray, M. M. (1982). Adv. Biochem. Eng. 16, 158187.

FA0 (1968). “Amino Acid Content of Foods and Biological Data on Proteins,” FA0 (1970). ‘‘Amino Acid Content of Foods and Biological Data on Proteins,” Nutr. Stud. No. 24. Food Policy Food Sci. Serv., Nutr. Div., Food Agric. Organ., Rome. FA0 (1978). FA0 (1985). “Production Year Book,” Vol. 39. Food Agric. Organ., Rome. FA0 (1989). “Production Year Book,” Vol. 41. Food Agric. Organ., Rome. FAO/WHO (1965). “Protein Requirements,” Rep. Jt. FAO/WHO Expert Group, 4th Ed. Food Agric. Organ., World Health Organ., Geneva. FAO/WHO (1972). “Sixth Report of a Joint FA0 and WHO Expert Committee on Food Additives,” Tech. Rep. Semin. No. 505. Food Agric. Organ., World Health Organ., Geneva. FAO/WHO (1973). “Energy and Protein Requirements,” Rep. Jt. FAO/WHO Ad Hoc Expert Comm., Food Nutr. Meet. Rep. Ser. No. 52. Food Agric. Organ., World Health Organ., Geneva. Farcher, S. (1985). Holzforsch. Holzvenvert. 37(5),89. Farrell, R. L., Murtagh, K. E., Tien, M., Mozuch, M. D., and Kirk, T. K. (1989). Enzyme Microb. Technol. 11, 322-328. Fawer, M. S., Stierli, J., Cliffe, S., and Fiechter, A. (1991). Biochim. Biophys. Acta 1076, 15-22.

Feniksova, R. V., Ulezlo, I. V., and Pukit, N. Y. (1972). Prikl. Biokhim. Mikrobiol. 8, 337340.

Fenn, P., and Kirk, T. K. (1984). 1. Wood Chem. TechnoI. 4, 131-148. Fergus, C. L. (1978). Mycologio 70, 636-650. Fermor, T. R., and Wood, D. A. (1982). Mushroom J. 119, 388-391. Fernando, T.,Austor, S. D., and Bumpus, J. A. (1989). Chemosphere 19, 1387-1398. Fernando, T., Bumpus, J. A,, and Aust, S. D. (1990). Appl. Environ. Microbiol. 56, 16661671.

Ferrer, T., Dezotti, M., and Duran, N. (1991). Biotechnol. Lett. 13(3), 577-582. Ferri, F. (1972). Micol. Ital. 1, 61. Ferri, F. (1984). Mushroom Inf. 2, 47. Fitzpatrick, W. H., Esselen, W. B., Jr., and Weir, E. (1946).J. Am. Diet. Assoc. 22, 318-323. Fletcher, J. T. (1977). Mushroom J. 56, 252-266. Forney, L. J. (1982). Diss. Abstr. Int. B 43, 343-515. Forrester, I. T., Grabsky, A. C., Mishra, C., Kelley, B. D., Strickland, W. N., Leatham, G. K., and Burgess, R. R. (1990). Appl. Microbiol. Biotechnol. 33, 359-365. Frankland, J. C. (1982). Syrnp. Br. Mycol. SOC.4,241-261. Freer, S.N., and Detroy, R. W. (1982). Mycologia 74, 943-951.

346

SOMASUNDARAM RAJARATHNAM ET AL.

Friedel, K., Seidel, D., Moegling, R., and Funk, I. (1983). Wiss. Z . Wilhelm-Pieck-Univ. Rostock, Math.-Naturwiss. Reihe 32, 25-28. Fuentes, J. L., and Robert, M. (1988). Eur. Pat. 262040. Fujita, J., Kumori, H., Komemushi, S., and Yamagata, K. (1990). Lett. Appl. Microbiol. 11, 27-29.

Fujita, T., Komemushi, S., and Yamagata, K. (1990). Lett. Appl. Microbiol. 11, 27-29. Fukuzumi, T., Yotukura, A., and Hayashi, Y. (1983). Recent Adv. Lignin Biodegrad. Res., Proc. Int. Semin., 2nd, Kyoto pp. 246-256. Galliano, H., Gas, G., and Durand, H. (1988). Biotechnol. Lett. 10, 655-660. Gandy, D. G. (1974). Mushroom I. 23, 428-429. Gandy, D. G. (1985). In “The Biology and Technology of the Cultivated Mushroom” (P. B. Flegg, B. M. Spencer, and D. A. Wood, eds.), pp. 261-278. Wiley, N e w York. Gapinski, M. (1982). Ogrodnictwo (Warsaw) 19, 239-240. Garibova, L. V., Kozlova, R. G., Losyakova, L. S., and Safnova, N. V. (1982). Biol. Nauki [MOSCOW) 3, 79-84. Gavrilova, V. P., and Grigor’eva, N. K. (1983). Mikol. Fitopatol. 17, 127-131. Gavrilova, V. P., Goncharova, N. A., Shamolina, I. I., and Vol’f, L. A. (1980). Mikol. Fitopatol. 14, 328-331. Gerrits, J. P. G. (1969). Mushroom Sci. 7, 111-126. Ghosh, A. K., and Sengupta, S. (1981). j . Food. Sci. Technol. 18, 133-135. Gibson, D. T., and Subramanian, V. (1984). In “Microbial Degradation of Organic Compounds” (D. T. Gibson, ed.), pp. 181-252. Dekker, New York. Gilberton, R. L. (1980). Mycologia 72, 1. Ginterova, A. (1973). Folia Microbiol. (Prague) 18, 277. Ginterova, A., and Lazarova, A. (1987). Food Chem. 32,434-437. Ginterova, A., and Lazarova, A. (1989). Folio Microbiol. (Prague) 34, 141-145. Ginterova, A,, Janotkova, O., Zemek, J., Augustin, J., and Kunsik, L. (1980). Folia Microbiol. (Prague) 25, 318-323. Glenn, J. K., and Gold, M. H. (1983). Appl. Environ. Microbiol. 43, 1741. Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M., and Gold, M. H. (1983). Biochem. Biophys. Res. Commun. 114, 1077-1083. Glenn, J. K., and Gold, M. H. (1985). Arch. Biochem. Biophys. 242, 329-341. Gohl, B. (1981). “Tropical Feeds.” FAO, Rome. Gold, M. H., Cheng, T. M., Krisnangkura, K., Mayfield, M. B., and Smith, L. M. (1980). Lignin Biodegrad.: Microbiol., Chem., Potential Appl., Proc. Int. Semin., Madison, Wis., 1978 2, 65-71. Gold, M. H., Mayfield, M. B., Cheng, T. M., Krisnangkura, K., Shimada, M., Enoki, A,, and Glenn, J. K. (1982). Arch. Microbiol. 132, 115-122. Gold, M. H., Glenn, J. K., Mayfield, M. B., Morgan, M. A., and Kutsuki, H. (1983). Recent Adv. Lignin Biodegrad. Res., Proc. Int. Semin., znd, Kyoto pp. 219-232. Gold, M. H., Kuwahara, M., Chiu, A. A., and Glenn, J. K. (1984). Arch. Biochem. Biophys. 234, 353. Golovlev, E. L., Chermenskii, D. N., Okunev, 0. N., Brustavetskaya, T. P., Golovlera, L. A,, and Stryabin, G. N. (1983). Mikrobiofogiyo 52, 78-82. Goto, I., and Minson, D. J. (1977). Anim. Feed Sci. Technol. 2, 247. Gotoh, S.,Aoyama, M., and Abe, H. (1985). Nippon Eiyo Shakuryo Gakkaishi 38, 135139.

Grabbe, K. (1980). Ger. Pat. 3024737. Grabbe, K. (1983). Wiss. Umwelt. 4,221-226. Grabbe, K., and Domsch, K. H. (1976). Mushroom Sci. 9, 209-220.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

347

Gramss, G. (1977). Champignon 192, 18. Grandjean, J., and Huls, R. (1974). Tetrahedron Lett. 22, 1893-1895. Gregory, F. J., Healy, E. M., Agersbory, H. P. K., Jr.. and Warren, G. H. (1966).Mycologia 58, 80-90.

Gross, B., Yonnet, G., Picque, D., Brnnerie, P., Corrien, G., and Asther, M. (1990). Appl. Microbiol. Biotechnol. 34, 387-391. Gujral, G. S., Bisaria, R.,Madan, M., and Vasudevan, P. (1987). J. Ferment. Technol. 65, 101-105.

Gutierrez, I., Fernandez, N., and Lopez, P. (1987). Int. Sugar J. 89, 157-159. Guzman, G., and Martinez, D. (1986). Mushroom News Lett. Trop. 6, 7-10. Gyurk6, P. (1977). In “Soil Biology and Conservation of the Biosphere” Szegi, ed.), pp. 293-295. Akademiai Kiad6, Budapest. Haars, A., Majcherczyk, A., Trojanowski, J., and Huttermann, A. (1985). Comm. Eur. Communities [Rep.] EUR, 973-977. Hadar, Y.,and Cohen-Arazi, E. (1986). Appl. Environ. Microbiol. 51, 1352. Haemmerli, S. D., Leisola, M. S. A., Sanglard, D., and Fiechter, A. (1986). J. Biol. Chem.

u.

261, 6900-6903.

Haider, K., and Trojanowski, J. (1975). Arch. Microbiol. 105, 33. Haider, K., Ellwardt, P. C., and Ernst, L. (1981). Ekman-Days 1981, Int. Symp. Wood Pulping Chem., SPCI, Stockholm 3, 93-98. Hammel, K. E., and Tardone, P. (1988). J. Biochem. 27, 6563-6568. Hammel, K. E., Kalyanaraman, B., and Kirk, T. K. (1986). J. Biol. Chem. 261, 1694816952.

Hammel, K. E., and Moen, M. A. (1991). Enzyme Microbiol. Technol. 13(1), 15-18. Han, Y.H., Chen, K. M., and Cheng, S. (1977). Mushroom Sci. 9, 167. Han young, W., and Callihan, C. D. (1974). Appl. Microbiol. 27, 159. Hara, M., Yoshida, M., Morimoto, M., and Nakano, H. (1987).J. Antibiot. 40, 1643-1646. Harada, T., and Kanetaka, J. (1987). Kan-Tan-Sui 14, 327. Harsh, N. S. K., and Bisht, N. S. (1981). Int. Biodeterior. Bull. 17, 19. Harsh, N. S. K., and Bisht, N. S. (1984). Int. Biodeterior. Bull. 20, 253. Hartley,R. D., Jones,E.C., King,N. J., andsmith, G. A. (1974).J. Sci.FoodAgric. 25,430-437. Hartree, M., Yu, E. K. C., Reid, I. D., and Saddler, J. N. (1987).Appl. Microbiol. Biotechnol. 26, 120-125.

Harvey, P. J., Schoemaker, H. E., and Palmer, J. M. (1986). FEBS Lett. 195, 242-246. Harvey, P. J., Schoemaker, H. E., and Palmer, J. M. (1987). Plant, Cell Environ. 10, 709714.

Hashimoto, K., and Takahashi, 2. (1970). Toyo Shokuhin Kogyo Tanki Daigaku, Toyo Shokuhin Kenkyusho Kenkyu Hokokusho 60, 320-326. Hashioka, Y., and Arita, I. (1978). Mushroom Sci. 10, 127-135. Hatakka, A. I. (1983). Eur. J. Appl. Microbiol. Biotechnol. 18, 350-357. Hayes, W. A. (1972). Mushroom Sci. 8, 663-674. Hayes, W. A. (1975). Mushroom J. 30, 204-206. Hayes, W. A. (1978). In “Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), p. 220. Academic Press, New York. Hayes, W. A. (1981). Mushroom Sci. 11, 103-129. Hayes, W. A., and Lim, W. C. (1979). In “Straw Decay and Its Effect on Utilization and Disposal” (E. Grossbard, ed.). Wiley (Interscience), New York. Heaney, D. P., and Bender, F. (1970). For. Prod. J. 20, 98. Heltay, I. (1978). Mushroom Sci. 10, 463-481. Herrman, H. (1962). Naturwissenschaften 49, 542.

348

SOMASUNDARAM RAJARATHNAM ET AL.

Higgs, D. J., Morris, V. C., and Levander, 0. A. (1972). J. Agric. Food Chern. 20, 678. Higuchi, T. (1986). Wood Res. 73, 58-81. Higuchi, T. (1989). ACS Syrnp. Ser. No. 339, 485-502. Higuchi, T., Nakatsubo, F., Kamaya, Y., and Umezawa, T. (1983). Recent Adv. Lignin Biodegrad. Res., Proc. Int. Semin., 2nd, Kyoto pp. 209-218. Hira, A,, Barnett, S. M., Shiek, C. H., and Montecalvo, J,, Jr. (1978). AKHE Symp. Ser. No. 74, 17-20. Hirano, S., Matsuura, Y., Kusunoki, M., Kitagawaand, Y., and Katsube, Y. (1987). J. Biochem. (Tokyo) 102, 445-446. Hiroi, T. (1981). Mokuzai Gakkaishi 27, 684-690. Hitachi, K., and Kogyo, K. K. (1983). Jpn. Pat. 58 202,018; Jpn. Pat. Appl. 82 87,004. Ha, M. S., and Han, Y. S. (1978). Mushroom Sci. 10, 561-587. Hoffmann, W., and Huttermann, A. (1975). J. Biol. Chem. 250, 7420. Holtz, R. B., and Schister, L. C. (1971). Lipids 6 , 176. Hong, J. S. (1976). MisaengrnuJ Hakhoe Chi 14,99. Hong, J. S. (1978). Hanguk Nonghwa Hakhoe Chi 21,150. Hong, J. S., and Kim, D. H. (1981). Hanguk Nonghwa Hakhoe Chi 24, 7-14. Hong, J. S., Uhm, T. B . , Jung, G. T., and Lee, K. B. (1984). Hanguk Kyunhakhoechi 12,59. Hong, S . W., Shin, K., Yoon, Y., and Lee, W. (1986). Hanguk Kyunhakhoechi 14,189-194. Hongo, M., Hayashida, S., and Tanaka, Y. (1974). Jpn. Pat. 74 130,056 (CI91C9); Jpn. Pat. Appl. 73 44,206. Horton, G. M., and Steacy, G. M. (1979). J. Anim. Sci. 48, 1239. Hu, K. J., Song, S. F., and Liu, P. (1974). Mushroom Sci. 9, 687-691. Huang, B. (1982). Yaoxue Tongbao 17, 282-285. Huang, N. L. (1982). Mushroom News Lett. Trop. 2 , 2-4. Hugh, M. M. (1988). Can. Pat. 1,235,917 A l . Hughes, D. H. (1962). Mushroom Sci. 5, 540. Humphrey, A. E. (1974). Chem. Eng. 81(26), 98-112. Huynh, V. B. (1985). Tappi 68,98-102. Ibrahim, M.N. M., and Pearce, G. R. (1980). Agric. Wastes 2, 199-205. Ikari, Y., Yokoyama, S., Ohama, C., and Fukui, R. (1985). PCT Int. Pat. WO 85101512 A l ; PCT Int. Pat. Appl. WO 84JP 457. Ikeda, H. (1988). Jpn. Pat. 88 169,940 A2; Jpn. Pat. 63169940. Ikeda, H. (1990). Jpn. Pat. 90 31,651 A2; Jpn. Pat. 0231651. Ikekawa, T., Ikeda, Y., Yoshioka, Y., Nakanishi, K., Yokoyama, E . , and Yamazaki, E. (1982). J. Pharmacobio-Dyn. 5, 576-581. Inaba, K., Iizuka, Y.,and Koshijima, T. (1984). Bokin Bobai 12(2), 57-64. Isabel, L. V. (1984). Diss. Abstr. Int. B 44,2950-2951. Ishihara, M., Toyama, S., and Yonaha, K. (1985). Ryukyu Daigaku Nogakubu Gakujutsu Hokoku 32, 63-71. Iten, W., and Matile, P. (1970). J. Gen. MicrobioJ. 61, 301-309. Ito, K.. and Hitaka, H. (1981). Jpn. Pat. 81 76,401; Jpn. Pat. Appl. 79 153,159. Iwade, I. (1987). Jpn. Pat 87 87,095 A2; Jpn. Pat. 6287095; Jpn. Pat. Appl. 85 224,698. Iwahara, H., Yoshimoto, T., and Fukuzumi, T. (1981). Mokuzai Gakkaishi 27, 331-336. Iwai, K., Ikeda, M., and Fujino, S. (1977). 1. Nutr. Sci. Vitaminol. 23, 95. Izumi, Y., Furuya, Y., and Yamada, H. (1990a). Agric. BioJ. Chern. 54, 799-801. Izumi, Y.,Furuya, Y., and Yamada, H. (199Ob). Agric. BioJ. Chem. 54, 1393-1399. Jablonsky, I., and Schanel, I. (1979). Symp. Physiol. Ecol. Cultiv. Edible fungi, 3rd, Prague. Jafelice, L. R. S., Wiseman, A., and Goldfarb, P. (1988). Biochem. Soc. Trans. 16,369-370.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

