Advances in Applied Microbiology, Volume 9

ADVANCES IN

Applied Micro biology VOLUME 9

CONTRIBUTORS TO THIS VOLUME

R. F. Christman J. Ross Colvin

C. H. Driver G. F. Gause

I. H. Harrison Richard M. Hyde June Jenkins

L. Juragek Ralph E. Kunkee Birgitta Norkrans

R. T. Ogelsby Wesley 0. Pipes A. D. Russell

D. R. Whitaker

ADVANCES IN

Applied Microbiology Edited by WAYNE W. UMBREIT Department of Bacteriology Rutgerr, The State University N e w Brunswick, N e w Jersey

VOLUME 9

@

1967

ACADEMIC PRESS, New York and London

COPYRIGHT 6:

1967, BY ACADEMIC PRESS

INC.

ALL RIGHTS RESERVED. NO PART OF TIIIS BOOK MAY BE REPRODUCED IN ANY FORM,

BY PHOTOSTAT, MICROFILM,

on

ANY OTHER MEANS, WITHOUT

WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W . l

LIBRARYOF CONGRESS CATALOG CARDNUMBER: 59-13823

PRINTED IN T H E UNITED STATES O F AMERICA

CONTRlBUTORS Numbers in parentheses indicate the pages on which the authors' contxibutions begin. R.

F. CHRISTMAN, Departments of Civil Engineering and College of Forestry, University of Washington, Seattle, Washington (171)

J . ROSS COLVIN, Division of Biosciences, National Research Council, Ottawa, Canada (131) C.

Departments of Civil Engineering and College of Forestry, University ofwashington, Seattle, Washington (171)

H . DRIVER,

G. F .

CAUSE,

lnstitute of New Antibiotics, Academy of Medical Sciences, Moscow,

U.S.S.R. (69) I. H . HARRISON, Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain ( 1 ) M. HYDE, Department of Microbiology, School of Medicine, University of Oklahoma, Oklahoma City, Oklahoma (39)

RICHARD

Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain (1)

JUNE JENKINS,

L. JURASEK, Division of Biosciences, National Research Council, Ottawa, Canada (131)'

RALPH E. KUNKEE, Department of Viticulture and Enology, University of California, Davis, California (235) BIRGITTA NORKRANS, Department of Marine Botany, University of Goteborg, Goteborg, Sweden (91)

R. T. OGLESBY, Departments of Civil Engineering and College of Forestry, University of Washington, Seattle, Washington (171) WESLEY0. PIPES, Department of Civil Engineering, Northwestern University, Evans-

ton, Illinois (185) A. D. RUSSELL, Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain (1) D. R. WHITAKER, Division of Biosciences, National Research Council, Ottawa, Canada (131)

'Present address: Stitny drevarsky vyskumny ustav, LamaGska 5, Bratislava IX, Czechoslovakia. V

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PREFACE

The scope of this year’s volume is as broad as applied microbiology itself. Its problems range from the making of wine to the bulking of sludge, which may seem rather too wide to the uninitiated but which the professional will recognize as not entirely unrelated. Since cellulose comprises perhaps a third of the carbon in the world, we have devoted two separate papers to it. The knowledge of the transformation of lignin to humus is brought up-to-date - no one could say that the problem is solved. The preservation of pharmaceuticals, unfamiliar to many applied microbiologists, is brought to our attention. In addition, the present status of the changing relation between microbial metabolic research and the cancer problem is reviewed. This is the ninth volume of this serial publication, and for almost a decade I have watched and occasionally assisted its growth. The next volume will complete a decade and, as a matter of principle, one man ought not to edit a publication for a longer period of time particularly when he bears the sole responsibility for the work. Inevitably, the content will reflect his own interests and contacts, and these will not necessarily result in contributions containing the most important developments in the field. Therefore, for the next volume, Dr. David Perlman, of the University of Wisconsin will join me as coeditor, and in subsequent volumes he will be the sole editor. It has been evident that Advances in Applied Microbiology is a useful and valuable addition to the literature of the subject and, as editor, I am happy to have served this discipline.

W. W. UMBREIT

Rutgers University October, 1967

vii

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CONTENTS CONTRIBUTORS .......................................................................................... PREFACE................................................................................................... CONTENTSOF PREVIOUSVOLUMES ..............................................................

V

vii xi

The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. RUSSELL,JUNEJENKINS,AND I. H. HARRISON

I. Introduction ............................... ........... 11. Injections ....................._...........,............................ , ...... ..................... 111. Immunological Products ............., ....................................................... IV. Eye Drops ................................................................... istration ......, ....................... ........................ V. Medicines VI. Preparations for External Use ............................................................. References ................................... ..............

1 2 15 22 28 32 35

Antiserum Production in Experimental Animals RICHARDM. HYDE Introduction .... ................ .................................................................. Methods Employed in Antiserum Collection .......................... ............. . Animal Species Employed in Antiserum Production. Routes of Inoculation Employed in Antiserum Produ V. Factors Affecting Antibody Production ...................... ..... ..... ..... ............ VI. Specific Examples of Antiserum Production VII. Conclusion ............ ..................... ,.................... ....................... .... ...... graphy .................. ... ............

1. 11. 111. IV.

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

39 40 41 43 47

58 58 59 63

Microbial Models of Tumor Metabolism C. F. CAUSE I. Introduction

......, ....

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

11. Disturbance of Control Mechanisms in the Metabolism of Tumors

References .......................................................................................

69 70 77 88 88

Cellulose a n d Cel Iu lolysis BIRGITTANORKRANS I. Introduction .......... ............................................................................ 11. Cell Wall Morphology and Chemistry ................................................. 111. Cellulose Chemistry and Supramolecular Morphology .................. ......... IV. Biosynthesis of Cell Wall Polysaccharides ....................... ... ..................

ix

91 92 95 98

X

CONTENTS

V. Degradation of Cellulose by Bacteria and Fungi ................................... VI . Applications for Cellulases ,. .................. ... ........................ .................. References ....... ,...................................... .....................................'. .

.

101 124 125

Microbiological Aspeck of the Formation and Degradation of Cellulosic Fibers L. J u R A ~ E K ,J. Ross COLVIN, AND D. R. WHITAKER 131 131 141

155 166

The Biotransformation of Lignin to HumusFacts and Postulates R. F . CHRISTMAN, AND DRIVER ................................................... 1. Introduction ............... .... 11. Mechanisms of Lignin Bio n ...................... ................. . ............. Humification .... IV. The White-Rot Fungi .................... ............................................. sis and Humification V. Other Organisms .................................. References ....... ........ ...................

171 173 175 178 182 183

Bulking of Activated Sludge 0 . PIPES

...... ..................... . ...... .................. 185 189 ................................................................. 11. Types of Settling P 111. Superficial Aspects of Bulking ..................... . ........................ 204 IV. Fundamental Aspects of Filmentous Bulking ........................ 217 229 V. Summary ........................ ............. ......................... References ............ ...................... 232 I. Introduction

.........

I

Malo-lactic Fermentation RALPH E. KUNKEE

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

235 236 239 ................................ IV. Malo-lactic Bacteria ............ ............... ..... ... ............. .............. ... ......... 24 1 ......... ................. 259 V. Detection of Malo-lactic Fermentation 260 ........................ 270 VII. Control of Conclusions ...... ......................... 273 274 281 297 SUBJECT INDEX

.

CONTENTS OF PREVIOUS VOLUMES

A Commentary on Microbiological Assaying F . Kavanagh

Volume 1

Protected Fermentation

MiloH Herold and Jan NeEasek

Application of Membrane Filters

The Mechanism of Penicillin Biosynthesis Arnold L. Demain

Richard Ehrlich Microbiol Control Brewery

Preservation of Foods and Drugs by Ionizing Radiations

in

the

Gerhard J . Hass

W . Dexter Bellamy

Newer Development in Vinegar Manufactures

The State of Antibiotics in Plant Disease Control

Rudolph J . Allgeier and Frank M . Hildebrandt

David Pramer Microbial Synthesis of Cobamides

The Microbiological Transformation of Steroids

D. Perlman

T. H . Stoudt

Factors Affecting the Antimicrobial Activity of Phenols E. 0.Bennett

Biological Transformation of Solar Energy William J . Oswald and Clarence G .

Golueke

Germfree Animal Techniques and Their Applications Arthur W. Phillips andJames E . Smith

SYMPOSIUM ON ENGINEERING ADVANCES IN FERMENTATION PRACTICE

Insect Microbiology S . R. Dutky

Rheological Properties of Fermentation Broths

The Production of Amino Acids by Fermentation Processes

Fred H. Deindoerfer and John M . West

Shukuo Kinoshita

Fluid Mixing in Fermentation Processes J . Y. Oldshue

Continuous Industrial Fermentations Philip Gerhardt and M . C . Bartlett

Scale-up of Submerged Fermentations

W .H. Bartholemew

The Large-Scale Growth of Higher Fungi Radclife F. Robinson and R. S.

Air Sterilization Arthur E. Humphrey

Davidson AUTHOR INDEX-SUBJECT

Methods

INDEX

Sterilization of Media for Biochemical Processes

Volume 2

Lloyd L. Kempe

Newer Aspects of Waste Treatment

Nandor Porges

Fermentation Kinetics and Model Processes

Aerosol Samplers

Fred H. Deindoerfer

Harold W . Batchelor

xi

xii

CONTENTS OF PREVIOUS VOLUMES

Continuous Fermentation W. D. Muxon Control Applications in Fermentation George]. Fuld

AUTHOR INDEX- SUBJECT INDEX Volume 4

Induced Mutagenesis in the Selection of Microorganisms S. 1. Alikhanian

Volume 3

Preservation of Bacteria by Lyophilization RobertJ. Heckly

Sphuerotilus, Its Nature and Economic Significance Norman C . Doridero

The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . 1.Babel Applied Microbiology in Animal Nutrition Harlow H . Hall

Large-Scale Use of Animal Cell Cultures Donald 1. Merchant and C . Richard Eidam

Biological Aspects of Continuous Cultivation of Microorganisms T. Holme

Protection against Infection in the Microbiological Laboratory: Devices and Procedures Murk A . Chatigny

Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C . Morris

Oxidation of Aromatic Compounds by Bacteria Martin H . €logo8

Submerged Growth of Plant Cells L. G. Nickel1 AUTHOR INDEX- SUBJECT INDEX Volume 5

Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, Jr., und Robert F . Pittillo

Correlations between Microbiological Morphology and the Chemistry of Biocides Adrien Albert

The Classification of Actinoinycetes in Relation to Their Antibiotic Activity Elio Baldacci

Generation of Electricity by Microbial Action 1.3. Davis

The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus

Microorganisms and Biology of Cancer G . F . Gause

Intermediary Metabolism and Antibiotic Synthesis /. D. Bu’Lock

Rapid Microbiological with Radioisotopes Gilbert V. Levin

Methods for the Determination of Organic Acids A . C . Hzrlme

The Present Status of the 2,S-Butylene Glycol Fermentation Sterling X . Long and Roger Patrick

the

Molecular

Determinations

...

Xlll

CONTENTS OF PREVIOUS VOLUMES

Aeration in the Laboratory W . R. Lockhart and R. W. Squires Stability and Degeneration of Microbial Cultures on Repeated Transfer

Fritz Reusser Microbiology of Paint Films Richard T . Ross The Actinomycetes and Their Antibiotics

AUTHOR INDEX-SUBJECT

INDEX

Volume 7

Microbial Carotenogenesis

Alex Ciegler Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility M . Alexander

Selman A. Waksman Fuse1 Oil

A. Dinsmoor Webb and John L. lngraham AUTHOR INDEX - SUBJECT INDEX Volume 6

Global Impacts of Applied Microbiology: An Appraisal Carl-Goran Heden and Mortimer P.

Starr Microbial Processes for Preparation of Radioactive Compounds D. Perlman, Aris P. Bayan, and Nancy

A. Giufre

Cold Sterlization Techniques John B . Opfell and Curtis E . Miller Microbial Production of Metal-Organic Compounds and Complexes

D. Perlman Development of Coding Schemes for Microbial Taxonomy S. T. Cowan Effects of Microbes on Germfree Animals

Thomas D. Luckey Uses and Products of Yeasts and Yeastlike Fungi Walter J . Nickerson and Robert G.

Brown Secondary Factors in Fermentation Processes

P. Margalith Nonmedical Uses of Antibiotics Herbert S. Goldberg Microbial Aspects of Water Pollution Control K . Wuhrmann Microbial Formation and Degradation of Minerals Melvin P. Silverman and Henry L.

Ehrlich Enzymes and Their Applications

Irwin W . Sizer A Discussion of the Training of Applied Microbiologists B . W . Koft and Wayne W . Umbreit

Microbial Amylases

Walter W . Windish and Nagesh S. M ha tre The Microbiology of Freeze-Dried Foods

Gerald J. Silverman and Samuel A. Goldblith Low-Temperature Microbiology ]udith Farrell and A. H . Rose AUTHOR INDEX-SUBJECT INDEX Volume 8

Industrial Fermentations and Their Relations to Regulatory Mechanisms Arnold L. Demain Genetics in Applied Microbiology S. G. Bradley

xiv

CONTENTS OF PREVIOUS VOLUMES

Microbial Ecology and Applied Microbiology Thomas D . Brock The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers and Norman A . Clarke The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods Stephen Alan Kollins

Oral Microbiology Heiner Hoffman Media and Methods for Isolation and Enumeration of the Enterococci Paul A . Hartmun, George W . Reinbold, und Deui S. Saruswut Crystal-Forming Bacteria Pathogens Martin H . Rogoff

as

Insect

Mycotoxins in Feeds and Foods Emanuel Borker, Nino F . Insalata, Colette P . Leui, and John S . Witzeman AUTHOR INDEX-SUBJECT

INDEX

ADVANCES IN

Applied Micro biology VOLUME 9

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The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. RUSSELL,JUNE

JENKINS, AND

I. H. HARRISON

Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain I. Introduction 11. Injections ...................... A. Introduction ............................... B. Methods of Sterilization ......................................... C. Vehicles ............................................................... D. Containers and Closures E. Selection of Bactericides ...... 111. Immunological Products .............................................. A. Types of Immunity ................................................ B. Products Requiring a Preservative IV. Eye Drops .................................................................. A. Preparation and Sterilization .................................. B. Preservatives ........................................................ V. Medicines for Oral Administration A. Introduction ........................ B. Preservatives in Common Use ................................ VI. Preparations for External Use ....................................... A. Introduction ......................................... References .................

1 2 2 3 6 7 9 15 15 16 22 22 23 28 28 31 32 32 33 35

I. Introduction Certain pharmaceutical products require the inclusion of an antimicrobial substance in their formulation. This term antimicrobial substance may itself be interpreted as having a wide meaning, and may be thought to include such groups as the antibiotics and sulfonamides in addition to other antibacterial and antifungal substances. However, this paper deals only with those substances which are deliberately included in pharmaceutical formulations ( a ) for reasons of preservation, or ( b ) when combined with heat, as a method of sterilization. Such substances thus do not comprise the active medicament(s) of the products, and in fact, the concentration of the agents is usually much smaller. The choice of a preservative is governed by the type of product and its chemical and physical properties, because of the possibility of interaction occurring between the agent and the other constituents of the preparation. Incompatibility may result in the inactivation of the 1

2

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

drug and/or the agent or in undesirable changes in the nature of the product, e.g., precipitation, coagulation, or separation. Although in many cases it is possible to predict that a given preservative will be unsuitable for use in a certain product, the converse is less easily predictable. Moreover, data on the antimicrobial activity of a preservative obtained from studies on nutrient media may be misleading. It is therefore important to test the final formulation thoroughly to ensure that it will produce the required results and that the preservative present will in fact protect the product against all the microorganisms likely to contaminate it in practice. To detect changes which occur slowly, prolonged storage tests under a variety of storage conditions are necessary. Some of the problems encountered in this field and methods for determining the efficacy of preservatives have been discussed by Rdzok et al. (1955). Products that require the inclusion of an antimicrobial compound include injections, immunological products, eye drops, creams, and other two-phase systems, tinctures, liquid extracts, and certain mixtures. Although normally administered parenterally, immunological products are considered separately from injections because of their special nature of preparation and composition. These groups of products, together with the antimicrobial substances which may be incorporated into their formulation, are discussed below. In each section, brief details of the types and preparation of the constituent products are given; it is hoped that such introductory data will be of some use to those microbiologists not engaged in this type of project. The abbreviations used throughout this paper are as follows: B.P. (British Pharmacopoeia), B.P.C. (British Pharmaceutical Codex), U.S.P. (United States Pharmacopoeia), U.S.N.F. (United States National Formulary) and A.N.F. (Australian National Formulary). II. Injections

A. INTRODUCTION

1 . Definition The International Pharmacopoeia (I.P.) defines injections as “a class of sterile pharmaceutical solutions, emulsions or suspensions, packaged in containers which will maintain sterility, and intended for parenteral administration, i.e., under or through one or more layers of skin or mucous membrane.” The parenteral route is used for several reasons: the drug may be destroyed when given orally, or it may be

ANTIMICROBIAL AGENTS IN PHARMACEUTICALS

3

inactive when given by that route. In addition, drugs given by injection often act more quickly: they may be used to produce a rapid localized effect, or in some cases to produce a prolonged action.

2. Routes Injections may be subdivided according to the route of administration. The British Pharmaceutical Codex (1963) lists seven, which Cooper and Gunn (1965) have defined. Intradermal or intracutaneous injections are made into the skin, between the dermis and the epidermis. Subcutaneous or hypodermic injections are made under the skin, into the subcutaneous tissue; the term hypodermic has also been used more generally, as in hypodermic syringe, but such syringes are not limited to subcutaneous injections. Intramuscular injections are made into the muscle tissue itself. Injections made into a vein, that is, intravenous injections, may include large volume injections, more specifically called infusion fluids. The three remaining, intrathecal, intracisternal, and peridural injections, are made into the cerebrospinal fluid.

3. Types Injections may be obtained in single dose or multidose containers, depending upon a number of factors, which include the nature of the active constituent and the requirements of both patient and doctor. However, the physical form of the injection varies more widely. The majority are aqueous solutions, but in other cases the medicament is dissolved in a suitable oil, ester, or alcohol. Some injections are formulated as suspensions or emulsions, while in the case of a watersoluble, but readily hydrolyzable drug, the formulation may be presented in two ampoules, a powder ampoule and an ampoule containing a solvent, the contents of the one to be dissolved in the other immediately before use. If the extended definition of the B.P. (1963) is considered, it will be noted that certain injections used in radiography, e.g., iodized oil fluid injection, may be introduced into the body cavities-the lungs, uterus, or urethra, as a contrast medium. For the purposes of this paper, these latter injections will not be considered.

B. METHODS OF

STERILIZATION

Alin and Diding (1949) demonstrated that the autobactericidal properties of a number of solutions for injection are insufficient to give a sterile product, and all injections must therefore be subjected

4

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

to a suitable, efficient sterilization procedure. Nonsterile injections are a potential danger to the patient, especially since he may already be suffering from a condition which has weakened the body's natural defenses. The introduction of such contaminated injections into blood, tissue, or spinal fluid provides an opportunity for the rapid growth of microorganisms, and must at all times be avoided. Furthermore, it has been reported (Kedzia et al., 1961) that the presence of bacteria in a solution, albeit not necessarily of a pathogenic species, may cause the breakdown of the drug, with resultant loss of activity. A number of different methods of sterilization are available, and the choice of method is determined by the nature of the active constituent of the injection, which must not be adversely affected b y the sterilization procedure selected. Methods may be physical, chemical, or physicochemical. Sykes (1963a) gives a useful commentary upon the methods of sterilization commonly used, but the following are the most important in the preparation of injections.

1 . Methods Involving Heat a . Dry Heat This method is suitable for oily solutions and suspensions, and for powders which are to be dissolved in sterile solvents before use. T h e choice of suitable temperature and times is influenced b y the stability of the product, and some variation is found in official recommendations (Table I).

OFFICIAL

TABLE I RECOMMENDATIONSFOR DRYHEAT STERILIZATION

Reference

Temperature ("C.)

B.P. (1963) I.P. U.S.P. (1965)

Duration

150 1 hour 150 2 hours General recommendations

b. Heating in an Autoclave. Moist heat is generally accepted as being a more efficient sterilizing agent than dry heat, and steam under pressure provides a convenient and effective method of sterilization. This method, when carried out correctly, is the most suitable for aqueous injections of thermostable substances. Suggested sterilization times and temperatures are given in the B.P. (1963), U.S.P. (1965), and I.P., but large volumes will

ANTIMICROBIAL AGENTS IN PHARMACEUTICALS

5

require increased periods of exposure to the sterilization temperature to ensure that the whole of the product has been subjected to the correct temperature for the required time. The B.P. (1963) recommends a sterilizing temperature of 115" to 116"C., while the U.S.P prefers the higher temperature of 121°C. with a correspondingly shorter exposure time. The B.P. (1963), U.S.P. (1965), and I.P. all state that when a container is sealed so as to permit the withdrawal of successive doses on different occasions, the solution or preparation should contain sufficient amounts of a suitable bactericide to prevent the growth of microorganisms (Table 11). The term bactericide, in this context, TABLE I1 BACTERIOSTATS RECOMMENDED IN DIFFERENTPHARMACOPOEIAS Reference B.P. (1963)

I.P.

U.S.P. (1965)

Recommended bacteriostat Phenol, 0.5% w./v. Cresol, 0.5% w./v. Chlorocresol, 0.1% w./v. Phenylmercuric nitrate, 0.001% w./v. Phenol, 0.5% w./v. Cresol, 0.3% w./v. Chlorbutanol or its hydrate, 0.5% w./v. Phenylmercuric nitrate, 0.001% w./v. Phenylmercuric borate, 0.001% w./v. General recommendations

indicates a substance in a bacteriostatic concentration, and will hereafter be referred to as a bacteriostat. Bacteriostats must not be included in any injection for intrathecal, intracisternal, or peridural use (these should be packed in single-dose containers), or in an intravenous injection of dose-volume greater than 15ml. Furthermore, a bacteriostat is unnecessary if the medicament itself has an antibacterial effect: leptazol is an example of this (Gilbert and Russell, 1963). Other commonly used injections are also claimed to exert some bacteriostatic effect (though not sufficient to preclude the addition of a bacteriostat) and Kohli et al. (1950) and Sen Gupta (1951) have studied these with a view to designing suitable sterility tests for them. It may be noted, in passing, that a number of injections containing ratioactive materials, although they may be packed in multidose containers, do not contain a bacteriostat. The reason for this omission has

6

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

not yet been satisfactorily established but research by the British Pharmacopoeia Commission (1966) in this field is continuing.

c . Heating w i t h a Bactericide.

The solution or suspension is prepared using a vehicle containing a given percentage of a suitable bactericide, and heated for a time which ensures that the whole of the product in each container is maintained at 98" to 100°C. for 30 minutes. The choice of bactericides is smaller than that of bacteriostats but neither bactericide nor bacteriostat should interfere with the therapeutic efficacy of the drug, nor cause a turbidity. In the B.P. (1963) the two recommendations are 0.2% w./v. chlorocresol and 0.002% w./v. phenylmercuric nitrate, in each case, twice the recommended bacteriostatic strength. The U.S.P. (1965) does not mention this process per se. The I.P. permits the two bactericides already mentioned, arid also 0.002% w./v. phenylmercuric borate.

2. Filtration Solutions which are sterilized by filtration may contain enough suitable bacteriostat to prevent the growth of microorganisms, although when such solutions are to be used for intrathecal, intracisternal, or peridural injection, or when they are to be used for intravenous injection in doses exceeding 15 ml., the bacteriostat is to be omitted. Solutions prepared by this method are packed in their final containers using aseptic technique, and after sealing, each batch must b e subjected to a test for sterility with which they must comply. The value of this method lies in the fact that it can be used for thermolabile medicaments, but it is a technique requiring skill and must be carried out by trained operators. Sterilization b y heating methods, or by filtration are the most commonly employed; however, other methods are available, including gaseous and radiation sterilization, but will not be considered. C. VEHICLES

1 . Water for Injection The majority of injections are aqueous solutions and the nature of the water used in their preparation must be considered. Potable water contains a wide range of contaminants, depending upon its source, subsequent treatment, and storage, (Taylor and Burman, 1956) and the degree of bacterial contamination, although not normally high,

ANTIMICROBIAL AGENTS IN PHARMACEUTICALS

7

may be sufficient to render it unsuitable for use in the preparation of injections. Dissolved chemicals may cause incompatibilities with the medicament, resulting in turbidity, discoloration, precipitation, or inactivation. Water for injection (B.P. 1963; sterile water for injection, U.S.P., 1965) is potable water distilled from a special still and sterilized in its final containers by heating in an autoclave, or by filtration, without the addition of a bacteriostat. Saunders and Shotton (1956) have examined the preparation of water for injection, and indicate that deionized water is not a suitable substitute for freshly distilled potable water, since the resins used in a deionizing column frequently become contaminated with bacterial colonies, and the water obtained from such columns, although free from chemical substances, may contain microorganisms. It is, however, conceivable that deionized water may, at some time in the future, be used as a vehicle in the preparation of injections.

2 . Nonaqueous Vehicles Aqueous solvents are the vehicles of choice in the preparation of injections, but certain formulations, for reasons of stability, solubility, or usage must be made in nonaqueous vehicles. Oils or fatty acid esters may be used to give a depot effect, while alcohols, propylene glycol, benzyl benzoate, and other substances are used to aid solution of a drug or to preserve its activity. The requirement to add a suitable bacteriostat to injections sealed so as to permit the withdrawal of successive doses also applies to oily injections, although the B.P. (1963) states that “bactericides are ineffective in oils.” Their inclusion would appear to be as a safeguard rather than as having a proved value.

D. CONTAINERS AND CLOSURES 1 . Suitable Materials

Injections may be packed in single-dose or multiple-dose containers, although the former are generally regarded as more desirable, since in the withdrawal of successive doses from a multidose container, there is the continued risk of the introduction of contaminants. Kohan et al. (1962) in a study of 490 multiple-dose vials, found that 13 had become contaminated during use. However, Ravnick and Yatoko (1962) working with 141 vials, found that none had been contaminated. The presence of a bacteriostat does deter the multiplication of microorganisms, but obviously a single-dose container is

8

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

more satisfactory in this respect, although it might not be as convenient in administration. Single dose containers may be ampoules or cartridges; large-volume infusion solutions may be packed in graduated bottles suitable for use in a transfusion apparatus. Multidose injections which have the advantage of flexibility of dose volume, are usually packed in small bottles of the Clinbritic type. The containers should be made of glass which has been tested for its suitability for packing injections: certain types of glass have a high extractive, and yield, among other substances, alkali, which may substantially alter the pH of the injection, with consequent deleterious effects upon the medicament and added substances. Tests for the limit of alkalinity of glass may be of the “whole-container” type (B.P., 1963, I.P.) or of the “crushed-glass” type (U.S.P., 1965). Materials other than glass are permitted for use as containers, provided that they do not react with the medicament, affect its therapeutic properties, or yield small solid particles. Amber glass may be used for injections which must be protected from light.

2. Absorption of Bactericides by Rubber

The ideal method of sealing an injection is by fusion of glass, as in ampoules, but since such a sea1 is broken to withdraw the dose, it can only be used for single-dose containers. Multidose containers are sealed by a rubber closure which will permit the withdrawal of successive doses. A plastic or metal oversea1 must be applied to prevent the removal of the closure before or during use. Ideally, the rubber used in the preparation of the closures should not contribute anything to the solution (Wing, 1958), should not absorb substances from it (Royce and Sykes, 1957) nor allow volatilization of contents through it, or absorption of air and moisture by the contents. Careful formulation of the rubber can reduce to a minimum the possibility of the closure contributing to a solution. Synthetic rubbers have been examined and some types are found to be comparatively inert, but because of expense, poor tensile strength, and other properties, are not always suitable for use in pharmaceutical closures. However, the problem of rubber absorbing bactericides from an injection solution is less easily overcome. Royce and Sykes (1957) have shown how common preservatives will be distributed between two immiscible solvents-that is, water and rubber- according to their partition coefficients. Their experiments showed that rubber can absorb from 15% (benzyl alcohol) to more than 95% (phenylmercuric nitrate) of an injection preservative when a solution of bacteriostatic strength is studied. The figures quoted are at equilibrium and may

ANTIMICROBIAL AGENTS IN PHARMACEUTICALS

9

not be reached for several weeks, but it is evident that, during storage, a high percentage of preservative may be removed from an injection, with subsequent risk of bacterial contamination. To minimize extraction from injections, rubber closures should be subjected to a treatment which will ensure that they are saturated with the preservatives to be used, before use. The B.P. (1963) recommends that the caps, which should be made of good quality compounded, natural or synthetic rubber, should, after thorough washing, be autoclaved at 10 p.s.i. for 30 minutes in a solution of the bactericide of concentration at least twice that to be used in the preparation. The closures are then stored in the autoclaved solution for at least 7 days. If sodium metabisulfite is to be used as an antioxidant in the injection formulation, 0.1% w./v. should be included in the solution in which the caps are stored. At the moment, no such treatment is indicated in the U.S.P. ( 1965). The volatilization of contents through rubber closures can be reduced by several methods (Royce and Sykes, 1957) but when possible, it it better to use one of the less volatile preservatives. In general, the B.P. (1963) recommends that a multidose container should not contain an excessive number of doses (U.S.P., 1965 limits the volume of a multidose container in most cases to 30 ml.), nor should the period of time between the withdrawal of the first and last doses be unduly prolonged. These precautions help to ensure that the period during which an injection is being used (and thus the period during which it is most liable to contamination) is not long. In certain cases, an injection may have a recommended shelf life, e.g., injection of heparin (B.P., 1963): if kept in a container sealed with a rubber closure, it may not maintain a satisfactory concentration of bactericide for more than 3 years.

E.

SELECTION OF

BACTERICIDES

1 . Desirable Properties The number of bactericides available is considerable, but those suitable for use in injectable products are limited by several factors, the most important being suitability for injection into the body, and good antibacterial activity. Guillot (1950) points out that the choice and concentration of bactericide is governed by the type and number of contaminants likely to occur, their resistance to the bactericide, the duration of contact, and the physical and chemical properties of the injection, e.g., pH, osmotic pressure. In addition, the official requirements indicate that the chosen bactericide should not interfere with

10

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

the therapeutic efficacy of the drug, nor cause a turbidity. Felix (1964) suggests that the ideal bactericide should be compatible with a wide range of medicaments, effective over a reasonable p H range, against a wide range of microorganisms. It should also be soluble over normal and refrigerated temperatures in the concentration used and should remain stable during the preparation and shelf life of the injection. Finally, it should not be adversely affected by the closure or container, should be nontoxic and nonirritant.

2 . Choice and Evaluation of Bactericides

The range of official bacteriostats and bactericides has already been indicated, but this does not cover all the substances used in this field. The wide range of substances used shows that, as yet, there is no substance which can be regarded as suitable for use in every injection. In the production of new formulations, it is necessary to confirm bactericidal effect after storage tests, for it cannot be assumed that a bactericide previously effective in one set of conditions will remain so in another. Moreover, studies carried out by Loosemore and Russell (1963), Russell and Loosemore (1964), Davison (1951), Klarmann (1959), and other workers on the recovery of spores after treatment by certain bactericides have shown that substances in concentrations originally thought to be bactericidal are, in actual fact, bacteriostatic, and in suitable recovery media, organisms can be recovered from supposedly “bactericidal” solutions. Thus it is apparent that tests carried out on a bactericide should ensure its effectiveness against both microorganisms and their spores. Sykes (1958) has pointed out the profound effect that concentration has upon the bactericidal properties of certain commonly used substances. Substances at even half their recommended strengths have considerably reduced or in some cases practically no lethal powers. The margin between the effective lethal concentration and the minimum inhibitory Concentration may be very narrow. Bearing in mind the readiness with which some substances are absorbed by rubber, it will be seen how quickly the originally bactericidal strength may be reduced below an effective level. Hess (1965), who emphasizes that the concentration exponent of the phenolic bactericides is very high, also draws attention to the fact that bacteriostats commonly used do not have the same degree of activity, e.g., 0.1% chlorocresol is significantly more active than 0.3%cresol. The ideal antimicrobial compound is effective against all microorganisms and should kill bacteria, molds, fungi, viruses, and also spores. However, it is manifestly impossible to test a bactericide

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11

against the wide range of organisms which may contaminate an injection. The normal practice is to subject a representative sample of the batch to a test for sterility by subculturing a suitable sample into media which provide optimum growth conditions for as large a number of types of microorganism as possible. The design of sterility tests varies in different pharmacopoeias but it is interesting to note that the World Health Organization Report (1960) suggests the setting up of two tests, with the incubation of one at 35" to 37"C., and the other at 15" to 22°C. to facilitate the growth of bacteria and molds. Davies and Fishburn (1946) suggested an alternative type of sterility testthe filtration of the solution through a membrane filter, followed by subculture of the membrane. This method is especially valuable when the injection contains substances which may inhibit the growth of bacteria, as the membrane can be eashed free of any such substance. An alternative to the test for sterility used to assess the effectiveness of a bactericide is used by Rdzok et al. (1955) who described a standardized test whereby the preservative effectiveness of a formulation was challenged by the deliberate introduction of common contaminants. This method was developed by Kenny et al. (1964) who applied it to a wide range of products, including a multidose injection preserved with benzyl alcohol. It is noteworthy that while this product maintained a satisfactorily low leveI of contamination for 28 days, it was found that at the end of this period, the preservative had been completely lost, apparently via the rubber closure. It is evident that any contaminant introduced during the subsequent use of the injection could multiply without hindrance. It is difficult to determine the most likely cause of contamination of parenteral products. Contamination during preparation is always possible and the need for extreme care at this stage cannot be overemphasized. In certain cases, organisms may be introduced during storage, and the use of the oversea1 in multidose containers plays a large part in preventing this. However, hairline cracks in containers, which may arise during sterilization processes, can allow the ingress of organisms. The possibility of the introduction of Contaminants during the use of a multidose container has already been indicated and the inclusion of a bacteriostat reduces the risk of such organisms developing.