349

Jafelice, L. R. S., Wiseman, A., and Goldfarb, P. (1990).Biochem. SOC.Trans. 18,644-645. Jandaik, C. L., and Kapoor, J. N. (1976). Mushroom Sci. 9, 667. Janshekar, H., and Fiechter, A. (1983). Adv. Biochem. Eng./Biotechnol. 27, 119. Janssen, F. N., and Ruelius, H. W. (1968). Biochim. Biophys. Acta 167, 501. Janssen, F. W., and Ruelius, H. W. (1975). Methods Enzymol. 41, 170. Jeffries, T. W., Choi, S., and Kirk, T. K. (1981). Appl. Environ. Microbiol. 42, 290-296. Jilek, R., Zezula, J,, and Vodickova, M. (1979). Mushroom Sci. 10, 303-309. Johnson, M. A., and Carlson, J. A. (1978). Biotechnol. Bioeng. 20, 1063. Johnsrud, S. C. (1987). Nordic Pulp Pap. Res. J., Spec. Issue B Steenberg 75, 47. Johnsrud, S. C., and Eriksson, K. E. (1985). Appl. Microbiol. Biotechnol. 21, 320-327. Jong, S. C., and Donovick, R. (1989). Adv. Appl. Microbiol. 34, 183-244. Jonsson, L., Johansson, T., Sjostrom, and Nyman, P. 0. (1987). Acta Chem. Scand., Ser. B B41, 766. Jung, H. G., and Fahey, G. C., Jr. (1983). J. Anim. Sci. 57, 206-219. Jurasek, L., and Paice, M. G. (1988). Biomass 15, 103. Kabir, Y., Yamaguchi, M., and Kimura, S. (1987). J. Nutr. Sci. Vitaminol. 33, 341-346. Kadam, K. L., and Drew, S. W. (1986). Biotechnol. Bioeng. 28, 394-404. Kajuno, C., Nishijima, M., Miura, H., and Kamoi, I. (1991). Nippon Shokuhin Kogyo Gakkaishi. 38(2), 107-115. Kamaya, Y., and Higuchi, T. (1983). Mokuzai Gakkaishi 29, 789-794. Kamra, D. N., and Zadrazil, F. (1985). Biotechnol. Lett. 7, 335-340. Kamra, D. N., and Zadrazil, F. (1986). Agric. Wastes 18, 1-17. Kanda, T., Noda, I., Wakabayashi, K., and Nisizawa, K. (1983). J. Biochem. (Tokyo) 93, 787-794. Kaneda, M., Seza, K., Nishi, T., and Tominaga, N. (1985). Kagoshima Daigaku Rigakubu Kiyo, Sugaku, Butsurigaku, Kagaku 18, 53-57. Kaneda, T., and Tokuda, S. (1966). J. Nutr. 90, 371-376. Kaneda, T., Arai, K., and Tokuda, S. (1964). J. Jpn. SOC.Food Nutr. 16, 106-108. Kaneshiro, T. (1976). Dev. Ind. Microbiol. 18, 591-597. Kang, C. Y., Shim, M. J., Choi, E. C., Lee, Y. N., and Kim, B. K. (1981). Hanguk Saenghwa Hakhoe Chi 14(2), 101-112. Kantelinen, A., Waldner, R., Niku-Paavola, M. L., and Leisola, M. S. A. (1988). Appl. Microbiol. Biotechnol. 28, 193. Kashiwagi, Y., Magae, Y., and Sasaki, T. (1985). Shokuhin Sogo Kenkyusho Kenkyu Hokoku 46, 145-147. Kawai, S., Umezawa, T., and Higuchi, T. (1988). Arch. Biochem. Biophys. 262, 99-100. Kawakami, H. (1979). Bokin Bobai 7, 505. Kern, H. W. (1983). Holzforschung 37, 287-292. Kern, H. W. (1990). Appl. Microbiol. Biotechnol. 35, 582-588. Kerrigan, R. W., and Ross, I. K. (1989). Mycologia 74, 569. Kersten, P. J., Kalyanaraman, B., Hammel, K. E., Reinhammar, B. R., and Kirk, T. K. (1990). Biochem. J. 268,475-480. Kewalramani, N., Kamra, D. N., Lall, D., and Pathak, N. N. (1988). Biotechnol. Lett. 10, 369-372. Keyser, P., Kirk, T. K., and Zeikus, J. G. (1978). J. Bacteriol. 135, 790-797. Khan, S. M., and Ali, M. A. (1981). Mushroom Sci. 11, 691-696. Khanna, P., and Garcha, H. S. (1983). Taiwan Mushrooms 7(1), 18. Khanna, P., and Garcha, H. S. (1986). Mushroom News Lett. Trop. 6 , 17. Kiguchi, T., and Yanagi, S. 0. (1985). Appl. Microbiol. Biotechnol. 22, 121.

350

SOMASUNDARAM RAJARATHNAM ET AL.

Kihlberg, R. (1972). Annu. Rev. Microbiol. 26, 427. Kikumoto, S., Yamamoto, O., Komatsu, N., Kobayashi, H., and Kamasuka, T. (1978). U.S. Pat. 4,098,661. Kim, J. H., Hosobuchi, M., Koshimoto, M., Seki, T., Yoshida, T., Taguchi, H., and Ryu, D. D. Y. (1985). Biotechnol. Bioeng. 27, 1445-1450. Kim, Y. L., Lee, C. O., Shim, M. J., Kim, S. W., Choi, E. C., and Kim, B. K. (1984).Han’guk Kyunhakhoechi 12(1), 35-43. Kinoshita, S., Chua, J. W., Kato, N., Yoshida, T., and Taguchi, H. (1986). Enzyme Microb. Technol. 8, 691-695. Kinoshita, S., Okuno, K., Sawamura, K., and Yoshida, T. (1991).J. Ferment. Bioeng. 71(3), 151-155.

Kirin Brew. Co., Ltd. (1980). U.K. Pat. 2,023,131. Kirk, T. K. (1973). Phytopathology 63, 1504-1507. Kirk, T. K. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 2515. Kirk, T. K. (1983). In “The Filamentous Fungi” (J. E. Smith, D. R. Sarry, and B. Kristaisen, eds.), p. 266. Arnold, London. Kirk, T. K. (1988). FEMS Symp. 43, 315-332. Kirk, T. K., and Chang, H. M. (1981). Enzyme Microb. Technol. 3, 189-196. Kirk, T. K., and Chang, H. M. (1990). “Biotechnology in Pulp and Paper Manufacturer.” Butterworth, Stoneham, Massachusetts. Kirk, T. K., and Eriksson, K. E. (1990). In “World Pulp and Paper Technology, the International Review for the Pulp and Paper Industry” (R. Frank, ed.), pp. 23-28. Sterling, London. Kirk, T. K., and Farrell, R. L. (1987). Annu. Rev. Microbiol. 41,465-505. Kirk, T. K., and Kelman, A. (1965). Phytopathology 55, 739-745. Kirk, T. K., and Moore, W. E. (1972). Wood Fiber 4, 72-79. Kirk, T. K., and Tien, M. (1983). Recent Adv. Lignin Biodegrad. Res., Proc. Int. Semin., 2nd, Kyoto pp. 233-245. Kirk, T. K., and Yang, H. H. (1979). Biotechnol. Lett. 1, 347. Kirk, T. K., Connors, W. J., and Zeikus, J. G. (1976). Appl. Environ. Microbiol. 32, 192194.

Kirk, T. K., Mozuch, M. D., and Tien, M. (1985). Biochem. J. 226, 455-460. Kirk, T. K., Croan, S., Tien, M., Murtagh, K. E., and Farrell, R. L. (1986). Enzyme Microb. Technol. 8, 27-32. Kirk, T. K., Tien, M., Kersten, P. 7.. Kalyanaraman, B., Hammel, K. E., and Farrell, R. L. (1990). Methods EnzymoJ. 188, 159-171. Kirkpatrick, N., and Palmer, J. M. (1989). Appl. Microbiol. Biotechnol. 30, 305-311. Kirkpatrick, Reid, I. D., Ziomek, E., and Paice, M. (1990). Appl. Microbiol. Biotechnol. 33, 105-108.

Klesov, A. A., Bolobova, A. V., Ostrovskaya, M. V., and Poglazov, B. F. (1985). FEBS Lett. 187, 101-104.

Klysov, A. A., Mitkevich, 0. V., and Sinitsyn, A. P. (1986a). Biochemistry 25, 540-542. Klesov, A. A., Bolobova, A. V., Ostrovskaya, M. V., and Poglazov, B. F. (1986b). Biokhimiya (Moscow) 51, 188-194. Kolankaya, N., and Saglam, N. (1983). Doga, Ser. A 7, 513-517. Kolfstein, T. J. (1978). J. Anim. Sci. 46,841. Komai, T., Mizutani, S., Gocho, S., and Hatae, S. (1986).Jpn. Pat. 86 293,367 A 2; Jpn. Pat 61293367; Jpn. Pat. Appl. 85 134,384. Komatsu, N., Okubo, S., Kikurnoto, S., Kimura, K., Saito, G., and Sakai, S. (1969). Gann. 60, 137-144.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

351

Komatsu, N., Nikumoto, S., Kimura, K., Sakai, S., Kamasuka, T., Momaki, Y., Takada, S., Yamamoto, T., and Sugayama, J. (1973). U.S. at. 3,759,896. Kovdcs-Ligetfalusi, I. (1977). In “Soil Biology and Conservation of the Biosphere” 0. Szegi, ed.), pp. 287-291. Akademiai Kiadb, Budapest. Kozlik, I. (1980). Drev. Vysk. 25, 94-99. Kubo, K., and Nisizawa, K. (1983). J. Ferment. Technol. 61, 383-389. Kubo, K., and Nisizawa, K. (1984). Gakujutsu Kenkyu Hokoku 41, 9-14. Kurek, B., and Odier, E. (1990). Appl. Microbiol. Biotechnol. 34, 264-269. Kurosawa, S., Sugahara, T., and Hayashi, J. (1982). Nutr. Rep. Int. 26, 167. Kurosawa, S., Hayashi, M., and Ishizawa, H. (1984). Agric. Biol. Chem. 48, 299. Kurosawa, S., Shimabuku, A. M., Ishizawa, H., and Sen, K. (1990).Agric. Biol. Chem. 54, 587-592.

Kurtzman, R. H. (1976). Abstr., Am. Chem. SOC.Meet., San Francisco, Calif. Kurtzman, R. H. (1978a). Mushroom Sci. 10, 427. Kurtzman, R. H. (1978b). Mushroom Sci. 10, 429-436. Kutsuki, H., and Gold, M. H. (1982). Biochem. Biophys. Res. Commun. 109, 320-327. Kuwahara, M., and Asada, Y. (1987). Colloq. INRA 40, 171-176. Kvachadze, L. L., Chkhartishvili, D. A., Milkhlin, E. D., and Kvestiadze, G. I. (1985).Prikl. Biokhim. Mikrobiol. 21, 624-630. Kvestiadze, G. I., Kvachadze, L. L., Elisashvili, V. I., and Kachlishvili, E. T. (1988). VlT Symp. 88,131-140. Kwan, H. S., and Chang, S. T. (1981). Mushroom Sci. 11, 585-594. Lamaison, J. L., Pourrat, H., and Pourrat, A. (1980). Phytochemistry 19, 1021-1023. Lange, M., and Hora, F. B. (1963). In “A Guide to Mushrooms and Toadstools” (E. P. Button, ed.), p. 257. New York. Lankinen, V. P., Inkeroinen, M. M., Pellinen, J., and Hatakka, A. I. (1991). Water Sci. Technol. 24. 189-198. Lanzi, G. (1991). Mushroom Inf. 8(1/2), 18-27. Leal-Lara, H., and Eger-Hummel, G. (1982). Theor. Appl. Genet. 61, 65-68. Leatham, G. F. (1982). For. Prod. J. 332, 29-35. Leatham, G. F., and Griffin, T. J. (1984). Appl. Microbiol. Biotechnol. 20, 360-363. Leatham, G. F., and Kirk, T. K. (1983). FEMS Microbiol. Lett. 16, 65-67. Leatham, G. F., Craford, R. L., and Kirk, T. K. (1982). Appl. Environ. Microbiol. 46, 191. Leatham, M. J. (1979). In “Straw Decay and Its Effect on Utilization and Disposal” (E. Grossbard, ed.), pp. 131-137. Wiley (Interscience), New York. Lee, B., Pometto, A. L., Fratzke, A., and Bailey, T. B. (1991). Appl. Environ. Microbiol. 57(3), 678-685. Lee, J. S., Lee, U. J., and Suh, D. S. (1985). Sanop Misaengmul Hakhoechi 13, 65. Leisola, M. S. A. (1983). Comm. Eur. Communities [Rep.] EUR EUR-8641, 23-25. Leisola, M. S. A., and Garcia, S. (1989). In “Enzyme Systems for Lignin Degradation” (M. P. Coughlan, ed.), pp. 89-99. Elsevier Applied Science, London and New York. Leisola, M. S . A., Ulmer, D. C., Waldner, R., and Fiechter, A. (1984a).J. Biotechnol. 1,5-6. Leisola, M. S. A., Ulmer, D. C., and Fiechter, A. (1984b). Arch. Microbiol. 137, 171-175. Leisola, M. S. A., Meussdoerffer, F., Waldner, R., and Fiechter, A. (1985).J. Biotechnol. 2, 379-382. Leonard, T. J. (1971). J. Bacteriol. 106, 162-167. Leong, P. C. (1982). In “Tropical Mushrooms: Biological Nature and Cultivation Methods” (S. T. Chang and T. H. Quimio, eds.), pp. 349-361. Chinese Univ. Press, Hong Kong. Leonowicz, A., and Trojanowski, J. (1975). Microbios. 13, 167. Le Roux, P., and Danglot, Y. (1972). Mushroom Sci. 8, 471-474.

352

SOMASUNDARAM RAJARATHNAM ET AL.

Leung, W. T. W., Butrum, R. R., and Chang, F. H. (1972). “Food Composition Table for Use in East Asia. Part I: Food Policy and Nutrition Division.” Food Agric. Organ., Rome. Levonen-Munoz, E., and Bone, D. H. (1985). Biotechnol. Bioeng. 27, 382-387. Levonen-Munoz, E., Bone, D. H., and Daugulis, A. J. (1983a). Eur. J. Appl. Microbiol. Biotechnol. 18, 120-123. Levonen-Munoz, E., Bone, D. H., and Daugulis, A. J. (1983b). “Studies on Fractionation of Lignocellulosics of Oat Straw by Basidiomycetes,” Tech. Rep. 83-1. Dep. Chem. Eng., Queen’s Univ., Kingston, Jamaica. Li, J., and Zhu, 2. (1987). Fujian Nongxueyuan Xuebao 16, 220-223. Lin, J., Wang, H. Y., and Hickey, R. F. (1991). Biotechnol. Bioeng. 38, 273-279. Lin, J. N., Zheng, C. R., Zhang, Q. L., and Liu, Q. M. (1982). Mushroom News Lett. Trop. 2, 14-15.

Lindenfelser, L. A., Detroy, R. W., Ramstack, J. M., and Worden, K. A. (1979). Dev. Ind. Microbiol. 20, 541-551. Linko, Y . Y., Leisola, M., Lindholm, N., Troller, J., Linko, P., and Fiechter, A. (1986). J. Biotechnol. 4, 283-291. Lintzel, W. (1943). Chem. Ztg. 67,33-34. Livernoche, D., Jurasek, L., Desrochers, M., Dorica, J., and Valiky, I. A. (1983). Biotechnol. Bioeng. 25, 2055-2065. Lobarzewski, J., Trojanwski, J., and Woytas-Wasilewska, M. (1982). Holzforschung 36, 173.

Lucas, E. H. (1959). Centen. Rev. 3, 173-188. Lyr, H. (1958). Arch. Mikrobiol. 28, 310-324. Lyr, H.(1963). Phytopathol. Z. 47, 73-83. Machida, Y.,and Nakanishi, T. (1984). Agric. Biol. Chern. 48, 2463. Madan, M., and Bisaria, R. (1983). Biotechnol. Lett. 5,601-608. Madan, M., Vasudevan, P., and Sharma, S. (1987). Biol. Wastes 22, 241-250. Maeda, H., Choji, Y.,Hibino, Y . ,Yasumura, S., Masumi, A., and Sugano, N. (1983). Congr. Fed. Asian Biochem., 3rd, Bangkok. Maga, J. A. (1976). J. Agric. Food Chem. 16, 517. Maggioni, A,, and Renosto, F. (1970). Ind. Conserve 45, 311-314. Mandelstam, J. (1976). Proc. R. SOC.London, Ser. B 193,89-106. Manning, K.,and Wood, D. A. (1983). J. Gen. Microbiol. 129, 1839-1847. Manu-Tawaiah, W., and Martin, M. (1986). J. Sci. Food Agric. 37, 833-838. Margaritis, A,, and Creese, E. (1981). Biotechnol. Lett. 3, 471-476. Margaritis, A., Merchant. R., and Yaguchi, M. (1983). Biotechnol. Lett. 5, 265-270. Martinez, D., Quirate, M., Sato, C., Salmones, D., and Guzman, G. (1984). Biol. Soc. Mex. Micol. 19, 207. Martinez, D., Guzman, G., and Soto, C. (1985). Mushroom News Lett. Trop. 6, 21-28. Matsubara, H., and Feder, J. (1971). In “The Enzymes” (P. D. Boyer, ed.), 3rd Ed., Vol. 3, pp. 721-795. Academic Press, New York. Mc Hale, M. (1970). “Ecological Context.” Braziller, New York. Mc Leod, M. N., and Minson, D. J. (1980). Anirn. Feed Sci. Technol. 5, 347. Mc Nally, K. J., and Harper, D. B. (1991). J. Gen. Microbiol. 137, 1029-1032. Mel’nichuk, G.G., and Danilyak, M. I. (1981). Ukr. Bot. Zh. 38, 55-58. Memuna, H., and Chakrabarti, C. H. (1982). Indian J. Nutr. Diet. 19, 203. Mes-Hartree, M., Yu, E. K. C., Reid, I. D., and Saddler, J. N. (1987). Appl. Microbiol. Biotechnol. 26, 120-125. Michel, F., Balachandra, S., Grulke, E., and Adinarayana Reddy, C. (1991). Appl. Environ. Microbiol. 57(8),2368-2375.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

353

Michel, F. C., Jr., Grulke, E. A., and Reddy, C. A. (1990).J. Ind. Microbiol. 5, 103-112. Mickey, D. D., Bencuya, P. S., and Foulkes, K. (1989).Int. J. Immunopharmocol. 11,829838. Mileski, G. J., Bumpus, J. A., Jurek, M. A., and Aust, S. D. (1988).Appl. Environ. Microbiol. 54, 2885-2889. Milgram, L. (1985).New Sci. May 16,p. 16. Miller, M. W., and Jong, S. C. (1987).Dev. Crop Sci. 10,421-426. Millett, M. A., Baker, A. J., Feist, W. C., Mellenberger, R. W., and Satter, L. D. (1970). J. Anim. Sci. 31, 781. Millett, M. A., Baker, A. J., and Satter, L. D. (1976).Biotechnol. Bioeng. Syrnp. 6,125-153. Milstein, 0. A., Vered, Y., Sharma, A., Gressel, J., and Flowers, H. M. (1983).Appl. Environ. MicrobioI. 46, 55-61. Milstein, 0.A., Vered, Y., Sharma, A., Gressel, J., and Flowers, H. M. (1986).Biotechnol. Bioeng. 28, 381-386. Mishra, C., Forrester, I. T., Kelley, B. D., Burgess, R. R., and Leatham, G. F. (1990).Appl. Microbiol. Biotechnol. 33, 226-232. Miyao, K. (1989).Jpn. Pat. 89 199,525A2; Jpn. Pat. 01199525;Jpn. Pat. Appl. 88 22,756. Molitoris, H. P. (1978).Mushroom Sci. 10, 243-263. Moore, D. (1975).Trans. Br. Mycol. SOC.65,134. Moore, N. W., and Walker, C. H. (1964).Nature (London) 201, 1072. Moo-young, M., Chahal, D. S., Swan, J. E., and Robison, C. W. (1977).Biotechnol. Bioeng. 19, 527. Mora, F., Comtat, J., Barnoud, F., Pla, F., and Noe, P. (1986).J. Wood Chem. Technol. 6 , 147-165. Mori, K., Toyomasu, T., Nanba, H., and Kuroda, H. (1987).Mushroom News Lett. Trop. 7 , 121-126. Morinaga, T., Kikuchi, M., and Nomi, R. (1985).Agric. Biol. Chem. 49,523-524. Morohoshi, N., Shibuya, Y., Murayama, A., Katayama, Y., and Haraguchi, T. (1989). Mokuzai Gakkaishi 35, 342-347. Mouri, T. (1976).Kanzume Jiho 55,104-118. Mourin, T., Seki, H., Hasegawa, K., and Seki, Y. (1981).Toyo Shokuhin Kenkyusho Kenkyu Hokokusho 14,113-125. Mudgett, R. E., and Paradis, A. J. (1985).J. Enzyme Microb. Technol. 7, 150-154. Mueller, H. W., and Trosch, W. (1986).Appl. Microbiol. Biotechnol. 24, 180-186. Mueller, H. W., Trosch, W., and Kulbe, K. D. (1984).Eur. Congr. Biotechnol. 3, 89-94. Mueller, H. W., Trosch, W., and Kulbe, K. D. (1988).FEMS Microbiol. Lett. 49,87-93. Mueller, J. (1987).Mushroom J. Trop. 7, 89-96. Mueller, J. C.,Gawley, J. R., and Hayes, W. A. (1984).Mushroom News Lett. Trop. 5, 3. Mueller, J. C., Gawley, J. R., and Hayes, W. A. (1985).Mushroom News Lett. Trop. 6,15-20. Mukherjee, K., and Sengupta, S. (1985).Can. J. Microbiol. 31, 773-777. Musilek, V., Cerna, V. J., Sasek, V., Semerdzieva, M., and Vondracek, M. (1969).Folia Microbiol. (Prague) 14, 377-387. Myers, G. C., Leatham, G. F., Wegner, T. H., and Blanchette, R. A.(1988).Tappi 71,105108. Nakamatsu, T., Akamatsu, T., Miyajima, R., and Shiio, I. (1975).Agric. Biol. Chern. 39, 1803. Nakamura, T., Yabuuchi, M., Kato, K., Masuda, M., and Akanuma, H. (1988).Eur. Pat. 261,591. Nanci Togyo Co., Ltd. (1979).Jpn. Pat. 80 96,090(ClClZN 1/14);Jpn. Pat. Appl. 79/617. Natalaya, S. (1978).Mushroom Sci. 10,555-559.

354

SOMASUNDARAM RAJARATHNAM ET AL.

Netzer, U. V. (1979). Mushroom Sci. 10, 703-711. Nicolini, L., Von Hunolstein, C., and Carilli, A. (1987). Appl. Microbiol. Biotechnol. 26, 95-98. Nigam, P., and Prabhu, K. A. (1988). Biotechnol. Lett. 10, 919-920. Nizkovskaya, 0. P., Pan’kov, I. M., Kochetova, G. A., and Manukovskii, N. S. (1981). Mikol. Fitopatol. 15, 398-401. Noda, S., and Matsui, S. (1987). Jpn. Pat. 6279,781 (87 79,781) (ClC12N9/08); Jpn. Pat. Appl. 85 218,610. Noe, P., Chevatier, J., Mora, F., and Comtat, J. (1986). Wood Chem. Technol. 6, 167-184. Odier, E., and Roch, P. (1983). Recent Adv. Lignin Biodegrad. Res., Proc. Int. Semin., Znd, Kyoto pp. 188-194. Odier, E., Mozuch, M. D., Kalyanaraman, B., and Kirk, T. K. (1988).Biochimie 70, 847852.

Odile, 0. K., and Elisabeth, G. (1989). Mutot. Res. 226, 121-126. Ogawa, K., and Toyama, N. (1982). Miyazaki Daigaku Nogakubu, Kenkyu Hokoku 29, 239-247. Ohtsuka, S., Uneo, S., Yoshikumi, C., Hiroshi, F., Ohmura, Y., Wada, T., Fujii, T., and Takahashi, E. (1977). U.S. Pat. 4,051,314. Olah, G. M., Desbiens, O., and Reisinger, 0. (1979). Mushroom Sci. 10, 437-450. Ololade, B. G., Mowat, D. N., and Winch, J. E. (1970). Can. J. Anim. Sci. 50, 657. Orth, A. B., Denny, M., and Tien, M. (1991). Appl. Environ. Microbiol. 57(9), 2591-2596. Ortega Cerrillia, M. E., Can Acosta, B., Herrera Patino, F., and Perez-Gil Romo, F. (1986). Arch. Latinoam. Nutr. 36, 345-350. Osaka Gas Co., Ltd. (1984). Jpn. Pat. 59,198,987 (84 198,987) (ClC12P19114); Jpn. Pat. Appl. 83 73,455. Oser, B. L. (1959). In “Protein and Amino Acid Nutrition” (A. A. Albanese, ed.), p. 281. Academic Press, New York. Otjen, L., Blanchette, R., Effland, M., and Leatham, G. (1987). Holzforschung 41,343-349. Paice, M. G. (1989). Tappi 72, 217. Pal, A., Das, A., and Roy, A. (1986). Folio Microbiol. (Prague) 31, 203-206. Paszczynski, A., Huynh, V. B., and Crawford, R. (1986). Arch. Biochem. Biophys. 244, 750-765. Paturau, J. M. (1982). “Byproducts of the Cane Sugar Industry.” Elsevier, Amsterdam. Pellinen, J. (1988). Tappi 71, 191. Penn, C. A. (1976). Mushroom J. 37, 13-16. Perie, F., and Gold, M. H. (1991). Appl. Environ. Microbiol. 57(8), 2240-2245. Perry, F. G. (1987). Mushroom J. 171, 97-103. Peyton, T. 0. (1984). Enzyme Microb. Technol. 6, 146. Phillip, B., Dan, D. C., Fink, H. P., Eriksson, K. E., and Pettersson, B. (1984). Acta Biotechnol. 4, 333-345. Pidgen, W. J., and Heaney, D. P. (1969). Adv. Chem. Ser. No. 95, 245-261. Pilat, A. (1935). “Atlas des champignons de 1’Europe. Tome Ili: Pleurotus Fries.” Charles Kamia et Albert Pilat, Prague. Pirazzi, R., Cavalcaselle, B., and Ricci, G. (1978). Cellul. Carta 29, 9. Plassard, C. S., Mousain, D. G., and Salsac, L. E. (1982a). Phytochemistry 21, 345-348. Plassard, C. S., Mousain, D. G., and Salsac, L. E. (1982b). Colloq. INRA 13, 373-379. Platt, M. W., Chet, I., and Henis, Y . (1981). Eur. J. Appl. Microbiol. Biotechnol. 13, 194195. Platt, M. W., Chet, I., and Henis, Y. (1982). Mushroom J. 120, 425-427. Platt, M. W., Trojonowski, J., Chet, I., and Huttermann, A. (1983). Mirobiol. Lett. 23, 1921.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

355

Platt, M. W., Hadar, Y., and Chet, I. (1984). Appl. Microbiol. Biotechnol. 20, 150-154. Platt, M. W., Hadar, Y., and Chet, I. (1985). Appl. Microbiol. Biotechnol. 21, 394-396. Popp, J. L., Kalyanaraman, B., and Kirk, T. K. (1990). Biochemistry 29, 10475-10480. Pyysalo, H. (1978). Mushroom Sci. 10, 669-675. Qin, S., Zhang, H., Ren, L., and Yan, X. (1989). Huanjing Kexue 9,76-78. Quackenbush, F. W., Peterson, W. H., and Steenbock, H. (1935). J. Nutr. 10, 625-643. Quimio, T. H., and Abilay, L. E. (1983).Mushroom News Lett. Trop. 3,6-7. Rabinovich, M. L., Savickiene, R., Gerasimas, V., Melnik, M. S., Novikova, T.V., Steponavicius, I., Dienys, G., and Klesov, A. A. (1985). Bioorg. Khim. 11, 1330-1342. Raeder, U., Thompson, W., and Broda, P. (1987). Philos. Trans. R. SOC.London 321,475483.

Rajarathnam, S.(1981). Ph.D Thesis, Univ. of Mysore, Mysore, India. Rajarathnam, S., and Zakia Bano. (1987a). Indian Mushroom Sci. 2, 296-304. Rajarathnam, S.,and Zakia Bano. (1987b). CRC Crit. Rev. Food Sci. Nutr. 26, 157-223. Rajarathnam, S., and Zakia Bano. (1988). CRC Crit. Rev. Food Sci. Nutr. 26, 243-311. Rajarathnam, S., and Zakia Bano. (1989). CRC Crit. Rev. Food Sci. Nutr. 28, 31-113. Rajarathnam, S., and Zakia Bano. (1991). In “Advances in Applied Mycology. 3: Foods and Feeds” (D. K. Arora and E. H. Marth, eds.), pp. 241-292. Dekker, New York. Rajarathnam, S., Zakia Bano, and Muthu, M. (1977). Mushroom J. 57, 294-296. Rajarathnam, S.,Wankhede, D. B., and Patwardhan, M. V.(1979a). Eur. J. Appl. Microbiol. Biotechnol. 8, 125-134. Rajarathnam, S., Singh, N. S., and Zakia Bano. (1979b). Ann. Appl. Biol. 92, 323-328. Rajarathnam, S.,Zakia Bano, and Narasimham, P. (19831. Mushroom News Lett. Trop. 3, 3-11.

Rajarathnam, S., Zakia Bano, and Patwardhan, M. V. (1986). J. Hortic. Sci. 61, 223-232. Rajarathnam, S., Wankhede, D. B., and Zakia Bano. (1987).J. Chem. Technol. Biotechnol. 37, 203-214. Rajarathnam, S., Zakia Bano, Berger, R. G., and Drawert, F. (1991). Chem. Mikrobiol. Technol. Lebensm. 12, 145-150. Rajiv, K. (1991).Appl. Environ. Microbiol. 57(6),1735-1739. Ramaswamy, K., Kelley, R. L., and Reddy, C. A. (1985). Biochem. Biophys. Res. Commun. 131, 436-441. Randle, P. E. (1983). Crop. Res. 23, 51-69. Rangaswamy, K., and Kandaswamy, T. K. (1976). Indian J. Mushrooms 2, 8-11. Raper, C. A. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 83-118. Academic Press, New York. Rauter, W. (1975). 2. Lebensm-Unters.-Forsch. 159, 149-151. Rawal, P. P., Singh, R. D., and Khander, R. R. (1981). Indian J. Mushrooms 7, 14-17. Reade, A. E., and Mc Queen, R. E. (1983). Can. J. Microbiol. 29, 457-463. Reese, E. T., Sir, R. G . , and Levinson, H. S. (1950). J. Bacteriol. 59, 485-497. Reid, I. D. (1983a). Appl. Environ. Microbiol. 45, 830-837. Reid, I. D. (1983b). Appl. Environ. Microbiol. 45, 838-842. Reid, I. D. (1985). Appl. Environ. Microbiol. 50, 133-139. Reid, I. D. (1989). Enzyme Microb. Technol. 11,786-803. Reid, I. D. (199la). Appl. Environ. Microbiol. 57(10),2834-2840. Reid, I. D. (1991b). Can. J. Bot. 69, 156-160. Reid, I. D. ( 1 9 9 1 ~ )Biotechnol. . Tech. 5, 215-218. Reid, I. D., and Seifert, K. A. (1980). Can. I. Microbiol. 26, 1168-1171. Reid, I. D., and Seifert, K. A. (1982). Can. J. Bot. 60, 252-260. Reid, I. D., Chao, E. E., and Dawson, P. S. S. (1985). Can. J. Microbiol. 31, 88-90.

356

SOMASUNDARAM RAJARATHNAM ET AL

Reid, I. D., and Deschamps, A. M. (1991). Can. J. Bot. 69, 147-155. Rexen, F. P. (1969). Feed Stuffs 51, 33. Riaz, M., Wilke, C. R., Yamanaka, Y., and Carroad, P. A. (1977). Proc. Symp. Res. Appl. Notl. Needs 2, 108-115. Riondel, J., Beriel, H., Dardas, A., Carraz, G., and Oddoux, L. (1981).Arzneh-Forsch. 31, 293-299.

Robbins, S. R. J. (1981). Trop. Prod. Inst. [Rep.] (G.B.) No. 6171. Roch, P., Buswell, J. A., Cain, R. B., and Odier, E. (1989).Appl. Microbiol. Biotechnol. 31, 587-591.