3. Bactericides and Bacteriostats in Common Use

a. Phenols and Cresols. Phenol itself has been used as a bacteriostat (strength 0.5%w./v.)

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A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

in injections for many years. It is accepted as a standard in the B.P. (1963) which states that, apart from the list of bacteriostats recommended, other substances may be used, provided that they have an activity not less than 0.5% w.lv. phenol in solution in water for injection. This strength solution also possesses considerable bactericidal properties, although Sykes (1958) points out that it is not rapid in action. This author also mentions that spores may survive treatment with phenol, a fact which has been confirmed by many subsequent workers. Phenol (and indeed, all phenolic bacteriostatics), has a high concentration exponent; Hess (1965) quotes in example the fact that a 1% w./v. solution of phenol kills a certain inoculum 50 times faster than a 0.5% w.lv. solution. It is fairly free from incompatibilities, although Cooper and Gunn (1965) point out that it reacts unfavorably after sterilization in injections of aneurine hydrochloride, hexamethonium, and quinine hydrochloride, and in injection of procaine penicillin. In the case of hexamethonium, the incompatibility is therapeutic in nature. Phenol also has the disadvantage that it is prone to oxidation and discoloration on storage, and that it is volatile. Royce and Sykes (1957) have demonstrated that its distribution between rubber and water is 25 : 75, a fact which makes its high concentration exponent the more significant. Acid conditions enhance its activity, as is shown by the fact that in injection of insulin, which is of p H 2.5 to 3.5, both the B.P. (1963) and the U.S.P. (1965) permit a lesser percentage of phenol as bacteriostat than is normally used. Conversely, its activity in alkaline solution is reduced or entirely lost. Finally, it has the advantage that its activity in the presence of serum is not markedly reduced. Cresol B.P., which has been deleted from the current U.S.P., is a mixture of cresols and other phenols. It is used as a bacteriostat in strengths up to 0.5% w./v. and has the virtue of ease of solubility and a wide range of activity. Cooper and Gunn (1965) mention only three cases of incompatibility (carbachol, ergometrine, and quinine hydrochloride). Cresol is of some value in immunological products, since its activity is not extensively reduced in the presence of organic matter. It is also used as a bacteriostat in certain insulin injections. In the case of isophane insulin injection, both the B.P. (1963) and the U.S.P. (1965) specify that meta-cresol should be used. Royce and Sykes (1957) state that the distribution between rubber and water is 33 : 67. It is generally found that the halogenated cresols are more active than their parent compounds and Hess (1965) states that a 0.1%w.lv. solution of chlorocresol (the recommended bacteriostatic strength in

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13

the B.P. 1963) has significantly greater activity than 0.3%w./v. cresol. Chlorocresol, which is used both as bacteriostat and bactericide (0.2% w./v.) is frequently used in the sterilization of injections, despite the fact that it is incompatible with a number of medicaments. The solid form is subject to oxidation and discoloration and the resulting impurities may account for some of these incompatibilities. In addition, the bactericidal solution is virtually a semisaturated one (chlorocresol is soluble 1 in 260) and strong solutions of medicaments may cause salting out of the bactericide or even of the bacteriostat. McEwan and MacMorran (1947) have pointed out that strong solutions of sodium chloride cause the precipitation of chlorocresol, although it has also been suggested that low concentrations of sodium chloride will potentiate the action of chlorocresol. Chlorocresol is volatile, and is preferentially dissolved in rubber to a high degree, the distribution ratio between rubber and water being 85: 15 (Royce and Sykes, 1957). Davies and Davison (1947) used a filtration technique in the study of injection contamination and found spores of B . subtilis survived the B.P. process of heating with a bactericide using 0.2% w./v. chlorocresol. However, the degree of contamination used in the study (5000 spores/ml.) is not normally met with in injection, which should always be prepared in clean conditions. Another bacteriostat whose use in the preservation of injections is indicated in the U.S.P. (1965) and the B.P. (1958) is chlorbutol 0.5% w./v. Hopkins (1960) points out that chlorbutol is compatible with a wide range of drugs, and that it has some analgesic properties. Up to 90%may be absorbed by rubber vaccine caps (Royce and Sykes, 1957). It is volatile and difficult to dissolve. Its main disadvantage lies in the fact that it hydrolyzes rapidly above p H 3, the hydrochloric acid released in the reaction lowering the pH and causing loss of bactericidal activity. In neutral and alkaline solutions it is ineffective (Felix, 1964; Nair and Lach, 1959).

b. Mercurial s The most commonly used of the mercurial compounds are the phenyl mercuric salts, phenyl mercuric nitrate and phenyl mercuric acetate being the general choice, although the I.P. also permits the use of the borate. The main difference in these salts lies in their solubility, the nitrate being relatively insoluble (1 in 1500 at room temperature) while the acetate and borate are more soluble. Hess (1965) points out that the salts are more soluble in alkaline than in acid solution but Hopkins (1960) indicates that the changes in p H do

14

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

not significantly affect activity. Phenyl mercuric nitrate is official (B.P., 1963) as a bacteriostat (0.001%w./v.) and bactericide (0.002% w./v.). One of the virtues of these compounds is that they have a low concentration exponent and are not readily diluted out, but unfortunately, as demonstrated by Royce and Sykes (1957), up to 95% may be absorbed by rubber vaccine caps. For this reason, these authors do not recommend the use of phenylmercuric salts as bactericides or bacteriostats in multidose containers. Hess (1965) also states that the mercurials may react with certain types of rubber, and numerous instances of incompatibilities may be found in the literature (Hadgraft and Short, 1947); McEwan and MacMorran, 1947).Hopkins (1960) states that phenylmercuric acetate is a satisfactory preservative for antigens. Thiomerosal (thiomersal or Merthiolate) is also used in certain kinds of injections, mainly biological products, and further reference to it will be found under that section. It has both bacteriostatic and fungistatic properties and is used in strengths ranging from 0.01% w.lv. to 0.02%w.lv.

c . Other Substances Alchols also possess bactericidal properties, and of these, the one most commonly used in injections is benzyl alcohol. It has bacteriostatic rather than bactericidal properties and in addition, has some local anesthetic effect. It is included in compound injection of benzocaine, B.P.C. (1954) (5% w./v.), and injection of ethanolamine oleate, B.P. (1963) (2% w./v.). P-phenylethyl alcohol in strengths of 0.7 to 1.5%w.lv. is also bacteriostatic, but its greater use lies in the field of ophthalmic preparations. A 0.1% w./v. solution of benzoic acid has both bacteriostatic and bactericidal properties, but above pH 5, only the bacteriostatic property is retained. Due to this difficulty, the acid is not suitable for use in injections, but the esters of the hydroxylated acid (methyl p hydroxybenzoate and propyl p-hydroxybenzoate) have been used for their bacteriostatic properties in insulin zinc suspensions. In general, however, the esters are not used in injections. The quaternary ammonium compounds are useful in the formulation of injection suspensions, e.g., procaine penicillin injection. Being surface-active agents and having bactericidal properties, they serve a dual purpose, but care must be taken to avoid any incompatibility. From what has been written, it can be seen that, at present, there is no ideal bactericidal substance which can be used in all injections;

ANTIMICROBIAL AGENTS I N PHARMACEUTICALS

15

many of the bactericides in use, of which by no means all have been discussed, present problems of incompatibility, instability, absorption b y rubber, and other difficulties. It is difficult to foresee the development of the ideal bactericide and so pharmacists have to apply their knowledge to the improvement of substances at present in use. I I I. lmmu no1og ica I Products A. TYPESOF IMMUNITY Immunity may be defined as the resistance presented b y the host to an infecting organism. Without taking account of racial and species immunity, it is conveniently subdivided into:

1 . Passive Immunity a. Natural Immunity This is brought about by the passage of antibodies from mother to child.

b. Arti$cial Acquired Immunity This type of immunity involves no work on the part of the body defense mechanisms, since antibody is produced in another animal, usually the horse. Immunity to a specific organism is immediate, but is short-lasting. Products which confer this type of immunity are antibacterial, antitoxic, and antiviral sera, the last-named including sometimes the gamma-globulins.

2. Active Zmmunity a. Natural Zmmunity

This is exemplified by cases where a single attack of a disease confers increased resistance against a second attack.

b. Arti$cial Acquired Immunity An antigen is administered with the specific intention of inducing an antibody response in the recipient. Immunity is slow to develop, but is relatively long-lasting, and may easily be restored. The antigen may consist of living or dead bacteria, rarely of toxins, of toxoids, of living or inactivated virus particles, and of dead rickettsiae. A further group of products consists of diagnostic reagents. This group includes the tuberculins, and the Schick and Dick test toxins and their controls.

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A. D.

RUSSELL, J. JENKINS,

AND 1. H. HARRISON

B. PRODUCTS REQUIRINGA PRESERVATIVE

The U.S.P. (1965) emphasizes that the choice of antibacterial agents for use in immunological products requires especial care. It is desirable that such an agent should suppress microbial growth, be compatible with the product, b e stable at normal temperatures, and b e nontoxic; in addition, there should be relative freedom from inactivation of the therapeutic principle, and no absorption into the rubber used in multiple-dose containers. A substance that is sometimes used for preservation of some vaccines and other biological preparations is thiomersal (thimerosal, sodium ethylmercurithiosalicylate, Merthiolate); however, it has been found that its concentration diminishes with storage, presumably due to its absorption by rubber (Birner and Garnet, 1964a) and not to the presence of zinc released from the rubber (Birner and Garnet, 1964b). Nevertheless, thiomersal occupies an important place in the preservation of immunological products. The products that require the addition of a preservative are: 1 . Antisera a . Antibacterial Sera These are prepared against certain bacteria which do not produce exotoxins, e.g., Leptospira antisera.

b . Antitoxic sera These sera are prepared against specific exotoxins, which are first converted into toxoids, by means of formaldehyde. The toxoids are injected into horses and the blood collected and processed. c. Antiviral Sera Apart from rabies antiserum, which is obtained from animals injected with the specific virus, antiviral sera are usually obtained from human convalescents, from adults who have had the disease in the past, or from persons who have been artificially immunized. Antisera exist in liquid form. A suitable antibacterial substance may be included; this is essential when antisera are present in multidose containers. No preservative is specifically named, but phenol is suitable.

2 . Vaccines a . Bacterial Vaccines Vaccines are defined (B.P., 1963) as being suspensions of living

ANTIMICROBIAL AGENTS IN PHARMACEUTICALS

17

organisms (bacteria, rickettsiae, or viruses), sterile suspensions of the organisms, or toxoids. Thus, toxoids are included in the official definition of vaccine. For convenience, however, toxoids are here considered separately. Two factors that need to be considered in preparing killed bacterial vaccines are the method employed in killing the bacteria, and the choice of a preservative. In neither case must damage occur to the antigenic potency (antigenic identity) of the organism. Cohen and Wheeler (1946), for example, described a method of preparing pertussis vaccine from Phase I cultures in which the organisms were killed with 0.01% w.lv. thiomersal, which was also present as a preservative in the final vaccine. This had no deleterious effect on the antigenic identity of Bordetella pertussis. More recently, Gardner and Pittman (1965) investigated the stability of pertussis vaccine in the presence of different preservatives, and showed that the potency of the vaccines preserved with 0.01%w./v. thiomersal was more stable than that of vaccines preserved with 0.0025% w.lv. benzethonium chloride (Phemerol) or a mixture of 0.15% w./v. and 0.02% w./v., respectively, of methyl and propyl p-hydroxybenzoates. The potency of vaccines containing no preservative was more stable than those containing benzethonium or parabens, but less stable than those containing thiomersal. Olson et aZ. (1964) had just previously shown that the saturation of negative sites on the pertussis cell with cations prior to the addition of benzethonium chloride prevented its uptake b y the cell and stabilized the antigenic potency of the vaccine. The histamine-sensitizing factor is also unstable in the presence of benzethonium chloride or the parabens. Formaldehyde, also, has an adverse effect on the potency of pertussis vaccine (Pittman and Cox, 1965). Further applications of these findings are considered in Section 111,2,d. At present, whole cells of B. pertussis are used in the preparation of vaccines. However, it has been found (see, e.g., Sutherland, 1963) that cell wall material from B. pertussis, prepared by Mickle disruption followed by purification by various chemical treatments, is a good protective agent. van Hemert et al. (1964) considered that chemical solubilization of the outer layers of the cell was a better approach in isolating the cell fraction responsible for immunizing man against whooping cough. This type of research opens up interesting possibilities for the future; at present, however, there is no information available as to a suitable preservative for this type of product, although it is not unreasonable to suggest that thiomersal would be of use.

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A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

The preparation of cholera vaccine has been described by Dick (1959) and by the World Health Organization (1959b), which points out that considerable differences occur in various countries in the choice of strains, their virulence, method of killing, and use of preservatives. It further stipulates that a suitable preservative be incorporated into cholera vaccines issued in multiple-dose containers and that such a preservative “shall have been demonstrated, to the satisfaction of the national control laboratory, not to affect the antigenicity and safety of the vaccine.” A suitable preservative appears to be 0.5%phenol (Dick, 1959). Typhoid-paratyphoid A and B vaccine (TAB vaccine) consists of Salmonella typhi and S . paratyphi A and B. In addition to the 0 and H antigens, it contains the Vi antigen in the case of S . typhi. Typhoidparatyphoid A, B, and C vaccine (TABC vaccine) consists of s. typhi and S . paratyphi A, B, and C. In addition to the 0 and H antigens, it contains the Vi antigen in the case of S . typhi and S . paratyphi C . The vaccines may be either alcohol-killed alcohol-preserved, or heatkilled phenol-preserved (phenolized). It had been found by Felix (1941) that the former type of vaccine conferred greater protection on mice, and that phenol tended to destroy the Vi antigen. However, alcoholized vaccines were later shown to be disappointing, since the protection obtained in the mouse was not reproduced in man (Parish and Cannon, 1963, 1964; Parish, 1965). Controlled clinical trials later confirmed that the phenolized vaccine gave better protection than the alcoholized one (Parish and Cannon, 1963), thus indicating that the importance of the Vi antigen was less than originally thought. In fact, Morgan (1958) has stressed that it had not been demonstrated that it played an essential role in protection. The U.S.P. (1960) lists 0.5% phenol or 0.4% cresol as a suitable preservative for TAB vaccine. An interesting development in the prevention of typhoid would appear to concern the finding that glycine-induced spheroplasts of S . typhi possessed a far higher immunogenic ability, and much lower toxicity, than intact cells (Diena et al., 1964). Difficulties of storing the spheroplasts for long periods could be discounted, since lysed spheroplast suspensions were as efficient as intact spheroplasts in conferring protection in mice. No indication of a suitable preservative for use in a multidose container is given, but 0.02% w./v. thiomersal was used to kill the spheroplasts, so that in the event of spheroplasts or their lysates becoming an accepted method of vaccine preparation, it is conceivable that this antibacterial agent would be investigated for its effect over longer periods.

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b. Toxoids Gram-positive bacteria generally produce the so-called “soluble” toxins, or exotoxins, whereas most Gram-negative bacteria produce endotoxin (see Luderitz et al., 1966). Exotoxins, but not endotoxins, can be toxoided by means of formaldehyde to give the group of immunological products known as toxoids. An example of such products is diphtheria vaccine, which occurs in five different forms: formol toxoid, toxoid-antitoxin floccules, alumprecipitated toxoid, purified toxoid aluminum phosphate, and purified toxoid aluminum hydroxide. With the last three, phenols and cresols adversely affect the antigenic potency (B.P., 1963), and a suitable preservative is thiomersal. Phenol (0.5% w./v.) may be used as a preservative for the toxoid-antitoxin floccules.

c. Viral Vaccines These vaccines may be subdivided into living vaccines, e.g., poliomyelitis (Sabin) and smallpox vaccines, and inactivated vaccines, e.g., poliomyelitis (Salk) vaccine. Pivnick et al. (1963) and Tracy et al. (1964) have studied the use of differentpreservatives in polio (Salk) vaccine. Several antimicrobial agents may be present in this vaccine, e.g., streptomycin, neomycin, and polymycin may be employed to inhibit bacterial contamination of tissue culture (in Britain, penicillin and streptomycin are sometimes, but not necessarily, used); formaldehyde is added as a virucidal agent, although it is normally neutralized by sodium metabisulfite before the blending of individual components into the trivalent vaccine; and preservatives added to the finished vaccine. The World Health Organization (1959a) states that any preservative incorporated must have no deleterious effect on the product. Benzethonium chloride is a suitable agent (McLean, 1957; Schuchardt et al., 1960)and it has been recommended that it should preferably be used in vaccines to which sodium metabisulfite had not been added. Stable antibiotics contribute considerable antibacterial activity, and formaldehyde (if not neutralized) is active against fungi as well as bacteria (Pivnick et al., 1963). However, Pivnick et al. (1963) considered that benzethonium added little if any antibacterial activity, but did have some antifungal activity. The parabens are not harmful to poliomyelitis vaccine, and their addition to inactivated vaccine containing nonneutralized formaldehyde was found to give a mixture of preservatives inhibitory against high numbers of both bacterial or fungal contaminants.

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A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

Mercurials appear to be deleterious to the potency of inactivated polio vaccine (Gardner and Pittman, 1965) and thus cannot normally be employed as preservatives in this case. However, Davisson et al. (1956) have shown that thiomersal may be used as such a preservative when ethylenediaminetetraacetic acid (EDTA) is present. The chelating agent protects the thiomersal from breakdown to products which affect the antigenicity of the vaccine. Since polio viruses are labile agents, oral (living) vaccines must be handled carefully to prevent inactivation of virus, and a consequent loss of vaccine potency. Sabin vaccine stabilized with magnesium chloride was found by Melnick and Wallis (1964) to retain its potency for much greater periods than control vaccines (MgC12 absent). In addition to protecting the live virus, the concentration of magnesium salt used, 1to 2 M , was inhibitory to most bacteria and fungi. Smallpox vaccine is prepared by growing the virus (i) in the skin of living animals, (ii) in the chick embryo, or (iii) in tissue culture. It is a living vaccine; however, vaccine prepared by method (i) is likely to contain extraneous microorganisms, which must be reduced to < 10OO/ml. (also certain specific bacteria must be absent). If this vaccine is issued in liquid form, treatment consists of the addition of glycerol with or without another antibacterial substance, e.g., 1% w./v. phenol. In vaccines prepared by methods (ii) and (iii), extraneous organisms will be absent, but the World Health Organization (1959~) recommends the addition of glycerol with or without an antibacterial substance as a precaution against later contamination. Smallpox vaccine (liquid) is issued in single- or multidose containers. Each container of dry vaccine should be issued with an ampoule of sterile reconstituting fluid, which may contain glycerol with or without an antibacterial substance. A suitable antibacterial preservative for this (in addition to glycerol, which itself possesses bacteriostatic properties) is 0.5% w./v. phenol. Antibiotics may be used in the preparation of smallpox vaccine b y method (iii) but the World Health Organization ( 1 9 5 9 ~ )recommends that the use of antibiotics as additives to smallpox vaccine should be discouraged. The U.S.P. (1965) proposes that smallpox vaccine contains 40 to 60% glycerol (or sorbitol) with not more than 0.5% w./v. phenol as a preservative. Glycerol itself has been used as a preservative for over a century; its advantages, e.g., valuable bacteriostatic properties, good dispersing agent, and its disadvantages, e.g., fairly rapid rate of inactivation of virus particles, have been considered by Amies (1962), who has described an improved smallpox vaccine containing 0.4% w./v. phenol as preservative, except when the vaccine is lyophilized.

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Other viral vaccines, e.g., influenza and rabies vaccines, must contain a suitable preservative. The U.S.P. (1960) stipulates in the case of influenza vaccine that if formaldehyde is used for inactivation, the finished vaccine contains > 0.02% w./v. free formaldehyde. Thiomersal, 0.013%, is used in Britain as a preservative in influenza (inactivated) vaccine.

d . Combined Prophylactics Diphtheria, tetanus, and pertussis vaccine (DTP/Vac) has, in recent years, been combined with polio vaccine (inactivated) to give a “quadruple antigen” vaccine, DTPP. Pittman (1962) and Edsall et al. (1962) have independently found that in this combination, the pertussis vaccine was unstable. In addition to containing the poliomyelitis component, DTPP differed from DTP in having a different preservative. Thiomersal has been used as a preservative in pertussis vaccine, and in DTP. However, mercurials are deleterious to the potency of polio vaccine (inactivated) and thus when the conditions in DTP were adjusted so as to be optimal for the less stable polio vaccine, the pertussis component became unstable. (Gardner and Pittman, 1965). McLean (1957) and Schuchardt et al. (1960) showed that benzethonium was not harmful to polio vaccine, and benzethonium thus replaced thiomersal as preservative when DTP was combined with polio vaccine to form DTPP. It has also been found, however, that benzethonium chloride causes a loss of potency of the pertussis vaccine component (Gardner and Pittman, 1965; Olson et al., 1964) and was thus responsible for the instability of pertussis in DTPP. Likewise, the parabens, which do not reduce the antigenic potency of polio vaccine, could not be used as a preservative in DTPP, as in their presence, the pertussis component was again unstable (Gardner and Pittman, 1965). Other combinations of pertussis, e.g., pertussis and tetanus vaccine, and pertussis and diphtheria vaccine, may all be preserved with thiomersal. Phenol (0.5%w./v.) is a suitable preservative for typhoidparatyphoid A and B and tetanus vaccine, and for typhoid-paratyphoid A and B and cholera vaccine.

3. Diagnostic Reagents These reagents include Dick and Schick test toxins (and their controls), old tuberculin and old tuberculin, purified protein derivative (PPD). PPD contains 0.5% phenol as a preservative (B.P., 1963; Landi, 1963). When used in the Mantoux test, this stock solution is diluted

22

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

to contain graded amounts. The potency of diluted PPD solutions is, however, reduced by phenol (see Pivnick et al., 1965), and a diluent for PPD that contained 8-hydroxyquinoline sulfate was devised by Magnusson et al. (1958).However, because of reports that 8-hydroxyquinoline was ineffective as a preservative in PPD, Pivnick et al. (1965) reinvestigated its antimicrobial activity, and found that it was an effective preservative against contamination of PPD with certain yeasts and molds, and that it had a bactericidal action against Pseudornonas aeruginosa provided that not more than 100 viable cellsiml. were present. Although results of its activity against Staphylococcus uureus are not clear-cut, other work has shown that low concentrations of 8-hydroxyquinoline inhibit the growth of strains of this organism. Old tuberculin contains, as a result of its preparation, a high concentration of glycerol, which itself exerts a bacteriostatic effect. In diluted preparations, which are stable for 3 months, phenol or thiomersal may be employed as a preservative. IV. Eye Drops

A. PREPARATION AND STERILIZATION It is widely agreed that, at the time of dispensing, eye drops should b e sterile. The B.P.C. (1963), U.S.P. (1965), and U.S.N.F. (1960) list methods for preparing and sterilizing eye drops. More recently, the B.P.C. Addendum (1966)has made important and far-reaching amendments to these methods as a result of further work on the part of research groups both in Britain and abroad. In addition, eye drops need no longer be made isotonic with the lachrymal secretions. Basically, the methods of preparation and sterilization listed by the B.P.C. Addendum (1966) are as follows: the medicament is dissolved in the aqueous vehicle containing one of the prescribed antimicrobial substances, and the solution is sterilized by heating in an autoclave at 115"C.,by heating at 98" to 10o"C.,or by filtration through a bacteriaproof filter, the actual method depending to a great extent on the stability to heat of the active constituent. In the case of oily e y e drops, an aseptic technique is used. As is the case with the rubber caps used in multidose injection containers (see Section 11, D2), the rubber teats used in eye-drop bottles may absorb antimicrobial substances from the solutions, and they must thus be impregnated with the selected agent, with which they must be compatible.

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0. PRESERVATIVES

1. The Ideal Preservative The B.P.C. Addendum (1966) stipulates that aqueous eye drops must contain a vehicle that is bactericidal and fungicidal, and lists the following as suitable substances: 0.002% w./v. phenylmercuric nitrate (PMN), 0.002% w./v. phenylmercuric acetate (PMA), 0.01% w./v. benzalkonium chloride, and 0.01% w./v. chlorhexidine acetate. These replace the parabens which were used in many B.P.C. (1959) eye-drop formulations, then deleted from, but later brought back to, several B.P.C. (1963)formulations. The ideal antimicrobial compound for use in eye drops should have the following properties (based in part on Foster, 1965): (i) possess a wide antimicrobial spectrum, (ii) be compatible with the active medicament, (iii) be nontoxic and nonirritant, (iv) be stable to heat, moisture, and on storage, (v) show little or no absorption into rubber teats. To these may be added: (vi) a sterilizing time of < 1 hour, since Kohn et al. (1963b) propose that an antibacterial substance that has a sterilizing time of > 1 hour may be arbitrarily considered too slowacting for use as a preservative in multidose ophthalmic solutions; (vii) no reduction of activity due to composition of the container: as recently pointed out (Editorial, 1966), the use of plastic containers is still limited to some extent b y incomplete information on interactions between drugs, preservatives, and plastics; (viii) the antimicrobial compound should not significantly alter the pH and toxicity of ophthalmic solutions (Lawrence, 1955a). The ideal antimicrobial agent for use in eye drops has not yet been discovered, however (Hopkins, 1966),and the search for such a substance continues. The U.S.P. (1965)and the U.S.N.F. (1960)have stated that eye-drop preparations for use in surgical procedures on an injured eye should not contain an added antimicrobial agent, since this may be irritating to tissues lining the anterior chamber and should be in single-dose containers. Ophthalmic solutions may be packed in multiple-dose containers when intended for use on eyes with intact corneal membranes, and the most frequently used preservative is 0.01% w./v. benzalkonium chloride. Other preservatives that may be used are 0.5% w./v. chlorbutanol, 0.5% w./v. phenylethyl alcohol, 0.05%w./v. chlorocresol, and PMN at a concentration of 0.004%(U.S.N.F., 1960) or 0.001% (U.S.P., 1965). The Australian National Formulary (A.N.F.) requires all eye drops to be sterile, and states that 0.01%w./v. chlor-

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A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

hexidine is a suitable bactericide; in cases where difficulty of formulation is experienced, this may be replaced by 0.5% w./v. chlorbutanol. These and other substances which have been, or are, used as antimicrobial substances in e y e drops are considered in more detail below. A major difficulty in their use is presented by the abnormally high resistance of Pseudornonas aeruginosa (P. pyocyanea) to chemical substances; furthermore, some species of B . subtilis and C. welchii are implicated as eye pathogens (Brown and Norton, 1965; see also Beloian and Koski, 1964).

2 . Substances in Use a . Parebens Hugo and Foster (1964a) found that P . aeruginosa NCTC 7244, a strain originally isolated from an infected human eye, would grow readily without previous adaptation in solutions of esters of p-hydroxybenzoic acid, with or without mineral supplementation. The concentrations of esters conformed to those (methyl, 0.02%w./v. propyl, 0.01% w./v.) used in solution for eye drops (B.P.C., 1959). This paper was criticized by Montgomery and Halsall (1964a,b) who pointed out that no variation in ester concentration had been made, that the manufacturers of the esters recommended a total ester concentration of at least O.OS%, and that in their personal experience this concentration was at the least bacteriostatic and probably bactericidal against P . aeruginosa. These findings were confirmed by McIver (1964) who also found 0.08% w./v. Nipasept (a mixture of methyl, ethyl, and propyl p-hydroxybenzoates) to be effective. These criticisms were answered by Foster (1964a,b) who stated that a 0.08% (Me:Pr ratio of 2 : 1) total ester concentration was just bacteriostatic for P.aeruginosa, and that a total concentration of 0.2% w./v. was needed for a bactericidal action within 30 minutes at room temperature; at this concentration, the esters were too irritant. Confirmation of these results was later produced (Hugo and Foster, 1964b). Other workers have also produced evidence to show that the parahens are virtually inactive against P . aeruginosa (Lawrence, l955a; Kohn et at., 1963a; Brown et al., 1964).

+

b. Chlorocresol This replaced solution for eye drops in the B.P.C. (1963), but before this became official, chlorocresol was deleted as an eye drop preservative, and solution for eye drops reinstated. The reasons for this apparently hasty action have been described (Editorial, 1963): a

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report was received to the effect that a solution of normal saline containing 0.1% w.lv. chlorocresol had caused damage to the corneal epithelium when used in a series of operations involving the anterior chamber of the eye, and the Faculty of Ophthalmologists requested that action be taken to find an alternative substance to maintain the sterility of eye drops. However, chlorocresol is not unduly irritant to intact noninflamed eyes, and is known to be an effective antibacterial agent. Furthermore, it has been used for some years in America as a preservative in e y e drops intended to be used on eyes with intact corneal membranes, and it is thus reasonable to propose that the possibility of using it for this purpose in Britain could well be reinvestigated,

c. Mercury Compounds The three mercury compounds that may be used as eye-drop preservatives are PMN, PMA, and thiomersal. There is conflicting information about the activity of PMN against P . aeruginosa, e.g., Lawrence (1955a) found that PMN was considerably more active than thiomersal in its effects against various strains of P . aeruginosa, but Kohn et al. (1963a) showed that thiomersal and PMN individually required 6 hours to exert a bactericidal effect. Anderson et al. (1964a) found that, in fluorescein eye-drop preparations, PMN failed to inhibit P . aeruginosa in one formulation, and both Pseudomonas and Proteus sp. in another, but a concentration of 0.004% w.lv. PMN was effective in a third formulation. Differences in technique could account for some of the discrepancies noted by various authors in the effectiveness of various antimicrobial agents (Brown and Norton, 1965), e.g., in the use of a suitable inactivator or neutralizing agent (Russell, 1964). It has been shown that organic mercurials were less active in the presence of active medicaments of eye drops than in their absence (Lawrence, 1955a) and Kohn et al. (1963a) did not recommend mercury compounds as being suitable preservatives. Also, although PMN is bactericidal in low concentrations against vegetative organisms, it is only slowly sporicidal (see review by Russell, 1965). However, the U.S.N.F. (1960) recommends its use at a concentration of 0.004%. Foster (1965) considers that thiomersal may prove to be a suitable eye-drop preservative; some of the above findings may suggest otherwise. With regard to the toxicity of organic mercury compounds, Abrams (1963) showed that mercurialentis (deposition of mercury on the lens) may result from exposure of the e y e to 0.004% w.lv. PMN over

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A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

a long period of time; this did not occur with thiomersal (Abrams et al., 1965). Foster (1965), however, has stated that mercurialentis is a rare event, may be innocuous, and should not be allowed to detract from the usefulness of PMN.

d . Benzalkonium Chloride Various authors have shown that benzalkonium chloride is an effective preservative in eye-drop formulations. Thus, Lawrence (1955a,b) found it to be the most effective of seven commonly used chemicals in destroying Pseudomonas and Proteus contaminants. Benzalkonium is a quaternary ammonium compound, and possesses lower activity against Gram-negative than against Gram-positive bacteria. Also, the activity of quaternary ammonium compounds may be reduced in the presence of certain metallic ions. The chelating agent, EDTA, has been found to reduce the resistance of P . ueruginosa to a quaternary ammonium compound (MacGregor and Elliker, 1958), and recently it has been shown that the activity of polymyxin B sulfate, chlorhexidine diacetate, and benzalkonium chloride against P . aeruginosa NCTC 8203 was substantially increased in the presence of EDTA (Brown and Richards, 1965). This potentiating action was blocked by Mg++ and Ca++,and it was postulated that EDTA was synergistic with these antibacterial agents by a mechanism involving removal of Ca++ or Mg++ions or both from the bacterial cytoplasmic membrane. This potentiation by EDTA finds application in eye drops of prednisolone sodium phosphate (B.P.C., 1963); also the U.S.N.F. (1960) states that resistant strains of P . aeruginosa are made more sensitive to benzalkonium in the presence of 0.01 to 0.1% EDTA. I n addition, a combination of 0.01% w./v. benzalkonium and 1000 USP units/ml. of polymyxin B sulfate may be effective against resistant strains of this organism and yet be sufficiently nonirritating to eye tissues (USP, 1965). A concentration of 0.02% w./v. is rapidly bactericidal against some strains of P . aeruginosa (Kohn et al., 1963a); Foster (1965) considers this concentration to be rather high, and the B.P.C. Addendum (1966) and the U.S.P. (1965), in fact, recommend a concentration of 0.01% w./v. Thus, the evidence in favor of benzalkonium chloride being the most reliable of the preservatives available (Brown et al., 1964) appears to be overwhelming. However, some dissension from this has been voiced by Anderson et al. (1964b), who find benzalkonium to be almost inactive against P . aeruginosa, and who quote the results obtained by Riegelman et al. (1956) in support of their claims; how-

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ever, it would appear from the work of Kohn et al. (1963a) that Riegelman’s findings have now been disproved. The question of whether the quaternary ammonium compounds are toxic to the e y e has led to some confusion. Klein et al. (1954) in an otherwise interesting paper, misquoted the results of Ginsberg and Robson (1949) and in consequence reported that these substances could prove harmful by solubilizing the intercellular cement of the corneal epithelium. In fact, Ginsberg and Robson did not use a quaternary ammonium compound, and the quaternary ammonium compounds (including high concentrations of benzalkonium chloride) do not cause loss of intercellular cohesion (Buschke, 1949). Benzalkonium chloride suffers from the disadvantage that some rubber teats may be incompatible with it, and the B.P.C. Addendum (1966) thus recommends the use of silicone rubber teats for those eye drops in which this antimicrobial substance is included.

e. Chlorhexidine Anderson et al. (1964a) found that chlorhexidine (bis-p-chlorophenyldiguanohexane) was an efficient bacteriostatic agent in 69 out of 75 eye-drop formulations tested. Later (Anderson et al., 1964b), they again claimed its superiority as an eye-drop preservative over benzalkonium and cetrimide. The A.N.F. use chlorhexidine as such a preservative, and it has recently been advocated for this purpose in the B.P.C. Addendum (1966). Unfortunately, chlorhexidine is incompatible with some active medicaments of e y e drops, such as borates, phosphates, sulfates, fluorescein, and physostigmine. Chlorhexidine is not listed by the U.S.P. (1965) as being a suitable eyedrop preservative.

f. Chlorbutanol This is usually employed at a 0.5% w./v. concentration as a preservative in e y e drops. Provided that it is permitted to act upon the organisms for a sufficient length of time (several hours), it is effective in destroying Pseudomonas and Proteus cultures (Lawrence, 1955a,b; Kohn et al., 1963a). It is thus doubtful whether chlorbutanol has any significant value in eye-drop formulations. In addition, it hydrolyzes to hydrochloric acid, thus causing a decrease in pH, which occurs rapidly during heating and slowly at room temperature.