Rohtagi, K., Kuhad, R. A., and Johri, S. N. (1986). J. Microbiol. Biotechnol. 1, 91. Rolz, C., De Leon, R., De Arriola, M. C., and De Cabrera, S. (1986). Appl. Environ. Microbiol. 52, 607-611. Rolz, C., De Leon, R., De Arriola, M. C., and De Cabrera, S. (1987). Appl. Microbiol. Biotechnol. 25, 535-541. Rolz, C., De Leon, R., and De Arriola, M. C. (1988). Acta Biotechnol. 8, 211-223. Rouau, X., and Foglietti, M. (1985). Biochem. SOC.Trans. 13, 451-452. Roughan, P. G., and Holland, R. (1982). J. Sci. Food Agric. 28, 1057-1064. Rouland, C., Civas, A., Renoux, J., and Petek, F. (1988). Comp. Biochem. Physiol., B: Comp. Biochem. SlB, 449-458. Royse, D. J., and May, B. (1982). Mycologia 74, 569. Royse, D. J., and May, B. (1989). Agric. Biol. Chem. 53, 2861-2866. Royse, D. J., Schisler, L. C., and Diehle, D. A. (1985). Interdiscip. Sci. Rev. 10, 329-335. Rudder, V. (1984). Ger. Pat. 3,246,265 A1. Ruelius, H. W., Kerwin, R. M., and Janssen, F. W. (1968).Biochim. Biophys. Acta 167,493. Ryan, T. P., and Bumpus, J. A. (1989). Appl. Microbiol. Biotechnol. 3, 302-307. Rypacek, V. (1966). “Biologie Holzerstorenda Pilze.” Fischer, Jena. Sachs, I. B., Leatham, G. F., and Myers, G. C. (1989). Wood Fiber Sci. 21, 331-342. Samajpati, N. (1979). Mushroom Sci. 10,695. Samuelsson, L., Mjoberg, P. J., Hartler, N., Vallander, L., and Eriksson, K. E. (1980). Sven. Papperstidn. 83, 221-225. Sanglard, D., Leisola, M. S. A., and Fiecht, A. (1986). Enzyme Microb. Technol. 8, 209212.

Sarkanen, S., Razal, R. A., Piccariello, T., Yamamoto, E., and Lewis, N. G. (1991). J. Biol. Chem. 266, 3636-3643. Sato, E., Aoyagi, Y., and Sugahara, T. (1985). Nippon Shokuhin Kogyo Gakkoishi 32, 509. Satyawati, S., Mira, M., and Padma, V. (1989). J. Ferment. Bioeng. 68, 296-297. Schalch, H. (1989). Mol. Cell. Biol. 9, 2743-2750. Schanel, L., Herzig, I., Dvorak, M., and Veznik, M. (1966). Zlepsovaci Novrh C 13, 1-14 (Pat. CSSR). Schisler, L. C., and Patton, T. G . , Jr. (1978). Mushroom Sci. 10,173-185. Schmidt, O., and Kebernik, U. (1987). Mater. Org. 22, 237-255. Schmitz, H., and Eger, H. G. (1981). In “Advances in Biotechnology” (M. Moo-young and C. W. Ribonson, eds.), pp. 505-509. Pergamon, Oxford. Schreiner, R. P., Stevens, S. E., Jr., and Tien, M. (1988). Appl. Environ. Microbiol. 54, 1858-1860.

Schuchardt, F., and Zadrazil, F. (1986). COST-84 bis EG Workshop. Schurz, J. (1978). In “Bioconversion of Cellulosic Substances into Energy Chemicals and Microbial Protein” (T. K. Ghose, ed.), p. 37. Thomas Press, New Delhi. Schwalb, M. N. (1975). Biochem. Biophys. Res. Commun. 67,478-482. Scott, R. W., Millett, M. A., and Hajny, G. J. (1969). For. Prod. J. 19, 14-18.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

357

Scrimshaw, N. S. (1975). In “Single Cell Protein 11” (S. R. Tannebaum and D. I. C. Wang, eds.). MiT Press, Cambridge, Massachusetts. Semichaevskii, V. D. (1988). Itogi Nauki Tekh., Ser. Biotekhnol. 10, 97-132. Semichaeviskii, V. D., Butovich, I. A., and Tsarevich, N. V. (1984). Dopov. Akad. Nauk Ukr. RSR, Ser. B: Geol., Khim. Biol. Nanki 10, 75. Semichaevskii, V. D., Dudchenko, L. G., and Melnichuk, G. G. (1985). Mikrobiol. Zh. 47, Sengupta, S., and Sengupta, S. (1990). Enzyme Microb. Technol. 12, 309-314. Sengupta, S., Naskar, A. K . , and Jana, M. L. (1984). Biotechnol. Bioeng. 26, 188-190. Senior, D. J. (1988). Biotechnol. Lett. 10, 907. Sermanni, G. G., and Luna, M. (1981). Mushroom Sci. 11, 485-496. Sermanni, G. G., Basile, G., and Luna, M. (1979). Mushroom Sci. 10, 37-49. Setliff, E. C., and Eudy, W. W. (1980). Lignin Biodegrad.: Microbiol., Chem., Potential Appl., Proc. Int. Semin., Madison, Wis., 1978 1, 135-149. Shangguan, Z. (1988). Shipin Kexue (Beijing) 103, 28-30. Shannon, L. J., and Stevenson, K. E. (1975a). J. Food Sci. 40, 826-829. Shannon, L. 7.. and Stevenson, K. E. (1975b). 1. Food Sci. 40, 830-832. Sharma, H. S. S. (1978a). Appl. Microbiol. Biotechnol. 26, 358-362. Sharma, H. S. S. (1978b). Appl. Microbiol. Biotechnol. 13, 194-195. Sharma, H. S. S. (1987). Appl. Microbiol. Biotechnol. 25, 542-546. Shiio, T., Okunishi, M., and Okumura, S. (1974). Mushroom Sci. 9, 799-808. Shimada, M. (1980). Lignin Biodegrad.: Microbiol., Chem., Potential Appl., Proc. Int. Semin., Madison, Wis., 1978 1, 195-213. Shimada, M. (1984). Mokuzai Kenkyu Shiryo 17, 21-53. Shimada, M., Nakatsubo, F., Kirk, T. K., and Higuchi, T. (1981). Arch. Microbiol. 129, 321-324.

Shirohata, A., Mori, K., Kishida, K., Maruyama, I., and Suzuki, K. (1989). Abstr. Pap. Annu. Meet. AIDS Kenkynukai, Mastue p. 63. Shivrina, A. N. (1965). “Biologically Active Compounds of Higher Fungi.” Izd. Nauka, Moscow. Sims, P., Brown, A., James, C., Raeder, U., Schrank, A,, and Broda, P. (1988). FEMS Symp. 43, 365-373.

Sinden, J. W. (1971). Annu. Rev. Phytopathol. 9, 411. Singh, N. S., and Rajarathnam, S. (1977). Curr. Sci. 46, 617. Skryabin, G. K., Golovleva, L. A., Mal’tseva, 0. V., and Myasoedova, N. M. (1985). Izv. Akad. Nauk SSSR, Ser. Biof. 3, 330-338. Smith, A. H. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 3-34. Academic Press, New York. Smith, R. E., Osothsilp, C., Bicho, P., and Gregory, K. F. (1986). Biotechnol. Lett. 8, 31-36. Smith, J. F., Fermor, T. R., and Zadrazil, F. (1987). In “Treatment of Lignocellulosics with White-Rot Fungi” (F. Zadrazil and P. Reiniger, eds.), pp. 3-13. Elsevier, New York. Solov’ev, V. A., Malysheva, 0. N., Saplina, V. I., Maleva, I. L., and Strel’mikova, L. L. (1982). Mezhvuz. Sb. Nauchn. Tr., Ser. Ekol. Zashch. Leca 7, 128-134. Sommer, A., Skultetoyova, N., and Ginterova, A. (1978). Pol’nohospodarstvo 24, 152158.

Sonnenberg, A. S., Wessels, J. G., and Van Greinsven, L. J. (1988). Curr. Microbiol. 17,285. Stanek, M., and Rysava, J. (1971). Mykol. Sb. 8, 59. Stevanovic-Janezic, T., Bujanovic, B., and Miric, M. (1988). Bull. Liaison Groupe Polyphenols 14,161-164. Streeter, C. L., and Horn, G. W. (1980). Okla., Agric. Exp. Stn., Res. Rep. No. 795.

358

SOMASUNDARAM RAJARATHNAM ET AL

Streeter, C., Conway, K. E., Horn, G. W., and Mader, T. I. (1982). J. Anim. Sci. 54, 183. Sugahara, T., Arai, S., Aoyagi, Y., and Kunisaki, N. (1975). J. Jpn. Soc. Nutr. Food Sci. 28, 477-483. Sugano, N., Hibono, Y., Choji, Y., and Maeda, H. (1982). Cancer Lett. 17, 109. Sugano, N.,Choji, Y., Hibino, Y., Yasumura, S., and Maeda, H. (1985). Cancer Lett. 27, 1. Sujan, A.,Tan, A. G., and Stevens, M. (1980). Meded. Fac. Landbouwwet., Rijksuniv. Gent 45, 1255-1260. Sundstol, F., Coxworth, E., and Mowat, D. N. (1978). World Anim. Prod. 26, 13. Suzuki, H., Okubo, A., Yamazaki, S., Suzuki, K., Mitsuya, H., and Toda, S. (1989). Biochem. Biophys. Res. Commun. 160,367. Iiyama, K., Yoshida, O., Yamazaki, S., Yamamoto, N., and Toda, S. (1990). Suzuki, H., Agric. Biol. Chem. 54, 479-487. Szudyga, K. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 559-571. Academic Press, New York. Taguchi, T., Ohawaki, K., and Okuda, J. (1985). J. Appl. Biochem. 7, 289. Tai, D.,Terasawa, M., Chem, C. L., and Chang, H. M. (1983). Recent Adv. Lignin Biodegrad. Res., Proc. Int. Semin., 2nd, Kyoto pp. 44-63. Tai, D., Terazawa, M., Chen, C., and Chang, H. (1990). Holzforschung 44,185-190. Takama, F., Sasaki, M., and Nunomura, N. (1978). Mushroom Sci. 10, 677-684. Takahashi, Z.(1976). Kanzume Jiho 55, 153-157. Tam, S.C., Yip, K. P., Fung, K. P., and Chang, S. T. (1986). Biotechnol. Bioeng. 38, 11551161. Tamada, M., Kasai, N., Kumakura, M., and Kaetsu, I. (1986). Biotechnol. Bioeng. 28, 1227-1232. Tamaki, H., Kishihara, S., Fujii, S., Komoto, M., Arita, A. I . , and Hiratuka, N. (1986). Nippon Shokuhin Kogyo Gakkaishi 33, 270-273. Tamura, S., and Arai, (1986). Jpn. Pat. 86 177,986. Tapia, G., Curotto, E., O’Reilly, S., and Gonzalez, G. (1981). FEBS Lett. 130, 205-207. Terada, M., Minami, J., Abe, N., Sato, M., Horie, Y., and Komiyama, M. (1985). Jpn. Pat. 85 217,899 A2; Jpn. Pat. 60217899; Jpn. Pat. Appl. 84 75,505. Terashita, T., and Kono, M. (1984). Kinki Daigaku Nogakubu Kiyo 17, 113-119. Terashita, T., and Kono, M. (1989). Kinki Daigaku Nogakubu Kiyo 22, 5-12. Thayumanavan, B. (1979). Farm Sci. 6, 33. Thayumanavan, B. (1982). Madras Agric. J. 69, 132-134. Thayumanavan, B.,and Manickam, A. (1980). Indian J. Nutr. Diet. 17, 140. Thomas, B., Roughan, J. A,, and Walters, E. D. (1972). J. Sci. Food Agric. 23, 1493. Thomas, D., and Mullins, J. T. (1967). Science 156,84-85. Thorn, R. G., and Barron, G. L. (1984). Science 224, 76-78. Tien, M. (1987). CRC Crit. Rev. Microbiol. 15, 141-168. I‘ien, M., and Kirk, T. K. (1983). Science 221,661-663. Tien, M.,and Kirk, T. K. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 2280-2284. Tien, M., Kirk, T. K., Bull, C., and Fee, J. A. (1986). J. Biol. Chem. 261, 1687-1693. Timofeeva, S. S . , and Ustyuzhina, G. S. (1983). Deposited Doc. VINITI No. 3283. Timofeeva, S. S., and Vatyuzhina, G. S. (1984). U.S.S.R. Pat. SU 1,130,540 (ClCOZ,F3/32); U.S.S.R. Pat. Appl. 3,504,972. Tochikura, S. T., Nakashima, H., Ohashi, Y., and Yamamoto, N. (1988). Med. Microbiol. Immunol. 177, 235. Tokimoto, K., and Kawai, A. (1975). Rep. Tottori Mycol. Inst. 12, 25. Tokimoto, K., and Komatsu, M. (1978). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes. eds.), pp. 445-473. Academic Press, New York. Tolentino, P. R. (1981). Mushroom Sci. 11, 577-584.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

359

Tominaga, Y. (1978). In “The Biology and Cultivation of Edible Mushrooms” ( S . T. Chang and W. A. Hayes, eds.), pp. 683-697. Academic Press, New York. Tonomura, H. (1978). In “The Biology and Cultivation of Edible Mushrooms” ( S . T. Chang and W. A. Hayes, eds.), pp. 409-421. Academic Press, New York. Tonon, F., and Odier, E. (1988). ~ p p l Environ. . Microbiol. 54, 466-472. Toyama, N., and Ogawa, K. (1974). Mushroom Sci. 9, 745-760. Tressl, R., Bahri, D., and Engel, K. H. (1982). J. Agric. Food Chem. 30, 89. Trojanowski, J., and Leonowicz, A. (1985). Polish Patents PL 126830B1 and PL 227367. Uniwersylet Marii Curie-Sklodowsklej. Trutneva, I. A. (1978). Ukr. Bot. Zh. 35, 625. Tsang, L., Reid, I. A., and Coxworth, E. (1987). Appl. Environ. Microbiol. 53, 1304-1306. Tsao, G. T. (1978). Annu. Rep. Ferment. Processes 2, 1. Tschierpe, H.J. (1959). Mushroom Sci. 4, 211. Tschierpe, H. J., and Hartman, K. (1977). Mushroom J. 60, 404-416. Tsuruta, T., and Kawai, M. (1983). Nippon Kingakkai Kaiho 24, 65. Turner, E. M., Wright, M., Ward, T., Osborne, D. M., and Self, R. (1975).J. Gen. Microbiol. 91,167-176.

Tyler, G. (1980). Trans. Br. Mycol. SOC.74, 41. Uchimoto, I. (1988). Jpn. Pat. 135 597/88. Uden, P. (1984). Dev. Anim. Vet. Sci. pp. 533-556. Ulmer, D., Leisola, M., Puhakka, J., and Fiechter, A. (1983). Eur. J. Appl. Microbiol. Biotechnol. 18, 153-157. Umezawa, T. (1988). Wood Res. 75, 21-79. Umezawa, T., and Higuchi, T. (1987). FEBS Lett. 218, 255-260. Umezawa, T., Umezawa, T., Nakatsubo, F., and Higuchi, T. (1982). Arch. Microbiol. 131, 124-128. Umezurike, G. M. (1970). Ann. Bot. 34, 217. Urushadze, T. R., Khomasuridze, T. S., and Gogiblashvili, L. Z. (1989). Soobshch. Akad. Nauk Gruz. SSR 133, 153-156. Ushiyama, R., and Nakai, Y. (1977). Rep. Tottori Mycol. Inst. 15, 1. Valmaseda, M., Almendros, G., and Martinez, A. T. (1990). Appl. Microbiol. Biotechnol. 33, 481-484.

Vetter, J. (1983). Ukr. Bot. Zh. 40, 74. Viesturs, U., Leite, M., and Strikauska, J. (1985). In “Monitoring and Control of Plant Raw Material Bioconversion” (M. L. Niku-paavola, ed.), pp. 103-124. Tech. Res. Cent. Finland, Espoo. Viikari, L., and Linko, M. (1977). Process Biochem. 12, 17. Volc, J., Sedmera, P., and Musilek, V. (1978). Folia Microbiol. (Prague) 23, 292. Wang, C. W. (1982). In “Tropical Mushrooms-Biological Nature and Cultivation Methods” (S. T. Chang and T. H. Quimio, eds.), pp. 167-186. Chinese Univ. Press, Hong Kong. Wang, C. W. (1985). Mushroom News Lett. Trop. 5, 4-8. Wang, Y., and Wang, Y. (1989). Weishengwuxue Tongbao 16, 137-140. Wariishi, H., Valli, K., and Gold, M. H. (1989). Biochemistry 28, 6017-6023. White, P. F. (1985). In “The Biology and Technology of the Cultivated Mushroom” (P. B. Flegg, D. M. Spencer, and D. A. Wood, eds.), pp. 279-336. Wiley, Chichester, England. Wicklow, D. T., Detroy, R. W., and Jessee,B. A. (1980).Appl. Environ. Microbiol. 40,169170.

Wicklow, D. T., Langie, R., Crabtree, S . , and Detroy, R. W. (1984). Can. J. Microbiol. 30, 632-636.

360

SOMASUNDARAM RAJARATHNAM ET AL.