3. Conclusions At present, no one substance fills all the requirements of the ideal eye-drop preservative. As to the future, it might be more rewarding to

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attempt to understand the nature of the resistance of P . aeruginosa to chemical inactivation rather than to investigate new agents (Brown and Norton, 1965).Certainly the work to date on the effects of EDTA and of Tween 80 (Richards and Brown, 1964) in reducing the resistance of P . aeruginosa to chemical substances is a start in this direction. V. Medicines for Oral Administration

A. INTRODUCTION Liquid medicines for oral use are dispersions of the therapeutically active drug(s), either solid or liquid, in a liquid which is called the vehicle. The vehicle is usually aqueous, either water itself or an aqueous solution of a flavoring agent, etc. Some vehicles possess therapeutic properties while others are pharmacologically inert. The dispersion may be a solution, a suspension, or an emulsion. The medicines are of several types and often have names indicative of their particular use or formulation, e.g., a “linctus” is a thick syrupy product intended to be sipped and swallowed slowly so as to exert a soothing effect on the lining of the throat; and “elixir” is a sweetened and flavored preparation containing a potent or nauseating drug and may contain a high proportion of ethanol, glycerol, or propylene glycol. The substances used in liquid medicines may be incorporated as such, e.g., sodium bicarbonate, morphine sulfate, or may be incorporated in the form of a “galenical” or other preparation. A galenical is a preparation made by macerating or percolating a crude drug with a solvent, e.g., ethanol, of an appropriate strength. The object is to extract the active principles of the drug as completely as possible while leaving behind the unwanted substances. Among the most common galenicals are tinctures (these are alcoholic preparations of vegetable or animal drugs containing rather low concentrations of active principles); and fluid extracts (these are similar to tinctures but contain high concentrations of active ingredients; usually 1 ml. of fluid extract contains the active principles from 1 gm. of drug). Other preparations of drugs used to make medicines include syrups (concentrated solutions of sucrose containing therapeutic or flavoring substances), spirits (alcoholic solutions of volatile substances), and solutions (aqueous solutions of solids, liquids, or gases, often containing sufficient ethanol to inhibit microbial growth). The galenicals and other preparations per se are usually resistant to microbial growth, either because of their ethanol content, or their high osmotic pres-

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sure due to their sucrose concentrations (syrup). However, on dilution with water they frequently become ideal media for microbial growth. The addition of a relatively small amount of water may make all the difference; the B.P.C. 1963 states that solutions of sucrose containing less than 65% w.lw. will ferment, whereas those containing 66.7% w.lw. seldom permit microbial growth. The simplest mixture that could be made would consist of a solution or suspension of a solid drug in water. In many cases such a preparation would not support microbial growth and hence would not require the addition of a preservative. Certain drugs in aqueous solution, however, will support microbial growth; cocaine hydrochloride is an example (Extra Pharmacopoeia, 1958). Few mixtures consist solely of a drug and water since it is frequently necessary to include other substances to make a stable and palatable product. The pH of a mixture may have to be adjusted so as to achieve optimal stability of the drug or to obtain its optimal therapeutic effect; the resulting product may encourage microbial growth. If the drug has an unpleasant taste, it is usual to disguise this by the addition of suitable flavoring and/or sweetening agents. Probably the most commonly used sweetening agents used in pharmaceutical products are sucrose, sorbitol (especially suitable for preparations for diabetics), glycerol, and propylene glycol. Barr and Tice (1957a) studied the inhibition of growth of several bacteria ( S . aureus, B . subtilis, P . aeruginosa) and molds (Aspergillus niger, P . notatum, and Monilia (Candida) albicans) in aqueous solutions of either glycerol or propylene glycol containing various concentrations of sorbitol. Of the microorganisms studied, M . albicans was the most resistant but its growth was inhibited by 50% w.lv. glycerol and by 30% w.lv. propylene glycol. When more than 10% w./v. sorbitol was present, the inhibitory concentrations of glycerol and propylene glycol were decreased. Barr and Tice attributed the inhibition to the osmotic pressure of the solutions. In a subsequent paper (Barr and Tice, 1957b) they reported on the inhibitory effects of other sugars, including dextrose, levulose, and invert sugar. They found that 60% w./w. invert sugar solution inhibited Aspergillus niger, but solutions containing 50% w./w. of either levulose or dextrose did not. Stable solutions of these last two substances containing more than 55% w.lw. could not be made. It is evident, therefore, that unless mixtures are made in almost pure syrup, or contain very high concentrations of glycerol or propylene glycol, the presence of a preservative in sweetened aqueous preparations is essential.

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A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

The physical stability of many suspensions and emulsions is enhanced by the presence of suspending and/or emu1sifying agents. Some of these agents, e.g., acacia, tragacanth, are carbohydrates and are liable to microbial attack. Various cellulose derivatives, e.g., methyl cellulose, are also used as suspending or emulsifying agents. Although these were formerly believed to be resistant to microbial growth (Mueller and Deardorff, 1956), there is now evidence that under certain conditions suitable microorganisms do attack them and destroy their pharmaceutical usefulness (Brown, 1961). Moreover, methyl cellulose has been shown to form complexes with a number of common preservatives, notably the parabens (Tillman and Kuramoto, 1957). The synthetic nonionic surface-active agents of the fatty acid ester type (e.g., sorbitan esters and their polyoxyethylene derivatives) are also widely used in pharmaceutical products as emulsifying or solubilizing agents. Barr and Tice ( 1 9 5 7 ~showed ) that various microorganisms, e.g., P. aeroginosa, A. niger, P. notatum, and M . albicans, could grow in solutions and dispersions of such substances and produce esterases which split the ester linkages. The adverse effects of this splitting of the surface-active molecules on the physical properties of the product are obvious as is the need for the inclusion of an effective preservative. In another paper Barr and Tice (1957d) described the results obtained when they screened over 50 antimicrobial substances and combinations for effectiveness in preserving both solutions of and preparations containing these nonionic agents. Among the effective preservatives were sorbic acid (0.1 to 0.2%), the phenylinercuric salts (about 0.01%), hexylene glycol (3.0%),and benzalkonium chloride (0.1%).The phenols were ineffective, probably due to complexation with the surface-active agent. As to whether a particular formulation will, or will not, support microbial growth, only thorough testing in a laboratory will show. However, by a suitable choice of ingredients, galenicals, etc., it may be possible to achieve the desired result without adding an antibacterial agent as such. This is the preferred procedure because it avoids the possibility of interaction between the antibacterial and the drugs, etc., present in the mixture. The use of a pleasantly flavored tincture in sufficient quantity to provide a final ethanol content of about 15% should prevent microbial attack especially if the pH is low (Gabel, 1921). Another important factor is the length of time the preparation is required to “keep.” Medicines made extemporaneously for a given patient are usually consumed within a few days, and preservation of

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these is relatively simple. The problem becomes more difficult when a manufacturer wishes to prepare a product having a shelf life of perhaps 2 years. The storage conditions required by the drug are also important. Some drugs are only stable in mixtures when refrigerated; such mixtures would not require additional preservatives. The desirable properties of an antimicrobial for use in oral preparations include freedom from an unpleasant taste and smell, in addition to the usual pharmaceutical requirements of compatibility, absence of toxicity, etc.

B.

PRESERVATIVES IN COMMON U S E

1 . Benzoic Acid Benzoic acid is a useful preservative for mixtures having a p H of 5 or less; about 0.1%w.lv. of the acid is sufficient to inhibit microbial growth under these conditions. In the United Kingdom the acid is normally used in the form of benzoic acid solution B.P.C. (which contains 5.5% w.lv. benzoic acid and 75% propylene glycol in water) because the acid itself is difficult to dissolve in water. This solution is used in a number of pediatric mixtures included in the B.P.C.

(1963). 2. Esters of p-Hydroxybenzoic Acid (Parabens) These esters are tasteless, stable, and nontoxic, but are incompatible with certain substances used in medicines, e.g., nonionic surfaceactive agents. The parabens have been widely studied over many years. Neidig and Burrell (1944) comprehensively reviewed the extensive literature appertaining to their use as preservatives up to the early 1940’s. This work covered bacterial studies, toxicity studies, and the preservation of cosmetics, food, and pharmaceutical products (170 references to the latter). Subsequent work by Aalto e t al. (1953) and by Husa and co-workers (Littlejohn and Husa, 1955; Schimmel and Husa, 1956) and others, have confirmed the original findings that these esters are useful preservatives for many pharmaceutical products, especially syrups and medicines containing syrups or gums. Prickett e t al. (1961) demonstrated the potentiation of the preservative action of the parabens on the addition of small amounts (2%or 5%)of propylene glycol. None of the elixirs, linctuses, or mixtures mentioned in the current B.P.C. contain these esters as preservatives though some manufacturers use them in their proprietary products, e.g., Lederle Laboratories use several combinations in their antibiotic mixtures.

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3. Chloroform This has been widely used for many years as a preservative for oral medicines because of its sweet and pleasant taste. In addition, its carminative action is useful in certain preparations. The chief disadvantage of chloroform is its volatility which leads to its loss from a medicine unless the container is well closed. Sykes (1963b) stated that 0.15 to 0.25% chloroform is sufficient to inhibit or even kill bacteria and molds, but below 0.1%it is ineffective. Chloroform per se is seldom used in preparing medicines because of the small amount required and the difficulty of dissolving it in water. The B.P. contains several preparations which overcome these difficulties. Chloroform water contains 0.25%v./v. chloroform and is used as the vehicle for a number of mixtures, e.g., chalk and opium mixture B.P.C. Strong solutions of certain salts sometimes throw the chloroform out of solution, but, apart from this, chloroform water is compatible with most ingredients of medicines. Sometimes for purely pharmaceutical reasons, more concentrated chloroform preparations are preferred, e.g., chloroform spirit B.P. (15% chloroform in 90% v./v. ethanol) or chloroform emulsion B.P. (5%chloroform emulsified in water with quillaia tincture).

4 . Sorbic Acid (2,4-hexadienoic acid) This has been reported by Puls et al. (1955) to be an effective fungistat for use in mucilages of vegetable gums and in diluted syrups. About 0.2% w./v. will prevent the growth of most molds. It does not appear to be used much in the preservation of oral pharmaceutical products, though it is used in various foodstuffs.

5. Sulfur Dioxide and Sulfites Sulfur dioxide, either as such, or present in the form of sulfites, can

be used to preserve fruit syrups and other preparations. Recently, the 1966 Supplement to the B.P.C. 1963 increased the recommended sulfur dioxide content of raspberry syrup from 350 to 420 1J.p.m. because the former has been found to be ineffective in preventing mold growth. VI. Preparations for External Use

A. INTRODUCTION Included in this section are lotions, liniments, creams, ointments, and pastes for application to the skin, and various “drops,” douches, and other preparations intended for insertion into body cavities, e.g.,

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ear, nose. Ophthalmic preparations have been dealt with separately (Section IV). The various preparations may be liquid or semisolid, and may be made with either aqueous or oily vehicles. Those made with oily bases, e.g., ointments, pastes, and some liniments, seldom permit microbial contaminants to flourish and hence do not usually require the inclusion of a preservative. Many of the products are designed to treat local infections and consequently contain antimicrobial agents as their chief active ingredients. However, it is occasionally necessary to add another agent to extend the antimicrobial spectrum of the product to inhibit possible contaminants. Sometimes, an antimicrobial substance is included in a preparation primarily to exert some other effect. An example of this is phenol, which is included in some lotions to exert a mild local anesthetic effect and alleviate itching; the concentration required for this purpose (about 1%)is generally sufficient to prevent microbial growth in the lotion. SimiIarly, alcohol is often included to provide a cooling effect on the skin and again the concentration used is normally sufficient to inhibit the growth of microorganisms. €3. TYPESOF PRODUCT

In addition to the usual pharmaceutical requirements of compatibility, etc., the preservatives used in preparations for external use should be nonirritant and nonsensitizing. Hjorth and Trolle Larsen (1962) have reported on the latter aspect with regard to the parabens and sorbic acid. The antimicrobial agents commonly used in preparations for external use include most of those used in parenteral, ophthalmic, or oral products, viz., parabens, phenols, chlorinated phenols, organic mercurials, quaternary ammonium compounds, etc. The aqueous preparations can be divided into four types: solutions, suspensions, emulsions, and jellies. It will be convenient to discuss the preservation of each type of product separately.

I. Solutions T h e preservation of these is normally a relatively simple matter. A wide variety of suitable agents is available and the choice is usually governed by questions of compatibility and cost. The most commonly used agents include chlorocresol, alcohol, phenylmercuric salts, and quaternary ammonium compounds.

2. Suspensions The preservation of suspensions is complicated by two possible factors, the adsorption of the antimicrobial agent onto the suspended

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material and/or inactivation of the agent by the suspending agent. Harris (1961) showed that cationic antiseptics including cetrimide and chlorhexidine, were inactivated by suspensions containing 5% bentonite.

3. Emulsions The literature pertaining to the preservation of emulsions has been thoroughly reviewed and discussed by Wedderburn (1964). Persons interested in this topic are strongly recommended to study this article which covers in detail the many factors involved, and lists the preservatives most commonly used in cosmetic and pharmaceutical emulsions.

4 . Jellies Jellies are used in medicine for a variety of purposes. Some are formulated for application to the skin or to a body cavity, e.g., nose or vagina, while others are formulated for lubricating surgical instruments prior to insertion into a body cavity. Basically, a jelly consists of approximately 1 to 5% of a gel-forming substance, e.g., tragacanth, methyl cellulose, gelatin, sodium alginate, up to about 10% of a dispersant (e.g., glycero1 or propylene glycol), the active drug, and sufficient water to make 100%. Most gel-forming substances are prone to microbial attack especially when a lot of water is available, so a preservative is almost invariably essential. Jellies used as lubricants for surgical instruments must, of course, be sterile, but sterility is usually less important in the jellies for application to the skin. Interactions between various preservatives and gel-forming substances have been reported. Tillman and Kuramoto (1957), using a spectrophotometric analysis technique, found evidence for complex formation between methyl cellulose 400 and methyl-, propyl-, and butylparabens. Miyawaki et al. (1959), also using physical methods, found that methyl- and propylparabens interacted to some extent with methyl cellulose, polyvinylpyrrolidone, and gelatin. They found no evidence for a significant degree of interaction between methylparaben and carboxymethyl cellulose or tragacanth. They concluded that in the concentrations of polymers generally used, binding is insufficient to prohibit the effective application of the parabens as preservatives. Eisman et al. (1957) found that in jellies containing 3% tragacanth and buffered at pH 7 the bactericidal activity of a number of preservatives, e.g., chlorbntanol, benzalkonium chloride, methyl- and

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propylparabens, was markedly reduced. The properties of phenol, Merthiolate, and PMA were less affected. The test organism employed was Staphylococcus uureus. Taub et ul. (1958) studied jellies containing 2% tragacanth and 5% propylene glycol. Jellies were prepared having specific p H values in the range pH 3 to 7 , and one of the following preservatives added: 0.2% benzoic acid, 0.5% chlorbutanol, and a combination of 0.2% methyl- and 0.05% propylparabens. The jellies were sterilized and then inoculated with the following microorganisms: S . aweus, B . subtilis, Escherichia coli, and Candida albicans. Samples of the jellies were removed at various time intervals and inoculated into culture media, care being taken to ensure that bacteriostatic concentrations of the preservatives were not carried over into the plating medium. The results obtained showed that the combination of parabens sterilized the jellies over the pH range 3 to 7 except when the contaminant was B . subtilis, though even with this organism the bacterial population was much reduced. Almost identical results were obtained with jellies which had been stored for 28 days prior to inoculation and testing, showing that no inactivation of the preservative occurred during storage. As might be expected, the results obtained with benzoic acid as preservative varied with the pH of the jelly, being satisfactory below pH 5. Chlorbutanol exerted some preservative action but was inferior to the parabens. Fiedler and Lee (1955)found a heavy black mold growth on samples of ephedrine sulfate jelly N.F. (containing 1%tragacanth). The jelly also suffered from a number of other imperfections, and thus a better formulation was sought. It was shown that when the tragacanth was replaced by 4% sodium alginate, a clear jelly was formed which could be preserved using 0.2% sodium benzoate. A mixture of methyl- and propylparabens would also preserve this new formulation but the resulting jelly was cloudy. More recently, Swafford (1960) suggested the use of 2% Carbopol934 (B. F. Goodrich Chemical Co., Cleveland, Ohio, U.S.A.) as the gel-forming agent in ephedrine sulfate jelly. Carbopol 934 is a synthetic hydrophilic gum and appears to have a number of advantages over the natural products, not least of these being its ability to resist bacterial and fungal attack. REFERENCES Aalto, T. R., Firman, M . C., and Rigler, N. E. (1953).J . Am. Pharm. Assoc. Sci. Ed. 42, 449-457. Abrams, J. D. (1963). Trans. Ophthalmol. Soc. U . K . 83, 263.

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Abrams, J. D., Davies, T. G., and Klein, M. (1965). Brit. J. Ophthalmol. 49, 146-147. Alin, K., and Diding, N. (1949). F a m . Reuy 48, 545. Amies, C. R. (1962). J . H y g . 60,473-481. Anderson, K. F., Lillie, S., and Crompton, D. 0.(1964a).Pharm.J.192,593-594. Anderson, K. F., Lillie, S., and Crompton, D. 0.(1964b). Pharm.J.193,165. Barr, M., and Tice, L. F. (1957a).J. Am. Pharm. Assoc. Sci. E d . 46,217-218. Barr, M., and Tice, L. F. (1957b).J. Am. Pharm. Assoc. Sci. E d . 46,219-221. J . Am. Pharn~Assoc. Sci. E d . 46, 442-445. Barr, M., and Tice, L. F. (1957~). Barr, M., and Tice, L. F. (1957d).J. Am. Pharm. Assoc. Sci. E d . 46, 445-451. Beloian, A., and Koski, T. (1964).J. Assoc. Ofic. Agr. Chemists 47,804-807. Birner, J., and Garnet, J. R. (1964a).J. Pharm. Sci. 53,1264-1265. Birner, J., and Garnet, J. R. (1964b).]. Pharm. Sci.53,1266-1267. British Pharmaceutical Codex (1954). British Pharmaceutical Codex (1959). British Pharmaceutical Codex (1963) British Pharmaceutical Codex Addendum (1966). British Pharmacopoeia (1958). British Pharmacopoeia (1963). British Pharmacopoeia Commission (1966). Personal communication. Brown, M. R. W., and Norton, D. A. (1965). J. SOC. Cosmetic Chemists 16, 369-387. Brown, M. R. W., and Richards, R. M. E. (1965). Nature 207, 1391-1393. Brown, M. R. W., Foster, J. H. S., Norton, D. A., and Richards, R. M. E. (1964). Pharm. J. 192, 8. Brown, W. R. L. (1961). Pharm. J. 187,221-222. Buschke, W. (1949). J . Cellular Comp. Physiol. 33, 145-149. Cohen, S. M., and Wheeler, M. W. (1946). Am . ]. Public Health 36,371-376. Cooper, J. W., and Gunn, C. (1965). “Dispensing for Pharmaceutical Students,” 11th Ed. Pitman, New York. Davies, G. E., and Davison, J. E. (1947). Quart J . Pharm. Pharmacol. 30, 212-218. Davies, G. E., and Fishburn, A. G. (1946). Quart. J . Pharm. Pharmacol. 29, 365-372. Davison, J. E. (1951). J. Pharm. Phamacol. 3,734-740. Davisson, E. O., Powell, H. M., MacFarlane, J. O., Hodgson, R., Stone, R. L., and Culbertson, C. G. (1956). j . Lab. Clin. Med. 47, 8-19. Dick, G. W. A. (1959). Practitioner 183,305-312. Diena, B. B., Wallace, R., and Greenberg, L. (1964). Can. J . Microbiol. 10, 555-560. Editorial (1963). Pharm. J. 192, 587-588. Editorial (1966). Pharm. J . 196, 159-160. Edsall, G., McComb, J. A,, Wetterlow, L. H., and Ipsan, J. (1962). New Engl. J. Med. 267,687-689. Eisman, P. C., Cooper, J., and Jaconia, D. (1957). J . Am. Pham. Assoc. Sci. E d . 46, 144-147. Extra Pharmacopoeia (1958). Pharmaceutical Press, London. Felix, A. (1941). Brit. Med. J. i, 391. Felix, R. I. (1964).J. SOC. Cosmetic Chemists 16, 1-12. Fiedler, W., and Lee, C. 0. (1965). J . Am. Pham. Assoc. Pract. Pharm. E d . 16, 101. J. 192,429. Foster, J. H. S. (1964~).Pha~m. Foster, J. H. S. (1964b). Pharm. J. 192,461. Foster, J. H. S. (1965). Mfg. Chemist 36, Pt. 5 , 45-50; Pt. 6, 43-46. Gabel, L. F. (1921). I . Am. Pharm. Assoc. 10,767-768. Gardner, R. A., and Pittnr,iii. \ I . (1965). Appl. Microbiol. 13, 564-569.

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Gilbert, R. J., and Russell, A. D. (1963). Pharm. J. 190, 111-112. Ginsberg, M., and Robson, J. M. (1949). Brit. J . Ophthalmol. 33,574-579. Guillot, M. (1950).J . Pharm. Pharmacol. 2, 345-360. Hadgraft, J. W., and Short, P. (1947). Pharm. J. 158,202. Harris, W. (1961). Australasian J. Pharm. 42,583-584, 587-588. Hess, H. (1965). Pharm. Weekblad 100, 764-774. Hjorth, N., and Trolle Larsen, C. (1962). Am. Pe$umer 77, 43-45. Hopkins, S. J. (1960). M . B . Pharm. Bull. Hopkins, S . J. (1966). Pharm. J. 196, 141-142. Hugo, W. B., and Foster, J . H. S. (1964a).J. Pharm. Pharmacol. 16,209. Hugo, W. B., and Foster, J. H. S. (1964b). J . Pharm. Pharmacol. 16, 124T-126T. Kedzia, W., Lewon, J., and Wisniewski, T. (1961). J. Pharm. PharmacoZ. 13,614-616. Kenney, D. S., Grundy, W. E., and Otto, R. H. (1964). Bull. Parenteral Drug. Assoc. 18,lO-19. Klarmann, E. G. (1959).Am.J . Pharm. 131,86-91. Klein, M., Millwood, E. G., and Walther, W. W. (1954).J. Pharm. Pharmacol. 6,725-732. Kohan, S . , Carlin, H., and Whitehead, R. (1962).Am . ]. Hosp. Pharm. 19,83. Kohli, J. D., Chopra, I. C., and Chander, K. (1950). ZndianJ. Med. Res. 33, 413-416. Kohn, S . R., Gershenfeld, L., and Barr, M. (1963a).J. Pharm. Sci. 52,967-974. Kohn, S . R., Gershenfeld, L., and Barr, M . (1963b).J. Pharm. Sci. 52,1126-1129. Landi, S. (1963). Appl. Microbiol. 11, 408-412. Lawrence, C. A. (1955a).J. Am. Pharm. Assoc. Sci. Ed. 44,457-464. Lawrence, C . A. (1955b). Am. J . Ophthalmol. 39,385-394. Littlejohn, 0. M., and Husa, W. J. (1955). J . Am. Pharm. Assoc. Sci. Ed. 44, 305-308. Loosemore, M., and Russell, A. D. (1963).J . Pharm. Pharmacol. 15, 558. Luderitz, O., Staub, A. M., andwestphal, 0. (1966). Bacteriol. Rev. 30,192-255. McEwan, J. S., and MacMorran, G. H. (1947). Pharm.]. 158,260-262. MacGregor, D. R., and Elliker, P. R. (1958). Can.J.Microbiol. 4,499-503. MvIver, A. K. (1964). Pharm. J. 192,429. McLean, I. W. (1957). US.Patent No. 2,763,160. Magnusson, M., Guld, J., Magnus, K., and Waaler, H. (1958). Bull. WorldHealth Organ. 19, 799, 828. Melnick, J. L., and Wallis, C. (1964). Clin. Med. 71, 2053-2067. Miyawaki, G. M., Patel, N. K., and Kostenbauder, H. B. (1959). J. Am. Pharm. Assoc. Sci. Ed. 48, 315-318. Montgomery, W. F., and Halsall, K. G. (1964a). Pharm. J . 192,407. Montgomery, W. F., and Halsall, K. G. (196413). P h a m . J. 192,461. Morgan, H. R. (1958). In “Bacterial and Mycotic Infections of Man” (R. J. Dubos, ed.), 3rd Ed. Pitman, New York Mueller, W. H., and Deardo& D. L. (1956).J. Am. Pharm. Assoc. Sci. Ed. 45,334-341. Nair, A. D., and Lach, J. L. (1959).J. Am. P h a m . Assoc. Sci. Ed. 48,390-395. Neidig, C. P., and Burrell, H. (1944). Drug. Cosmetic Znd. 54,408-410,481-489. Olson, B. H., Eldering, G., and Graham, B. ( 1 9 6 4 ) ~Bacteriol. . 87,543-546. Parish, H. J. (1965). “A History of Immunisation.” Livingstone, Edinburgh and London. Parish, H. J . , and Cannon, D. A. (1963). Practitioner 190, 75-80. Parish, H. J., and Cannon, D. A. (1964). “Antisera, Toxoids, Vaccines and Tuberculins,” 6th Ed. Livingstone, Edinburgh and London. Pittman, M. (1962).J . Am. Med. Assoc. 181,25-30 Pittman, M., and Cox, C . B. (1965). AppE. Microbiol. 13, 447-456. Pivnick, H., Tracy, J. M., and Glass, D. G. (1963). J . Pharm. Sci. 52, 883-888.

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Pivnick, H., Siebenmann, C. D., Landi, S., and Ashford, W. R. (1965).J . Pharm. Sci. 54, 640-642. Prickett, P. S., Murray, H. L., and Mercer, N. H. (1961).J . Pharm. Sci. 50, 316-320. Puls, D. D., Lindgren, L. B., and Cosgrove, F. P. (1955).J.Am. Pharm. Assoc. Sci. Ed. 44,85-87. Ravnick, A,, and Yatoko, J . (1962).Am. J . Hosp. Pharm. 19,469-471. Rdzok, E. J., Gmndy, W. E., Kirchmeyer, F. J . , and Sylvester, J. C. (1955).J . Am. Pharm. Assoc. 44,613-616. Richards, R. M. E., and Brown, M. R. W. (1964).J . Pharm. Pharmacol. 16, 360-361. Riegelman, S., Vaughan, D. G., and Okumoto, M. (1956).J . Am. Pharm. Assoc. Sci. Ed. 45, 93-98. Royce, A,, and Sykes, G. (1957).J . Pharm. Phavmacol. 9, 814-822. Russell, A. D. (1964). Lab. Pruct. 13, 114-122. Russell, A. D. (1965).Mfg. Chemist 36, 38-45. Russell, A. D., and Loosemore, M. (1964).App2. Microbiol. 12,403-407. Saunders, L., and Shotton, E. (1956).J. Pharm. Pharmacol. 8,832-847. Schimmel, J., and Husa, W. J . (1956).J.Am. Pharm. Assoc. Sci. Ed. 45,204-208. Schuchardt, L. F., Munoz, J., and Verwey, W. F. (1960). Am. J . Public Health, 50, 321-328. Sen Gupta, P. N. (1951). IndianJ. Med. Res. 40, 115-119. Sutherland, I. W. (1963). Immunology 6, 246-254. Swafford, W. B. (1960).Am. J . Pharm. 132,383-384. Sykes, G. (1958).J . Pharm. Pharmacol. 10,40T-46T. Sykes, G. (1963a).Practitioner 190,52-57. Sykes, G . (196313).“Dissinfection and Sterilisation,” p. 366. Spon, London. Taub, A., Meer, W. A., and Clausen, L. W. (1958).J. Am. Pharm. Assoc. Sci. Ed. 47, 235-239. Taylor, E. W., and Burman, N. P. (1956).J . Pharm. Pharmacol 8,817-831. Tillman, W. J., and Kuramoto, R. (1957).J . Am. Phurm. Assoc. Sci. Ed. 46, 211-214. Tracy, J . M., Glass, D. G., Nicholson, M. J., and Pivnick, H. (1964).J . Pharm. Sci. 53, 659-663. U S . Natl. Formulary (1960). U.S. Pharmacopoeia (1960)XVIth Revision. U.S. Pharmacopoeia (1965).XVIth Revision. van Hemert, P., van Wezel, A. L., and Cohen, H. H. (1964). Nature 203, 774-775. Wedderburn, D. (1964).Aduan.Pharm. Sci. 1,195-268. Wing, W. T. (1958).Proc. Inst. Rubber Ind. (Trans. Inst. Rubber Ind.) 5, 67-72. World Health Organization (1959a).Tech. Rept. Ser. 178. World Health Organization (195913).Tech. Rept. Ser. 179. World Health Organization (1959~).Tech. Rept. Ser. 180. World Health Organization (1960). Tech. Rept. Ser. 200.

Antiserum Production in Experimental Animals

RICHARD M. HYDE Department of Microbiology, School of Medicine University of Oklahoma, Oklahoma City, Oklahoma

I. Introduction ............................................................. 11. Methods Employed in Antiserum Collection ................ 111. Animal Species Employed in Antiserum Production ....... IV. Routes of Inoculation Employed in Antiserum Production ................

39 40 41

43 47 47 50 C. Presence of Specific Antibody ............................... 52 D. Age of Animal ..................................................... 54 ................................... ......... 56 F. Other ................................... ................. 57 VI. Specific Examples of Antisemm Production ................. 58 VII. Conclusion ............................................................... 58 VIII. Selected Bibliography.. ....................... 59 References ................. ............................. 63 V.