Woggon, H., and Bickerich, K. (1978). Nahrung 22, K13. Wojfas-Wasilewska, M., Trojanowski, J., and Luterek, 1. (1980). Acta Microbiol. Pol. 29, 353-364. Wojfas-Wasilewska, M., Trojanowski, J., and Luterek, J. (1983). Acta Biochim. Pol. 30,291. Wolters, B., and Eilert, V. (1982). Z. Naturforsch., C: Biosci. 37C, 575-583. Wood, D. A. (1979). Biotechnol. Lett. 1, 255-260. Wood, D. A. (1980). J. Gen. Microbiol. 117, 327-338. Wood, D. A. (1984). J. Chem. Technol. Biotechnol. 34(B), 232-240. Wood, D. A. (1986). Mushroom J. 164, 257-265. Wood, D. A., and Fermor, T. R. (1981). Mushroom Sci. 11, 63-71. Wood, D. A,, and Goodenough, P. (1977). Arch. Microbiol. 114, 161-165. Wood, D. A., and Smith, J. F. (1988). Mushroom J. 188, 665-674. Woranisrakul, C., Kinoshita, S., and Taguchi, H. (1988).J. Sci. SOC.Thailand 14,219-223. Xia, E., Wang, S., and Chen, Q. (1987). Zhongguo Yaoke Daxue Xuebao 18, 141-143. Xua, W., Wang, S., and Chen, Q. (1987). Zhongguo Yaoke Daxue Xuebao 18,45-47. Yadav, J. S. (1988). Biotechnol. Bioeng. 31, 414-417. Yamada, O.,Magey, Y., Kashiwagi, Y., Kakimoto, Y., and Sasaki, T. (1983). Eur. J. Appl. Microbiol. Biotechnol. 17, 298. Yamamura, Y., and Cochran, K. W. (1974). Mushroom Sci. 9, 495-507. Yanagi, S. O., and Takebe, I. (1984). Appl. Microbiol. Biotechnol. 19, 58. Yanagi, S. O., Monma, M., Kawasumi, T., Hino, A., Kito, M., and Takebe, I. (1985). Agric. Biol. Chem. 49,171. Yasui, A., Tsutsumi, C., Takasaki, M., and Mori, T. (1988). Nippon Shokuhin Kogyo Gakkaishi 35, 160-165. Yomeishu Seizo Co., Ltd. (1984). Jpn. Pat. 84 166,076 A2; Jpn. Pat 59166076; Jpn. Pat. Appl. 83 38,264. Yoshida, H., Sugahara, T., and Hayashi, J. (1983).J. Jpn. SOC.Food Sci. Technol. 30, 375378. Yoshioka, Y., Emori, M., Ikekawa, T., and Fukuoka, F. (1975). Carbohydr. Res. 43, 305320. Young, J., Griffin, E., and Russell, J. (1986). Biomass 10, 9. Yu, H. S., and Eriksson, K. E. (1985). Sven. Papperstidn. 88, R57-R60. Zadrazil, F. (1973). Champignon 13, 3. Zadrazil, F. (1976a). Mushroom Sci. 9, 621-652. Zadrazil, F. (1976b). Z. Aker. Pflanzenbou 142, 44-52. . Forsch., Sonderh. 32, 153. Zadrazil, F. ( 1 9 7 6 ~ )Landwirtsch. Zadrazil, F. (1977). Eur. J. Appl. Microbiol. 4,273-281. Zadrazil, F. (1978a). In “The Biology and Cultivation of Edible Mushrooms” (S. T. Chang and W. A. Hayes, eds.), pp. 521-557. Academic Press, New York. Zadrazil, F. (1978b). Mushroom Sci. 10, 529-538. Zadrazil, F. (1979a). In “Straw Decay and Its Effect on Utilization and Disposal” (E. Grossbard, ed.), pp. 139-146. Wiley (Interscience), New York. Zadrazil, F. (1979b). Mushroom Sci. 10,231-239. Zadrazil, F. (1980a). Eur. J. Appl. Microbiol. Biotechnol. 9, 243-248. Zadrazil, F. (1980b). Eur. J. Appl. Microbiol. Biotechnol. 9, 31-35. Zadrazil, F. (1981). Adv. Biotechnol. 2, 369-374. Zadrazil, F. (1985a). In “Degradation of Lignocellulosics in Ruminants and in Industrial Processes” (J. M. Van Der Meer, M. P. Rijkens, and B. A. Ferrante, eds.), pp. 55-62. Elsevier, New York. Zadrazil, F. (1985b). Angew. Bot. 59, 433-452.

BIOPOTENTIALITIES OF BASIDIOMACROMYCETES

361

Zadrazil, F., and Brunnert, H. 11979). Z . Pflanzenernaehr. Bodenkd. 142,446. Zadrazil, F., and Brunnert, H. (1980).Eur. J. Appl. Microbiol. Biotechnol. 9, 37-44. Zadrazil, F., and Brunnert, H. (1981). Eur. J. Appl. Microbiol. Biotechnol. 11, 183-191. Zadrazil, F., and Brunnert, H. (1982). Eur. I. Appl. Microbiol. Biotechnol. 16, 45-51. Zadrazil, F., and Grabbe, K. (1983). In “Biotechnology” (H. Dellweg, ed.), Vol. 3, pp. 145165. Verlag Chemie, Weinheim. Zadrazil, F., and Kurtzman, R. H. (1982). In “Tropical Mushrooms-Biological Nature and Cultivation Methods” (S. T. Chang and T. H. Quimio, eds.), pp. 277-298. Chinese Univ. Press, Hong Kong. Zadrazil, F., and Peerally, A. (1986). Biotechnol. Lett. 8, 663-666. Zadrazil, F., and Schliemann, J. (1977). Mushroom Sci. 9, 199. Zadrazil, F., and Schneidereit, M. (1972). Champignon 12, 25. Zadrazil, F., Grinbergs, J., and Gonzslez, A. (1982). Eur. J. Appl. Microbiol. Biotechnol. 15, 167-171.

Zafar, S. I., Kausar, T., and Shah, F. H. (1981). Folia Microbiol. (Prague) 26, 394-397. Zakhary, J. W., El-Mahdy, A. R., Abo-Bakr, T. M., and El Tabey-Shehata, A. M. (1984).Food Chem. 13, 265-276. Zakia Ban0 (1967). Ph.D. Thesis, Univ. of Mysore, Mysore, India. Zakia Bano, and Rajarathnam, S. (1982a). Mushroom J. 115, 243-245. Zakia Bano, and Rajarathnam, S. (1982b). In “Tropical Mushrooms-Biological Nature and Cultivation Methods” (S. T, Chang and T. H. Quimio, eds.), pp. 363-382. Chinese Univ. Press, Hong Kong. Zakia Bano, and Rajarathnam, S. (1988). CRC Crit. Rev. Food Sci. Nutr. 27, 87-158. Zakia Bano, Narasimham, P., and Singh, N. S. (1975). Indian J. Microbiol. 15, 68-74. Zakia Bano, Rajarathnam, S., and Nagaraja, N. (1978). Mushroom Sci. 10, 597-608. Zakia Bano, Rajarathnam, S., and Muthu, M. (1981). Mushroom Sci. 11, 541-547. Zakia Bano, Rajarathanam, S., and Narasimhamurthy, K. (1986). Mushroom News Lett. 6, 11-16.

Zemek, J., and Kunaik, L. (1978). Czech. Pat. 186,059. Zetelaki-Hovrath, K. (1984). Biotechnol. Bioeng. 26, 389-396.

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INDEX

A

Acer pseudoplatanus, hydrodynamic shear stress, 212 Achromobacter, biphenyl degradation, 138 Achromobacter xerosis, biphenyl degradation, 152 Acinetobocter, biphenyl degradation, 139, 141-143, 145, 150 Actin, hydrodynamic shear stress, 169, 201, 206-208 Agaricus applications of spent substrate, 326328 biology, 236, 238, 241-242 growth substrate changes, 291, 293, 295, 300-302, 307, 310,313, 315 lignocellulosic substrates, 258, 261 lignocehlosic wastes, 270-272, 274, 280-281, 284, 287 mycelium, 331-332, 334 values, 246 Agrobacterium rhizogenes, hydrodynamic shear stress, 215 Agrocybe growth substrate changes, 290, 307 mycelium, 332 values, 245 Air-liquid interface, hydrodynamic shear stress, 186, 190 Alcaligenes, biphenyl degradation, 139141, 147, 150-151, 154 Aldehydes, haloperoxidases, 75 Algae haloperoxidases, 42, 90, 92 reactions, 59-60, 64-65, 72, 75, 79 mechanisms of, 82, 86 sources, 45, 52 nitroaromatic compounds, 2

Alkenes, haloperoxidases, 56-63 Alzheimer’s disease, Ganoderma, 123 Amanita, biology, 236 Amines, haloperoxidases, 75-77 Amino acids basidiomacromycetes applications of spent substrate, 318, 326 growth substrate changes, 291-292, 301, 307, 310 lignocellulosic substrates, 268 values, 242-244, 246-247 Ganoderma, 102,109-110,114,119 haloperoxidases, 42, 45-46, 75-78 2-Amino-3-chloropyridine, haloperoxidases, 71 Aminonitrotoluene, nitroaromatic compounds, 3-4 2-Aminopyridine, haloperoxidases, 71 Ammonium, nitroaromatic compounds, 6-7 Ampicillin, haloperoxidases, 78 Anchorage-dependent cells, hydrodynamic shear stress, 195-202, 218 Anchorage-independent cells, hydrodynamic shear stress, 202-208 Angiotensin-converting enzyme, Ganoderma, 114,122 Anthranilate, nitroaromatic compounds, 9 Antibiotics, Ganoderma, 102, 123 Antibodies Ganoderma, 118-119 hydrodynamic shear stress, 201 Antibody labeling, hydrodynamic shear stress, 189 Antiflammatory activity, Ganoderma, 119-120 Antigens, Ganoderma, 118 Antimutagen, Ganoderma, 124

363

364

INDEX

Antitumor action, Ganoderma, 108-112 Antitumor activity, basidiomacromycetes, 248-250 Antitumor preparations, Ganoderma, 121-122 Aplysia, haloperoxidases, 42, 60 Arabidopsis thaliana, haloperoxidases, 79 Arachidonic acid Ganoderma, 122 haloperoxidases, 60 Aroclors, biphenyl degradation, 144, 146, 155-156 Arthrobacter biphenyl degradation, 136, 141-143, 147-148 nitroaromatic compounds, 8 Ascophyllum nodosum, haloperoxidases, 92 reactions, 56, 59, 65, 72, 78, 81 mechanisms of, 86-87, 89 sources, 46-47, 52 Ash, basidiomacromycetes, 295, 327 Aspergillus fumigatus, hydrodynamic shear stress, 194 Aspergillus niger, biphenyl degradation, 154 Assay methods, haloperoxidases, 53-56 Atherosclerosis, Ganoderma, 116 Auricularia biology, 238, 240, 242 growth substrate changes, 301 1ignoceh.dosic wastes, 270, 272, 285 mycelium, 333 values, 249 Azotobacter agilis, nitroaromatic compounds, 4

B B cells, Ganoderma, 118 Bacillus brevis, biphenyl degradation, 138 Bacteria basidiomacromycetes, 318, 320-321, 332 biphenyl degradation, 136, 154-155, 157 anaerobic degradation, 144

bacteria growth, 141-143 bioremediation trials, 146 metabolic pathway in bacteria, 136139 polychlorinated biphenyls, 139, 141 haloperoxidases, 90 reactions, 68, 70, 79 sources, 48-50 hydrodynamic shear stress, 167, 194, 215, 217 nitroaromatic compounds, 1, 3-4 stormwater, 22-26, 28-32, 34-36 Bacteroides fragilis, stormwater, 26 Barbituric acid, haloperoxidases, 71, 8486

Basidiomacromycetes, 234-236, 336-340 applications of spent substrate antiviral activity, 328-329 biogas production, 3 2 1 deodorization, 325 pulping agents, 321-324 recycling for mushroom culturing, 326-327 ruminant feed, 316-321 saccharification enzymes, 325-326 silica, 327 single-cell protein, 3 2 8 soil ameliorants, 327-328 biology geographic distribution, 238-241 morphology, 236-238 world production, 241-242 growth substrate changes biochemical, 297-316 chemical, 285-297 lignacellulosic substrates fermentation, 259-270 wastes, 250-259 lignocellulasic wastes biomass conversion efficiencies, 282-285 cultural conditions, 271-282 preparation of substrates, 270-271 mycelium decolorization, 332-333 fungal elicitors, 335 hair growth, 335 heavy metal accumulation, 331-332 low-alcohol wines, 335 metabolic substances, 336

365

INDEX oxidation of pollutants, 329-331 peroxidase production, 331 polyethylene, 336 polymer production, 334 pyranose oxidase, 334 retting of flax fibers, 335 tenderization of mushroom stipes, 334 test organisms, 336 values chemical composition, 242-246 medicinal properties, 247-250 nutrition, 246-248 Basidiomycetes, see also Basidiomacromycetes Ganoderma, 102,110,121-122,124 Bath preparation, Ganoderma, 124 Bathers, stormwater, 30, 35 Beijerinckia, biphenyl degradation, 136 Benzimidazoles, nitroaromatic compounds, 4 Benzoates, biphenyl degradation, 135, 138, 143, 146, 149, 152-154, 156 Bifidobacteria, stormwater, 26 Biogas production, basidiomacromycetes, 321

Biological efficiency (BE),basidiomacromycetes, 281, 284-285 Biological energetic efficiency (BEE), basidiomacromycetes, 291 Biological oxygen demand (BOD), basidiomacromycetes, 328, 332 Biomass conversion efficiencies, basidiomacromycetes, 282-285 Biomass yield, basidiomacromycetes, 287 Bioremediation, biphenyl degradation, 145-147, 156 Biphenyl degradation, 135-136, 155-157 chromosomal genes, 150-154 derivatives, 147-149 metabolic pathway in bacteria, 136139 metabolism, 154-155 plasmids, 149-150 polychlorinated biphenyls, 139-141 anaerobic degradation, 143-145 bacteria growth, 141-143 bioremediation trials, 145-147 Biphenyls, polychlorinated, see Polychlorinated biphenyls

Bleach plant effluent (BPE), basidiomacromycetes, 333 Bone marrow, Ganoderma, 120 Bromination biphenyl degradation, 147, 151 haloperoxidases, 91-92 alkenes, 57, 59-60 assay methods, 53, 55-56 heterocyclic compounds, 68-69, 71, 75 reactions, 75, 78, 81 mechanisms of, 82, 86-87 Bromohydrin, haloperoxidases, 56-57, 59-60, 63, 75 Bromoperoxidases, 90-92 alkenes, 59-60 aromatic compounds, 64 assay methods, 56 heterocyclic compounds, 68-69, 7172, 74-75 reactions, 75-76, 78-79, 81 mechanisms, 82, 86-89 sources, 43, 45-48, 50, 52-53 Bubble column, hydrodynamic shear stress, 185-186, 208-209, 212 Bubble effects, hydrodynamic shear stress, 204-205, 210

C Cadmium, basidiomacromycetes, 331-332 Ca1cium basidiomacromycetes, 295, 326 Ganoderma, 119 haloperoxidases, 44 hydrodynamic shear stress, 189, 201, 211, 217 Caldariomyces fumago, haloperoxidases, 90 alkenes, 56, 59-60, 65 heterocyclic compounds, 68, 71-73, 75 reactions, 77, 79, 81 mechanisms of, 82, 85, 87, 89 sources, 43-45, 50 Cantharellus cibarius, basidiomacromycetes, 243, 245 Carbohydrate, basidiomacromycetes applications of spent substrate, 317, 319

366

INDEX

growth substrate changes, 291, 299 lignocellulosic substrates, 258 mycelium, 329 values, 242-243, 246 Carbon basidiomacromycetes, 245, 336-337 applications of spent substrate, 326327 growth substrate changes, 303, 313315 lignocellulosic wastes, 285, 290 mycelium, 329-330, 333 biphenyl degradation, 135-136, 138139, 156 anaerobic degradation, 143 bacteria growth, 142 chromosomal genes, 153 derivatives, 147-148 haloperoxidases, 56, 72, 77, 91 nitroaromatic compounds, 1, 7-9, 11, 14 Carbon cycle, basidiomacromycetes, 234, 252, 269 Carbon dioxide basidiomacromycetes, 234, 339 applications of spent substrate, 317, 326 growth substrate changes, 286, 313315 lignocellulosic substrates, 252, 261, 269 lignocellulosic wastes, 280 mycelium, 329-331, 336 biphenyl degradation, 144, 146, 155 nitroaromatic compounds, 8, 11-12 Carboxymethylcellulase, basidiomacromycetes, 298-299 Cardiovascular system, Ganoderma, 114118 Catechol, biphenyl degradation, 138, 153 Catharanthus roseus, hydrodynamic shear stress, 212-213, 216 Cavitation, hydrodynamic shear stress, 210

cDNA, haloperoxidases, 48 Cell damage, collision-related, hydrodynamic shear stress, 181-185 Cell poker, hydrodynamic shear stress, 208