I. Introduction The production of specific antibodies in experimental animals has become a problem of practical importance to investigators in many areas of biology. Thus, the endocrinologist may wish to obtain an antibody to a specific hormone in order to study the role of the hormone in the physiology of the animal, or perhaps to ascertain the cellular site of production of the hormone. The need for such an antibody requires that the investigator understand the basic concepts of immunology. It is the purpose of this review to discuss certain aspects of immunization practices which will guide scientists wishing to produce specific antiserum to substances of biological interest. Numerous reviews have been written concerning immunization methods and vaccines available for the prevention of human disease (Menzin, 1961; Edsall, 1963, 1965; Hilleman, 1964; Riley, 1966), hence this .subject will not be dealt with here. No attempt will be made to review the methods employed for the induction of delayed hypersensitivity in experimental animals. Individuals interested in this particular facet of the immune response are referred to the excellent articles by Arnason and Waksman (1964) and Chase (1965). Certain aspects covered in this review have been considered recently by White (1963) and Edsall (1966). The reader is referred to these sources for supplementation of information presented herein. 39

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II. Methods Employed in Antiserum Collection Detailed procedures for bleeding rabbits, chickens, guinea pigs, rats, and mice b y both cardiac and venous puncture are described by Campbell et at. (1964).An even more extensive discussion of the techniques employed for collection of blood from experimental animals is presented by Moreland (1965).The latter included in his discussion methods for the collection of lymph and other body fluids from experimental animals. In addition, the author presents methods employed for the collection of blood, etc., from dogs, cats, monkeys, various birds, and ruminants, as well as the animal species considered by Campbell et al.. In general, it is desirable to fast the animal for approximately 18 hours prior to bleeding, thus reducing the amount of lipid in the serum. Occasionally, excessive hemolysis presents a problem. This may be minimized by collecting the blood specimen in an anticoagulant solution, centrifuging to remove the erythrocytes, and recalcifying the plasma supernatant. Alternately, the blood may be drawn into a chilled vacutainer tube (Becton, Dickinson, and Co.) and kept at a low temperature (to prevent clotting) until the erythrocytes have been spun down. After centrifugation of the chilled blood, the fibrin can be broken away from the glass and, after recentrifugation, the clear serum can be aspirated off. The latter method has the further advantage that the serum can be harvested from the blood less than 1 hour after the specimen has been removed from the animal. The success of either of these methods of avoiding hemolysis during the collection of blood will depend upon knowledge of the appropriate amount of anticoagulant to employ or, alternately, the clotting time of the blood in question. Information of this nature can be readily obtained from the Biology Data Book (Altman and Dittmer, 1964). A discussion of some of the steps involved in processing of blood and preparation of glassware and reagents can be found in Tocantins and Kazal (1964). The volume of blood removed from an animal is determined solely by the blood volume of that animal and consideration for the amount that can safely be withdrawn without jeopardizing the life of the donor. Bleeding has apparently no adverse effect on the antibody content of the serum. Smolens et al. (1957) subjected a group of human volunteers to plasmaphoresis (the removal of plasma while returning the cellular portion of the blood to the donor) for a period of 1 year, during which time 5200 ml. of plasma was removed from each individual. They reported that the bacterial and viral antibody content

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of the serums persisted at peak levels throughout the period of the experiment and for 1 year after the cessation of plasmaphoresis. In fact, several observers have recorded a stimulation of antibody production as a result of excessive or repeated bleedings. This observation is discussed by Wilson and Miles (1964). 111. Animal Species Employed in Antiserum Production Many practical considerations must be evaluated in the selection of the animal to be used as the source of antiserum, e.g., the amount of antiserum desired, the ease of bleeding the animal, the cost of purchase and maintenance of the animal, etc. No attempt will be made here to recommend guidelines in this regard, as each investigation will have its own requirements and limitations. However, a few comments of a general nature may be appropriate. The ability to synthesize antibodies in response to antigen exposure appears to be restricted to vertebrate animals. Attempts to induce antibody formation in various invertebrates such as sea anemones (Phillips and Yardley, 1960), earthworms (Triplett et al., 1958), caterpillars (Bernheimer et al., 1952), and insects (Stephens, 1959) have met with failure. The body fluids of the invertebrate animals under study failed to react in serological tests with the antigens employed in immunization. In lower vertebrates the immune response appears to increase in intensity as one ascends the phylogenetic scale. Papermaster et al. (1964) reported that the hagfish appeared to be completely devoid of any inducible immune responses. The lamprey possessed a low level of immunologic competence, while the capacity to form circulating antibody and demonstrate immunologic memory was found to be reasonably well developed in the elasmobranchs and lower bony fishes. The phylogeny as well as the ontogeny of the immune responce has been reviewed recently by Good and Papermaster (1964). Antigens must be foreign to the circulation of the experimental animal. In general, the greater the phylogenetic disparity of the antigen and the experimental animal, the greater will be the immune response the antigen induces. Thus, human serum albumin is a stronger antigen for the chicken than is duck serum albumin (Ivanyi and Valentova, 1966). Tempelis (1965) studied an even closer phylogenetic relationship when h e examined the antibody response of chickens to gamma-globulin fractions of turkey and goose serum. Ninety-seven percent of the birds produced antibody to the later antigen, whereas only 82% had a demonstrable immune response when injected with

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the turkey gamma-globulin. Furthermore, the goose gamma-globulin induced the production of approximately twice the amount of antibody in the chickens as did a comparable quantity of the phylogenetically more closely related turkey gamma-globulin. The ability of an animal to produce a specific antibody may be influenced by subtle genetic factors. It has been known for many years that mice (Avery and Goebel, 1933) and humans (Francis and Tillett, 1930; Felton et al., 1935) respond to the injection of pneumococcal polysaccharide with the production of circulating antibodies, while rabbits (Avery and Goebel, 1933; Downie, 1937a) are apparently unable to do so. Fink and Quinn (1953) presented evidence suggesting that there is a genetic variation in the ability of inbred mice to produce demonstrable antibody to either egg albumin or pneumococcal polysaccharide. Ipsen (1954) studied the ease of immunization of 10 inbred mouse strains with tetanus toxoid. Three of the strains examined were relatively easy to immunize against the lethal effects of the toxin, whereas the remaining seven strains required from 10 to 30 times more toxoid to achieve the same degree of protection. In a later study, the same investigator found that the strength of the anamnestic response to a second injection of antigen correlated with the ease of primary immunization. Strains of mice that were more efficiently immunized b y a primary injection of the toxoid also demonstrated a greater anamnestic response upon booster injection (Ipsen, 1959). When random-bred animals are employed in antiserum production it is common to obtain a wide range of antibody titers. Ipsen (1961), studying the primary immune response of rabbits to tetanus toxoid, observed that antitoxin was demonstrable in 73 of 79 immunized animals, with a geometric mean titer of 0.06 unitslml. However, the titers ranged from less than 0.005 antitoxin unitslml to 1.6 unitslml. Burnet (1964) states that even when immunizing genetically uniform inbred mice it is common to obtain a wide range of antibody titers. Hyde et al. (1965) employed immunoelectrophoresis to evaluate the antibody response of rabbits to human serum antigens. Of 14 animals injected with human serum, four failed to produce detectable antialbumin antibodies, whereas only one animal did not respond to transferrin, thus demonstrating that the percentage of animals producing antibody to a particular antigen will vary with the antigen employed. Better antigens apparently induce a higher percentage of reactive sera. Experiments conducted recently in several laboratories have shown that some strains of animals are genetically unable to respond to certain antigens. Pinchuck and Maurer (1965a) examined the anti-

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43

genicity of synthetic polypeptides in inbred strains of mice and found that three inbred strains produced antibodies to the antigen under study, while four strains were unable to do so. All seven strains tested were able to respond to two other synthetic polypeptides differing from the first terpolymer only in the percentage of the three amino acids present in each. They also tested the antigenicity of the first polypeptide in random-bred Swiss mice and observed that 47%of the animals were able to respond to the antigen. Through mating studies in this random-bred line they were able to obtain evidence which suggested that the antibody response to the polymer was controlled by a codominant Mendelian factor. Similar evidence for a genetic control of antibody response was obtained by McDevitt and Sela (1965) who examined the sera of two strains of inbred mice immunized with branched, multichain synthetic polypeptides. Arquilla and Finn (1965) have observed a similar genetic control of antibody production in inbred guinea pigs. They found that although both strain 2 and strain 13 animals produced antibodies to alum-precipitated insulin, they did not produce antibodies of identical specificity, i.e., strain 2 guinea pigs produced antibodies to one determinant group on the insulin molecule while strain 13 animals produced antibodies to a second (different) determinant group. Thus, both strains were able to recognize insulin as an antigen, but they did not recognize the same region of the molecule. Pinchuck and Maurer (1965a) discussed a similar observation in strain 2 and strain 13 guinea pigs. They found that strain 13 animals were completely unable to produce antibodies to a synthetic polypeptide copolymer, while all of the strain 2 animals immunized with the same material responded with antibody production. Further, they state that only 35% of the Hartley strain guinea pigs (a random-bred animal) recognized the polypeptide as antigenic. IV. Routes of Inoculation Employed in Antiserum Production The most common routes of parenteral inoculation of antigenic materials are intravenous (IV), intradermal (ID), subcutaneous (SC), intramuscular (IM), and intraperitoneal (IP). Descriptions of the techniques involved in these methods can be found in Campbell et al. (1964) and in Moreland (1965). In general, antigens in suspension are inoculated via the IV or IP routes, while antigens in solution are given by one of the other routes. This is an oversimplification and, as will be developed later in this section, the route selected will often affect the level of antibody attained in the animal. Better immuniza-

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tion usually results if the antigen is given in a manner that stimulates the entire antibody-synthesizing apparatus of the host and not just the lymphatics draining the site of injection. For example, when employing the SC route of immunization, it is desirable to place aliquots of the inoculum at several different sites on the body, and thus involve a larger portion of the lymphatic system in the ensuing antibody response. Special methods of immunization may be required to solve particular problems. For example, if the investigator is interested in studying the antibodies that might be concerned with immunity to a particular infectious agent, it may be desirable to expose the animal to the antigen by use of the natural route of infection for the agent under study. Similarly, if the study is designed to establish a method for immunization of large population groups, the investigator may wish to follow a procedure such as that used for jet injections. An excellent review on the latter method of immunization has been written recently by Hingson et al. (1963).A brief discussion of some of the routes of inoculation for specialized studies follows:

1 . Oral

The Sabin polio vaccine is an excellent example of the efficiency of this route of immunization. However, the oral administration of antigen is not always effective. Mascoli et al. (1966)attempted immunization of humans by oral feeding of live rhinoviruses in enteric-coated capsules. None of the individuals involved in the study developed neutralizing antibodies against the viruses ingested. Attempts to induce protective immunity to helminths by parenteral injection of worm extracts or dead worms have not been successful, with the exception of certain cestodes (Soulsby, 1962). Numerous workers in the past 20 to 40 years have shown that, in most cases, infection via the natural route does provide protection against a subsequent challenge with an animal parasite. In many instances, however, the immunizing infection debilitates the host or causes significant weight loss. In the case of Dictyocaulus vipiparous (lung worm of cattle) and Ancylostoma caninum (hookworm of dogs) the debilitating effects can be avoided by immunizing with irradiated, but still viable worms (Jarrett and Sharp, 1963; T. A. Miller, 1966). A rise in antibody titer after natural infection can be detected by most conventional serologic techniques. However, with the exception of the cestodes, attempts to demonstrate protective antibody via passive transfer experiments have shown either no protection or only

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slight protection. Such findings suggest that cellular elements must play an important role in this immunity.

2. Ocular The results of numerous studies indicate that the introduction of soluble antigens into the ocular tissues of various laboratory animals induces the production of circulating antibodies (Thompson et al., 1957; Breebaart and James-Witte, 1959; Fernando, 1960). Parks et al. (1961) inoculated rabbits with identical doses of bovine gammaglobulin via either the intracorneal, intravitreous, intravenous, or intramuscular route and measured the effect that the route of inoculation had upon the development of antibodies. They reported that antibody was produced more consistently and in higher titer following primary inoculation of antigen into ocular tissues than via the conventional parenteral routes. The highest titers were obtained after intracorneal injection, followed by intramuscular injection of the antigen incorporated in incomplete Freund’s adjuvant. Intravitreous injection resulted in the production of demonstrable antibody in every animal tested. Only two of six animals receiving intramuscular injections of the antigen in the absence of adjuvant produced detectable antibody, while none of the six animals receiving antigen via the intravenous route produced enough antibody to be detected by the passive cutaneous anaphylaxis assay. Experimental herpetic keratoconjunctivitis has been induced in rabbits to study the production of precipitating antibodies against the virus. Mantyjarvi (1965) reported the production of seven to eight precipitin lines in his diffusion-in-gel detection system when he analyzed the serum from the infected animals. Pinkerton and Webber (1964) devised a method of injecting small laboratory animals by the ophthalmic plexus route. When done with care, this method facilitates the introduction of materials into the circulation of the animal, and offers at least an alternate route when the tails of the animal being immunized have become so scarred as to make further injections impossible. Further, they report that leakage of the inoculum is not a problem, nor does the procedure impair the eyesight of the animal.

3. Respirutory The efficacy of active immunization against airborne infections by inhalation of attenuated microorganisms has been demonstrated in experimental animals and man. Middlebrook (1961) has reviewed

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some of the immunologic aspects of the host’s reactions to inhaled antigens. Vaccination by the aerogenic route has also been discussed by Jemski and Phillips (1965) and was considered in the Second International Conference on Aerobiology (Lepper and Wolfe, 1966), hence this subject will not be dealt with in any detail here. Yamashiroya et al. (1966) compared the aerosol method of immunization with the more conventional subcutaneous route. They immunized guinea pigs with fluid tetanus toxoid b y one or the other of these two routes and concluded that, although the serum antibody titers were lower after aerosol vaccination, definite immunity was conferred after inhalation of the toxoid. Interestingly, the anamnestic response in the animals surviving toxin challenge was greater in the aerosol-immunized group than in the animals that received a subcutaneous injection of the toxoid.

4 . Other Essentially all areas of the body can be, and have been, employed as routes of immunization. Perhaps one of the most inclusive studies was that of Draper and Sussdorf (1957) who studied the nature of the hemolysin response following the injection of heated sheep erythrocyte stromata by various routes. Compared to the mean log peak titers in intravenously injected rabbits, the titers were: (a)higher following injection into the liver or femoral bone marrow; ( b )lower after injection into the appendix, kidney fat, or subcutaneous tissues of the lumbar region; and ( c ) not different after injection into the peritoneal cavity, thigh muscle, mesenteric lymph nodes, spleen, or hind footpads. The intravaginal route of immunization has been employed by Menzoian and Ketchel (1966) to test the antigenicity of homologous and heterologous seminal fluid. Antigen was placed deep into the vagina of the test rabbit by means of a glass tube with a polished end, care being taken to avoid trauma to the vaginal lining. Tuba1 ligations were performed on some of the rabbits to prevent absorption of antigen through the peritoneum. The rabbits produced antibody to heterologous bovine seminal plasma but not to homologous seminal plasma. Further, these workers found that it took approximately 10 times more antigen to induce detectable antibody by the intravaginal route than by the subcutaneous or intravenous routes.

5. Effects of Route of Immunization on Antibody Production The effect of the route of immunization on both the quantity and the quality of the antibody response has been well established. Webster

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(1965) studied the influence of route and schedule of vaccination on the time course of the immune response in a group of rabbits injected with influenza virus antigens. H e found, as expected, that these two variables markedly influenced the maximal level of serum antibody attained. The routes tested, ranked in order of decreasing efficiency, were; intravenous, intraperitoneal, and subcutaneous. Secondary stimulation with antigen (first booster injection) caused an 8- to 10fold rise in maximal antibody titers by all routes of inoculation. Tertiary stimulation (second booster injection) of all treatment groups served only to return the maximal titers of antibody to those obtained in the secondary response, but did not stimulate higher levels of antibody. The use of an improper route of immunization may even result in the complete absence of antibody formation. Sulzberger (1929) found that he was able to induce skin hypersensitivity to arsphenamine in guinea pigs if he injected the animals intradermally. However, if he administered a second aliquot of the antigen intracardially within 24 hours of the primary injection, the animals failed to develop any allergic manifestations upon subsequent challenge with arsphenamine. The significance of this antigen-induced refractory state remained obscure until Chase and others (reviewed by Chase, 1959) reinvestigated the phenomenon. This type of antigenic inertness is now considered to be an example of route-dependent immunologic tolerance.

V. Factors Affecting Antibody Production A. QUALITYOF THE ANTIGEN Antigenicity (immunogenicity) can be defined as the ability of a molecule to induce the production of homologous antibodies when introduced into an animal. There are certain qualities, or characteristics, intrinsic to a molecule which will determine its ability to initiate antibody synthesis. These characteristics are: (a) foreignness (F); (b) size (S); and (c) chemistry (C). Thus, the antigenicity (A) of a molecule can be expressed as an algebraic equation, A=F+S+C 1 . Foreignness

In general, the greater the phylogenetic distance between the source of the antigen and the animal that will be used to produce the antiserum, the greater will be the antigenicity of the material. For example, human serum albumin is a better antigen in chickens than is duck serum albumin (Ivanyi and Valentova, 1966).

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2. Size

The significance of particle size in determining the antigenicity of a molecule is indicated by the observation that nonantigenic or weakly antigenic substances can sometimes be made antigenic by absorbing them onto particulate carriers such as colloidion particles or charcoal (Landsteiner and Jacobs, 1932). Further, high molecular weight dextrans are antigenic in man while dextrans below 50,000 molecular weight appear to be nonantigenic (Maurer, 1965). The minimum molecular weight of protein antigens is usually accepted to be approximately 10,000. Generally, proteins of high molecular weight (above 40,000) are considered to be good antigens, while molecules below 10,000 are relatively ineffective in inducing antibody production. However, some low molecular weight polypeptide materials such as the pancreatic hormone glucagon (MW 3485) and synthetic polymers (MW 5000) can function as effective antigens (Unger et al., 1959; Maurer, 1963). The state of aggregation of the antigen also influences its ability to induce antibody formation. Nossal et al. (1963) studied the immune response of rats to various flagellar preparations from Salmonella adelaide and observed that the antigenicity of the materials increased with their complexity. Thus, the intact, isolated flagella induced the best antibody response; the low molecular weight protein, flagellin, prepared by acid treatment of the flagella, was the poorest antigen; and a polymer prepared from flagellin proved to be of intermediate antigenicity. A similar observation was made by Biro and Garcia (1965) who injected rabbits with either aggregated or aggregate-free human gamma-globulin (HGG). They found that the animals injected with aggregated HGG produced large amounts of precipitating antiHGG antibodies, whereas those injected with aggregate-free HGG produced no detectable antibodies and, in fact, became inimunologically unresponsive to HGG. A procedure has been developed recently whereby bovine serum albumin (BSA) can be rendered insoluble without concomitant alterations in its characteristic determinant groups (Hirata and Campbell, 1965). This finding led Hirata and Sussdorf (1966) to a comparison of the immunogenicity of insolubilized BSA and its native counterpart. They found that the use of the insoluble protein as immunizing antigen in rabbits resulted in both a larger number of responding animals and in markedly higher serum antibody levels than those obtained with soluble BSA. All of these observations may be related to the suggestion made by

ANTISERUM PRODUCTION IN EXPERIMENTAL ANIMALS

49

Frei et al. (1965) and Nossal (1965) that phagocytosis of the antigen is a crucial step in the induction of an immune response. In fact, Medawar (1963) has proposed that the outcome of a direct encounter between immunologically competent lymphoid cells and antigen may be the specific loss of antibody-synthesizing ability, whereas antigen presented “uia the proper diplomatic channels” (i.e., after processing by phagocytic cells) results in the production of antibodies. It is known that serum proteins which have been aggregated are very actively cleared from the circulation b y phagocytic cells of the reticuloendothelial system (Thorbecke et al., 1960). If, indeed, phagocytosis is an essential prerequisite of antibody synthesis, then the influence that particle size and physical state have upon antibody formation would be relatively easily explained.

3. Chemistry The chemical nature of a molecule markedly influences its antigenicity. In general, proteins are better antigens than carbohydrates, while nucleic acids and lipids are not antigenic in themselves but can function as haptens when attached to appropriate carrier molecules. For example, it has been found that denatured deoxyribonucleic acid becomes immunogenic after it has formed a complex with methylated bovine serum albumin (Plescia et al., 1964). These same investigators have recently summarized some pertinent data dealing with the antigenicity of nucleic acids (Plescia et al., 1965). Unfortunately, the question that remains is why it is necessary to couple such complex materials to carrier molecules in order to induce the synthesis of antibodies specific for determinants of the nucleic acids. Another interesting, but as yet poorly understood observation on the effect of the carrier molecule on antihapten antibodies was made recently by Siskind et al. (1966).These investigators were studying the antibody response of guinea pigs to dinitrophenol hapten conjugated to various protein carriers and observed that the carrier affected not only the amount of antibody synthesized by the animal but also affected the avidity of the resulting antibody. For example, when bovine gamma-globulin was compared with bovine serum albumin as a hapten carrier, it was found that the animals produced more than twice as much antibody if the dinitrophenol were conjugated with the former carrier. There appears to be a minimum degree of complexity which a molecule must possess in order to be able to function as a complete antigen. This was demonstrated by the work of Pinchuck and Maurer

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(1965b) who observed an apparently consistent nonimmunogenicity in mice of synthetic polymers of two amino acids, and the appearance of immunogenicity upon the introduction of a few mole percent of a third amino acid, without regard to the nature of the third amino acid. There are many other facets of the chemistry of a molecule which influence its antigenicity, e.g., the presence of charged determinant groupings, the shape of the molecule, susceptibility to the hydrolytic enzymes of the host, etc. Recent investigations on the structural requirements for antigenicity have been reviewed by Eisen and Pearce (1962), Maurer (1964, 1965), and by Sela (1966) and will not be discussed further here. In addition to these intrinsic characteristics of the molecule which determine its antigenicity, there are extrinsic factors which influence the quantity of antibody produced in response to a given antigen. In the following sections a brief discussion of some of these quantitative determinants of antigenicity will be presented. B. QUANTITYO F ANTIGEN Over 50 years ago, Smith and St. John-Brooks (1912) suggested that a direct mathematical relationship exists between the amount of antigen injected into an experimental animal and the peak antibody response obtained. Stevens (1956) reviewed several sets of published data on the relationship of dose to response and concluded that the relationship is a logarithmic one with a straight-line function. H e also stated that protein antigens appeared to be approximately 50 times as effective as polysaccharide antigens in inducing a comparable increase in antibody levels. That is to say, to obtain a 10-fold increase in homologous antibody titer, it would require 50 times more polysaccharide antigen than protein antigen. However, this straight-line relationship between dose and response may not always occur. Stille et al. (1959) have reported that the relationship of antibody response to increasing amounts of influenza vaccine will vary with the route of injection employed. They obtained a straight-line relationship by using the intracutaneous route of injection and a sigmoid curve when the subcutaneous route was used. This difference in the antibody response may have been due to differences in the rate of antigen absorption from the site of inoculation, but it in no way alters the fact that the amount of antigen employed plays a major role in determining the extent of the immune response. Normally, two effects follow the first injection of an antigen: (a) the production of specific antibodies; and ( b ) the development of in-

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creased responsiveness to a second injection of the same antigen (the anamnestic response). However, under appropriate conditions, e.g., animal immaturity or excessive amounts of the antigen, the first injection may reduce or eliminate both of these responses with the resultant development of immune tolerance. This state, also referred to as immunological unresponsiveness, is specific for the antigen which induced it and is not comparable to the general immunologic anery which follows administration of immunosuppressive drugs (cf. Section V, F.). The phenomena of immunologic paralysis and antigen overload seen with carbohydrate and protein antigens, respectively, are examples of immune tolerance induced in adult animals. They will not be discussed here as this subject has been amply reviewed elsewhere (Smith, 1961; Hasek et al., 1962; Makinodan et al., 1965; Crowle, 1966). The minimum amount of antigen necessary to induce a demonstrable immune response will vary with the quality of the antigen under study, the method of immunization employed, as well as other factors discussed in this review. Some representative studies of the minimum amounts of antigen needed to induce an immune response are presented in Table I. Another variabIe which will influence the TABLE I MINIMUMAMOUNTSOF ANTIGEN NEEDEDTO INDUCE

Antigen Bovine serum albumin Bovine gammaglobulin Poliovirus type I Poliovirus type I1 Pneumococcal pol ysaccharide type I Flagellin

Micrograms injected

AN

IMMUNE RESPONSE

Route

Animal

Reference

60.0

IV

Rabbit

Farr and Dixon (1960)

6.0

IV

Rabbit

Farr and Dixon (1960)

4.0 0.8 0.05

IM IM IP

Man Man Mouse

Charney et al. (1961) Charney et al. (1961) Neeper and Seastone (1964)

0.001

SC

Rat

Nossal et al. (1964)

amount of antigen necessary to induce an immune response is the sensitivity of the assay method employed to detect the antibody produced. Obviously, if a very sensitive method of antibody detection is used, the quantity of antigen needed to induce a detectable immune response may appear lower, as it will take less antibody to reach the

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threshold of the assay system. This fact makes it difficult to compare results such as those presented in the table, since different methods of assay were employed. In general, repeated injections of small amounts of antigen are employed in the production of antiserums. The range of amounts over which effective antigenicity can be demonstrated is extremely wide, Thus, Downie (1937b) found that he could effectively induce immunity to pneumococcal infection with injections of as little as 0.01 to 1 micrograms of polysaccharide, while he did not reach inhibitory levels until he employed 0.1 to 1 milligram amounts. Repeated injections of antigen, however, may not always be successful in inducing an anamnestic response. Heidelberger et al. (1946) failed to obtain a rise in antibody titer when they administered a booster injection of pneumococcal polysaccharide to human volunteers 2 years after their primary immunization. A similar absence of anamnesis was observed by Maurer (1957) in his studies on the antibody response of human volunteers to polyvinylpyrrolidone. In both of these studies the investigators were unable to elicit a rise in the titer of an antibody which was already present in the serum of the experimental subjects. A different type of refractoriness was observed by Cryan et al. (1966) in their attempts to produce antiserums specific for tumor antigens. They found that the appearance of antibody was transient and was unresponsive to booster inoculation. Antibody was detectable in one or two bleedings, but it subsequently disappeared and did not reappear following booster injection.

c. PRESENCE O F

SPECIFIC ANTIBODY

The three possible effects that specific antibody may have on immunization are: ( a ) suppression of the antibody response; ( b ) enhancement of antibody production; and ( c )no apparent effect. The influence that antibody exerts will depend upon the ratio of antigen to antibody as well as upon the immunologic status of the animal being used for antiserum production. Most of the recent investigations on the effect of antibody on immune responses have dealt with the primary immune response (antibody production following the first injection of antigen), since secondary antigenic stimulation is reasonably refractory to the presence of specific antibody, particularly when the amounts of antigen are increased, as they commonly are in booster injections. In general, large amounts of antibody inhibit a primary immune response while small amounts may enhance it. More specifically, if the ratio of antigen to antibody molecules in the immune precipitate

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53

favors the antigen, antibody synthesis will ensue; whereas, if the ratio favors antibody then de novo production of antibodies may be inhibited (Leskowitz, 1960).The enhancing effect of low levels of specific antibody has been studied by Segre and Kaeberle (1962). They found that 3-week-old piglets deprived of colostrum (and hence possessing very little gamma-globulin) gave a markedly suppressed response to diphtheria and tetanus toxoid as compared to conventionally reared pigs. Further, they obtained successful antitoxin responses in the colostrum-deprived animals by mixing the toxoid with a minute amount of specific antitoxin, suggesting that the active principle in the colostrum was specific antibody which had been passively acquired from the mother by the colostrum-fed animals. This conclusion was substantiated by the observation that the enhancing effect of the antiserum could b e removed by specific absorption with toxoid. A similar enhancement of the antibody response by small amounts of specific antibody was observed in mice by Terres and Wolins (1961). In a subsequent publication, Terres and Stoner (1962) reported the results of their studies on the specificity of the antibody-mediated enhancement and showed that the augmentation of antibody production was demonstrable only to the antigens found in the specific precipitate. The mice demonstrated enhanced antibody production to both the antigen present in the precipitate as well as to the rabbit gamma-globulin that was employed as a heterologous antiserum source. However, the animals responded poorly to extraneous antigens incorporated in the injection materials. The suppressive effect of large amounts of passively acquired antibody on the primary immune response is well known (Osborn et al., 1952; Talmage et al., 1956; Perkins et al., 1958). However, the mechanism of this inhibition remains obscure. It is not known whether the effect is due to diversion of the antigen from its normal pathway to the induction of antibody synthesis or to a more direct effect that preformed antibody may have on the antibody-producing cells themselves. Uhr and Baumann (1961) found that antibody from heterologous species was more efficient in the inhibition of de novo antibody synthesis than was homologous antiserum. Moeller (1964) noted that antibody synthesis was not inhibited b y incubation of normal lymphoid cells prior to their transfer to, and stimulation in, X-irradiated recipients. Inhibition occurred if the mice were passively immunized prior to injection of the antigen or if the antiserum was added to the antigen prior to its injection into the animals. Tao and Uhr (1966) examined the capacity of enzyme-digested antibody preparations to

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inhibit active antibody formation and concluded that inhibition was due to the fragments of the antibody molecule which contain the combining sites for antigen. All of the above observations support the concept that inhibition is brought about through interaction with the antigen, rather than through direct action on the antibody-forming cells themselves. Specific antibody appears to be without effect in an animal that has already received one injection of the antigen. Rowley and Fitch (1964) studied the effect of passive antibody on the hemolysin response of rats immunized with sheep erythrocytes. Although they noted inhibition of the primary response to the antigen, the secondary response (antibody production following booster injection) was unaffected by the presence of passively administered antibody. Wigzell (1966) employed the Jerne plaque technique to study antibody-induced suppression of hemolysin response at the level of the lymphoid cells actively synthesizing the antibody. He concluded that the inhibitory action of antibody was not due to direct suppression of cells already producing antibody, but was an effect mediated by removal from the system of some stimulus (possibly antigen) required for the maintenance of antibody production. The use of antigen-antibody precipitates in immunization procedures may offer certain advantages. For example, it is possible to employ such precipitates to produce antisera to the antigen portion of the complex when this portion is too toxic for injection in the uncombined form. Copeman et al. (1922) used underneutralized mixtures of diphtheria toxin and antitoxin for human immunization before toxoid had been developed. Specific precipitates have also been employed to obtain antiserums of high specificity. Thus, Treffers and Heidelberger (1941) obtained antibody to horse gamma-globulin b y immunizing rabbits with a precipitate of pneumococcal polysaccharide (nonantigenic in rabbits) and its corresponding horse antibody. Similarly, Pace and Pappenheimer (1959) obtained a highly specific antistreptococcal diphosphopyridine nucleotidase rabbit serum by using precipitates of the enzyme and its homologous rabbit antibody as the immunizing agent.

D. ACE

OF

ANIMAL

The immune response of most animals is poorly developed at birth but rapidly matures during infancy and reaches maximum efficiency during adulthood. Usually, a decrease in the efficiency of antibody production is seen during senescence. The absence of antibody forma-

ANTISERUM PRODUCTION IN EXPERIMENTAL ANIMALS

55

tion in the fetus is probably due to the placental barrier which shields the offspring from exogenous antigenic stimuli. The ability of various animal species to produce antibodies in fetal life, or as neonates, has been reviewed recently by J. F. A. P. Miller (1966)and will not be discussed further here. The process of aging is commonly accompanied by a decrease in immune responsiveness. Goullet and Kaufmann (1965) examined the ability of young (3 months of age) and old (22 months of age) rats to produce antibodies to bovine serum albumin. They concluded that antibody production in the “aged” animals was definitely inferior to that obtained in the younger animals. A similar conclusion was reached b y Aoki and Teller (1966) who compared the ability of young and old mice to reject histoincompatible tumor homogenates. They also observed a decreased ability of the older mice to form cytotoxic and opsonic antibodies. However, not all animal species may show this age-associated decrease in immunologic competence. Wolfe et al. (1957) were unable to demonstrate a significant decrease in precipitin production by chickens up to 8 years of age. Comprehensive studies of the age-associated change in the immune system of mice have been conducted to ascertain the physiologic basis of this observation. Makinodan and Peterson (1964) studied the primary antibody-forming potential of spleen cells from mice ranging in age from 1 to 126 weeks. They concluded that the growth and aging of the immune system of this animal are due to an increase and decrease, respectively, in the number of potential antibody-forming cells rather than to a change in the efficiency of the cells. The relative number of potential antibody-forming cells increased approximately 600-fold by the fortieth week of life. After 40 weeks of age the number decreased gradually. At 120 weeks it was only 25% of that of the 40-week-old mice, thus suggesting that the immune potential of an individual does not remain at a plateau level after young adulthood but actually decreases. If this is the case, then the common practice of adjusting the dose of antigen in terms of body weight is of questionable validity. Wigzell and Stjernsward (1966) examined the immune responsiveness of mice ranging from neonates to animals 36 months of age. They observed a rapid exponential increase in immunologic reactivity immediately after birth which was followed by a slower rise. The peak of reactivity was reached when the animals were about 6 months of age. Although they noted that immunologic reactivity declined after this age, no evidence was obtained to indicate that individual anti-

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body-forming cells from senile mice differed from those of younger animals. They concluded, as did Makinodan and Peterson, that the decline in immunologic responsiveness seen with increasing age is due to a decrease in the relative and absolute numbers of potential antibody-forming cells.