Cellulases, basidiomacromycetes applications of spent substrate, 323, 325-326 growth substrate changes, 296-300, 303 Cellulose, basidiomacromycetes applications of spent substrate, 317319, 321, 325-326 growth substrate changes, 287, 290, 292-294, 297-298, 310, 312-313 lignocellulosic substrates, 251-252, 257, 268-269 lignocellulosic wastes, 281, 284 mycelium, 335 Central nervous system, Ganoderma, 113 Cereal straws, basidiomacromycetes, 254256, 283, 337 Chemical disinfection, stormwater, 32-33 Chlamydomonas reinhardi, hydrodynamic shear stress, 193 Chloramines, stormwater, 33 Chloramphenicol, nitroaromatic compounds, 1-2 Chlorella, Ganoderma, 125 Chlorine basidiomacromycetes, 329-3 32 biphenyl degradation, 135, 142, 151 anaerobic degradation, 143-145 polychlorinated biphenyls, 139-141 haloperoxidases, 90-92 alkenes, 56, 59-60 aromatic compounds, 64-65 assay methods, 53 heterocyclic compounds, 68-72, 75 reactions, 75-78 mechanisms of, 82-85, 89 sources, 47-48, 50 stormwater, 32-33 Chloroacetophenones, biphenyl degradation, 156 4-Chloroaniline, nitroaromatic compounds, 4 Chlorobenzoates, degradation, 135, 155156 anaerobic degradation, 144 bacteria growth, 141-143 bioremediation trials, 146 chromosomal genes, 152-154 polychlorinated biphenyls, 140-141

367

INDEX 3-Chlorobiphenyl, degradation, 142, 156 4-Chlorobiphenyl, degradation, 135, 138, 156

bacteria growth, 141-143 chromosomal genes, 152-153 plasmids, 150 4-Chlorocatechol, nitroaromatic compounds, 8-9 Chloroforms, Ganoderma, 119 4-Chloronitrobenzene, nitroaromatic compounds, 4 Chloronitrophenol, nitroaromatic compounds, 8 Chloroperoxidases, 90, 92 alkenes, 56, 59-60 aromatic compounds, 64-65 reactions, 68-73, 75-76, 78-81 mechanisms of, 82, 84-85, 87-89 Chlorophenols, biphenyl degradation, 141, 144 Chlorophyll, haloperoxidases, 71, 85 CholesteroI, Ganoderma, 115-1 16 Chromatography, see also Gas chromatography-mass spectroscopy; Highpressure liquid chromatography; Thin-layer chromatography biphenyl degradation, 140-141 Ganoderma, 108 Chromosomes, biphenyl degradation, 135, 150-154, 156 Chronic bronchitis, Ganoderma, 123 Cinnamic acid, haloperoxidases, 59 Citrobacter, stormwater, 23-24 Citrobacter freundii, nitroaromatic compounds, 4 Citronella bagasse (CB), basidiomacromycetes, 284, 294, 297 Clones biphenyldegradation, 136,150,152-153 haloperoxidases, 44, 46, 52-53 nitroaromatic compounds, 14 Clostridiurn perfringens, stormwater, 26 Coffee pulp (CP], basidiomacromycetes, 284 Coliform, stormwater, 22-35 Collision frequency, hydrodynamic shear stress, 173 Collision-related cell damage, hydrodynamic shear stress, 181-185

Collision theory, hydrodynamic shear stress, 194-195, 2 2 1 Combined sewer overflows (CSO), stormwater, 26, 32-36 Concanavalin A, Ganoderma, 118-119 Congeners, biphenyl degradation, 145, 147, 153 Contaminants, basidiomacromycetes, 281-282 Coprinus, 337 biology, 236, 241 growth substrate changes, 297, 301 lignocellulosic wastes, 272, 280-281, 284-285 mycelium, 331 values, 245, 248, 250 Corallina pi1ulifera, haloperoxidases alkenes, 60 aromatic compounds, 64, 66 heterocyclic compounds, 71, 74-75 reactions, 81, 87 sources, 52 Corallina vancouveriensis, haloperoxidases, 65, 87 Coriolus applications of spent substrate, 322, 326

growth substrate changes, 293, 303, 315-316 mycelium, 334 values, 249 Corynebacterium, biphenyl degradation, 140 Couette viscometer. hydrodynamic shear stress, 194, 203, 207-208, 213 Crude protein, basidiomacromycetes, 235 Cudrania tricuspidata, hydrodynamic shear stress, 212 Cultivation, basidiomacromycetes, 272273, 280 Cunninghamella echinulata, biphenyl degradation, 154 Cunninghamella elegans, biphenyl degradation, 154 Curvularia inaequalis, haloperoxidases, 50, 56, 79, 88-89, 92 Cyanide, nitroaromatic compounds, 3 Cyanobacteria, biphenyl degradation, 154-155

368

INDEX

Cyathus, basidiomacromycetes, 317-318, 327 Cytochalasin, hydrodynamic shear stress, 205,208 Cytochrome P-450 haloperoxidases, 44-45 nitroaromatic compounds, 3 Cytodex microcarriers, hydrodynamic shear stress, 196-197 Cytoplasm, hydrodynamic shear stress, 189,223 cell architecture, 166, 169 sensitivity of biocatalysts, 195,197, 211, 217 Cytoskeleton, hydrodynamic shear stress, 187,189,223 cell architecture, 166, 169 sensitivity of biocatalysts, 200-202, 205,207-208

D Dechlorination, biphenyl degradation, 142-144 Decolorization, basidiomacromycetes, 332-333 Deformation, hydrodynamic shear stress, 168,223 fluid mechanics, 175,182 sensitivity of biocatalysts, 207-208,217 Degradation basidiomacromycetes, 339 applications of spent substrate, 317318,320,323,327 biochemical growth substrate changes, 297-316 chemical growth substrate changes, 285-297 lignocellulosic substrates, 260-261, 268-270 lignocellulosic wastes, 283-284 mycelium, 332,336 biphenyl, see Biphenyl degradation of nitroaromatic compounds, see Nitroaromatic compounds, microbial degradation of Degradatory enzymes, basidiomacromycetes, 306-310, 339

Deodorization of waste gases, basidiomacrornycetes, 325 Z’-Deoxyuridine, haloperoxidases, 74 Desulfovibrio desulfuricans, biphenyl degradation, 149 Detoxicant actions, Ganoderma, 113-114 Dibromobarbituric acid, haloperoxidases, 72 Dichlorobiphenyls, degradation, 136,143, 151 Dichlorodimedone, haloperoxidases, 45, 47 Dichlorophenol, nitroaromatic compounds, 7,12 2,4-Dichlorophenoxyaceticacid, Ganoderma, 120 N,N-Dichloropiperazine, haloperoxidases, 77 Dichomitus squalens applications of spent substrate, 322 growth substrate changes, 316 lignocellulosic substrates, 261 Digestible dry matter, basidiomacromycetes, 319 Dihalides, haloperoxidases, 56 Dihydroxybiphenyls, degradation, 136, 148-149,151-152 Dimethyl sulfoxide, Ganoderma, 112 Dinitroaniline, nitroaromatic compounds, 4,9 1,3-Dinitrobenzene, nitroaromatic compounds, 3-4,9-10 4,6-Dinitro-Z-methylphenol, nitroaromatic compounds, 3 2,4-Dinitrophenol, nitroaromatic compounds, 3-4 Dinitrotoluene, nitroaromatic compounds, 3-4 2,4-Dinitrotoluene, nitroaromatic compounds, 3,s Dioscorea deltoidia, hydrodynamic shear stress, 212 Dioxygenases, biphenyl degradation, 135, 138 chromosomal genes, 151-152 derivatives, 147,149 polychlorinated biphenyls, 139-141 Diseases, basidiomacromycetes, 281-282 Disinfection, stormwater, 21-22,31-34

369

INDEX DNA basidiomacromycetes, 249, 330 biphenyl degradation, 150-1 54 Ganoderma, 1 1 2 haloperoxidases, 44, 71 hydrodynamic shear stress, 203 DNA probes, stormwater, 26 Dry matter digestibility, basidiomacromycetes, 296 Dye exclusion test, hydrodynamic shear stress, 189

E Electron acceptors, biphenyl degradation, 156 Electron microscopy, hydrodynamic shear stress, 189, 203, 211 Endothelial cells, hydrodynamic shear stress, 198, 200-202, 215 Energy dissipation, hydrodynamic shear stress, 171, 180, 186 Energy losses, basidiomacromycetes, 291 Enterobacter, stormwater, 23 Enterobacteria, biphenyl degradation, 150 Enterococci, stormwater, 24-25, 27, 2930 Enteroviruses, starmwater, 26-28 Environmental pollutants, basidiomacromycetes, 329-331 Environmental Protection Agency, stormwater, 23-26, 32, 34 Enzymes, see also Haloperoxidases basidiomacromycetes, 339-340 applications of spent substrate, 317, 319, 325-326 growth substrate changes, 296-310, 312 lignocellulosic substrates, 253-254, 269-270 lignocehlosic wastes, 271-272 mycelium, 329-331, 333-335 values, 245 biphenyl degradation, 135-136, 138140, 156 Ganoderma, 108,114,122-123 hydrodynamic shear stress, 166, 189, 224

plant cells, 213, 215, 217 sensitivity of biocatalysts, 190-191, 198, 207, 218-219 nitroaromatic compounds, 3, 7-8 Eosinophil peroxidase reactions, 8 2 sources, 46 Erythrocytes Ganoderma, 119, 123 hydrodynamic shear stress, 206-207, 210 nitroaromatic compounds, 2 Escherichia coii biphenyl degradation, 136, 152-153, 157 haloperoxidases, 52 nitroaromatic compounds, 4-5 stormwater, 23, 25, 29-30 Essential amino acids, basidiomacromycetes, 246-247 Eukaryotes basidiomacromycetes, 330 hydrodynamic shear stress, 169, 172 insect cells, 211 sensitivity of biocatalysts, 191-195, 208, 217 EX-FERM, basidiomacromycetes, 257 Extensin, hydrodynamic shear stress, 211

F Fat, basidiomacromycetes, 243-244, 318 Fatty acids basidiomacromycetes, 244, 246 Ganoderma, 113 Fecal coliform (FC), stormwater, 22-31, 33,35 Fecal contamination, stormwater, 23, 2526, 29, 31 Fecal streptococcus, stormwater, 22, 2627 Fermentation, basidiomacromycetes, 271, 290 applications of spent substrate, 321,328 lignocellulosic substrates, 259-270 mycelium, 333-335 Fibroblasts, hydrodynamic shear stress, 197

3 70

INDEX

Flammulina velutypes, 339 biology, 238-239, 242 growth substrate changes, 299, 301 lignocellulosic substrates, 258 lignocellulosic wastes, 270, 272, 285 mycelium, 332 values, 249-250 Flax fibers, basidiomacromycetes, 335 Flaxobacterium, nitroaromatic compounds, 9 Flow cytometry, hydrodynamic shear stress, 189 Fluorescein diacetate test, hydrodynamic shear stress, 189, 214 Fluorimetry, hydrodynamic shear stress, 189 Forest plant residues, basidiomacromycetes, 258-259 Fucus distichus, haloperoxidases, 75-76 Fungal elicitors, basidiomacromycetes, 335 Fungicides haloperoxidases, 68 nitroaromatic compounds, 5 Fungus, see also Basidiomacromycetes biphenyl degradation, 141, 154-156 Ganoderma, 116,120, 125 haloperoxidases, 48 nitroaromatic compounds, 3-4, 7, 111 2 , 14 stormwater, 22 Fusarium, basidiomacromycetes, 302, 325 Fusarium oxysporum, nitroaromatic compounds, 5

G Galactose, Ganoderma, 116, 1 2 1 , 123 Ganoderal, Ganoderma, 103, 105, 114 Ganoderans, Ganoderma, 116-117, 121, 123 Ganoderenic acids, Ganoderma, 103, 105 Ganoderic acids, Ganoderma, 103-108, 113-116,119, 1 2 2 Ganoderma, medicinal benefits of, 1011 0 2 , 125-126 antitumor action, 108-112 cardiovascular system, 114-118 chemical composition, 102-108

immunomodulatory action, 118-120 liver protection, 113-114 muscular dystrophy, 120 nervous system regulation, 113 patents, 1 2 1 Alzheimer’s disease treatment, 123 antibiotics, 123 antimutagen, 124 antitumor preparation, 121-122 bath preparation, 124 beverages, 124-125 chronic bronchitis treatment, 123 hair tonics, 124 hypocholesterolemic preparations, 122

hypoglycemic preparations, 123 hypotensive preparations, 1 22 immunomodulatory agents, 1 2 3 liver function stimulants, 1 2 2 skin preparation, 124 protein synthesis, 112 radiation protection, 120-121 respiratory system, 118 toxicity, 1 2 1 Ganoderma applanata applications of spent substrate, 319 mycelium, 336 Ganoderma applanatum, medicinal benefits of, 108-110, 118, 121, 124 Ganoderma boninense, medicinal benefits of, 1 2 1 Ganoderma capense, medicinal benefits of, 102, 113, 120 Ganoderma japonicum, medicinal benefits of, 102, 110, 114, 120 Ganoderma lucidum lignocellulosic wastes, 285 medicinal benefits of, 102-119, 121125 values, 250 Ganoderma tsugae, medicinal benefits of, 121

Ganodermic acids, Ganoderma, 103, 108, 115 Ganoderols, Ganoderma, 103, 105, 114 Ganodosterone, Ganoderma, 1 22 Ganolucidic acid, Ganoderma, 104-106 Gas chromatography biphenyl degradation, 145, 155 Ganoderma, 108

371

INDEX Gas chromatography-mass spectroscopy biphenyl degradation, 140-141 nitroaromatic compounds, 4, 11 Gastrointestinal illness (GI), stormwater, 23-25, 29-31 Gene expression, hydrodynamic shear stress, 219, 224 Genetics, basidiomacromycetes, 336-337, 339-340 Gloeophyllum, basidiomacromycetes, 300, 331 Glucans basidiomacromycetes, 249-250 Ganoderma, 109-112, 117, 120-123 D-Glucopyranosyl residues, Ganoderma, 109-110 Glucose basidiomacromycetes applications of spent substrate, 322, 325-326 growth substrate changes, 293, 300301, 313 mycelium, 334 values, 249 Ganoderma, 111-112,116-117,121 nitroaromatic compounds, 11-12 Glucosidase, basidiomacromycetes, 297299, 325-326 Glucuronide, biphenyl degradation, 154 Glycans, Ganoderma, 116 Glycosylation, haloperoxidases, 45 GrifoJa growth substrate changes, 313 values, 249 Guardia, stormwater, 26

H

Haake viscometer, hydrodynamic shear stress, 181 Hair growth stimulants, basidiomacromycetes, 335 Halide ions, haloperoxidases, 41-42, 50, 81

Halohydrins, reactions, 56 Halometabolites, 42-43, 90-91 Haloperoxidases, 41-43, 90-92 reactions, 53 alkenes, 56-63

amines, 75-77 aromatic compounds, 63-66 assay methods, 53-56 heme-containing enzymes, 82-85 heterocyclic compounds, 66-75 immobilized enzymes, 79-81 mechanisms, 82, 89 substrates, 77-79 vanadium-containing enzymes, 8689 sources, 43 known sources, 43-49 new sources, 49-53 Heat of combustion, basidiomacromycetes, 290-291 Heavy metal accumulation, basidiomacromycetes, 331-332 Helicostyleium piriforme, biphenyl degradation, 154 Heme-containing enzymes, haloperoxidases, 82-85 Hemicellulase, basidiomacromycetes, 299-300 Hemicellulose, basidiomacromycetes, 281, 335 applications of spent substrate, 318, 322, 325-326 growth substrate changes, 292-294, 300, 312-313 lignocellulosic substrates, 251-252, 257 Hemoglobin, hydrodynamic shear stress, 206 Herbicides biphenyl degradation, 147 nitroaromatic compounds, 2, 4, 8 Heterocyclic compounds, haloperoxidases, 66-75 Heterokaryons, basidiomacromycetes, 236, 326, 339 Hexosamine, basidiomacromycetes. 290 High-pressure liquid chromatography haloperoxidases, 84 nitroaromatic compounds, 9 Histamine, hydrodynamic shear stress, 200

Homokaryons, basidiomacromycetes, 236, 337,339 Homology basidiomacromycetes, 305, 307

372

INDEX

biphenyl degradation, 143, 150-151, 153-154,156 HOPDA, biphenyl degradation, 138, 152 Horseradish peroxidase, 91 reactions, 55, 85 sources, 48 Hudson River, biphenyl degradation, 144 Human immunodeficiency virus, basidiomacromycetes, 329 Hybridization, biphenyl degradation, 142, 150, 152-153, 156 Hybridomas, hydrodynamic shear stress, 219, 223 fluid mechanics, 187 sensitivity of biocatalysts, 202-205, 208, 211 Hydrocarbons biphenyl degradation, 136, 141, 145, 150, 154-155 nitroaromatic compounds, 11 Hydrodynamic shear stress, 165-166 assessment, 187-1 90 biological effects, 222-224 cell architecture, 166-170 fluid mechanics, 185-187 nomenclature, 225-226 physical effects, 219-222 sensitivity of biocatalysts comparative study, 2 17-2 19 enzymes, 190-191 insect cells, 208-211 lower eukaryotes , 191-1 95 mammalian cells, 195-208 nematodes, 217 plant cells, 211-217 prokaryotes, 191 stirred tank reactors, 170-171 collision-related damage, 181-185 turbulent regime, 171-181 Hydrodynamic stress proteins, hydrodynamic shear stress, 217 Hydrogen, haloperoxidases, 42, 55 Hydrogen peroxide biphenyl degradation, 154 haloperoxidases, 41-42, 91 reactions, 60, 76, 80-81, 85, 87 sources, 50 Hydrolysis, basidiomacromycetes, 253254, 300, 317, 325