E. ADJUVANTS Adjuvants may be defined as substances which, when mixed with the antigen to be injected, enhance antigenicity and increase the amount of antibody produced over that obtained by injection of the antigen alone. To site a specific example, Dixon et al. (1966) reported that the incorporation of hernocyanin in incomplete Freund’s adjuvant enhanced its immunogenicity approximately 1000-fold. Many materials have been reported to have adjuvant activity, e.g., zymosan (Cutler, 1960), crystalline silica particles (Pernis and Paronetto, 1962), branched-chain, saturated, noncyclic hydrocarbons (Wilner et al., 1963),aliphatic nitrogenous bases (Gall, 1966),lipid (Youngnerand Axelrod, 1964), acrylamide gel (Weintraub and Raymond, 1963), cellulose (Olovnikov and Gurvich, 1966), saponin (Gill, 1965), endotoxin (Munoz, 1964), and calcium alginate (Amies, 1959). Infection of mice with the lactic dehydrogenase virus has even been found to greatly enhance their immune response to human gamma-globulin (Notkins et ul., 1966).Aluminum salts and water-in-oil emulsions are the most widely used adjuvants and they have been discussed extensively in other reviews (White, 1963; Edsall, 1966; Freund, 1947, 1951, 1956; Berlin, 1963), hence only brief consideration of them will be given here. The effect of the concentration of antigen in water-in-oil adjuvants on the initiation of antibody synthesis was studied by Farr and Dixon (1960). They noted that the critical factor in eliciting an antibody response in rabbits appeared to be the concentration of antigen in the aqueous phase. By maintaining the concentration of antigen constant, it was possible to reduce the total amount of antigen injected by a factor of 10 without significantly reducing the proportion of animals producing antibody. The use of adjuvants may make it possible to convert an apparently nonantigenic substance into an effective antigen. Maurer and Lebovitz (1956) demonstrated that modified fluid gelatin was antigenic in rabbits only when it was injected in water-in-oil emulsions. No antibody production was noted when the antigen was injected as a saline solution or as an alum precipitate.

ANTISERUM PRODUCTION IN EXPERIMENTAL ANIMALS

57

A substance may demonstrate adjuvant activity with one antigen and yet be completely devoid of any ability to enhance the immune response to a second antigen. Gamble (1966) studied the effect of phytohemmagglutinin on the primary antibody response of mice and noted that, while a significant enhancement of antibody production occurred to rat erythrocytes, there was an unexpected suppression of antibody formation to human gamma-globulin.

F. OTHER Any factor which upsets the physiologic balance of the host can potentially interfere with the immune response; although, in general, a severe imbalance must occur before marked inhibition of antibody synthesis is noted. For example, Northey (1965)reported that shaved rabbits, maintained at 4" C. prior to and during the period of immunization with various protein antigens, failed to demonstrate any appreciable impairment of antibody production. In an earlier study, Trapani (1960)had observed a slight decrease in the immune response of rabbits similarly shaved and housed at -15°C. The hormonal balance of the host may play a significant role in determining the outcome of immunization. Sang and Sobey (1954) reported that both pregnant and lactating rabbits have a significantly decreased response to diphtheria or tetanus toxoid. Likewise, the nutritional status of the animal may influence the immune response. Panda and Combs (1963) studied the agglutinin response of 4-weekold chicks fed diets deficient in vitamin A, pantothenic acid, or riboflavin and noted an impairment of antibody production which, when compared with that obtained with control animals fed a complete diet, was significant at the 0.01 level. The inhibition of antibody production by immunosuppressive drugs has been amply reviewed elsewhere (Berenbaum, 1965; Schwartz, 1965; Makinodan et al., 1965), as has the competition of antigens occasionally observed when animals are injected with a mixture of several antigenic materials (Adler, 1959, 1964), consequently these subjects will not be considered here. The effect of the time interval between primary and secondary injections of antigen on the immune response has also been investigated. Brown et al. (1964) immunized 373 human infants with diphtheria, tetanus, pertussis, and polio vaccines and found that the second injection was more efficient if the interval between primary and secondary injection was increased from 1 month to 2 months. The efficiency was evidenced by higher antibody titers after secondary

58

RICHARD M. HYDE

injection, as well as by a greater response to booster injections administered 1 year later. The variation in secondary antibody response as a function of the interval between injections has also been studied by Fecsik et al. (1964).These investigators employed a subcutaneous injection of diphtheria toxoid in mice as their test system and found that, as the interval between two antigen injections lengthens, the magnitude of the secondary response increases. The optimal time for secondary stimulation of mice with the dose of antigen employed was found to be between 20 and 40 days. The duration of the capacity to respond to a booster injection has also been investigated. An apparently unimpaired capacity to respond to booster injections of tetanus toxoid has been found as long as 9 to 11 years after primary immunization (Turner et al., 1954; Peterson et at., 1955). The anamnestic reactivity of rabbits to sheep erythrocyte hemolysin has been reported to persist for at least 2.5 years (Taliaferro and Taliaferro, 1966). VI. Specific Examples of Antiserum Production

The selected bibliography (Section VIII) has been included in this review to serve as a guide to immunization procedures currently in use. Appropriate review articles have been cited whenever possible. The review by Ouchterlony (1962) is also recommended as a source of references for the production of specific antiserums. It includes an antigen-index bibliography which allows the investigator to obtain a reference to any particular antigen employed as an immunizing agent. VII. Conclusion Because of the variation in antibody response seen in experimental animals, at least two precautions should be taken when establishing an immunization schedule. First, sufficient numbers of animals should be used to assure that, even if the proportion of animals producing antibody is low, some of them will produce the antibody desired. Second, periodic bleedings should be done during the immunization period and the serum should be examined for the presence of antibody. When the antibody level has reached a plateau, the animal can be exsanguinated or bled successively over a period of days. From the preceding brief discussion of some of the variables that influence the outcome of an immunization procedure, it is obvious that there is no universal vaccination procedure that will guarantee maximum antibody production. How, then, does the investigator who

ANTISERUM PRODUCTION IN EXPERIMENTAL ANIMALS

59

wishes to produce an antiserum proceed? One possibility is to search the literature and obtain a reference article that is appropriate to the needs of the experiment. Alternately, an immunization procedure can be determined empiricaIIy. It is hoped that some of the information contained in this review will be helpful, regardless of the approach taken. ACKNOWLEDGMENTS The preparation of this article has been assisted by U.S.P.H.S. Training Grant 5TlAI162. The author is grateful to Mrs. Kay Christiansen for excellent clerical assistance and to Dr. Robert A. Patnode for his critical reading of the manuscript.

VI II. Selected Bib1iogra phy

A. BLOOD

1 . Cellular Elements Dietrich, F. M. (1966). The immune response to heterologous red cells in mice. Immunology 10,365-376. Gray, J. G., Monaco, A. P., Wood, M. L., and Russell, P. S. (1966). Studies on heterologous anti-lymphocyte serum in mice. I. In vitro and in uiuo properties. I. Immunol. 96,217-228. Harris, S., and Harris, T. N. (1966). Suppression of rabbit lymph node cells by rabbit anti-leucocyte serum demonstrated in vitro by the antibody plaque test.]. Immunol. 96,478-487. Kochan, I., Christopher, J. A., and Kupchyk, L. (1966). Study on the cellular factor of delayed hypersensitivity.]. Allergy 38,280-289. Ovary, Z. (1964). Antigenicity of hemoglobin and its constituents. I. Antigenicity of human adult hemoglobin and its structural units (alpha and beta chains). fmmunochemistry 1,241-248. Reichlin, M., Hay, M., and Levine, L. (1965). Immunochemical studies on inter-species molecular hybrids of hemoglobin. lmmunochemistry 2,337-350. Stone, W. H., and Irwin, M. R. (1963). Blood groups in animaIs other than man. Aduan. Immunol. 3,315-350.

2 . Plasma (Serum) Berglund, G. (1965). Preparation of antiserum to an antigen of low molecular weight (fibrinopeptide).Nature 206,523-524. Bergmann, F. H., Levine, L., and Spiro, R. G . (1962). Fetuin: Immunochemistry and quantitative estimation in serum. Biochim. Biophys. Acta 58,41-51. Bergquist, L. M., Carroll, V. P., Jr., and Searcy, R. L. (1961). Evaluation of a specific antiserum for serum-P-lipoprotein estimations. Lancet 1,537-538. Fudenberg, H. H. (1965).The immune globulins. Ann. Rev. Microbiol. 19,301-338. Gitlin, D. (1966). Current aspects of the struchire, function and genetics of the immunoglobulins. Ann. Reu. Med. 17,l-22. Matheson, A., Jensen, R. S., and Donaldson, D. M. (1966). Serologic relationships between P-lysins of different species.]. Immunol. 96,885-891.

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Robbins, K. C., and Summaria, L. (1966). An immunochemical study of human plasminogen and plasmin. lmmunochemistry 3,29-40. West, C. D., Davis, N. C., Forristal, J., Herbst, J., and Spitzer, R. (1966). Antigenic determinants of human PlC- and P l G . globulins.]. Immunol. 96,650-658. Williams, R. C., Jr. (1964). Anti-albumin antibodies after immunization with whole and and enzyme-digested autologous rabbit albumins.]. Immunol. 93,850-859.

B. TISSUESA N D

ORGANS

Beernink, K. D., Courcon, J., and Grabar, P. (1965). Immunochemical studies on bone marrow of the rat. Immunology 9,377-390. Clarke, W. M., and Fowler, I. (1960). The inhibition of lens-inducing capacity of the optic vesicle with adult lens antisera. Deuelop. Biol.2,155-172. Dumonde, D. C. (1966).Tissue-specific antigens. Aduun. Immunol. 5,245-412. Flickinger, R. A. (1962).Embryological development of antigens. Aduun. Immunol. 2, 310-366. Harvie, N. R., and Elberg, S. S. (1961). Observations on cytotoxic effect of antihistiocyte serum. PTOC.Sac. Exptl. Biol.Med. 107,8-11. Hook, W. A., Muschel, L. H., and Faber, J. E. (1966).Antibodies to X-irradiated rabbit testes. Immunology 10,245-248. Inoue, K. (1961).Precipitin reactions and developmental arrest by antisera in amphibian embryos. Develop. Biol. 3,657-683. Kurata, Y., and Okada, S. (1966). Immunological studies of insoluble lipoproteins. I. Antigenic analysis of thyroidal lipoproteins. Intern. A d . Allergy Appl. lmmunol. 29,495-509. LeVeen, H. H., Falk, G., and Schatman, B. (1961). Experimental ulcerative colitis produced by anticolon sera. Ann. Surg. 154,275-280. Loewi, C., and Muir, H. (1965).The antigenicity of chondromucoprotein. Immunology 9,119-128. Milgrom, F., Kasukawa, R., and Calkins, E. (1966). Studies on antigenic composition ofamyloid.]. Immunol. 96,245-252. Myers, J., Frei, J. V., Cohen, J . J., Rose, B., and Richter, M. (1866).Basement membrane specific antisera produced to solubilized tissue fractions. Immunology 11,155-162. Rajam, P. C., Bogoch, S., Rushworth, M. A., and Forrester, P. C . (1966). Antigenic constituents of basic proteins from human brain. Immunology 11,217-222. Rothbard, S., and Watson, R. F. (1965). Immuiiologic relations among various animal collagens.]. Exptl. Med. 122,441-454. Smith, D. E., and Lewis, Y. S. (1961). Preparation and effects ofan anti-mast cell serum. ]. Exptl. Med. 113,683-692. Spragg, J., Austen, K. F., and Haber, E. (1966). Production of antibody against bradykinin: demonstration of specificity by complement fixation and radio-immunoassay. ]. Zmmunol. 96,865-871. Tamanoi, I., Yagi, Y., and Pressman, D. (1961). Rate of localization of anti-rat lung antibody. Proc. Sac. Exptl. Biol. Med. 106,769-772. Warren, B., Johnson A. G . , and Hoobler, S. W. (1966). Characterization of the reninantirenin system.]. Exptl. Med. 123,1109-1128. Yakulis, V. J., and Heller, P. (1962). The detection of myoglobin by means of imniunologic technics. Am.]. Clin. Puthol. 37,253-256.

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ANIMALS

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C. BIOLOGICAL FLUIDS 1 . Hormones Bourdel, G. (1961). Effect of rabbit antiserum to sheep pituitary interstitial-cell stimulating hormone in immature female rats. Gen. Comp. Endocrinol. 1,375-380. Geschwind, I. I. (1963). The chemistry and immunology of gonadotropins. In “Gonadotropins” (H. H. Cole, ed.). Freeman, San Francisco, California. Goodfriend, T., Fasman, G., Kemp, D., and Levine, L. (1966). Immunochemical studies of angiotensin. lmmunochemistry 3,223-232. Levi-Montalcini, R., and Angeletti, P. U. (1966). Immunosympathectomy. Pharmacol. Rev. 18,619-629. Levy, R. P., and Sampliner, J. (1962). Prolactin, immunologic evidence of species specificity. Proc. Soc. Exptl. Biol. Med. 109,672-673. McGarry, E. E., and Beck, J. C. (1963). Some studies with antisera to human FSH. Fertility Sterility 14,558-564. Pope, C. G. (1966).The immunology of insulin. Aduan. Immunol. 5,209-244. van Hell, H., Goverde, B. C., Schuurs, A. H. W. M., D e Jager, E., Matthijsen, R., and Homan, J. D. H. (1966). Purification, characterization, and immunochemical properties of human chorionic gonadotropin. Nature 212,261-262. Wallace, A. L. C., and Sobey, W. R. (1965). Immunological studies of a bovine growth hormone preparation. J . Endocrinol. 32,321-327. Weigle, W. 0. (1965). The induction of autoimmunity in rabbits following injection of heterologous or altered homologous thyroglobulin. J . Exptl. Med. 121,289-308. Wolstenholme, G. E. W., and Cameron, M. P., eds. (1962). “A Ciba Symposium: Immunoassay of hormones.” Churchill, London. Zimmering, P. E., Beiser, S. M., and Erlanger, B. F. (1965). Purification and some properties of anti-testosteroneantibodies. J . Immunol. 95,262-272.

2. Enzymes Arnon, R., and Schechter, B. (1966). Immunological studies on specific antibodies against trypsin. Immunochemistry 3,451-462. Barrett, J. T. (1965). An immunochemical study of procarboxypeptidase A and its enzyme. Immunology 8,129-135. Barrett, J. T., and Thompson, L. D. (1965). Immunochemical studies with chymotrypsinogen A. Immunology 8,136-143. Cinader, B. (1957).Antibodies against enzymes. Ann. Reu. Microbiol. 11,371-390. Cinader, B. (1963). Antibodies to enzymes-a three-component system. Ann. N . Y. Acad. Sci. 103,493-1154. Glimp, H. A., and Tillman, A. D. (1965). Effect of jackbean urease injections on performance, anti-urease production and plasma ammonia and urea levels in sheep. J . Animal Sci. 24,105-112. Lehrer, H. I., and van Vunakis, H. (1965). Iminunochemical studies on carboxypeptidase A. Immunochemistry 2,255-262.

3. Other Abelli, G. (1962). Immunoelectrophoretic studies on colostrum and milk using specific immune sera. Panminerua Med. (EnglishEd.)4,181-184.

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Abuelo, J. G., and Ovary, Z. (1965). Dinitrophenylated bacitracin A as an antigen in the guineapig.J. Zmmunol. 95,113-117. Beard, R. L. (1963). Insect toxins and venoms. Ann. Reu. Entomol. 8,1-18. Halloran, M. J., and Parker, C. W. (1966). The production of antibodies to mononucleotides, oligonucleotides and DNA.]. Zmmunol. 96,379-385. Hochwald, G. M., and Thorbecke, G. J, (1962).Use ofanantiserumagainstcerebrospinal fluids in demonstration of trace proteins in biological fluids. PTOC.Soc. E x p t l . Biol. Med. 109,91-95. J O I I S S O I ~ ,J., and Paleus, S. (1966). Studies on the antigenic capacity of chicken atrtl bovine cytochrome c. Intern. Arch. Allergy A p p l . Zmmunol. 29,272-277. Kaminski, M. (1965). The analysis of the antigenic structure of protein molecules. P‘TOgT. Allergy 9,79-157. Katsh, S., and Katsh, G . F. (1965). Perspectives in immunological control of reproduction. Paci$c Med. Surg. 73, Suppl. l A , 28-43. Keegan, H. L., Whittemore, F. W., Jr., and Flanigan, J. F. (1961). Heterologous antivenin in neutralization of North American coral snake venom. Public Health Rept. (U.S.)76,540-542. Mange, A. P., and Stone, W. H. (1962). Tests on immune sera produced against calf thymus chromosome. Proc. Soc. E x p t l . Biol. Med. 109,42-44. Rohbins, K. C., Wu, H., and Hsieh, B. (1966). Physical, chemical, and immunochernical studies on a low ragweed pollen antigen. Zmmunochemistry 3 , 7 1 4 0 . Rumke, P., and Sluyser, M. (1966).Antigenicity of histones. Bi0chem.J. 101,1C-2C. Stevens, K. M., and Fost, C. A. (1964). Sperm and antibody production in rabbits following immunization with sperm and semen. PTOC. Soc. E x p t l . B i d . Med. 117, 125-127.

D.

MALIGNANCIES

Boyle, W., and Davies, D. A. L. (1966). Antigens of the surface of mouse ascites tumor cells. I. Studies with rabbit anti-mouse cell sera. Immunology 11,353-360. Caso, L. V. (1965). The relation of the immune reaction to cancer. Aduan. Cancer Res. 9,47-141. Day, E. D. (1965). “The Immunochemistry of Cancer.” Thomas, Springfield, Illinois. Garb, S., Stein, A. A., and Sims, G. (1962). The production of anti-human leukemic serum in rabbits. J. Zmmunol. 88,142-152. Klein, G. (1966).Tumor antigens. Ann. Reu. Microbiol. 20,223-252. Old, L. J., and Boyse, E. A. (1964).Immunology of experimental tumors. Ann. Reu. Med. 15,167-186. Pilch, Y. H. (1964). Immunogenicity of cell free extracts of neoplastic tissue. S U T ~ . F O ~ U V15,348-350. ~

E. MICROORGANISMS Barber, C., Vladoianu, I. R., and Dimache, G. (1966). Contributions to the study of Salmonella immunological specificity of proteins separated from Salmonella typhi. Immunology 11,287-296. Basaca-Sevilla, V., Pesigan, T. P., and Finkelstein, R. A. (1964). Observations on serological responses to cholera immunization. Am. /. Trop. Med. H y g . 13, 100107.

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Blyth, W. A., Reeve, P., Graham, D. M., and Taverne, J. (1962). The production of antisera that neutralize inclusion blennorrhoea virus. Brit. ]. Exptl. Pathol. 43, 340343. Cuadrado, R. R. (1966). Agar gel diffusion method for the study of antigenic components of mumps virus. J. Immunol. 96,892-897. Daniel, T. M. (1965). Observations on the antibody response of rabbits to mycobacterial antigens. J. Immunol. 95,100-108. Eaton, M . D. (1965). Pleuropneumonia-like organisms and related forms. Ann. Reu. Microbial. 19,379-406. Estrup, F., and Santer, M. (1966).Immunological analysis of the proteins of Escherichia coli ribosomes.]. Mol. Biol. 20,447-452. Houba, V., and Hana, I. (1966).The difference in immunological characteristics of two streptokinases. Immunology 11,387-398. Hsiung, C. D., Chang, P. W., Cuadrado, R. R., and Isacson, P. (1965). Studies of parainfluenza viruses. 111. Antibody responses of different animal species after immunization. J. Immunol. 94,67-73. Kwapinski, J. B. (1966). Serological and chromatographic characterization of exoantigens of the Dermatophilus. Australian]. E x p t l . Biol.Med. Sci. 44,87-92. Levine, L., Wasserman, E., and Murakami, W. T. (1966). Immunochemical studies on bacteriophage DNA. VI. Renaturation of T, DNA. Immunochemistry 3,41-50. McCarty, M., and Morse, S. I. (1964). Cell wall antigens of Cram-positive bacteria. Advan. Immunol. 4,249-286. Matthews, R. E. F. (1957). “Plant Virus Serology.” Cambridge Univ. Press, London and New York. Nell, E. E., and Hardy, P. H. (1966). Studies on the chemical composition and immunologic properties of a polysaccharide from the Reiter treponeme. lmmunochemistry 3,233-246. Norris, J. R. (1962). Bacterial spore antigens: A review. J . Gen. Microbiol. 28, 393408. Salvin, S. B. (1963).Immunologic aspects of the mycoses. Progr. Allergy 7,213-331. van Heyningen, W. E., and Arseculeratne, S. N. (1964). Exotoxins.Ann. Reu. Microbiol. 18,195-216. Weidanz, W. P., Jackson, A. L., and Landy, M. (1964). Some aspects of the antibody response of rabbits to immunization with enterobacterial somatic antigens. Proc. Soc. E x p t l . Biol. Med. 116,832-837. REFERENCES Adler, F. L. (1959).In “Mechanisms of Hypersensitivity” (J. H. Shaffer, G. A. LoGrippo, and M. W. Chase, eds.), p. 539. Little, Brown, Boston, Massachusetts. Adler, F. L. (1964).Progr. Allergy 8,41-57. Altman, P. L., and Dittmer, D. S. (1964). “Biology Data Book.” Federation Am. SOC. Exptl. Biol., Washington, D.C. Amies, C. R. (1959).]. Pathol. Bacterial. 77,435-442. Aoki, T., and Teller, M. N. (1966).Cancer Res. 26,1648-1652. Arnason, B. G., and Waksman, B. H. (1964).Aduan. Tuberc. Res. 13,l-97. Arquilla, E. R., and Finn, J. (1965).]. E x p t l . Med. 122,771-784. Avery, 0.T., and Goebel, W. F. (1933).]. Exptl. Med. 58,731-755. Berenbaum, M. C. (1965).Brit. Med. Bull. 21,140-146.

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Berlin, B. S. (1963).Ann. Allergy 21,82-90. Bernheimer, A. W., Caspari, E., and Kaiser, A. D. (1952).J.E x p t l . Zool.119,23-35. Biro, C. E., and Garcia, G. (1965). Immunology 8,411-419. Breebaart, A. C., and James-Witte, J. (1959).A m . ] . Ophthalmol. 48,37-47. Brown, G . C., Volk, V. K., Gottshall, R. Y., Kendrick. P. L., and Anderson, H. D. (1964). Public Health Rept. ( U S . )79,585-602. Burnet, M. (1964).Nature 203,451-454. Campbell, D. H., Carvey, J. S., Cremer, N. E., and Sussdorf, D. H. (1964). “Methods in Immunology.” Benjamin, New York. Charney, J., Tytell, A. A., Machlowitz, R. A,, arid Hilleman, M. R. (1961).J. Am. Med. A S ~ O177,591-595. C. Chase, M . W. (1H50).Attii. RUG.Microbiol. 13, 349-376. Chase, M. W. (1965). Med. Clin. N . Am. 49,1613-1646. Copeman, S. M., O’Brien, R. A., Eagleton, A. J., and Glenny, A. T. (1922). B r i t . ] . E x p t l . Puthol. 3,42-50. Crowle, A. J. (1966).Ann. Allergy 24, 195-214. Cryan, W. S., Hyde, R. M., and Garb, S. (1966).Cancer Res. 26,1458-1465. Cutler, J. L. (1960).J.Immunol. 84,416-419. Dixon, F. J., Jacot-Guillarmod, H., and McConahey, P. J. (1966).J . Immunol. 97, 350355. Downie, A. W. (1937a).J.Pathol. Bacteriol. 45,149-155. Downie, A. W. (1937b).J.Pathol. Bacteriol. 45,131-147. Draper, L. R., and Sussdorf, D. H. (1957).]. Infect. Diseases 100,147-161. Edsall, G. (1963). In “Modern Trends in Immunology” (R. Cruickshank, ed.). Butterworth, London and Washington, D.C. Edsall, G . (1965).Med. Clin. N.Am. 49,1729-1743. Edsall, G. (1966).Ann. Reu. Med. 17,39-48. Eisen, H. N., and Pearce, J. H. (1962).Ann. Reu. Microbiol. 16,101-126. Farr, R. S., and Dixon, F. J., Jr. (1960).J.Immunol. 85,250-257. Fecsik, A. I., Butler, W. T., and Coons, A. H. (1964).J.E x p t l . Med. 120,1041-1049. Felton, L. D., Sutliff, W. D., and Steele, B. F. (1935).J.Infect. Diseases 56,101-110. Fernando, A. N. (1960).A.M.A. Arch. Ophthalmol. 63,515-539. Fink, M. A., and Quinn, V. A. (1953).]. Immunol. 70,61-67. Francis, T., Jr., and Tillett, W. S. (l930).J.E x p t l . Med. 52,573-585. Frei, P. C., Benacerraf, B., and Thorbecke, G. J. (1965). Proc. Natl. Acad. Sci. U . S. 53, 20-23. Freund, J. (1947).Ann.Reu. Microbiol. 1,291-308. Freund, J. (1951).A m . ] . Clin. Pathol. 21,645-656. Freund, J . (1956).Advan. Tuberc. Res. 7,130-148. Gall, D. (1966).Immunology 11,369-386. Gamble, C. N. (1966). Intern. Arch. Allergy Appl. Immunol. 29,470-477. Gill, B. S. (1965).]. Comp. Pathol. Therap. 75,233-240. Good, R. A., and Papermaster, B. W. (1964).Aduan. Zmmunol. 4,l-115. Goullet, P., and Kaufmann, H. (1965).Experientia 21,46-47. Hasek, M., Lengerova, A,, and Vojtiskova, M. (1962). “Mechanisms of Immunological Tolerance.” Academic Press, New York. Heidelberger, M., Macleod, C. M., Kaiser, S. J., and Robinson, B. (1946).]. E x p t l . Med. 83,303-320. Hilleman, M. R. (1964).An,. Rev. Respirat. Diseases 90,683-706.

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Hingson, R. A,, Davis, H. S., and Rosen, M. (1963).Military Med. 128,516-524. Hirata, A. A., and Campbell, D. H. (1965).lmmunochemistry 2,195-205. Hirata, A. A., and Sussdorf, D. H. (1966).J.Zmmunol. 96,611-613. Hyde, R. M., Bennett, A. J., and Garb, S. (1965). Intern. Arch. Allergy Appl. Immunol. 28,271-279. Ipsen, J., Jr. (1954).J . Zmmunol. 72,243-247. Ipsen, J., Jr. (1959).J . Immunol. 83,448-457. Ipsen, .I Jr. ., (1961).J . Zmmunol. 86,50-55. Ivanyi, J., and Valentova, V. (1966).Folia Biol. (Prague)12,36-48. Jarrett, W. F. H., and Sharp, N. C. C. (1963).]. Perasitol. 49,177-189. Jemski, J . V., and Phillips, G. B. (1965).In “Methods of Animal Experimentation” (W. I. Gay, ed.). Academic Press, New York. Landsteiner, K., and Jacobs, J. (1932).Proc. Soc. Exptl. B i d . Med. 29,570-571. Lepper, M. H., and Wolfe, E. K. (1966).Bacteriol. Reu. 30,485-698. Leskowitz, S . (1960).J . Immunol. 85,56-66. McDevitt, H. O., and Sela, M. (1965).J.Exptl. Med. 122,517-531. Makinodan, T., and Peterson, W. J. (1964).J . Zmmunol. 93,886-896. Makinodan, T., Albright, J. F., Perkins, E. H., and Nettesheim, P. (1965). Med. Clin. N . Am. 49,1569-1596. Mantyjarvi, R. (1965).Acta Pathol. Microbiol. Scand. 65,581-586. Mascoli, C . C., Leagus, M. B., Weibel, R. E., Stokes, J., Jr., Reinhart, H., and Hilleman, M. R. (1966).Proc. Soc. Exptl. Biol. Med. 121,1264-1268. Maurer, P. H. (1957).J.Immunol. 79,84-88. Maurer, P. H. (1963).J.Zmmunol. 90,493-504. Maurer, P. H. (1964).Progr. Allergy 8,l-40. Maurer, P. H. (1965).Med. Clin. N.Am. 49,1505-1516. Maurer, P. H., and Lebovitz, H. (1956).J.Zmmunol. 76,335-341. Medawar, P. B. (1963). In “Acquired or Natural Immune Tolerance Toward Defined Protein Antigens” (A. Bussard, ed.). C. N. R. S. Symposium, Academic Press, New York. Menzin, A. W. (1961).Arch. Internal Med. 107,409-429. Menzoian, J. O., and Ketchel, M. M. (1966).Nature 211,133-135. Middlebrook, G . (1961). Bacteriol. Reu. 25,331-346. Miller, J. F. A. P. (1966).Brit. Med. Bull. 22,21-26. Miller, T. A. (1966).J.Parasitol. 52,512-519. Moeller, G. (1964). Transplantation 2,405-415. Moreland, A. F. (1965). In “Methods of Animal Experimentation” (W. I. Gay, ed.). Academic Press, New York. Munoz, J. (1964).Aduan. Zmmunol. 4,397-440. Neeper, C. A,, and Seastone, C. V. (1964).J . Zmmunol. 93,867-871. Northey, W. T. (1965).J . Zmmunol. 94,649-657. Nossal, G . J . V. (1965).Australasian Ann. Med. 14,321-328. Nossal, G . J. V., Ada, G. L., and Austin, C. M. (1963).Nature 199,1257-1262. Nossal, G. J. V., Ada, G. L., and Austin, C. M. (1964). AustralianJ. Exptl. Biol. Med. Sci. 42,283-294, Notkins, A. L., Meryenhagen, S. E., Rizzo, A. A., Scheele, C., and Waldmann, T. A. (1966).J.E x p t l . Med. 123,347-362. Olovnikov, A. M., and Gurvich, A. E. (1966).Nature 209,417-419. Osborn, J. J., Dancis, J., and Julia, J. F. (1952).Pediatrics 10,328-334.

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Microbial Models of Tumor Metabolism

G. F. GAUSE lnstitute of New Antibiotics, Academy of Medical Sciences, Moscow, U.S.S.R.

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

I. Introduction

69

anisms in the Metabolism

of Tumors ....................................... A. Tumor Metabolism in the Evolutionar

70

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70 72 73

111. Disturbance of Control Mechanisms in the

Respiratory-Deficient Yeast ....................................... A. Metabolism of the Respiratory-Deficient Yeast B. Disturbance of Control Mechanisms in the Respiratory-Deficient Yea ......................... C. Distorted Organization of Respiratory-Deficient Yeast.. ...... IV. Summary ................... References ..............................................................

77 77 83 85 88 88

I. Introduction Theories are like living organisms that can survive only b y evolving in order to adapt themselves to new demands. If the concept of microbial models of cancer cells were restricted to its initial formulation, it would experience the fate of other theories and wither away, or at least become mummified. Fortunately, the concept is acquiring new life and becoming more fruitful of understanding, because its scope is being widened. At first focused on some resemblances between respiratory-deficient mutants of microorganisms and cancer cells, it is now taking cognizance of the multiplicity of similarities in the molecular organization of cancer cells and of their microbial equivalents. Seen from this broader point of view, microbial models of cancer cells may stimulate investigation of the nature of malignancy, and act as a creative force in setting goals for research (Gause, 1966). In recent times considerable attention has been paid in microbiology to biochemical correlates of respiratory deficiency in yeast and to cytoplasmic transformation, when mitochondria from normal yeast cells restore the respiratory capacity of the respiratory-deficient mutants. This research in microbiology now closely approaches one of the most fundamental problems of cancer research, namely, the search 69

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for an experimentally controlled reversion of the malignant cell to its “normal” counterpart (Pitot and Cho, 1965). There is a growing feeling among investigators concerned with analysis of control mechanisms in the normal and neoplastic cell that microbiology and cancer research have much to learn from each other. The purpose of this review is to summarize the available literature on disturbance of control mechanisms in the respiratory-deficient mutants of yeast, and to compare it with more recent data on disturbance of control mechanisms in the metabolism of tumors. II. Disturbance of Control Mechanisms in the Metabolism of Tumors

A. TUMORMETABOLISM IN

THE

EVOLUTIONARY PERSPECTIVE

It is reasonable to begin a discussion of tumor metabolism with a consideration of the contributions of Warburg, extended over almost half a century. His first paper on this subject was published in 1923, a book entitled “The Metabolism of Tumors” in 1926, and the most recent contribution to this series of investigations appeared in 1965. Two features are characteristic of Warburg’s approach: the first is his consideration of tumor metabolism in the evolutionary perspective, and the second is his continued reference to microbial models, and particularly to yeast cells, for better understanding of the essence of tumor metabolism. The transformation of the normal cell into a malignant one, or the process of malignization, can be considered as a transition to an evolutionarily more primitive type of metabolism, d u e to impaired respiration and greater dependence upon “life without oxygen,” observed b y Pasteur in yeast cells almost a century ago. Respiration is a much more complicated and subtle process than fermentation, and it is more vulnerable to the effect of injurious carcinogenic agents, responsible for the malignization of cells. The discovery by Warburg in 1923 of the impaired respiration of tumor cells and of their greater dependence upon fermentation was an important turning point in the study of tumor metabolism. This phenomenon has various aspects. One of the consequences of the metabolic shift in tumors is the capacity of tumor cells to live and multiply under anaerobic conditions for a long period of time, which differentiates them from normal cells. This characteristic feature of tumor cells can be demonstrated with the aid of different techniques, including a very elegant microbiological experiment of Malmgren and Flanigan (1955). It is well known that tetanus bacteria belong to

MICROBIAL MODELS OF TUMOR METABOLISM

71

anaerobic microorganisms; tetanus spores germinate and grow only in the absence of oxygen. When injected intravenously into the body of normal or of pregnant mice, tetanus spores do not germinate anywhere in the organism, as far as complete anaerobiosis is practically excluded in the body. However, Malmgren and Flanigan (1955) observed that the injection of washed tetanus spores, which was without untoward effect in normal animals, uniformly resulted in tetanic death in the tumor-bearing host in approximately 48 hours, regardless of the tumor size. The production of a lethal toxin in the vegetative state makes determination of germination of these spores both simple and conclusive. On the basis of examination of comparable tumors in untreated animals prior to spore injection, it appears likely that necrotic areas were absent. It is therefore possible to conclude that tetanus bacilli can grow and multiply only among cancer cells in the tumors. This experiment clearly demonstrates that cancer cells, and cancer cells alone, grow and multiply in the organism under anaerobic conditions. However, growth under anaerobic conditions may occur only temporarily in the life of tumor cells. Warburg et al. (1965) compared it with an anaerobically growing yeast, which afterward can be brought into contact with oxygen and continue to grow aerobically. In the same way, metastasizing tumor cells migrate into the new areas of the body provided with oxygen. The observations of Warburg were for several decades the subject of controversy, which to a very considerable extent depended upon insufficient development of theoretical oncology and the absence of proper “control” cells for the evaluation of metabolic shifts in tumors. This situation has improved considerably only recently, with the wide availability of a new class of transplantable and primary tumors of animals that were named “minimal deviation” tumors by Potter and his colleagues (Potter et al., 1960). They were discovered in the course of a systematic search for liver tumors, induced by feeding animals with various carcinogens, and differing as little as possible from normal liver. The first example of this type of tumor was described by Morris et al. (1960), and designated the Morris hepatoma 5123. Following the discovery of this hepatoma, a systematic search for other representatives of the class was made, and a growing list of minimal deviation hepatomas is now available. Different strains of minimal deviation hapatomas in the rat grow with different speed: some of them double their weight in 2 weeks, while others do it in 10 months. The rate of growth may be taken as an index of malignancy: the better tumor cells grow, the more malignant

72

6. F. GAUSE

they are. It is very remarkable therefore that Burk and his colleagues (Burk et al., 1965, 1967) observed in this series of tumors a significant correlation between malignancy and glycolysis, which is shown in Table I. The initial rates of anaerobic and aerobic glycolysis of TABLE I CONNECTION BETWEENGLUCOSE FERMENTATION AND GROWTH W T E IN THE SPECTRUMOF MORRISRAT HEPATOMAS" ~~

Anaerobic glycolysis* Tissue Normal liver Host liver Slowest-growing hepatomas (subminimal deviation) Slow-growing hepatomas (minimal deviation) Intermediate-deviation hepatomas Fast-growing (advanced) hepatomas

Minimum

Maximum

Average

No. of specimens studied

0.18 0

0.50 0.68

0.32 0.33

6 26

0.40

1.00

0.69

9

0.58

1.80

1.03

13

2.00

6.40

4.10

15

4.5 11.8

8.0 17.5

6.7 13.7

3 6

From Burk et ul. (1967).

'Qco2 N20.1to 0.5% glucose

in medium.

minimal deviation hepatoma cells are up to several-fold higher than those of liver tissue from the host animal or from hepatoma-free animals. These metabolic differences between normal and tumor cells gradually increase with the increase of malignancy and rate of growth of tumors, and they clearly confirm observations made by Warburg on other strains of tumor tissue some years ago.

B. DISTURBANCE O F CONTROL MECHANISMS IN METABOLISM OF TUMORS

THE

A comparison of cancer cells with anaerobic yeasts is illuminating, but it is only partially valid. What is characteristic for a cancer cell is the disturbance of cellular control mechanisms on various levels of its organization (Pitot and Cho, 1965). Chance and Hess (1959)in the rapid measurements of intracellular events with the aid of spectroscopic methods observed that the respiratory system of tumor cells is not used to the extent of its capabilities. They suggested that there is a control mechanism imposed upon the respiratory activity of the

MICROBIAL MODELS OF TUMOR METABOLISM

73

intact tumor cell. Another manifestation of the work of this control mechanism can be seen in the fact that the oxidative rate of tumors is fixed, and quite refractory to the stimulation seen when excess carbohydrate or fatty acid is added to normal tissues. It might be said, therefore, that the respiratory activity of neoplastic material is a maximal one, while normal cells have ample reserves. This aspect of tumor metabolism represents only a special case of deficiency in biochemical control mechanisms, which has been described by Potter (1964) in a wider biological perspective as the biochemical inflexibility of tumors. H e believes that cancer cells probably exhibit irreversible changes in regulatory genes. It is this disturbance of control mechanisms which makes the cancer cell different from anaerobic yeast. In the latter case the respiration is decreased and glycolysis increased due to low oxygen tension, but regulatory genes are not lost, and in response to a change of environment, metabolic regulations are observed. It is only in the respiratorydeficient mutants of yeast that metabolic regulations are also irreversibly lost, and these mutants can therefore be approached with some justification as potential microbial models for the study of some aspects of metabolism resembling the metabolism of tumors.

c. MOLECULARORGANIZATION OF TUMORMITOCHONDRIA Impaired respiration and increased glycolysis of tumors were discovered by Warburg in the early 1920’s, in the beginning of an era of luxurious flowering of research on biological oxidations, which occurred with relatively little attention to mitochondria. Analyzing the major events of this period Lehninger (1964) wrote: “Actually, very few biochemists concerned themselves with the possible importance of the fact that respiratory enzymes were found to be associated with particulate matter of cells and tissues. It was a part of the biochemical Zeitgeist that particles were a nuisance and stood in the way of purification of the respiratory enzymes. Yet it almost seems paradoxical that it was two biochemists who had many years earlier made important discoveries on the occurrence of biological oxidationreduction mechanisms in granular elements of the cell. Michaelis had shown in 1898 that mitochondria of unfixed cells reduced oxidation-reduction indicators such as Janus green. , . . It is interesting that Warburg in 1913 found respiration to be associated with granular, insoluble elements of cell structure, which he recovered b y filtration of tissues dispersions, but the significance of his observation was not developed further by cytologists of the day.”

74

C . F. CAUSE

Almost 40 years later, in the early 1950’s, the mitochondria were first recognized to be the “power plants” of cells. At that time the isolated mitochondria were found to carry out the oxidation, and, most importantly, oxidative phosphorylation, at a rate compatible with that of the intact cell. On the other hand, the isolated mitochondria failed to catalyze the reactions of glycolysis, indicating the specificity of respiratory enzyme localization in mitochondria. Since the early 1950’s, biochemical studies of the enzymic mechanisms of the oxidative cycles, electron transport, and oxidative phosphorylation have proceeded in confluence with studies of the structure and cell biology of the mitochondrion. The molecular structure and the dynamic function of the mitochondrion are now beginning to illuminate and define each other. In the mechanism of energy-coupled respiration may lie the secret of the ultrastructure of the mitochondrion and, conversely, in the molecular organization of the mitochondrion may lie the secret of electron transport. The central objectives in attaining a molecular description of the mitochondrion are the isolation of the enzymic components of the oxidative cycles, definition of their molecular structure and action, analysis of their participation in the multi-enzyme systems in the mitochondrion, and mapping of their location in the mitochondrial structure. Another characteristic of mitochondrial enzymology is the phenomenon of compartmentation of enzymes and substrates in the mitochondrial structure. These features are intricately involved in the control and channeling of enzymic pathways within the mitochondrion and in its metabolic interactions with the surrounding cytoplasmic matrix (Lehninger, 1964). If at the time of Warburg’s early work on the impaired respiration of tumors the concept of oxidative metabolism was entirely “scalar,” it is now much more “vectorial,” and therefore an analysis of a possible reflection of the malignant state in the distorted structure of mitochondria is of great interest. The molecular organization of tumor mitochondria is at present but little investigated, and in his excellent monograph entitled “The Mitochondrion,” Lehninger (1964)has only a few words to say on this subject. He notes that integration of mitochondrial respiration with extramitochondrial glycolysis is distorted in tumor cells, presumably in view of the genetic deletion of some enzymes. However, there is now significant evidence that mechanochemical activities of the mitochondrial membrane are different in normal and in tumor cells. Changes of mitochondrial shape and volume have recently been found to be coupled to the energy-conserving mech-

MICROBIAL MODELS OF TUMOR METABOLISM

75

anisms of the respiratory chain. When isolated mitochondria respire in the absence of phosphate acceptor, they undergo swelling with uptake of water (and solutes) from the medium. On the other hand, when respiration is poisoned or the mitochondria are kept anaerobic, they do not swell. The reverse process, the shrinkage or contraction of mitochondria, with extrusion of water, may be brought about by instituting phosphorylating respiration. It was found that mitochondria contain the contractile protein, which is a contraction factor. Ohnishi and Ohnishi (1962), as well as Neifakh and Kazakova (1963), by extraction of mitochondria of normal liver cells with KC1 solutions of high ionic strength, were able to separate a protein that was similar in many respects to the actomyosin of skeletal muscle. It is of considerable interest therefore that Neifakh et al. (1964) reported the failure of all attempts to extract the contractile protein from mitochondria of tumor cells. This observation does not mean that the contractile protein is absent from tumor mitochondria, particularly in view of the fact that the latter display marked contractility. Nevertheless, this observation clearly indicates certain anomalies in the structure and properties of contractile protein in tumor mitochondria which make impossible extraction by a procedure appropriate for normal cells. It is clear that the nature of mitochondrial contractile protein and of its distortion in the tumor cell deserve further study. Neifakh et al. (1965) suggested that mitochondria may play an important part in the control of glycolysis, and that distorted structure of contractile protein in tumor mitochondria may be related to impaired control of glycolysis in tumor cells. It was in fact shown that liver, heart, brain and tumor cell mitochondria release into incubation medium glycolysis-stimulating factors (Neifakh et al., 1964). One of these factors is nicotinamide-adenine dinucleotide (NAD), and in normal cells its excretion depends upon reversible changes in the structure of mitochondrial membranes. Preincubation of mitochondria with adenosine triphosphate (ATP), magnesium, or ethylenediaminetetracetate (EDTA), i.e., with substances that cause membranes to contract, results in a strong decrease in the release of NAD, as shown in Fig. 1. Preincubation with phosphate or potassium chloride, causing the relaxation and swelling of mitochondrial membranes, leads to intensified release of NAD. In the case of tumor mitochondria the results are quite different (Fig. 1). The liberation of NAD from tumor mitochondria does not depend upon their swelling or contraction, and is out of any control. It seems highly probable therefore that in the tumor cell, due to a genetic abnormality in the structure of contractile protein, mitochondrial membranes are exempt from control, so

76

G . F. GAUSE

mATP EDTA KCI

Mouse liver

Ehrlich ascites carcinoma

Fig. 1. T h e effect of ATP, EDTA, KCI, and phosphate upon the release of NAD from normal and tumor mitochondria (from Neifakh et al., 1964).

that they release the stimulating factors incessantly. Tumor mitochondira are “leaky” mitochondria. This may be the condition responsible for monotonous continuation of aerobic glycolysis, occurring at a high level in the tumor cell. Lehninger (1964, p. 201) concludes that the swelling-contraction cycle of mitochondria may be an important cybernetic mechanism permitting self-adjustment of respiration and phosphorylation in the intact cell. Because these mechanochemical changes are brought about by intermediate reactions of oxidative phosphorylation, they may give some important clues to molecular relationships between the mechanical changes and the enzymes of the respiratory chain. It is of considerable interest that certain defects in the structure and properties of the contractile protein in tumor mitochondria may be correlated with the impaired control of respiration and glycolysis in the tumor cell. One possible approach in the study of molecular organization of tumor mitochondria may be concerned with the study of their cytochromes. In fact, cytochromes are important members of the respiratory assembly of the highly integrated mitochondrion, that is, of an organized arrangement of macromolecules located in a geometrical pattern favorable for their interaction. It is only reasonable to expect that any distortion in the molecular organization of tumor mitochondria may be reflected by alterations of cytochrome pattern. Therefore it is of considerable interest that in the Zajdela ascites hepatoma of the rat, as well as in the Ehrlich ascites tumor of the

MICROBIAL MODELS OF TUMOR METABOLISM

77

mouse, the complete or nearly complete absence of cytochrome b has been recorded in a low-temperature spectrographic study (Monier et al., 1959). Although there is now considerable evidence for the role of mitochondria in metabolic regulation of both respiration and glycolysis, the concept of mutant mitochondria in tumors is supported not only by the data of bioenergetics. Graffi (1940) had shown many years ago that carcinogenic hydrocarbons are preferentially accumulated in mitochondria, and it appeared probable that the malignization might be based upon a mutation of mitochondria. The most recent data from the laboratory of the same author (Graffi et al., 1965) demonstrate significant differences of mitochondrial protein synthesis in vitro in tumor and normal cells. It was observed that the rate of incorporation of amino acids into the mitochondrial protein of malignant tumors is very low. These differences may finally depend upon differences in nucleic acids governing this process. This possibility is being investigated now in a number of laboratories. Fiala and Fiala (1966a) concluded that the tumor mitochondria differ from normal liver mitochondria by the depletion of the structural protein. These authors also conclude that the scarcity of mitochondria, impaired respiration, and increased anaerobic glycolysis are fundamental features of the “minimal deviation” hepatoma (Fiala and Fiala, 1966b). These observations are particularly interesting in the light of recent work on biogenesis of mitochondria, indicating that one group of proteins is synthesized inside mitochondria and the other group is synthesized on non-mitochondria1 cytoplasmic ribosomes and later transferred to the mitochondria (Haldar et al., 1966). 111. Disturbance of Control Mechanisms in the Respiratory-Deficient Yeast

A. METABOLISMO F T H E RESPIRATORY-DEFICIENT YEAST One advantage of using microorganisms in the study of biochemical systems is the possibility of isolating mutants and of employing these for better understanding of metabolic organization. Since yeast can acquire necessary energy for growth through fermentation, it can survive with lesions in the respiratory chain, making it possible to study mutants having various blocks in the cytochrome system. Mutants of this type were first observed by stier and Castor (1941), Whelton and Phaff (1947), and much studied by Ephrussi, Slonimski, and other investigators since 1949 (see Slonimski and Ephrussi, (1949). I n fact, more than one hundred papers were published be-

78

G . F. GAUSE

tween 1949 and 1965 dealing with this type of metabolic mutant (Sherman, 1965). Respiratory-deficient mutants of yeast in many cases exceed the parent culture in their glycolytic power, and in view of this combination of impaired respiration with enhanced glycolysis they have been repeatedly discussed as possible micorbial models for metabolic organization of the cancer cell. The literature in this field has been reviewed recently (Gause, 1966), and there is no need to repeat it here. Slonimski and Hirsch (1952) have measured the activities of some enzymes of the glycolytic, tricarboxylic acid cycle and electron transport mechanisms in normally respiring Saccharomyces cerevisiae and in a respiration-deficient mutant. Their findings indicated the mutant yeast lacked cytochrome oxidase, succinic dehydrogenasecytochrome b complex, and NADHz cytochrome c reductase. Only the cytochrome c component was present in normal concentrations in both yeasts. Kovachevich (1964) compared oxidation rates of reduced pyridine nucleotides (NADHZ and nicotinamide-adenine dinucleotide phosphate, NADPHZ) in both normal and respiratory-deficient mutant yeasts in the presence and absence of antimycin A. In normal yeast the bulk of NADHZ and NADPHz oxidations with concomitant cytochrome c reduction occurs via an antimycin A sensitive pathway. In contrast, mutant yeast cell-free extracts demonstrated very low oxidation rates of both NADHZand NADPHZ, as measured by cytochrome c reduction, and both systems were only slightly affected by antimycin A. The very low activity of NADHz cytochrome c reductase in the mutant yeast shown in Table I1 is consistent with observations made earlier by Slonimski and Hirsch (1952). A number of metabolic impairments is associated with respiration deficiency in yeast. Lomander and Gundersen (1963),as well as Avers et al. (1965a) noted, for example, that none of the respiratory-deficient mutants sporulated, while respiratory-competent cells sporulated readily. It is of considerable interest that respiratory-deficient mutants in Saccharomyces cerevisiae are not identical, but belong to a number of different types. The apparent uniformity of mutants, which indeed are much unlike their parents, for some time precluded the recognition of their variety. Avers et al. (1965b) supposed that the variety of mutants has been overlooked by earlier workers because of the lack of suitable methods for identification, and they recognized among the respiratory-deficient mutants of Saccharomyces cerevisiae four

79

MICROBIAL MODELS O F TUMOR METABOLISM TABLE I1 COMPARISON OF OXIDATION RATESOF REDUCEDPYRIDINE NUCLEOTIDES BY CELL-FREEEXTRACTSOF NORMALAND RESPIRATORY-DEFICIENT MUTANTYEAST Saccharomyces cerevisiaeU NADH, as substrate

NADPH2 as substrate

Without antimycin A With antimycin A (1.2 pg./ml.)

Normal yeast

Mutant yeast

Normal yeast

Mutant yeast

74.8*

1.9

30.0

7.4

10.5

1.6

18.6

7.0

From Kovachevich (1964). bActivities are expressed as mpM of ferrocytochrome c formed/minute/0.05 ml. of cell-free extract as measured at 549 mp. a

types differing in the character of alteration of their mitochondria1 enzymes: Mitochondrial cytochrome oxidase Mitochondria1 succinic dehydrogenase

Type 1

Type 2

Type3

Type4

Reduced

Reduced

Absent

Absent

Normal

High

High

Normal

The variety of types of mutants in the respiratory-deficient yeast can be recognized also b y the study of their aerobic glycolysis. Cause et al. (1957) observed a number of mutants in Saccharomyces cere-

visiae with respiration impaired quantitatively in the same degree, which differed in the intensity of their aerobic glycolysis: in some glycolysis was strongly increased, while in others it did not differ from that of their parents. Similar relations were also observed by Trenina et al. (1965) in another yeast, Torulopsis holmii, and these are shown in Table 111. Respiratory-deficient mutants in yeast can be induced by different mutagens, notably by acriflavine (Ephrussi et al., 1949), 5-fluorouracil (Moustacchi and Marcovich, 1963), carcinogenic agent 4-nitroquinoline N-oxide (Mifuchi et al., 1963), and a water-soluble carcinogen, Styryl 430 (Constantin, 1964). Biochemical mechanisms of action of these mutagens are different, and it has been shown, for example, that methylene blue, which by itself is ineffective in the induction of

80

G . F. GAUSE

respiratory-deficient mutants of Succhuromyces cereuisiue, strongly suppresses the induction of these mutants by acriflavine but does not TABLE 111 RESPIRATION AND AEROBIC GLYCOLYSIS IN THE PARENTCULTUREAND RESPIRATORY-DEFICIENT MUTANTSOF Torulopsis holmif'

Torulopsis holmii, strain 424 Mutant 8 Mutant 30

57.8 1.4 1.7

IN THE

125.1 216.6 125.2

From Trenina et al. (1965). TABLE IV AND AEROBIC GLYCOLYSIS IN THE PARENTCULTUREOF RESPIRATION Saccharomyces chevalieri 406 AND IN THE RESPIRATORY-DEFICIENT MUTANTS INDUCED BY VARIOUS MUTAGENS. AVERAGE DATAOF Foun SERIESOF EXPERIMENTS" Culture Parent Mutant A-1 Mutant A-2 Mutant A-3 Mutant A-4 Mutant A-4-2 Mutant A-5 Mutant A-7 Mutant A-8 Mutant A-10 Mutant A-13 Mutant F-1 Mutant F-3 Mutant F-5 Mutant F-5-1 Mutant F-7 Mutant F-8 Mutant F-11 Mutant F-12 Mutant F-19 Mutant F-20 Mutant N-3-3 Mutant N-3-5 Mutant N-4

Mutagen Acriflavine, 50 pg.flml. Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 12 pg./ml. Acriflavine, 12 pg./mI. Acriflavine, 6 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./niI. 5-Fluorouracil, 125 pg./ml. 5-Fluorouracil, 125 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml.

From Gause et al. (1967).

Qo

Qco

50.4 1.8 3.5 4.2 1.2 1.2 1.5 4.9 1.3 3.0 8.5 4.9 5.2 2.5 4.9 2.2 3.6 2.6 2.1 3.6 7.5 3.2 3.2 4.1

261.1 345.2 317.2 333.2 292.9 331.2 265.2 295.2 309.6 338.6 347.9 352.5 371.1 308.1 333.4 447.6 354.3 325.2 299.6 311.6 312.9 343.0 360.5 301.5

81

MICROBIAL MODELS O F TUMOR METABOLISM TABLE V RESPIRATIONAND AEROBICGLYCOLYSIS IN THE PARENTCULTUREOF Saccharomyces ouiformis 493 AND IN THE RESPIRATORY-DEFICIENT MUTANTSINDUCED BY VARIOUS MUTAGENS.AVERAGE DATAOF FOUR SERIES OF EXPERIMENTS" Culture

Mutagen

Qo 2

QcoB

50.8 5.2 3.4 2.7 2.6 2.9 2.5 2.8 1.2 6.3 2.6 3.7 3.6 3.0 1.8 3.3 1.9 1.3 1.5 2.3 1.2 2.0 2.7 3.7 3.0 4.8 3.4 4.3 5.6

72.7 246.6 248.1 260.7 246.4 282.9 217.9 201.4 210.6 237.8 268.0 255.4 205.3 235.0 201.6 262.3 225.9 233.0 229.1 250.1 252.2 260.3 330.9 270.1 241.2 248.4 238.1 232.9 220.6

~

Parent Mutant A-2 Mutant A-3 Mutant A-4 Mutant A-5 Mutant A-6 Mutant A-8 Mutant A-12 Mutant A-13 Mutant A-14 Mutant A-22 Mutant F-1 Mutant F-3 Mutant F-3-2 Mutant F-7 Mutant F-7-2 Mutant F-10 Mutant F-12 Mutant F-13 Mutant F-15 Mutant F-16 Mutant F-18 Mutant N-2 Mutant N-4 Mutant N-5-2 Mutant N-5-3 Mutant N-5-4 Mutant N-10 Mutant N-12

Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 6 pg./ml. Acriflavine, 6 pg./ml. Acriflavine, 6 pg./ml. Acriflavine, 12 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg,/ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 125 pg./ml. 5-Fluorouracil, 125 pg./ml. 5-Fluorouracil, 62 pg,/ml. 5-Fluorouracil, 62 pg./ml. 5-Fluorouracil, 62 pg./ml. 5-Fluorouracil, 31 pg./ml. .~ 4-Nitroquinoline N-oxide, 0.25 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml.

From Cause eta.?.(1967).

show any effect on the frequency of mutations induced by 4-nitroquinoline N-oxide (Morita and Mifuchi, 1965). Gause et aZ. (1967) investigated alterations of aerobic glycolysis in respiratory-deficient mutants of yeast induced by mutagens with different mechanisms of action. Experiments were made with three mutagens (acriflavine, 5-fluorouracil, 4-nitroquinoline N-oxide) employing two species of yeasts, Saccharomyces chevalieri and Saccharomyces oviformis. Tables IV and V show metabolic alterations in respiratory-deficient

82

G . F. CAUSE

mutants of yeast induced by different mutagenic agents. If the rate of aerobic glycolysis of the parent culture is taken for a unit, the degree of increase of glycolysis in mutants can be expressed in the relative form. The results of these calculations are shown on Fig. 2. It can be S. chevolieri

S. owformis

Increase of glycolysis-

FIG.2. The relative increase of aerobic glycolysis in respiratory-deficientmutants of Saccharomyces cheualied and Saccharomyces ouiformis induced by different mutagenic agents (from Gause et al., 1967).

seen that in each species of yeast mutagens of different nature produce approximately the same effect, as far as values of enhanced aerobic glycolysis in these mutants are of the same order of magnitude. At the same time there are very strong differences between different species. In mutants of Saccharomyces chevalieri the aerobic glycolysis is increased on the average by 30%, and in Saccharomyces ouiformis by 230%. This observation is confirmed by the work with another species of yeast, Saccharomyces formosensis, where aerobic glycolysis in mutants is increased by 450%, as can be seen from the data given in Table VI. The increase of aerobic glycolysis evidently depends upon some distortions in mitochondria of mutant yeasts, and it seems that in different species of yeast, mitochondria are differently susceptible to such distortions. These results of the work with microorganisms have much in common with the experience acquired in the study of the action of chemical carcinogens upon mammalian cells (Kaiser and Barton, 1966).

83

MICROBIAL MOLELS O F TUMOR METABOLISM

TABLE VI RESPIRATIONAND AEROBICCLYCOLYSIS IN THE PARENT CULTURE O F Saccharomyces fomosensis 428 AND IN THE RESPIRATORY-DEFICIENT MUTANTS INDUCED BY ACRIFLAVINE. AVERAGEDATAOF FOURSERIES OF EXPERIMENTS* Culture

Qo I

Qco

Parent Mutant A-9 Mutant A-10 Mutant A-11 Mutant A-16 Mutant A-17

42.9 0.8 1.0 1.9 1.5 1.9

47.0 285.8 266.2 232.5 244.7 266.9

a

From Cause et al. (1967).

B. DISTURBANCE OF CONTROL MECHANISMSIN RESPIRATORY-DEFICIENT YEAST

THE

It has been mentioned earlier in this review that a fundamental difference between parent cells and their mutants with the impaired respiration is related to the loss of regulatory mechanisms in mutant cells. This can be clearly seen in the work with respiratory-deficient mutants of Saccharomyces cerevisiae induced by acriflavine, which have been investigated by Gause et al. (1967). Table VII indicates TABLE VII RESPIRATIONAND AEROBIC GLYCOLYSIS IN THE PARENT CULTURE OF Saccharomyces cereuisiae AND IN THE RESPIRATORY-DEFICIENT MUTANTS PREVIOUSLY CULTURED UNDER AEROBIC AND ANAEROBIC CONDITIONS. AVERAGEDATAOF FOURSERIESOF EXPERIMENTS~ Qo

QW

Strain

Aerobic culture

Anaerobic culture

Aerobic culture

Anaerobic culture

Parent Mutant ST-4 Mutant ST-I0

37.5 2.6 3.1

26.1 3.7 3.2

194.9 349.8 313.0

270.8 327.7 304.3

From Cause et al. (1967).

that in the study of metabolism of parent cells previously grown under aerobic or anaerobic conditions, one can see that the respiration is

84

G . F. CAUSE

decreased and glycolysis increased due to low oxygen tension at the time of cultivation. These regulatory mechanisms are entirely lost in mutant cells. Their aerobic glycolysis is not only quantitatively increased but it is no longer regulated, and its intensity does not depend upon oxygen tension at the time of cultivation. Another manifestation of biochemical inflexibility of the respiratorydeficient yeast was revealed in the study of the adaptive formation of ubiquinone or coenzyme Q. As is well known, ubiquinone serves as a carrier in the electron transport system in mitochondria, although there is still considerable uncertainty about its exact role in this process (Lehninger, 1964).The ubiquinone content in the hepatoma is one-quarter to one-tenth of that of the normal liver (Sugimura et al., 1962),and this observation evidently reflects the impaired respiration in tumor cells. It was also recorded that in a respiration-deficient mutant of Saccharomyces cerevisiae induced by acriflavine, the ubiquinone content of aerobically cultured cells ranged from 1/5 to 1/15 of that of the normal parent cells (Sugimura et al., 1964). What is essential, however, is not only the low content of ubiquinone in the cells of the respiratory-deficient mutant yeast, but also a strongly impaired adaptive response of mutants in the synthesis of ubiquinone after aeration. Table VIII shows that after aeration the content of TABLE VIII INCREASE OF THE UBIQUINONE ( U Q ) CONTENT IN ANAEROBICALLY CULTURED CELLS OF Saccharomyces cereuisiae AFTER AERATION"

UQs Strain Parent Respiratory-deficient mutant T-5

a From

Treatment

(pg.1100 mg. N)

Increase

Before aeration After aeration

13 274

x 21.0

Before aeration After aeration

42

9 X

4.6

Sugimura et al. (1964).

ubiquinone in the cells of parent yeasts increases by 21 times, while under similar conditions the content of ubiquinone in the cells of mutants increases only by 4.6 times. The nature of the defect in respiratory-deficient yeast mutants, as it pertains to adaptive ubiquinone synthesis, appears to be complex and its elucidation mdst await further knowledge of the pathways and intermediates involved in the normal synthesis of ubiquinone.

MICROBIAL MODELS O F TUMOR METABOLISM

85

The delay of synthesis of various inducible enzymes has been repeatedly recorded in the work with respiratory-deficient mutants of yeast. Lindegren et al. (1957) noticed the slow adaptation to galactose or maltose fermentation in these mutants. Reilly and Sherman (1965) observed that if parent and mutant cells were first grown for 1 day in glucose medium, and then inoculated into melibiose or raffinose medium, prolonged delays in growth of mutant cultures could be seen. When compared with parents, delays of about 1 day before growth is initiated in melibiose medium, and of over 3 days in raffinose medium have been recorded for respiratory-deficient mutants of the yeast Saccharomyces cerevisiae. This situation is shown in Fig. 3. It is of considerable interest that many defects in synthesis of various inducible enzymes have been recorded also in neoplastic cells (Pitot and Cho, 1965).