Hydroxyarylamines, nitroaromatic compounds, 3-4 Hydroxybiphenyls, biphenyl degradation, 148-149, 154 Hypertension, Ganoderma, 115, 1 2 2 Hypocholesterolemic activity, Ganoderma, 115-116,121-122 Hypoglycemia, Ganoderma, 116-1 17, 121, 123 Hypotension, Ganoderrna, 114-115

I Immobilization haloperoxidases, 53, 65, 72, 79-81 hydrodynamic shear stress, 197, 215 Immunomodulatory action, Ganoderma, 118-120, 123,125 Impellers, hydrodynamic shear stress, 183-185, 202-203, 212 Incubation time, basidiomacromycetes, 269, 291, 293 Industrial residues, basidiomacromycetes, 257-258 Inertial-subrange turbulent stresses, hydrodynamic shear stress, 179-180 Inhibition biphenyl degradation, 141, 154 Ganoderma, 108-119, 1 2 2 , 124-125 hydrodynamic shear stress fluid mechanics, 187 insect cells, 210 plant cells, 2 1 2 sensitivity of biocatalysts, 190, 200203, 207-208 nitroaromatic compounds, 9 Inhibitors, basidiomacromycetes, 249250, 270, 320, 326, 329 Insect cells, hydrodynamic shear stress, 204, 208-211 Insecticides, nitroaromatic compounds, 2

Insulin, Ganoderma, 117 Interferon, Ganoderma, 118, 125 Intermediate filaments, hydrodynamic shear stress, 169 Iodide, haloperoxidases, 60, 79 Iodination, haloperoxidases, 71, 82

373

INDEX Iodoperoxidases reactions, 63 sources, 48 Ion channels, hydrodynamic shear stress, 169 Iron basidiomacromycetes, 244, 295 haloperoxidases. 42, 44-45, 89 Isotropic turbulence theory, hydrodynamic shear stress, 177, 182, 203

K Klebsiella, stormwater, 22-23 Klebsiella pneumoniae, biphenyl degradation, 150 Kolmogoroff microscale, hydrodynamic shear stress fluid mechanics, 172, 180-182, 186 sensitivity of biocatalysts, 195-197, 211, 218

Kolmogoroff stresses fluid mechanics, 179-180 sensitivity of biocatalysts, 203, 210, 215

L Laccase, basidiomacromycetes, 302, 305307, 335 Lactate dehydrogenase release, hydrodynamic shear stress, 189, 203-205 Lactoperoxidases, 43, 91 reactions, 60, 62-63, 79 sources, 48 Laminar flow, hydrodynamic shear stress, 203-204, 215, 220 Lanostanoids, Ganoderma, 107, 116 Laurencia nipponica, haloperoxidases, 60, 62-63 Lectin, hydrodynamic shear stress, 207 Lemon grass, basidiomacromycetes, 294, 297 Lentinan, basidiomacromycetes, 249 Lentinus, 339 applications of spent substrate, 319, 328

biology, 238-239, 241-242 growth substrate changes, 290, 299301, 307, 313 lignocellulosic substrates, 258-259, 268 hgnocehlosic wastes, 272, 274, 281, 284 mycelium, 331, 333-335 values, 248-250 Leukocytes, hydrodynamic shear stress, 207 Ligands, haloperoxidases, 44-45, 47 Light, basidiomacromycetes, 280 Lignin basidiomacromycetes applications of spent substrate, 316319, 322, 326-329 growth substrate changes, 290, 292295, 297, 302-305, 307, 310-316 lignocellulosic substrates, 251-254, 257, 268-269 lignocellulosic wastes, 271, 281, 283 mycelium, 330, 332-333, 335-336 biphenyl degradation, 135-136, 154156 nitroaromatic compounds, 11 Lignin peroxidase basidiomacromycetes, 304-307, 316, 326, 333 reactions, 59, 66, 82 Ligninase, basidiomacromycetes, 303, 305-306 Lignocellulosic substrates, basidiomacromycetes, 337, 339 applications of spent substrate, 322323, 325, 328 biomass conversion efficiencies, 282285 cultural conditions, 271-282 growth substrate changes, 285-286, 301, 312-316 preparation, 270-271 Lignocellulosic wastes, basidiomacromycetes, 234 applications of spent substrate, 317319

growth substrate changes, 287, 296297, 310 substrates, 250-259

3 74

IN1)EX

Lignolytic enzymes, basidiomacromycetes, 301-306, 330 Lignosulfonic acid, basidiomacromycetes, 313, 315 Lipids Ganoderma, 114 haloperoxidases, 78 hydrodynamic shear stress, 166 Liver, Ganoderma, 112-1 17, 121-1 22 Loss of organic matter (LOM), basidiomacromycetes, 286-287 Low-alcohol wines, basidiomacromycetes, 335 Lucidenic acids, Ganoderma, 103-106 Lucidone, Ganoderma, 103, 105-106 Lymphocytes, hydrodynamic shear stress, 202, 208 Lypophyllum, 249, 285 Lysosomes, hydrodynamic shear stress, 207

M Macrocystis pyrifera, haloperoxidases, 75-76 Macrophages, hydrodynamic shear stress, 207-208 Magnesium, basidiomacromycetes, 244, 295, 326 Mammalian cells, hydrodynamic shear stress, 195 anchorage-dependent cells, 195-202 anchorage-independent cells, 202-208 Manganese, haloperoxidases, 45 Manganese peroxidase, basidiomacromycetes, 304, 306-307, 316 Mass spectroscopy, see Gas chromatography-mass spectroscopy Mass transfer, hydrodynamic shear stress, 193, 213, 224 Maximum time-independent shear rate, fluid mechanics, 178 Mean shear rate, hydrodynamic shear stress, 178 Medicinal properties, basidiomacromycetes, 247-250 Mercury, basidiomacromycetes, 332 Merulius applications of spent substrate, 317 growth substrate changes, 303

Messenger RNA haloperoxidases, 44 hydrodynamic shear stress, 202, 223 Microbial degradation biphenyl, see Biphenyl degradation of nitroaromatic compounds, see Nitroaromatic compounds, microbial degradation of Microcarriers, hydrodynamic shear stress, 183, 185-186, 195-197, 212 Microfilaments, hydrodynamic shear stress, 169, 205-206, 208 Microtubules, hydrodynamic shear stress, 169,195, 201, 208 Mineralization biphenyl degradation, 141-143, 146, 154-156 nitroaromatic compounds, 12 Molasses pigment, basidiomacromycetes, 333 Monochlorodimedone, haloperoxidases, 90 reactions, 53, 55, 60, 68 mechanisms of, 83-84, 86 sources, 45, 50 Monoclonal antibodies, hydrodynamic shear stress, 202, 205 Monoculturing, basidiornacromycetes, 260-261 Moraxella, nitroaromatic compounds, 8 Morphology, basidiomacromycetes, 236238, 269 mRNA, see Messenger RNA Murex trunculus, haloperoxidases, 52 Mushroom, see Basidiomacromycetes; Ganoderrna, medicinal benefits of Mutagenesis, nitroaromatic compounds, 2-3, 1 2 Mutation basidiornacromycetes, 303, 306, 323, 326, 339 biphenyl degradation, 136, 152 Mycelium basidiomacromycetes applications of functions, 329-336 applications of spent substrate, 322, 326 biology, 236 growth substrate changes, 287, 293294, 296, 299, 302

375

INDEX lignocellulosic substrates, 261, 268269 lignocellulosic wastes, 271, 278, 280 values, 246, 250 Ganoderma, 103, 105, 107-108, 110111, 118-123 hydrodynamic shear stress, 193 Myeloma cells, hydrodynamic shear stress, 202, 204 Myeloperoxidase, 45, 71, 82, 91 Myosin, hydrodynamic shear stress, 201

N

Nematodes, hydrodynamic shear stress, 217 Nervous system, Ganodermo, 113 Neurospora crasso, nitroaromatic compounds, 7 Nicotiana tabacum, hydrodynamic shear stress, 216-217 Nitration, nitroaromatic compounds, 3 Nitrazepam, degradation of, 2 Nitrite, nitroaromatic compounds, 7-8, 11

Nitro groups biphenyl degradation, 141 degradation of, 9, 11 microbial reduction of, 3-6 removal of, 6-7 Nitroanilines, degradation of, 8-9 Nitroaromatic compounds, microbial degradation of, 1-3, 14 growth substrates chloronitrophenols, 8 1,3-dinitrobenzene, 9-10 nitroanilines, 8-9 nitrobenzene, 7 nitrobenzoates, 9 nitrophenols, 8 2,4,6-trinitrotoluene, 9, 11-14 nitro group reduction, 3-6 removal, 6-7 Nitrobenzene, degradation of, 2-4, 7 Nitrobenzoates, degradation of, 1-4, 6, 9 4-Nitrobenzyl alcohol, degradation of, 1 4-Nitrobiphenyl, biphenyl degradation, 138, 141

Nitrocatechol, degradation, 9 Nitrogen basidiomacromy cetes applications of spent substrate, 321, 326-328 growth substrate changes, 287, 292, 295, 301, 303, 311, 313 lignocellulosic substrates, 254, 268 lignocellulosic wastes, 270, 281 values, 242-243 biphenyl degradation, 155 haloperoxidases, 47 nitroaromatic compounds, 1, 7-9, 11, 14 4-Nitromandelate, degradation of, 1 4-Nitrophenols, degradation of, 1-4, 7-8 1-Nitropyrene, degradation of, 3 Nitroreduction, nitroaromatic compounds, 7 Nitrotoulenes, degradation of, 2, 4 Nocardia biphenyl degradation, 138, 154 nitroaromatic compounds, 7, 9 Normal stress, hydrodynamic shear stress, 174 Nucleotide release, hydrodynamic shear stress, 189 Nutrient supplementation, basidiomacromycetes, 281 Nutritional index (NI), basidiomacromycetes, 246-247 Nutritional value, basidiomacromycetes, 246-247

0 Oil seed crop residues, basidiomacromycetes, 255, 337 Oligomers, hydrodynamic shear stress, 206-207 Oscillatoria, biphenyl degradation, 155 Oxalic acid, basidiomacromycetes, 280, 291, 300 Oxidation basidiomacromycetes growth substrate changes, 297, 303305, 315 lignocellulosic wastes, 280 mycelium, 329-332, 334, 336

376

INDEX

biphenyl degradation, 141, 144, 151 Ganoderma, 103-104 haloperoxidases reactions, 55, 69, 76, 78-80, 87 sources, 48 nitroaromatic compounds, 7, 9, 14 stormwater, 32 Oxygen basidiomacromycetes, 280, 328, 334 growth substrate changes, 287, 292, 304-306, 311, 313-314 lignocellulosic substrates, 261, 269270 biphenyl degradation, 147, 149, 156 Ganoderma, 106, 114, 116 haloperoxidases, 47, 80, 87 hydrodynamic shear stress, 193, 215, 221

nitroaromatic compounds, 4, 8

P Paper manufacture, basidiomacromycetes, 321-324 Paper mill sludge, basidiomacromycetes, 258 Paraoxon, nitroaromatic compounds, 2 Parasites, basidiomacromycetes, 281 Parathion, nitroaromatic compounds, 2, 7 Pathogens, stormwater, 22, 26, 28, 31, 35 PCBs, see Polychlorinated biphenyls Pectin, basidiomacromycetes, 335 Pectin transeliminase (PTE),basidiomacromycetes. 300 Penicillin, haloperoxidases, 78 Penicillium Ganoderma, 102 lignocellulosic wastes, 271, 281 mycelium, 334 Penicillium chrysogenum, hydrodynamic shear stress, 192 Penicillus capitatus, haloperoxidases, 45, 47, 78, 82, 90 Pentachloronitrobenzene, nitroaromatic compounds, 5 Peptones, basidiomacromycetes, 301 Peroxidase, basidiomacramycetes, 302, 331, 397

Pesticides, nitroaromatic compounds, 1

Pests, basidiomacromycetes, 282 pH, basidiomacromycetes growth substrate changes, 291, 295299, 303, 306, 310 lignocellulosic substrates, 268 lignocehlosic wastes, 280 Phanerochaete chrysosporium, 339-340 applications of spent substrate, 322323, 326, 328 biphenyl degradation, 154-155 growth substrate changes, 293, 299, 301, 303-307, 311, 313-316 haloperoxidases, 48, 59, 66 lignocellulosic substrates, 261, 268269 mycelium, 329-334, 336 nitroaromatic compounds, 11 Phellinus applications of spent substrate, 322 mycelium, 336 Phenol oxidases, basidiomacromycetes, 301-306, 310-311 Phenolics, basidiomacromycetes growth substrate changes, 296, 299, 304, 306-307, 311 mycelium, 330 Phenols basidiomacromycetes, 280-281, 291292, 336 biphenyl degradation, 141,145,147,154 haloperoxidases, 64-65 Phenotype basidiomacromycetes, 339 biphenyl degradation, 142, 149-150 nitroaromatic compounds, 14 Phlebia applications of spent substrate, 323 growth substrate changes, 305, 313 mycelium, 331 Phlebia tremellosa growth substrate changes, 316 lignocellulosic substrates, 261, 268269 Pholiota, biology, 240 applications of spent substrate, 325 growth substrate changes, 307, 313 lignocehlosic wastes, 272, 285 Phospholipids basidiomacromycetes, 244, 305 Ganoderma, 115

INDEX Photosynthesis, basidiomacromycetes, 251-252, 254 Plant cells, hydrodynamic shear stress, 211-217 Plantation crop wastes, basidiomacromycetes, 255, 257, 337 Plasma membrane, hydrodynamic shear stress, 169, 182 biological effects, 223 sensitivity of biocatalysts, 197, 201202, 214 Plasmids, biphenyl degradation, 135, 143, 149-150,152-153.156 Platelet aggregation inhibition, Ganoderma, 117-118 Pleurotin, basidiomacromycetes, 248 Pleurotus, 339 applications of spent substrate, 318322, 325-328 biology, 236, 238-242 growth substrate changes, 286-287, 290-302, 306-307, 310-311, 313315 lignocellulosic substrates, 256-261, 268, 270 lignocehlosic wastes, 271-272, 274, 277-278, 280-282, 284-285 mycelium, 332-335 values, 243-250 P h o n i c polyols, hydrodynamic shear stress, 210 Phteus, lignocehlosic wastes, 280 Pollutants basidiomacromycetes, 329-331 biphenyl degradation, 139, 145, 150 stormwater, 21, 30-31 Polybrominated biphenyls, degradation, 147-149

Polychlorinated biphenyls (PCBs) degradation, 135-136,139-141, 155156 anaerobic degradation, 143-145 bacteria growth, 141-143 bioremediation trials, 145-147 chromosomal genes, 150-154 derivatives, 147 plasmids, 149-150 nitroaromatic compounds, 3, 11 Polycyclic aromatic hydrocarbons, nitroaromatic compounds, 11

377

Polyethylene, basidiomacromycetes, 336 Polygalacturonase (PG), basidiomacromycetes, 300 Polygalacturonase transeliminase (PGTE), basidiomacromycetes, 300 Polymer production, basidiomacromycetes, 334 Polymerase chain reaction, stormwater, 26 Polymerization basidiomacromycetes, 305, 315 hydrodynamic shear stress, 169,191, 205, 211 Polymethylgalacturonase (PMG), basidiomacromycetes, 300 Polyporus applications of spent substrate, 319, 322, 326 growth substrate changes, 299, 303 lignocehlosic wastes, 285 mycelium, 331, 332, 334, 335 values, 250 Polysaccharides basidiomacromycetes, 271, 335 applications of spent substrate, 316320 growth substrate changes, 290, 300, 306 values, 249-250 Ganoderma, 108-112, 118,120-121, 124-125 hydrodynamic shear stress, 167-168, 191-192 Porphyrins, haloperoxidases, 85 Potassium basidiomacromycetes, 244, 295, 336 hydrodynamic shear stress, 201 Prokaryotes, hydrodynamic shear stress, 172, 191, 195 Prostacyclin, hydrodynamic shear stress, 200-201 Proteases, basidiomacromycetes, 300301, 306-307, 310 Protein basidiomacromycetes applications of spent substrate, 326, 328 growth substrate changes, 292, 294, 301, 310 lignocellulosic substrates, 258