[

Melibiose

z f i n o s e

Hours

FIG.3. Adaptation to growth on melibiose and raffinose in normal cells of Saccharomyces cereuisiae (A) and in the respiratory-deficient mutant (B) (from Reilly and Sherman 1965).

c. DISTORTED ORGANIZATION O F MITOCHONDRIAIN T H E RESPIRATORY-DEFICIENT YEAST Respiratory-deficient mutants of yeast fail to form a substantial portion of the electron transport chain and are deficient in cytochromes a and b, ubiquinone, and associated enzymes. T h e simultaneous disappearance of these compounds suggests that the respiratory apparatus of the cell develops, at least in part, as a unit (Lascelles,

86

G . F. CAUSE

1965). It is also clear that the respiratory-deficient yeast represents a unique model for the study of organization of the highly integrated mitochondrion. Sherman and Slonimski (1964) pointed out that respiratory-deficient mutants of yeast lack cytochromes which are strongly particle-bound (cytochromes a and b) and retain cytochromes which are loosely bound (cytochrome c). This could be due to either a modification of a structural component common to several cytochromes, or to a common controlling mechanism. As suggested by Ycas (1956), the formation of one cytochrome could depend on the formation of another, e.g., the absence of cytochrome a may be only a secondary effect of the cytochrome b deficiency. It can be easily envisaged that the structural relationships between different bound cytochromes can take place at different levels of organization of the mitochondrion. For instance, cytochromes a and b could contain an identical polypeptide chain controlled by a single determinant; the formation or attachment of cytochromes a and b to the mitochondrial membrane may be conditioned by another protein. There is some evidence that a specific structural protein, which has precisely the property to form polymers with cytochromes a and b, and not with cytochrome c, does exist in mammalian mitochondria (Criddle ut al., 1962). If the structural protein is under genetic control, then it would be expected that the loss or alteration of this protein could result in the loss or diminution of several cytochromes. At a higher level of organization, multienzyme deficiencies could be due to the alterations in the lipoprotein matrix of the mitochondrial membrane. Whatever the exact nature of the lesions in these respiratory-deficient mutants, it is clear that there should be different ways of producing multienzyme deficiencies in such a highly organized organelle as a mitochondrion. Yotsuyanagi (1962) observed with the electron microscope that respiratory-deficient strains of yeast differed from normal strains by having a modified structure of the mitochondria. He suggests that as far as respiratory enzymes are arranged in the rigid and precise pattern on the mitochondrial membrane, the distorted architecture of mitochondria may represent the cause of the distorted electron transport. It is of great interest that alterations of mitochondrial structural protein in respiratory-deficient mutants of Neurospora have been reported (Woodward and Munkres, 1966). Schatz et al. (1963) studied the structure of mitochondria in the respiratory-deficient mutants of the yeast Saccharomyces cerevisiae and came to the following conclusions: (a) The number of mitochondria per cell in mutants is strongly reduced. ( b )Mitochondria of

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mutants sediment more slowly in the sucrose density gradient; this observation suggests an altered structure of mitochondria in mutants correlating with their loss of cytochromes a and b. ( c )Mitochondria of mutants are labile and more easily breakable in the preparation of a cell-free system. Mahler et al. (1964a) reported that respiratory particles prepared from respiratory-deficient mutants of the yeast Saccharomyces cerevisiae are devoid of cytochrome a and cytochrome b, are incapable of catalyzing the reduction of cytochrome c by succinate and the oxidation of reduced cytochrome c by oxygen, but exhibit a pattern of primary dehydrogenases similar to those of the wild type. The study of antigenic properties of respiratory particles (Mahler et al., 1964b) has shown that respiratory particles from respiratory-deficient mutants of yeast contain a cross-reacting material capable of binding (or reacting with) some components of sera prepared against particles from wildtype cells. An attempt has been made recently to investigate the molecular properties of mitochondrial DNA in the respiratory-deficient yeast (Moustacchi and Williamson, 1966). Density-gradient centrifugation of protoplast lysates of yeast cells indicates that a satellite component corresponding to low-density mitochondrial DNA is absent in the respiratory-deficient mutants of yeast. This problem was investigated in greater detail by Mounolou et al. (1966). They observed in the respiratory-deficient mutants of yeast a specific change in the buoyant density of mitochondria1 DNA. Whether this alteration in density corresponds to changes in base composition of mitochondrial DNA in mutants or to the presence of unknown components remains to be seen. A very important step in the study of respiratory-deficient mutants of yeast was reached with the demonstration of the possibility of cytoplasmic transformation (Tuppy and Wildner, 1965). In these experiments the mitochondria from a wild-type parent yeast Saccharomyces cerevisiae restored the respiratory capacity of the respiratory-deficient mutant yeast. The cells of mutants were converted into spheroplasts and the latter were treated with mitochondria isolated from normally respiring parent cells. A sizable portion of the treated cells acquired the ability to respire and to form normal colonies, indicating that normal mitochondria had been incorporated into the cells of mutants and in this way transformed mutants into parent cells. Very similar results were independently reached by Diacumakos et al. (1965) in the work with the fungus Neurospora crassa. In this case a mitochondrial fraction isolated from an abnormal strain of

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Neurosporu produced drastic changes in the rate of growth, morphology, reproductive characteristics, and cytochrome spectra of normal strains when single hyphal compartments were microinjected and isolated, whereas the mitochondria1 fractions of the wild type produced no effect. These results provide evidence for the transmission of biochemical and biological characters when mitochondria are transferred to new nucleocytoplasmic environments. This transformation of mutants into normal cells, which has been already achieved on microbial models, evidently opens the way for new attempts to investigate whether or not a reversion of the malignant process in mammalian cells is experimentally possible.

IV. Summary

It can be concluded from the discussion and examples presented on the preceding pages that the disturbance of control mechanisms in the respiratory-deficient yeast has much in common with the disturbance of control mechanisms in the metabolism of tumors. In particular, distorted organization of mitochondria in the respiratorydeficient yeast may be instructive as a model for understanding some aspects of molecular organization of tumor mitochondria. A number of similarities between respiratory-deficient mutants of yeast and cells of malignant tumors in the increased but uncontrolled glycolysis, in the defective synthesis of various inducible enzymes, and in some other respects indicate that microbiology and cancer research have much to learn from each other. REFERENCES Avers, C. J., Rancourt, M. W., and Lin, F. H. (1965a). Proc. Natl. Acud. Sci. U S . 54, 527-535. Avers, C. J., Pfeffer, C. R., and Rancourt, M. W. (1965b).J. Bucteriol. 90, 481-494. Burk, D., Woods, M., and Hunter, J. (1965). Proc. Am. Assoc. Cancer Res. 6,9. Burk, D., Woods, M., and Hunter, J. (1967).J. Natl. Cancer Inst. (in press). Chance, B., and Hess, B. (1959). Science 129,700-708. Constantin, T. (1964). Compt. Rend. Soc. B i d . 158,2263-2268. Criddle, R. S . , Bock, R. M., Green, D. E., and Tisdale, H. (1962). Biochemistry 1, 827-842. Diacumakos, E. G., Garnjobst, L., and Tatum, E. L. (1965). J. Cell B i d . 26, 427-443. Ephrussi, B., Hottinguer, H., and Chimenes, A. M. (1949). Ann. Inst. Pasteur 76, 351-362. Fiala, S., and Fiala, A. (1966a). Proc. Am. Assoc. Cancer Res.7,20. Fiala, S., and Fiala, A. (1966b).Naturwissenschaften 53,228. Cause, G . F. (1966). “Microbial Models of Cancer Cells.” Saunders, Philadelphia, Pennsylvania.

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Cause, G. F., Kochetkova, G. V., and Vladimirova, G. B. (1957). Dokl. Akad. Nauk SSSR 117,138-141. Cause, G. F., Kochetkova, G. V., Sarukhanova, L. E., and Vladimirova, G. B. (1967). Mikrobiologiya 36,340-352. Graffi, A. (1940). Z. Krebsforsch. 50,196-219. Graffi, A., Butschak, C., and Schneider, E. S. (1965). Biochem. Biophys. Res. Commun. 21,418-423. Haldar, D., Freeman, K., andWork,T. S. (1966).Nature211,9-11. Kaiser, H. E., and Barton, J. C. (1966). Federation Proc. 25 (2), 661. Kovachevich, R. (1964). Biochem. Biophys. Res. Commun. 14,48-53. Lascelles, J. (1965). In “Function and Structure in Micro-Organisms,” Proc. 15th Symp. Soc. Gen. Microbiol., London (M. Pollock and M. Richmond, eds.), pp. 32-56. Cambridge University Przss. Lehninger, A. L. (1964). “The Mitochondrion. Molecular Basis of Structure and Function.” Benjamin, New York Lindegren, C., Ogur, M., Pittman, D., and Lindegren, G. (1957). Science 126,398-399. Lomander, L., and Gundersen, K. (1963).J. Bacteriol. 86,956-965. Mahler, H. R., Mackler, B., Grandchamp, S., and Slonimski, P. P. (1964a).Biochemistry 3,668-677. Mahler, H. R., Mackler, B., Slonimski, P. P., and Grandchamp, S. (196413).Biochemistry 3,677-682. Malmgren, R. M., and Flanigan, C. C. (1955). Cancer Res. 15,473-478. Mifuchi, I., Morita, T., Yanagihara, Y., Hosoi, M., and Nishida, M. (1963). Japan J . Microbial. 7, 69-77. Monier, R., Zaidela, F., Chaix, P., and Petit, J . F. (1959). Cancer Res. 19, 927-934. Morita, T., and Mifuchi, I. (l965).JapanJ. Microbiol. 9,123-129. Morris, H. P., Sidransky, H., Wagner, B. P., and Dyer, H. M. (1960). Cancer Res. 20, 1252-1254. Mounolou, J. C., Jakob, H., and Slonimski, P. P. (1966). Biochem. Biophys. Res. Commun. 24,218-223. Moustacchi, E., and Marcovich, H. (1963). Compt. Rend. 256,5646-5649. Moustacchi, E., and Williamson, D. H. (1966). Biochem. Biophys. Res. Commun. 23, 56-61. Neifakh, S. A., and Kazakova, T. B. (1963). Nature 197,1106. Neifakh, S. A,, Gaitskhoki, V. S., and Kazakova, T. B. (1964).Acta, Unio-Intern. Contra Cancrum 20,1285-1287. Neifakh, S. A., Avramov, J. A., Gaitskhoki, V. S., Kazakova, T. B., Monakhov, N. K., Repin, V. S., Turovski, V. S., and Vassiletz, I. M. (1965). Biochim. Biophys. Acta 100,329-343. Ohnishi, Ts., and Ohnishi, To. (1962). J . Biochem. (Tokyo) 51,380-384. Pitot, H. C.,andCho, Y. S . (1965). Progr. Exptl. TumorRes. 7,158-223. Potter, V. R. (1964). In “Cellular Control Mechanisms and Cancer” (P. Emmelot and 0.Muhlbock, eds.), pp. 190-210. Elsevier, Amsterdam. Potter, V. R., Pitot, H. C., Ono, T., and Morris, H. P. (1960). Cancer Res. 20,1255-1261. Reilly, C., and Sherman, F. (1965). Biochim. Biophys. Acta 95,640-651. Schatz, G., Tuppy, H., and Klima, J. (1963). Z. Naturj‘orsch. 18b, 145-153. Sherman, F. (1965). Colloq. Intern. Centre Natl. Rech. Sci. (Paris) 124,465-479. Sherman, F., and Slonimski, P. P. (1964).Biochim. Biophys. Acta 90,l-15. Slonimski, P. P., and Ephrussi, B. (1949).Ann. Znst. Pasteur 77,47-53.

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Slonimski, P. P., and Hirsch, H.M . (1952).Compt. Rend. 235,741-743. Stier, T.,and Castor, J. (1941)~. Gen.Physiol. 25,299-233. Sugimura, T.,Okabe, K., and Baba, T.(1962).Gunn 53,171-181. Sugimura, T.,Okabe, K., and Rudney, H. (1964).Biochim. Biophys. Actu 82,350-354. Trenina, G . A.,Bibikova, M. V., and Samkhanova, L. E. (1965).Mikrobiologiya 34,300-

304. Tuppy, H., and Wildner, G. (1965).Biochem. Biophys. Res. Commun. 20, 733-738. Warburg, 0.(1923).Biochem. 2. 142,317-351. Warburg, 0.(1926).The Metabolism of Tumors. Constable Press, London. Warburg, O., Gawehn, K., Geissler, A., Kayser, D., and Lorenz, S. (1965).Klin. Wochschr. 43,289-293. Whelton, R., and Phaff, H. (1947).Science 105,44-45. Woodward, D.O.,and Munkres, K. D.(1966).PTOC.Nutl. Acud. Sci. U.S. 55,872-880. Ycas, M.(1956).E x p t l . Cell Res. 11, 1-6. Yotsuyanagi, Y. (1962).J . Ultrustruct. Res. 7 , 141-158.

Cel I ulose

CI

nd Cel lulolysis '

BIRGITTA NORKRANS Department of Marine Botany, University of Goteborg, Goteborg, Sweden

I. Introduction .............................................................. 11. Cell Wall Morphology and Chemistry

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

111. Cellulose Chemistry and Supramolecular Morphology ... IV. Biosynthesis of Cell Wall Polysaccharides .................... A. Cellulose Synthesis ............................................. B. Resynthesis of Cellulose ............................ ... V. Degradation of Cellulose by Bacteria and Fungi ........... A. Degradation of Cellulose by Bacteria ..................... B. Degradation of Cellulose by Fungi ........................ C. Cellulolytic Enzymes ........... D. Inhibition of Cellulases ........ VI. Applications for Cellulases ......................................... References ......

91 92 95 98 99 101 101 102 103 109 122 124 125

I . Introduction Cellulolytic microorganisms are involved in the deterioration of all types of cellulosic material, contributing to man's material needs. They are destructive to the raw materials and end products and detrimental in the manufacturing processes of wood, cotton, and other natural textile fibers. They cause damage to fruits and vegetables, and many species are phytopathogenic. The annual damage by woodrotting microorganisms in the United States amounts to 3 billion dollars. Furthermore, it has been reported that attacks solely on spruce by just one organism, the root-rotting Polyporous annosus, in Sweden cause an annual loss of 100-200 million Crowns. Even if similar economic losses caused by cellulolytic organisms are calculated for all parts of the world, it must be realized that this negative role is very small in comparison with their vital role as regulators of the dynamic equilibrium in nature. Calculations for the organic net production by the photosynthetic process indicate that about 3 x 1O'O tons of carbon in the form of COz are transformed yearly into plant materials over the earth as a whole. Approximately one third of the organic material produced is cellulose, which occurs in most plants as the skeletal 'The survey of literature pertaining to this review was concluded in November 1966. The references cited have partly been selected for the scope of information provided by summarizing reviews, lectures at Symposia, etc. than in recognition of original sources.

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substance of the cell. If microbial degradation of cellulose did not occur, the atmospheric carbon dioxide pool, which is generally the limiting factor for photosynthesis in nature, would -because of the equilibrium between carbon dioxide in the air, that dissolved in the sea, and carbonates in marine sediments (Sillen, 1963)-presumably not be consumed within decades as has been theorized, but dead vegetation would pile up and suppress new production. The consequences for a world population growing in a logarithmic scale can easily be imagined. In working toward the prevention of the disintegration of useful cellulosic material and toward facilitating the breakdown of dead plant debris, the enzymic process of the biological degradation of cellulose has to be clarified. Native cellulose is not simply a glucose anhydride chain, showing a characteristic insolubility in water at even very short chain lengths, but a high polymer arranged in supramolecular structures, laid down in cells and tissues and embedded in a noncellulosic matrix, often surrounded by lignin. These structural features hardly facilitate the understanding of the degradation processes. Several investigators have attacked the problem, and their work has been reviewed in extensive monographs and reviews (e.g., Cowling, 1958; Fghraeus, 1958; Gascoigne and Gascoigne, 1960; King, 1961; Seifert, 1962; Siu, 1951; Wood, 1960). Three years ago the present author compiled a review on the degradation of cellulose (Norkrans, 1963a). Since then, research work within this field has enlarged our knowledge about the micromorphology of wood decay and of the initial phase of degradation of native crystalline cellulose. It has brought new ideas to our concept of the structure of cellulose and its biosynthesis, which is of importance for our understanding of cellulose degradation. In the following discussion these topics will be dealt with in greater detail.

II. Cell Wall Morphology and Chemistry Microscopically, three fundamental parts associated with the plant cell wall can b e recognized: primary wall, defined as the part of the wall produced during surface growth; secondary wall, produced after the surface growth has ceased; and between adjacent cells in the tissue there exists the middle lamella. In the secondary wall of normal xylem fiber or tracheid, formed when successive polysaccharide layers are deposited next to each other, three distinct layers can be demonstrated. The outer layer, S,, is a thin transition layer, the middle S2 layer forms the bulk of the secondary wall, whereas S3, when

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present, is a thin layer adjacent to the cell lumen. In wood fiber, S z is 1-5 p thick, S1 and Ss 0.1-0.2 p. Chemical fractionation of the cell wall by means of dilute alkali (usually 17.5% NaOH) divides the polysaccharide compounds into an alkali-insoluble component, a-cellulose, and an alkali-soluble part comprising noncellulosic polysaccharides, i.e., “hemicelluloses” and pectic substances. Most chemical analyses deal with the overall composition of whole plant tissues, and deductions about the cell wall composition are made on the assumption that the polysaccharides and lignin are derived solely from these structures. With cambial and other soft tissues having no secondary thickening, the composition of the primary cell wall may be studied directly. Setterfield and Bayley (1961) and later Roelofsen (1965) summarize quantitative data from about thirty investigations dealing with primary wall material from different species and parts of plants. Generally, the a-cellulose constitutes only 20-35% of the dry wall materials. They also include investigations of cotton, the unicellular seed hair, which has about 50% cellulose in the primary wall (see Tripp et al., 1954), and a study of separate cambial cells of Acer pseudoplatanus (Northcote, 1963), which has been subjected to enzymic fractionation, instead of being analyzed by the usual chemical method (Lamport, 1965). With regard to the noncellulosic polysaccharides, it is more difficult to give precise figures for their percentage composition, mainly because of the difficulties involved in subgrouping. If, however, galactans, arabans, and galacturonides are grouped as a “pectic triad,” 10-20% are pectic substances, 3550% may be considered to consist of hemicelluloses. Proteins comprise 3-lo%, and lipids 2-7%. By far the most common analyses, however, deal with woody tissues from conifers (softwoods) or deciduous trees (hardwoods) in which xylematic cells with secondary thickening dominate. Thus the analytical values mainly reflect the composition of secondary walls. An average cellulose content of 43+2% for both hardwood and softwood, and 1-4% of pectic substances has been reported (Timell, 1965b). Hemicelluloses amount to about 40% in hardwood and 30%in softwood (Meier, 1964). The chemistry of wood hemicelluloses has recently been discussed in detail (Timell, 1964,1965a). By analyzing fibers from the outermost part of the xylem of birch, pine, and spruce at various stages of maturation, Meier and co-workers (Meier, 1964) have been able to deduce the proportions of the indi-

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vidual polysaccharides in the different layers of the fiber wall. For all these three woods, the cellulose and hemicellulose content is lowest in the middle lamella and primary wall layer (taken together at the analysis), increasing successively in the inner part of the cell to a maximum in the Sz-& layers. Pectic substances are mainly present in the middle lamella-primary wall layer. Figure l a illustrates this general trend. Microfibril (crystalline phase)

-

-

Continuous matrix (amorphous)

r

M I

1

I

I

I I

I

I I ~

Cellulose 50 I 55

I

YO

1

“Hemicellulose“

%

I

I

I

I I I

GDP-glucose

I

48 I 64

1 I

I 48 tI

24 I

P e ic substances -

B

I

Soluble pool of hexose phosphate

Glucose

2

60

-;j 35 24

UDP-glucose UDP-glucuronic acid UDP-xylose GOLGI BODY UDP-galactose UDP-galacturonic acid

P 46

FIG. 1. a. The cell wall, its different layers and their relative amounts (percentage) of chemical constituents in xylem fibers of pine (p)and birch (b). M, middle lamella; P, primary wall; S, secondary wall; S,, Sz, and S,, its different layers; S:% adjacent to cell lumen. The analytical data derive from Meier (1964), where values from pine and spruce were very much the same. Values for glucuronoxylan, by far the most important “hemicellulose” in hardwoods, have been given here as percentages of “hemicellulose” in birch. (A small percentage of glucomannan was also found in each layer.) For the same reason, the percentage glucomannan has been given as pine “hemicellulose.” In this case about 12% glucurono-arabino-xylan was also present in each layer.) Values for arabinan and galactan have been added and given as “pectic substances” (to which the nonanalyzed amounts of pectic acids should be added to get a complete account of pectic substances). In the Si pine value of 9% given, 8% derive from galactan, which might be due to the presence of the “hemicellulosic” galacto-glucomannan). b. Differences in (1) the nucleotide component of the precursors and ( 2 )possible sites of synthesis for the polvsaccharides formed by a I l l a i i t c,ell. nccordinq to tlw l r \ . l ~ o t l i c ~ s iII!,s Northcote and Pickett-Heaps (1966), when glucose was offered to the cell. Otherwise the plastid sugar will serve as donator to the hexose-phosphate pool.

CELLULOSE AND CELLULOLYSIS

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The final phase in the differentiation of fibers and tracheids is that of lignification. Basically, lignin consists of a three-dimensional polymer of phenylpropane units, present as guaiacylpropane in softwoods and guaiacyl- and syringylpropane in hardwoods. Lignin synthesis in vitro and in vivo has been treated by, e.g., Freudenberg (1964) and Neish (1964), and in a very readable article Stewart (1966) discusses the course of lignification during fiber senescence and the postmortem enzymic reactions. In softwoods, about 60% of the total lignin (amounting to 30%) is localized in the region of the lamella and primary wall, the corresponding figure for hardwoods, which contain 20% lignin, being about 90% (Meier, 1964). For some fiber strands in phloem of flax, hemp, jute, and ramie, the a-cellulose amounts to about 70% (Treiber, 1957), for cotton to about 90%. Cotton has about 5% noncellulosic polysaccharides, and no lignin. 111. Cellulose Chemistry a n d Supramolecular Morphology

Cellulose is a high polymer of p-1,4-linked D-glucose residues. All methods for the isolation, purification, and solubilization of cellulose seem to depolymerize the molecule more or less. However, by improved methods giving a minimum of depolymerization, higher values for the degree of polymerization (DP)are obtained. Goring and Time11 (1962) found DP values of 8000 to 10,000 for wood cellulose. Similar values were found for all the angiosperms, gymnosperms, and ferns tested. Species of Equisetum, Lycopodiurn, and Psiloturn, descendants of plants preceding the ferns in the course of evolution, had DP values of 2000-4000. A value as high as 15,000 was found for cellulose from unopened cotton balls, corresponding to a molecular weight of 2.4 x lo6and a chain length of about 7 p (unfolded chain). Marx-Figini and Schulz (1966b)obtained the same values for cotton, and are of the opinion that cellulose derived from the secondary wall of all higher plants will have a DP of about that magnitude. Native cellulose is an aggregate of well-defined partly crystalline microfibrils of indefinite length. Three different morphological models have been proposed. 1. From early X-ray diffraction studies on cellulose, its partially crystalline structure was proposed to fit into a model called the fringe micelle. When the concept of this morphological model was more developed, cellulose could be described as an aggregate of individual glucose anhydride chains arranged more or less parallel to

96

BIAGITTA NORKRANS

each other in the microfibril. This parallel arrangement occurs to a much lesser degree in amorphous and paracrystalline regions, whereas in crystallinic micelles (or crystallites) the chains are oriented strictly parallel by means of hydrogen bonds. Micelles measure at least 600 A along the fiber axes and about 50 to 60 A perpendicular to the axis, corresponding to a maximum of 100 in electron microscopical studies (RBnby, 1958);the cellulose molecules are believed to be long enough to pass through several micelles (Fig. 2a). The microfibril has been described as a flat ribbon, according to most authors 100 to 300 A wide, and 40 to 100 thick. Frey Wyssling (1959) and Muhlethaler (1960) have a somewhat different concept. They envisage “elementary fibrils” of 35 x 35 A, each having paracrystalline sheaths, which would occur either freely or aggregated. All experimental values for microfibrils found by them or others should thus be considered as multiples of 35

A

A

A.

a

b

C

-Y

FIG.2. Morphological models of native cellulose. a. The fringe micelle. Models with folded cellulose chains: b. Folded along the fiber axis according to Marx-Figini and Schulz (1966a,b). c. Molecules forming a flat ribbon by folding back and forth perpendicular to the ribbon axis, the ribbon being wound into a helix according to Manley (1964) (after Marx-Figini and Schulz, 1966b).

Since the concept of a folding process generally associated with the crystallization of macromolecules is gaining ground (cf. RBnby and Noe, 1961; Davidovits, 1966), this idea has been adopted even for the molecular morphology of cellulose (models 2 and 3).

CELLULOSE AND CELLULOLYSIS

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2. Manley (1964) made electron microscopical studies of aqueous suspensions of microfibrils from ramie, obtained by ultrasonic disintegration, by using the negative staining technique. The individual filament had a diameter of 35 and appeared to have a periodic variation along its length, giving it a beaded appearance. The same was observed in preparations from cotton, wood, and bacterial cellulose. According to Manley, the “beads” may possibly appear if the microfibril is composed of a flat ribbon wound into a tight helix. From X-ray diffraction studies it is known that the molecular chain axis is arranged parallel to the length of the microfibrils. In Manley’s folded model, this is achieved in the following way: the chain molecules form a flat ribbon by folding back and forth in a “concertinafashion,” nearly perpendicular to the ribbon axis, the ribbon then being wound into a tight helix (see Fig. 2c). Manley (1965a) suggested 4 to 5 glucose units in the fold and hypothesized that hydrolysis of cellulose involves preferential attack at the chain folds. When studying a number of regenerated celluloses, Manley (1965b) found that they were apparently also composed of filamentary structure elements, remarkably similar to those in native celluloses. The average length of the filaments reconstituted in vitro was found to increase with the molecular weight of the dissolved cellulose. It means that the elementary microfibril, characteristic of natural as well as of regenerated celluloses, is in fact a cellulose molecule. 3. Marx-Figini and Schulz (1966a) suggested a flat ribbon formed by the molecule chain folded along the ribbon axis (see Fig. 2b). Heyn (1966) made observations on cell walls of cotton, ramie, and jute fibers by high-resolution electron microscopy on negatively stained ultrathin sections. This new technique has advantages over the study of isolated fibers used hitherto in that the original arrangement of microfibrils is better preserved and their collapse into larger fibrillar units is prevented. Again, a certain periodicity could be observed, giving a beaded appearance to the microfibril, measuring about 35 A in diameter as determined by X-ray diffraction. Furthermore, it was found that the average distance between neighboring fibrils is equal to, or less than, the diameter of the microfibril, constituting the maximum space which can be occupied by amorphous material. These values, however, will naturally vary with the degree of swelling. Judging from the photomicrographs, Heyn’s material gives always a beaded appearance. When discussing the probability for a folded cellulose molecule, Muhlethaler (1965)points out that a few molecules might be folded, but it is unlikely that all cellulose

A

98

BIRGITTA NORKRANS

molecules are folded in the native material, because many of the physical properties, e.g., tensile strength, can only be explained if straight molecules are present. None of the fibril structures, however, has been definitely proved, and more work has to be done in order to prove or disprove them. The microfibrils are embedded in a continuous matrix of noncellulosic polysaccharides. Such an organization into microfibrils and matrix can be distinguished during the whole wall development. The microfibrils are oriented a t random in the primary wall, and in a more or less steep helix in the different layers of the secondary wall. The orientation of microfibrils has been extensively discussed (see Roelofsen, 1965). Whether or not any chemical linkage exists between cellulose and hemicelluloses, or between cellulose and incrusting material such as lignin, has been discussed several times. According to Lindberg (1960) and Meier (1964), linkages between cellulose and lignin seem improbable, although there are indications for linkages between hemicellulose and lignin. The same authors also point to the lack of evidence for chemical linkages between cellulose and hemicelluloses, or for the intermixing of, e.g., xylose residues with the glucose residues into the cellulose chain. The question of such chemical associations has arisen because of the difficulty in removing from a cellulose sample all of the hemicellulose components, chiefly xylose residues from hardwood, and mannose residues from softwood, and the difficulty in obtaining glucose as the sole sugar upon cellulose hydrolysis. Increasing knowledge of the biosynthesis of cellulose and other complex polysaccharides in the plant cell wall, however, should eventually yield definite information about the composition and structure of cellulose. IV. Biosynthesis of Cell Wall Polysaccharides Recently, Northcote and Pickett-Heaps (1966) presented a hypothesis for the localization and course of formation of cell wall polysaccharides. Their hypothesis is based on radioautographic studies of root tips of wheat, incubated with D- (1- or 6-) g l ~ c o s e - ~ H for short periods, followed by incubation with unlabeled glucose. The incorporation of labeled material was followed by means of electron microscopy on thin tissue sections and by chemical analysis. Within the Golgi apparatus (a cytoplasmic organelle consisting of a stack of about six cisternae, each 0.6 to 1p across, arranged on top of one another like a pile of plates and bound by a unit membrane) a pool of precursors for the synthesis of polysaccharides, containing galactose,

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galacturonic acid, and arabinose occurs. After the synthesis of these polysaccharides, which are probably identical with pectic substances, they are passed to the vesicles associated with the Golgi bodies, and transferred within these across the cytoplasmic membrane to be incorporated into the cell wall. Later on, a vesicle transport of polysaccharides from the hemicellulose fraction containing glucose, xylose, glucuronic acid, and mannose residues, may take place. The labeled glucose derivatives, which probably do not emerge from the Golgi body, are not immediately available for cellulose synthesis. From work by Hassid and co-workers on the synthesis of pectins and hemicelluloses by cell-free enzyme preparations (Villemez et al., 1965; Pridham and Hassid, 1966), it seems reasonable to assume that uridine-diphosphate sugars and sugar derivatives serve as precursors in these syntheses. In addition, Barber and Hassid (1965) demonstrated cellulose synthesis from guanosine-diphosphate-D-glucose, (GDP-glucose), with particulate enzyme preparations from cotton balls. Four to eight-day-old balls were the most active ones. Since GDP-glucose could not be replaced by other glucose nucleotides such as UDP-, ADP-, or CDP-glucose, both the nucleotide precursors and the site of the synthesis within the cell must be different for cellulosic and noncellulosic polysaccharides, which is illustrated in Fig. lb, modified from Northcote and Pickett-Heaps (1966). Ledbetter and Porter (1963) first demonstrated microtubules in plant cells. They suggested that these structures, situated just beneath the surface of the protoplast, and reflecting the orientation of the microfibrils in the adjacent cell wall, may be involved in fibril formation. Pickett-Heaps and Northcote (1966) and Pickett-Heaps (1966) further develop this idea and also propose the concept of the endoplasmic reticulum functioning in the transport and synthesis of cell wall material.

A. CELLULOSE SYNTHESIS Our knowledge of cellulose synthesis is very incomplete and the mechanisms for the formation of the supramolecular structure will not be understood until we know: (1)the biochemical pathway for the formation of the precursor of the glucose monomer and its polymerization into a p-1,4 glucose anhydride chain, (2) its transformation into a microfibril, and (3) the incorporation and orientation of the microfibrils into the material comprising the plant cell wall. Only a few years ago, almost all available information dealt with cellulose formed as a “by-product” by some bacteria, notably Acetabacter xylinum, studied mainly by Colvin and co-workers. Their concept of this

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synthesis (Colvin, 1964; Dennis and Colvin, 1965) is summarized by the following formulation: glucose, or a glucose precursor such as dihydroxyacetone, gluconate, or related compound, enters the cytoplasmic membrane, where it is activated. (The route by means of glucose 1-phosphate and UTP to UDPG could not be found, and neither guanosine, GDP, or GTP seemed to have any effect on the synthesis.) The glucose residue is then transferred to a lipid carrier, presumably a lipophilic 30-carbon long polyhydroxy alcohol, which diffuses across the cell wall and attaches the glucose residue presumably directly to the tip of the elongating microfibril, occurring free in the medium. The microfibril thus grows by end synthesis and is not produced by aggregation of preformed chains. Formation of the microfibril is, however, still a matter of dispute (cf. Ben-Hayyim and Ohad, 1965; for further references, see Horecker, 1966). It is probably synthesized directly in crystalline form (Sumi et al., 1963). Marx-Figini and Schulz (1966a,b) investigated cellulose synthesis by a series of mainly kinetic studies of cellulose formation in cotton balls, during a period of 24 to 72 days after pollination. According to these authors, cellulose synthesis takes place in two distinct phases, the first one proceeding slowly and yielding only a small amount of cellulose of nonuniform molecular size, D P 2000 to 6000. The second phase is rapid, resulting in the bulk of the cellulose being formed. Independent of time elapsed and amount of glucose available for synthesis, cellulose of a high and uniform degree of polymerization, D P about 14,000, is formed in this phase. The first phase, lasting up to 35 days, corresponds to the formation of primary wall, the second to that of the secondary wall. Low D P values of cellulose from the primary wall have been reported previously (Wardrop, 1962). In order to explain the formation of the uniformly large macromolecules, a template mechanism was suggested; the template could be situated inside the microtubules. The enzymic combination of activated glucose molecules with the cellulose chain along the template is associated with the folding of the chain. The synthesized, folded chain may be fixed at the growing end of the fibril by hydrogen bridges. If the synthesis of “secondary wall cellulose” is dependent on structures like endoplasmic reticulum, microtubules, and cytoplasmic membrane, many of the unsuccessful efforts to synthesize cellulose in uitro might be attributable to the lack of undamaged structures in the enzyme preparations. In the aforementioned in uitm synthesis by an enzyme system from young cotton balls (Barber and Hassid, 1965), not only GDP-glucose

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but also GDP-mannose was incorporated in the polysaccharide formed. According to Marx-Figini and Schulz this polysaccharide corresponds to “primary wall cellulose.” T h e difference between primary wall cellulose’’ and glucomannan, characterized as a hemicellulose, may perhaps be quantitative rather than qualitative and it brings to mind Preston’s (1961) statement from algal studies: “Cellulose is a mixed polysaccharide.” ‘I

B. RESYNTHESIS OF CELLULOSE Much of the aforementioned data is based on the assumption that cellulose is irreversibly deposited. This may be true for mature plant tissues; in young cells, however, breakdown of cellulose b y enzymes of endogenous origin has been discussed as a factor affecting the plasticity of the primary wall (Matchett and Nance, 1962; Maclachlan and Perrault, 1964) and involved in cell fusion processes (Sassen, 1965).Cellulases have been found in these cases, as well as in some plants studied by Reese and Mandels (1963). Evidence supporting the idea of reversible cellulose synthesis has been obtained by 14C studies on isolated wheat roots b y Margerie and Lenoel(l961). It has been shown later (Peaid-Lenoel and Axelos, 1965) that wheat roots previously depleted of sugars had a reduced capacity for synthesizing cellulose, a P-l,S-glucan, sophoran, not previously known from higher plants, being formed instead. Since Mandels and Reese (1959) have found sophorose to be a very active cellulase (C,) induced for Trichoderma virada, one might feel tempted to suggest a reuse of cellulose in starved cells by means of sophoraninduced cellulases. Besides the breakdown of cell wall material during seed germination (for refs. see Norkrans, 1963a), a resynthesis of cellulose may take place, which surely is not the case during the ripening process, where cellulases of endogenous origin are supposed to be involved (Hall, 1964; Dickinson and McCollum, 1964). V. Degradation of Cellulose by Bacteria and Fungi

Cellulase production is the common denominator for cellulolytic organisms. The cellulosic material occurring in nature, however, varies greatly. It ranges from heartwood and sapwood in living, more or less lignified trees, through nonliving trunks, stumps, felled timber, forest litter, nonwoody plants, soft tissues of fruits, to transformed debris of all this matter mixed in soil or water, material offering conditions for growth selective for microorganisms. Thus, even in cases of

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about equal cellulolytic potential per se for the organisms concerned, we will find them divided in specific groups specially adapted to compete on one or another of the natural substrata.