378

INDEX

lignocehlosic wastes, 281, 285 values, 241, 243-244, 246-248 Ganoderma, 102, 109-110, 112, 118119, 1 2 3 haloperoxidases, 42, 45, 48, 79 hydrodynamic shear stress assessment, 189 biological effects, 223-224 cell architecture, 168-169 denaturation, 186, 190 fluid mechanics, 187 insect cells, 210, 217 sensitivity of biocatalysts, 190-191, 193, 200, 205-208 Protoporphyrins, haloperoxidases, 42, 45-46 Protozoa, stormwater, 22-24, 26-27 Pseudomonas biphenyl degradation, 140-142, 147, 149,151, 153 growth substrate changes, 315 hgnocellulosic wastes, 271, 282 nitroaromatic compounds, 7-8 Pseudomonas aeruginosa biphenyl degradation, 142, 146, 150152 stormwater, 22, 26-29, 31 Pseudomonas cepacia, biphenyl degradat i m , 147 Pseudomonas cruciviae biphenyl degradation, 138 nitroaromatic compounds, 6 Pseudomonas ffuorescens, nitroaromatic compounds, 3 Pseudomonas paucimobilis, biphenyl degradation, 147, 151 Pseudomonas pseudoalcaligenes, biphenyl degradation, 150-151 Pseudomonas putida biphenyl degradation, 142-143, 146, 150, 152-153 nitroaromatic compounds, 7-8 Pseudomonas pyrrocinia, haloperoxidases, 90, 92 reactions, 56, 63-64, 68-70, 78, 81 mechanisms of, 88-89 sources, 50, 53 Pseudomonas testosteroni, biphenyl degradation, 143, 153 Pulping agents, basidiomacromycetes, 321-324

Pulse plant residues, basidiomacromycetes, 254-255 Pyranose oxidase (PROD], basidiomacromycetes, 334 Pyrazole, haloperoxidases, 70 Pyrolysis, biphenyl degradation, 135-136 2-Pyrone-6-carboxylic acid, biphenyl degradation, 147 Pyrrole, haloperoxidases, 68, 70 Pyrrolnitrin, haloperoxidases, 68-69, 90

R Radiation production, Ganoderma, 120121

Rainfall, stormwater, 29-30 Reactors, hydrodynamic shear stress biological effects, 224 cell architecture, 185-187 insect cells, 209-210 plant cells, 211-212, 215 sensitivity of biocatalysts, 203, 205, 217

Recycling, basidiomacromycetes, 326327 Relative humidity (RH), basidiomacromycetes, 272, 277-278 Resorcinol, haloperoxidases, 65, 81 Respiratory system, Ganoderma, 118-120 Reynolds number, hydrodynamic shear stress, 170-171, 173, 177 Reynolds shear stresses, 176, 180, 186, 203, 215 Rhodosporidium, nitroaromatic compounds, 4 Rice straw, basidiomacromycetes, 292293, 295, 298, 300-302 RNA basidiomacromycetes, 244, 249 Ganoderma, 112, 118 Ruminant feed, basidiomacromycetes, 316-321

S Saccharification enzymes, basidiomacromycetes, 325-326 Saccharomyces cerevisiae, hydrodynamic shear stress, 194-195

INDEX Saccharum, basidiomacromycetes, 284, 293 Salmonello, stormwater, 24, 26-29 Schizophyllym commune applications of spent substrate, 322 values, 249 ScIerotium rolfsii, growth substrate changes, 271, 334 Second messengers, hydrodynamic shear stress, 214, 217, 223 Sensitivity of biocatalysts to hydrodynamic shear stress, see Hydrodynamic shear stress Sequences biphenyl degradation, 143, 150-154 haloperoxidases, 42, 44, 48, 55 Sewers, stormwater, 21-22, 29, 32, 34-36 Shear stress, hydrodynamic, see Hydrodynamic shear stress Shigella, stormwater, 26, 28 Signal transduction, hydrodynamic shear stress, 222 Silica, basidiomacromycetes, 327 Single-cell proteins, basidiomacromycetes, 328 Sodium, Ganoderma, 114 Soil ameliorants, basidiomacromycetes, 327-328 Solid-state fermentation, basidiomacromycetes applications of spent substrate, 317318 growth substrate changes, 295-297 lignocellulosic substrates, 259-268 Solubility, basidiomacromycetes, 287, 290, 294 Spectrin, hydrodynamic shear stress, 206-207 Spectroscopy, haloperoxidases, 44, 46, 48, 55, 71 Spodoptera frugiperda, hydrodynamic shear stress, 208 Sporotrichum applications of spent substrate, 318, 326 growth substrate changes, 299-302, 313 lignocellulosic substrates, 259 Staphylococci, stormwater, 29-31 Staphylococcus aureus, stormwater, 2627

379

Stirred tank reactors, hydrodynamic shear stress, 170-171 collision-related damage, 181-185 turbulant regime, 171-181 Stormwater, treatment of, 21-22, 34-36 bacterial criteria development, 22-26 disinfection, 31 alternative techniques, 33-34 chemical, 32-33 human disease potential, 26-31 Streptococci, stormwater, 31 Streptomyces, nitroaromatic compounds, 3 Streptomyces aureofaciens haloperoxidases reactions, 64-65, 68-69, 78 mechanisms of, 88-89 sources, 53 nitroaromatic compounds, 5 Streptomyces griseus, haloperoxidases, 53, 68 Streptomyces lividans, haloperoxidases, 53 Streptomyces phaeochromogenes, haloperoxidases, 48 Streptomyces thermovulgaris, lignocellulosic wastes, 271 Streptomyces venezuelae, haloperoxidases, 53, 68 Streptomycin, biphenyl degradation, 142, 150 Stropharia applications of spent substrate, 319, 321 biology, 240 growth substrate changes, 292 lignocellulosic substrates, 269 Sugar basidiomacromycetes growth substrate changes, 287, 290292, 299-300 lignocellulosic substrates, 252, 268 hgnocellulosic wastes, 281 values, 241 Gonoderma, 115 haloperoxidases, 45-46 Sugarcane rubbish, basidiomacromycetes, 284 Sulfate, biphenyl degradation, 144, 149, 154 Sulfur, haloperoxidases, 44, 78

380

INDEX

Supplementation, nutrient, basidiomacromycetes, 281 Swimming-associated illnesses, stormwater, 22,24,26,30 T Tangential shear stress, hydrodynamic shear stress, 174-175 Temperature, basidiomacromycetes, 269, 306,319 lignocehlosic wastes, 272,277-278, 299 Termitomyces applications of spent substrate, 326 growth substrate changes, 299 Terpenoids, Ganoderma, 105-106 Tetrahymena, hydrodynamic shear stress, 187,194 Tetrazolium dye reduction test, hydrodynamic shear stress, 189 Thin-layer chromatography biphenyl degradation, 141 nitroaromatic compounds, 11 Thyroid, haloperoxidases, 63 Thyroid peroxidase, 42 reactions, 63 sources, 48 Time-dependent forces, hydrodynamic shear stress, 179-181 Time-independent forces, hydrodynamic shear stress, 177-179 TNT, see 2,4,6-Trinitrotoluene (TNT) Total coliform (TC), stormwater, 22-23, 25-27,29,31,33,35 Toxicity basidiomacromycetes, 320 Ganoderma, 113,121 Trametes applications of spent substrate, 320, 322 mycelium, 330,333 Trametes versicolor, growth substrate changes, 293,299,305 Tremella biology, 240,242 growth substrate changes, 301 values, 249 Trichoderma growth substrate changes, 298,300 lignocehlosic wastes, 281

Tricholoma biology, 240-241 values, 249-250 4-Trifluoromethyl-2,6-dinitroaniline, degradation, 2 Triglycerides, Ganoderma, 115 1,3,5-Trinitrobenzene, degradation, 9 2,4,6-Trinitrotoluene (TNT), degradation, 1-2,4,7,9,11-14 Triterpenes, Ganoderma, 103-104, 107, 114,116,119 Triterpenoids, Ganoderma, 103-107 Trypan blue, hydrodynamic shear stress, 189,209 Tumors, Ganoderma, 108-112.121-122, 125 Turbulence, hydrodynamic shear stress cell architecture, 166,168 fluid mechanics, 170,182,184 insect cells, 210 physical effects, 220 plant cells, 215 regime, 171-181 sensitivity of biocatalysts, 192,195, 197,203-205,218

U Ultraviolet light haloperoxidases, 84,87 stormwater, 33-34 Ultraviolet spectroscopy, haloperoxidases, 44,55,71 Urokinase, hydrodynamic shear stress, 198

V

Vanadium, haloperoxidases, 90-91 reactions, 65,75,86-89 sources, 46-47,52-53 Velocity, hydrodynamic shear stress, 170, 176,179,182,185,220 Velocity vector, hydrodynamic shear stress, 171,175 Virus, stormwater, 22 Viscometers, hydrodynamic shear stress, 181,188,223 insect cells, 208

383

INDEX plant cells, 213, 215 sensitivity of biocatalysts, 194, 202205, 207 Viscosity, hydrodynamic shear stress, 169 fluid mechanics, 170, 174, 176-179, 183-184 insect cells, 210 physical effects, 2 2 1 plant cells, 211 sensitivity of biocatalysts, 191, 194, 204, 206 Viscous stress, hydrodynamic shear stress, 174, 176 Vitamins, basidiomacromycetes, 244, 258, 318, 326 Volvariella applications of spent substrate, 327 biology, 236, 238-239, 241 growth substrate changes, 293, 298, 310 lignocellulosic substrates, 258 lignocehlosic wastes, 272, 274, 281, 284 Vortexing, hydrodynamic shear stress, 210

Wheat straw (WS), basidiomacromycetes applications of spent substrate, 317318, 320-321, 325 growth substrate changes, 290-297, 313, 315 lignocellulosic wastes, 284 White-rot reactions, basidiomacromycetes applications of spent substrate, 317320, 322-323, 327 growth substrate changes, 310-312, 315-316 mycelium, 330, 332-333

X Xanthoria parietina, haloperoxidases, 52 8

Y Yeast, hydrodynamic shear stress, 167, 169, 195

W Z Water retention value (WRV), basidiomacromycetes, 322

Zinc, basidiomacromycetes, 331

CONTENTS OF PREVIOUS VOLUMES Partition Affinity Ligand Assay (PALA): Applications in the Analysis of Haptens, Macromolecules, and Cells Bo Mattiasson, Matts Ramstorp, and Torbjorn G . I. Ling

Volume 27

Recombinant DNA Technology Vedpal Singh Malik Nisin A. Hurst The Coumermycins: Developments in the Late 1970s John C. Godfrey Instrumentation for Process Control in Cell Culture Robert 1. Flieschaker, James C. Weaver, and Anthony 1. Sinskey Rapid Counting Methods for Coliform Bacteria A. M. Cundell

Accumulation, Metabolism, and Effects of Organophosphorus Insecticides on Microorganisms Rup La1 Solid Substrate Fermentations K. E. Aidoo, R. Hendry, and B. 1.B. Wood Microbiology and Biochemistry of Miso (Soy Paste) Fermentation Sumbo H. Abiose, M. C. Allan, and B. 1. B. Wood

INDEX

Training in Microbiology at Indiana University-Bloomington L. S. McCJung Volume 29 INDEX

Stabilization of Enzymes against Thermal Inactivation Alexander M. Klibanov Production of Flavor Compounds by Microorganisms G. M. Kernpler

Volume 28

Immobilized Plant Cells P. Brodelius and K. Mosbach Genetics and Biochemistry of Secondary Metabolism Vedpal Singh Malik 382

New Perspective on Aflatoxin Biosynthesis I. W. Bennett and Siegfried B. Christensen

CONTENTS OF PREVIOUS VOLUMES Biofilms and Microbial Fouling W. G. Characklis and K. E. Cooksey Microbial Influences: Fermentation Process, Properties, and Applications Erick J. Vandamme and Dirk G. Derycke Enumeration of Indicator Bacteria Exposed to Chlorine Gordon A. McFeters and Anne K. Camper Toxicity of Nickel to Microbes: Environmental Aspects H. Babich and G. Stotzky

INDEX

Volume 30 Interactions of Bacteriophages with Lactic Streptococci Todd B. Klaenhammer Microbial Metabolism of Polycyclic Aromatic-Hydrocarbons Carl E. Cerniglia Microbiology of Potable Water Betty H. Olson and Laslo A. Nagy Applied and Theoretial Aspects of Virus Adsorption to Surfaces Charles P. Gerba Computer Applications in Applied Genetic Engineering Joseph L. Modelevsky

383

Volume 31 Genetics and Biochemistry of Clostridium Relevant to Development of Fermentation processes Palmer Rogers The Acetone Butanol Fermentation B. McNeiI and B. Kristiansen Survival of, and Genetic Transfer by, Genetically Engineered Bacteria in Natural Environments G. Stotzky and H. Babich Apparatus and Methodology for Microcarrier Cell Culture S . Reuveny and R. W. Thoma Naturally Occurring Monobactams WiIIiam L. Parker, Joseph O’Sullivan, and Richard B. Sykes New Frontiers in Applied Sediment Microbiology Douglas Gunnison Ecology and Metabolism of Thermomatrix thiopara Daniel K. Brannan and Douglas E. Caldwell Enzyme-Linked Immunoassays for the Detection of Microbial Antigens and Their Antibodies John E. Herrmann

Reduction of Fading of Fluorescent Reaction Product for Microphotometric Quantitation G. L. Picciolo and D. S . Kaplan

The Identification of Gram-Negative, Nonfermentative Bacteria from Water: Problems and Alternative Approaches to Identification N. Robert Ward, Roy L. Wolfe, Carol A. Justice, and Betty H. Olson

INDEX

INDEX

384

CONTENTS OF PREVIOUS VOLUMES Roam Control in Submerged Fermentation: State of the Art N. P. Ghildyal, B. K. Lonsane, and N. G. Karanth

Volume 32

Microbial Corrosion of Metals Warren P. Iverson Economics of the Bioconversion of Biomass to Methane and Other Vendable Products Rudy J. Wodzinski, Robert N. Gennaro, and Michael H. Scholla The

Microbial Production of Butanediol Robert J. Magee and Nain Kosaric

2,3-

Microbial Sucrose Phosphorylase: Fermentation Process, Properties, and Biotechnical Applications Erick J. Vandamme, Jan Van Loo, Lieve Machtelinckx, and Andre De Laports

Applications and Mode of Action of Formaldehyde Condensate Biocides H. W. Rossmoore and M. Sondossi Occurrence and Mechanisms of Microbial Oxidation of Manganese Kenneth H. Nealson, Bradley M. Tebo, and Reinhardt A. Rosson Recovery of Bioproducts in China: A General View Xiong Zhenping

INDEX

Antitumor Anthracyclines Produced by Streptomyces peucetius A. Grein Volume 34 INDEX

Volume 33

The Cellulosome of Clostridium thermocellum Raphael Lamed and Edward A. Bayer Clonal Populations with Special Reference to Bacillus sphaericus Samuel Singer Molecular Mechanisms of Viral Inactivation by Water Disinfectants R. B. Thurman and C. P. Gerba Microbial Ecology of the Terrestrial Subsurface William C. Ghiorse and John T. Wilson

What’s in a Name?-Microbial Secondary Metabolism J. W. Bennett and Ronald Bentley Microbial Production of Gibberellins: State of the Art P. K. R. Kumar and B. K. Lonsane Microbial Dehydrogenations of Monosaccharides Milo5 Kulhdnek Antitumor and Antiviral Substances from Fungi Shung-Chang Jong and Richard Donovick Biotechnology-The V. S. Malik

INDEX

Golden Age

CONTENTS OF PREVIOUS VOLUMES

385

Volume 35

Volume 36

Production of Bacterial Thermostable aAmylase by Solid-state Fermentation: A Potential Tool for Achieving Economy in Enzyme Production and Starch Hydrolysis B. K. Lonsane and M. V. Ramesh

Microbial Transformations of Herbicides and Pesticides Douglas J. Cork and James P. Krueger

Methods for Studying Bacterial Gene Transfer in Soil by Conjugation and Transduction G. Stotzky, Monica A. Devanas, and Lawrence A. Zeph Microbial Levan Youn W. Han

An Environmental Assessment of Biotechnological Processes M. S. Thakur, M. J. Kennedy, and N. G. Karanth Fate of Recombinant Escherichia coli K-12 Strains in the Environment Gregg Bogosian and James F. Kane Microbial Cyctochromes P-450 Xenobiotic Metabolism F. Sima Sariaslani

and

Review and Evaluation of the Effects of Xenobiotic Chemicals on Microorganisms in Soil R. J. Hicks, G. Stotzky, and P. Van Voris

Foodborne Yeasts T. Dedk

Disclosure Requirements for Biological Materials in Patent Law Shung-Chang Jong and Jeannette M. Birmingham

High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins Erik P. Lillehoj and Vedpal S. Malik

INDEX

INDEX

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