A. DEGRADATION OF CELLULOSE BY BACTERIA Most cellulolytic organisms are found among bacteria and fungi. In more or less anaerobic, closed environments, as in guts of herbivores, in the digestive juices of invertebrates, and in the rumen of cattle, bacteria are the outstanding cellulose decomposers. Our knowledge of this latter system, with a mixture of cellulolytic bacteria and protozoan is fairly good, as is evident from Hungate’s recent extensive monograph “Rumen and its Microbes” (1966). Anaerobic eubacterial cellulose decomposers are also to be found in soil (Enebo, 1954; Skinner, 1960), in addition to aerobic species belonging mainly to the genera Cellvihrio and Cellulomonas. Furthermore, some actinomycetes and myxobacteria, including the imperfect genera Sporocytophaga and Cytophaga, are known as strong cellulose decomposers in soil. Their cellulolytic activity and mucopolysaccharide formation have been studied thoroughly since the days of van Iterson and Winogradsky (Stanier, 1942; Fihraeus, 1947; Charpentier, 1965). Cytophaga species also play an important role in the unique cellulolytic marine bacterial flora treated by Kadota (1959). Cellulolytic activity ought to contribute to the ability of parasitic microogranisms to spread through the host plant tissues. Correlation between cellulolytic ability and pathogenicity, however, is presumed for only very few bacteria, and has been convincingly demonstrated for only one, namely the wilt bacterium Pseudomonas solanacearum, studied b y Kelman and co-workers (Kelman and Cowling, 1965; Husain and Kelman, 1959). By means of quantitative chemical analysis of experimentally diseased tomato stem tissue, in a way which has been frequently used in case of wood decay studies but seldom applied to phytopathological studies of nonwoody plants, it could be shown that the a-cellulose content, the ratio of a-cellulose to heniicellulose, and the DP of holocellulose decreased during pathogenesis. The cellulolytic activity tested in vitro, however, was weak in comparison with that of Trichoderma viride. Bacteria have always been considered to play an insignificant role, if any, in cellulolytic attack on wood. There have been recent findings of cellulose-decomposing hyphae-forming actinomycetes from the genus Micromonospora, but also of eubacteria, in softened wood from old foundation poles and logs (Harmsen and Nissen, 1965a,b).

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Anaerobic conditions may have existed. Attack on wood by Bacillus polymyxa has been experimentally shown (Greaves and Levy, 1965). The bacteria degraded rayparenkymatic tissues, and the permeability of the wood to liquids was greatly increased. Courtois (196613) has studied the bacterial attack on coniferous wood. The micromorphological cell wall deterioration is different from that caused by fungi. Irregular, ruffled corrosion starts from cell lumen and (or) the middle lamella. Close contact between bacteria and cell wall is necessary for decomposition, and the cell wall layers are destroyed successively one after another. In all cases, however, the bacteria show slow action within a restricted area.

B. DEGRADATION OF CELLULOSE BY FUNGI The strong cellulolytic attack of fungi might partly depend on their hyphal organization, which gives these organisms a penetrating capacity and presumably a continuous cellulase-releasing phase. No solely budding yeast of cellulolytic importance is known to the author. Ascomycetes and fungi imperfecti play an important role as decomposers of plant residues in soil, and may form a considerable part of the zymogenous microflora in soil sensu Winogradsky (1949). A closely allied flora is involved in the deterioration of cellulosic textile fibers, for which extensive lists of the genera and species involved have been presented by Reese and co-workers. Besides giving an idea about the succession of invaders in wood, a recent investigation on colonization of wood by soil fungi (Merrill and French, 1966) is quite revealing with respect to the soil flora population. Fusarium sotani was the dominating pioneer invader, followed by Trichoderma viride, Aspergillus ustus, and some species of Penicillium, all together accounting for more than 90% of the isolates from invaded wood. Furthermore, Rhizoctonia solani, previously known to competitively colonize and decompose cellulose in soil (Garrett, 1962), was found among the wood invaders. Even phycomycetes, otherwise known to produce cellulolytic enzymes only in their role as pathogens (e.g., Winstead and McCombs, 1961; Unestam, 1966) were found to be wood invaders. Their growth, however, may have been supported b y some easily available cytoplasmic nutrients. Correlation between cellulase activity and pathogenicity has been discussed for varieties of wilt-inducing Fusarium oxysporum (Husain and Dimond, 1960; Deese and Stahmann, 1962). Cellulolytic activity has been found in Rhizina undulata, a root rotter on young pine plants (Norkrans and Hammarstrom, 1963; Norkrans, 1967).

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1 . Wood Decay Ever since the time of Robert Hartig (1878), it has been known that fungi are responsible for decay of wood. Different fungi give rise to different types of wood rot, brown-, white- and soft-rot having been distinguished. The blue-staining fungi, also first recognized by Hartig, are associated with wood, although not causing decay. The composition of decaying wood has been studied in numerous investigations, early as well as recent ones (Seifert, 19661, and in some studies the wood analysis has been extended even to determinations of degree of polymerization, particle-size distribution, and X-ray diffraction (Cowling, 1961, 1963; Levi and Preston, 1965). Biochemical studies have dealt with the character of the enzyme systems involved (Lyr, 1959a,b; Lyr and Novak, 1962; Levi, 1964), and microstructural changes caused by all types of wood-inhabiting fungi have received much attention in recent microscopical and electron microscopical studies (Corbett, 1965; Courtois, 1963a,b, 1966a; Greaves and Levy, 1965; Levy and Stevens, 1966; Liese, 1966; Schmid and Liese, 1965; Wilcox, 1965). Some information obtained from these studies is briefly sunimarized below. In wood, the rays generally containing easily available nutrients for fungal growth afford initial penetration paths for fungi. This initial penetration is followed by a passive penetration via the pits until all the easily available nutrients have been utilized for the mycelial build-up. From this stage onward, active penetration with subsequent degradation of the cell wall material begins. a. Blue-Staining Fungi

The blue-staining fungi seem not to cause any enzymic breakdown of wall substances. Their ability to penetrate lignified cell walls has been attributed to their characteristic club-shaped hyphae, the heads of which are provided with a spearlike projection. T h e finding that these “transpressoria” are able to pass through metal foils suggests that mechanical forces are involved, although the possible presence of some lytic enzymes, localized to the spear, has not been excluded. The blue-staining fungi all belong to the Ascomycetes and Fungi Imperfecti. The above-mentioned results were obtained in studies of Aureobasidium pullulans (=Pullularia pullulans), Ceratocystis pilifera (=Ophiostoma coemleum), Ceratocystis piceae, and Phialocephala phycomyces (=Scopularia phycomyces). The flora of bluestaining fungi is known from extensive studies on wood pulp and timber by Meliri arttl co-workers (Lagerberg et at., 1927; Melin and

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Nannfeldt, 1934). Several of these fungi are dark-coloured per se, in other cases iron reactions between fungus and substrate give the discolored sap (Gadd, 1965).

b. Soft-Rot

The term soft-rot was coined (Savory, 1954) for the softening produced in the surface layers of wood by the action of Chaetomium globosum, isolated from the wood of water-cooling towers, when cylindrical cavities with conical ends appeared with certain constancy in the secondary cell wall of the decayed wood. The term soft-rot is now used whenever the characteristic cavity pattern is present, even if no softening of the wood surface has occurred. These cavities, first studied by Bailey and Vestal (1937), may be formed by Chaetomium globosum in the following way: Hyphae invade the tracheid lumen and align themselves parallel to the long axis. Hyphae within the lumen form some thin hyphae, which penetrate the wall of the tracheid laterally at right angles to its long axis. When reaching the S z layer, these penetration hyphae reassume their original thickness and branches out at right angles to its penetration path in a direction parallel to the tracheids’ long axis, thus assuming the shape of a T. The cellular substances around this hypha become decomposed, giving rise to an elongated cavity. Generally, a fine hypha grows out from the apex of this cavity, thus giving rise to another cavity. A chain of cavities, separated from the next by a short distance, is formed in this way and may be seen aligned in the same spiral arrangement as the cellulose microfibril in the S z layer. Where enzymic action has been considerable, the cavities have coalesced to form a large cavity. Courtois (1963a) has described in detail a series of somewhat similar decay patterns. Chemical analysis of beech wood undergoing attack b y Chaetomium globosum has shown that the marked cellulose depletion taking place in the early stages of the attack is accompanied by an initial increase in the high polymer fraction (Levi and Preston, 1965). This increase reaches a maximum at about 20% weight loss, whereafter it decreases slowly. Alkali solubility is regarded as a measure of the amount of degradation products present in excess of those not immediately being utilized by the attacking fungus. When red beechwood was decayed to 5396, both alkali solubility and soluble sugars amounted to zero (Seifert, 1966). In this respect soft-rot resembles white-rot. When the wood had decayed to about 80% weight loss, three-quarters of the residue was lignin. I n this respect soft-rot resembles brown-rot.

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Soft-rot is more common in deciduous wood than in coniferous; Courtois (196313) suggested that the reason for this is attributable rather to qualitative differences in the lignin than to quantitative differences, as has been previously supposed (Meier, 1955). The methoxyl content in hardwood lignin is generally higher (>21%) than that in conifers (ca. 14%). Like all other wood-destroying fungi, those responsible for soft-rot also remove methoxyl from lignin; this seems to be the main effect on lignin of soft-rotters as well as of brown-rotters. Accordirg to Levi and Preston, the longitudinal path for diffusion of the “lignin-modifying” or “cellulose-freeing” enzyme will lie between the microfibrils while the transverse path will lie through lignin deposited in the “amorphous” regions of the microfibrils. Assuming then that longitudinal diffusion is more rapid than transverse, a pathway having a conical pattern will be opened for the real cellulolytic enzymes producing the typical, conically ended cavities. This is a modification of Roelofsen’s mesh hypothesis (1956). Soft-rot is produced by ascomycetes and fungi imperfecti. Their importance is based not so much on their deterioration capacity as on their ability to grow under conditions too moist to favor growth of basidiomycetes and on their ability to survive during dry periods (Liese and Ammer, 1964). In a review dealing with soft-rot fungi, Levy (1965) makes mention of a list, compiled by Corbett (1963), containing all the fungi recorded as inducing soft-rot both in hardwoods and in softwoods in controlled cultures. Though the list records some forty organisms, it omits marine fungi, which have been given by Jones (1963).To a list of fungi isolated from wood in watercooling towers and later tested for soft-rot activity (Courtois, 1963b), some other rot-inducing organisms can be added: species of Paecilomyces, Chaetomium, Ceratocystis, Rhizoctonia, and Stysanus, these being the most active ones with respect to weight loss, formation of hyphae, and structural changes caused; they are much more aggressive than the strains of Trichoderma viride used. None of the phycomycetes isolated had any activity. Several species, listed above as bluestaining fungi, can also be found here. It has also been pointed out that the action of soft-rot fungi appears to be intermediate between that of blue-staining fungi and wood-rotting basidiomycetes, showing properties of both under certain conditions.

c . Brown-Rot and White-Rot Brown- and white-rot, first described by Falck (1926) as destruction and corrosion rot, respectively, are caused by basidiomycetes. The

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fungal hyphae are located within the cell lumina; they pass from one

cell to another through the pits or by producing bore holes. The brown-rotting fungi decompose cellulosic and noncellulosic polysaccharides. They depolymerize the cellulose more rapidly than the depolymerization products are utilized by the organism in the early stages of decay. Since they start their decaying process from hyphae within the cell lumen and break down the S2 and later on the S1 layer while the S3 layer, closest to the lumen, still is intact, d i h s i bility of the enzymes produced has been postulated. From electron microscopical studies of the attack of six different species on samples of sapwood from beech, spruce, or pine, however, Courtois (1966a) concluded that S3 was attacked before the S z layer. Certain difficulties may be associated with the interpretation of the different layers in electron microphotographs. Liese (1966) pointed to the observations of slime substances around the fungal hyphae which might previously have been attributed to the S3 layer. The white-rotting group is a more heterogenous collection of organisms having in common a capacity for decomposing lignin, and for producing enzymes oxidizing phenolic compounds -probably related to lignin-a fact which has been utilized in diagnostic color tests for identifying white-rotting fungi ever since it was first described (Bavendamm, 1928). The relative amounts of lignin and cellulose destroyed and utilized vary. A gradual thinning of the cell wall takes place, characterized by essentially complete removal of one wall layer at a time, beginning from lumen with Ss and progressing toward the middle lamella. Lignin solubilization is supposed to precede cellulose decomposition. The depolymerization of wood polysaccharides is slight, each constituent being depolymerized only as rapidly as the degradation products are metabolized. Among the investigations mentioned above, however, only that by Lyr and Ziegler (1959) relates the study of enzymes formed with decay produced. Two white-rotters, Phellinus igniarius Quel. and Collybia velutipes Curt. were studied by these authors. The attack on wood was polyenzymic, production of the different enzymes being simultaneous. The enzymes active against insoluble wood components were extracellular. The use of such heterogeneous a substrate as wood, however, presents a lot of difficulties and uncertainties. Information about the inducibility was later obtained from growth experiments on several wood-rotting and wood-inhabiting fungi, including the brown-rotting Coniophora cerebella, and subsequent examination of the extracellular polysaccharide-degrading enzymes

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produced. C . cerebella has recently been studied in a similar way b y King (1966) who used a highly active strain which produced rapid and extensive weight losses in the standard decay tests. Enzymes that degraded carboxymethyl-cellulose and precipitated cellulose (a-cellulose to only very limited extent), spruce xylan, and glucomannan, were found in the culture filtrates, besides starch-degrading glucoamylase and laminarinase [p-(1,3)-glucanase]. All were adaptive except the laminarinase, which however, may be a self-induced rather than a constitutive enzyme, associated with the lysis of older areas of mycelium (p-1,3-glucans have been found in the hyphal walls of fungi, see Clarke and Stone, 1963; Mitchell and Sabar, 1966). Approximately 2000 species of basidiomycetes have been classified as wood-decaying fungi. Approximately one-tenth, mostly polyporoid fungi, can attack the heartwood of living trees and a few of them are active exclusively at this site (Wagener and Davidson, 1954). Another group can attack living sapwood, whereas most of these basidiomycetes decompose dead trunks, stumps, and felled timber (Cartwright and Findlay, 1958). Some are also specially adapted to specific species of tree. The factors governing this specialization among the decay organisms are not well understood. It may be due, in part at least, to varying abilities of different organisms (1)to efficiently utilize bound nitrogen, present only in very small amounts in wood (the C : N ratio of wood varies from about 350: 1 to 1250: 1) and to reuse mycelial nitrogen (Merrill and Cowling, 1966) or, possibly, to fix atmospheric nitrogen, (2)to grow at low oxygen pressure (Gundersen, 1961) or at high wood moisture as well as at lack of moisture (Ammer, 1964), (3) to decompose adherent and incrusting material besides lignin resins, waxes, etc. (Hata et al., 1966), (4) to tolerate or eliminate substances in the wood which are toxic to the organism itself, as in the case of thujaplicine, pinosylvine, and other heartwood extractives (Lyr, 1962; Rudman, 1962) or which are inhibiting to its enzymes (cf. Reese, 1965). The ability to initiate attack on litter and other lignified plant material under aerobic conditions seems mainly to b e restricted to hymenomycetes, which exhibit differences among themselves with respect to the relative amounts of cellulose and lignin they decompose (Lindeberg, 1946; Haider et al., 1964).

2. Fiber Decay

The attack on a lignified fiber, i.e., jute fiber, has been studied by

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Basu and Ghose (1962a,b) who give details for the pattern of attack. Growth inside the lumen is not a universal feature although exhibited by nearly all the dark-colored ascomycetes and fungi imperfecti tested, e.g., Alternaria sp., Memnoniella echinata, Stachybotrys atra, and Myrothecium verrucaria. Internal damage along the lumen was noted in these cases. Photographs given for Chaetomium indicum (Basu and Ghose, 1962a) bring to mind those of soft-rot in wood, with thin penetration hyphae and attack in the secondary wall parallel to the fiber axis. Cracks across the fiber walls are a common feature. Complete breaks were also found as well as V-shaped corrosions and notches in the surface, and helical cracks. Fungal attack on cotton fiber usually involves growth of hyphae within the lumen, accompanied by complete or partial dissolution of wall material from within with transverse cracking and spiral fissures visible at the early stages. Since cotton is frequently used as substrate in the cellulase assays, the correlation between structural changes and enzymic attack will be discussed below. The following section will deal with more or less purified fungal and bacterial cellulase systems, their formation and mode of action. C. CELLULOLYTIC ENZYMES

1 . Extracellular Production As cellulose under physiological conditions is insoluble and cannot permeate cell membranes, the microbial cellulase has been supposed to be extracellular. In culture filtrates of cellulolytic fungi, cultivated under suitable conditions, extracellular cellulases can generally be demonstrated. According to Lyr and Sch6ni.l (1964), an active secretion into the culture medium is to be assumed; autolytic liberation can be excluded, since the activity of preparations from corresponding amounts of mycelia always were low. In replacement media, the cellulase synthesis could be shown to change with age of mycelium. For most fungi tested, the highest production was obtained from young mycelia and hyphal tips, which is surely of ecological importance for these wood rotters. Older mycelia, however, were more productive in, e.g., Fomes marginatus. Bacterial cellulases are more firmly bound to the cells, or to the cellulose, since culture filtrates of cellulolytic bacteria do not always

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contain cellulolytic enzymes. They can generally be obtained b y treatment with autolytic agents, though even such treatment did not release cellulases from C ytophaga cultures (FHhraeus, 1947). Hungate (1966) states that “the cellulase is quite firmly attached to the cellulose and unable to diffuse until the substrate has been digested.” King (1961) reports that there are indications that cellulases occur on the cell surface of rumen bacteria. The importance of culture conditions for cellulase production has often been pointed out (e.g., Whitaker and Thomas, 1963).This has to be stressed again, since apparently insignificant differences in the conditions may influence not only the quantity of enzymes produced but even the quality. Among the factors influencing cellulase production may be mentioned: composition of culture medium (quantity and quality of cellulose used, the amount of metal salts present), pH, temperature, the adequacy of the oxygen supply, and the way in which it is obtained (Norkrans, 1963b; Lyr, 1964).

2. Induced Synthesis Cellulolytic enzymes are generally considered to be formed only in the presence of cellulose. According to Mandels and Reese (196O), however, not cellulose itself but the soluble cellobiose, an hydrolysis product, is the true inducer of cellulase in cellulose cultures. Other compounds having P-glycosidic linkages, such as lactose and salicin, can serve a s inducers; sophorose, mentioned above, is 200 times more active than cellobiose. However, not all soluble compounds with P-glycosidic linkages are inducers. Glucose and cellobiose offered in amounts as large as those used in standard growth experiments generally depress the yield of cellulolytic enzymes (e.g., Norkrans, 1957a).

a. Inducer-Repressor Mechanism in the Relationship of Higher Plant and Fungus Recently, Horton and Keen (1966) studied the sugar repression on cellulase synthesis in the case of Pyrenochaeta terrestris, a fungus involved in the formation of onion pink root. P. terrestris has been observed to produce carboxymethyl cellulase in infected onion roots. The enzyme is also produced in quantities in cultures of the fungus containing cellulose. Studies on replacement media showed that cellulase synthesis was repressed to the basal level by glucose concentrations of ij x 10V’M or above. Krebs cycle intermediates had the same effect. By dilution of the medium, re-formation of a maximum cellulase level followed, identical with that obtained before sugar

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depression had occurred. This has been taken as evidence for the regulation of the cellulase synthesis by cellulolytic products through a repressor-inducer mechanism. From studies with toluene-treated cultures on various types of celluloses, differing in their susceptibility to enzymic hydrolysis, it appeared that hydrolytic liberation of more than 0.01 mg./ml. glucose per 48-hour periods invoked the repressorinducer regulation of cellulase synthesis (for the repressor-inducer system, see Jacob and Monod, 1963; Pollock, 1959). Thus for phytopathogenic microorganisms in general, it may be said that high sugar levels in the plants would delay development of disease through repression of cellulase synthesis, whereas low sugar levels would promote pathogenic invasion as a result of decreased repression of enzyme synthesis. Since the repression to basal level occurs at such a low concentration as 5 x 1 W M in the case of P. terrestris, cellulolytic enzymes may not be important in its pathogenesis, as sugar concentrations of that low level will exist only in very localized areas within the plants. Endopolygalacturanase repression in P. terrestris occurs first at hundred times higher sugar concentrations (Keen and Horton, 1966), hence this enzyme may be of more importance in connection with pathogenic invasion in the plant. In general, cellulolytic enzymes have been found less important than pectinolytic ones for phytopathogens, and the obligate parasites seem to be found among “high sugar” organisms, according to Horsfall and Dimond (1957). A similar sugar depression system has been suggested to work in a highly balanced symbiontic system, namely, in the formation of ectendotrophic mycorrhiza. Studies of different Tricholoma species (Norkrans, 1950b) revealed that, contrary to what had previously been supposed and later confirmed (Ritter, 1964), some mycorrhiza-forming species produced cellulolytic enzymes. Tricholoma f u m o s u m Fr. (non Pers.), a facultative mycorrhiza-former on pine, produced (when cultured on cellulose as the sole carbon source) cellulolytic enzymes in amounts as high as those produced by any of the litter decomposers. Other symbiontic species of TTichoZoma showed no cellulase production, except T. imbricatum (Fr.) Quel. and T. vaccinum (Pers. ex Fr.) Quel., in which very weak formation of cellulase was attained by adding some “start-glucose,” and indeed only after the glucose had been consumed. Studies by Melin (1923) and later by Melin and Nilsson (1957) and Bjorkman (1942, 1944) had shown that ectotrophic mycorrhizal fungi require soluble carbohydrates from trees. From these different facts, the following assumption was made: “The mycorrhizal mycelium obtains glucose (or soluble carbohydrates) from the plant. CelluIase production would be depressed as long as

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BIRGITTA NORKRANS

glucose or other soluble carbohydrates attacked by ‘constitutive’ enzymes are present. The cell wall, however, constitutes a cellulose substrate which will induce an increase in cellulase production when the plant, for some reason, no longer forms a surplus of soluble carbohydrates. This may furnish a possible explanation of the ectendotrophic mycorrhiza.” (Norkrans, 1950b).

3. Assay of Cellulolytic Enzymes and Purijicution Native cellulose is crystalline and possesses the complex molecular structure mentioned above. It is combined with noncellulosic material even in cotton fiber. These factors reduce its accessibility and susceptibility to cellulolytic degradation. Cellulase investigators have tried to obviate some of these difficulties by using well-purified, more or less modified celluloses. Some of the common substrates used, including the water-soluble substrates, namely, oligoglucosides, and some cellulose derivatives are presented in Table I, which also gives the methods of measuring the activity. Solubility greatly increases the accessibility of the linkages-providing that the degree of substitution (DS) of the derivatives is not too high-resulting in high TABLE I ASSAY CELLULASE

SUBSTRATES TO

Substrate Insoluble: Native cellulose: Cotton, undried Cotton, dried

“Native cellulose” Cotton, dewaxed

Physically modified cellulose (11): a-Cellulose from wheat straw Hydrocellulose

Determination

References

Morphological changes by: Microscopic observations Marsh (1957) Electron microscopic observations Porter et ul. (1960) Marsh et al. (1953) Tensile strength and alkaliswelling (weight increase) Reese and Gilligan (1854)

Microscopic observations Cellulose residue (gravimetrically or colorimetricall y)

Blum and Stahl(N52) Halliwell(lSl63) Selby (1961)

Formation of reducing groups

Grimes et ul. (1957)

Decrease in turbidity and Formation of reducing groups,

Norkrans (l950a) Li et ul. (1963)

c ~ ( ~ 1 I i i l oresidue w

__

.

113

CELLULOSE AND CELLULOLYSIS TABLE I (Continued) TO ASSAY CELLULASE SUBSTRATES Substrate

Determination ~

References ~

-

Cotton, swollen (HsP04)

Decrease in D P Formation of reducing groups

Walseth (1952) Gilligan and Reese (1951 Myers and Northcote (1959) Whitaker (1953)

Colloidal cellulose sol (alkali) DP 200-300

Decrease in turbidity and DP Formation of reducing groups

Norkrans (1950a,b) Norkrans and RQnby ( 1956)

Cellodextrin (acetolysis) (DP 24)

Formation of reducing groups; ratio moles oligosaccharides

Whitaker (1956)

Soluble: Chemically modified cellulose: Cellulose derivatives DS <1.0 Carboxymethyl cellulose Decrease in viscosity Formation of reducing groups

Broken bondslsecond (viscometrically ) Ethylhydroxyethyl cellulose Oligoglucosides: P-1,4-oligoglucosides

Reese et al. (1950) Toyama and Shibata (1961) Hanstein (1960-1961) Alminet al. (1967)

Decrease in viscosity

Lyr (1959a,b)

Formation of reducing groups and chromatographic separation

Whitaker (1954)

Methyl P-1,4-oligoglucosides

Hanstein and Whitaker (1963)

reaction rates. Since the upper limit for a suitable DS varies with different derivatives (for carboxymethyl cellulose it is
114

BIRGITTA NORKRANS

measure of the number of bonds split and the rate will vary with the initial viscosity-average DP of a given type of substrate. Almin and Eriksson (1967)and Almin et al. (1967)have developed the theoretical and semiempirical basis for a viscometric method of determining the activity of enzymes acting, preferably at random, in the degradation of polymers. Their method measures the activity in absolute terms, i.e., the number of bonds broken per unit time. By this method, the molecular activity of a purified sample of Penicillium notatum endoglucanase, molecular weight 78,000, acting on sodium-carboxylmethyl cellulose (DS 0.83) at 25°C has been determined to be 29 bonds broken per second. The degree of crystallinity seems to influence the reaction rate in a decisive manner. Though insoluble, the cellodextrins are amorphous, whereas all the other insoluble celluloses mentioned are more or less crystalline, including the precipitated cellulose sol. Hydrocellulose (Avicel, “micelle-solution”) is the depolymerized, highly crystalline, hydrophobic, aggregated residue of wood or cotton fibers obtained after boiling in hot mineral acids. The sol as well as the highly crystalline micelle solution (Norkrans, 1950a,b) and the hydrocellulose (Li et al., 1963) can be run in turbidimetrical tests. The change in alkali swelling (an increase in the uptake of alkali b y the attacked cotton fiber when placed in 18%NaOH) is detectable before any reducing sugar, loss in tensile strength, or change in DP can be observed (Marsh et al., 1953). The change is associated with an increase in fiber width. The thinly scattered microfibrillar network in the primary wall, orientated in directions nearly at right angles to the fiber axis, has been suggested to act restrictively on the alkali swelling. The fibers’ tensile strength may be attributable to microfibrils in the secondary wall, arranged more or less parallel to the fiber axis. Though it would have been desirable to present in Table I also the experimental conditions prevailing during the activity determinations, the great variations of these conditions make this impracticable. May it suffice to mention as an example that the carboxymethyl cellulase assay alone is performed at temperatures ranging from 25” to 50°C. and incubation periods ranging from 5 minutes to 24 hours, using various substrate concentrations and pH conditions, according to the test organism used. The reader is referred to the pertinent literature for the various experimental details. Gascoigne and Gascoigne (1960) give complete lists for cellulase sources. The agar diffusion technique also has been applied to cellulase

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assay in “cup” method (Toyama, 1963) and in a “mycelial-plug” method (Aschan and Norkrans, 1953). Almost all the techniques applied to general protein purification and separation have also been employed in the case of cellulolytic enzymes (cf. Hashimoto and Nisizawa, 1963; Whitaker et al., 1963). However, gel filtration with Sephadex preparations of different types offers a means of separating enzymes with little risk of deactivation and was first used for cellulolytic enzymes by Pettersson et al. (1963), Pettersson and Porath (1963).

4 . Mode of Action a. Cl-C, Concept

Some years ago, Reese et al. (1950) stated that many microorganisms are able to hydrolyze modified celluloses, but only some of them are able to attack native cellulose such as mature, dried cotton. They suggested that a C1-C, system should make the latter true cellulolytic organism capable of completely solubilizing native cellulose. Mandels and Reese (1964) have described their C1-Cx-concept in the following schematic way: cellulose

-T -T reactive

C,

cellobiose-

glucose

p-glucosidase

The hydrolytic C, is a P-1,4-glucanase7able to attack all celluloses in the range from soluble cellulose derivatives to celluloses “swollen” by alkali or acids or by means of mechanical treatments such as grinding. When the substrate does not offer hindrance to the approach of the enzyme molceule, and the end effects of very short chains are absent, the enzyme is of a random-splitting type. The work by Perlin and Reese (1963) has shown that the endoglucanase is not specific to the bond being broken but to the reducing end unit being split off. Thus, when acting on lichenin with alternate 1,3 and 1,4-linkages, cellobiose is produced but 1,3-linkages are broken. C, activity may also be attributed to the exoglucanases; some of their properties will be referred to below. Physically distinct multicomponents of C, have been referred to several times by Reese and others. They may be due to isoenzymes or carbohydrates complexing with the protein

116

BIRGITTA NORKRANS

moities of C,. Thus far a completely carbohydrate-free C, does not seem to have been isolated.

b. C ,-Hydrocellulase-Enxynw A True cellulolytic organisms, such as Myrothecium verrucaria, which used to be the organism of choice of cellulase investigators, could solubilize 80%or even more of undegraded cotton fibers, whereas cell-free culture filtrates or preparations from the same organisms were found notoriously incapable of more than about 3% degradation of native cotton. For this reason the C1-ghost has been hunted all these years, and until very recently without success. During the past few years, however, two species of Trichoderma, namely T . viride and T . koningii, were found to give cell-free preparations capable of solubilizing cotton fiber (Mandels and Reese, 1964; Halliwell, 1965b, 1966; Li et al., 1965). Halliwell obtained complete solubilization of cotton fibers with quantitative conversion to glucose within 19 days by a culture filtrate from T . koningii. In the first phase (7 days) short fiber fragments were produced and only minor quantities of soluble products. When soluble and insoluble products each constitute 40 to 50% of the weight of the initial substrate, the quantity of sugars increases at the expense of the insoluble short fragments. Enzyme preparations from Myrothecium verrucaria could not solubilize the short fragments formed. Cellulose powder was solubilized by Trichoderma filtrate at the same rate as cotton fibers (19 days), hydrocellulose required almost four times that period, and ground hydrocellulose only 21 hours. Fragmentation of fibers has been described several times by means of chemical acid hydrolysis. According to the model of native cellulose as a fringe micelle, this fact has been interpreted as a chain shortening in the amorphous parts in the same way as Amemura and Terui (1965)explain the enzymic attack by cellulase from PenicilZium uariabile. If we accept Manley’s model, less ordered parts and more accessible parts may be present along the microfibril between the crystalline molecules where the initial attack will occur. The initial attack will result in an increase in relative crystallinity, which has been found several times (Norkrans, 1950a,b; Walseth, 1952). Mandels and Reese worked with Trichoderma viride. After treating dewaxed cotton slivers for 45 days with culture filtrates from this organism, a weight loss of 60% was noted, which is 30 times or more the loss caused by Myrothecium verrucaria or Chaetomium globosum. Cotton was found to be the most resistant of the pure celluloses. Woody materials were d s o resistant unless they were thoroughly

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ground b y ball milling. Filter paper or newspaper showed considerable breakdown in 1 day, thus 50 gm. of newspaper gave rise to 12.8 gm. of sugars, mainly glucose, but also cellobiose and xylose, when treated with Trichoderma filtrate. After repeated chromatograms on DEAE-dextrans, Mandels and Reese succeeded in separating components having C1, C, , and 0-glucosidase activity. The p-glucosidase appeared very early and completely separated from C, and C1. Previously, Pettersson et al. (1963) found that P-glucosidase from Polyporus uersicolor, d u e to its high molecular weight, was separable from other cellulolytic components by gel filtration. Furthermore, it has been shown also for other comparable enzyme systems, such as mannanase-mannosidase and xylanase-xylosidase, that the endoenzymes capable of hydrolyzing insoluble high molecular polysaccharides are smaller molecules than their corresponding exoenzymes which bring about hydrolysis of soluble oligosaccharides (Ahlgren et al., 1967). Mandels and Reese have expressed the C1 activity in terms of loss in weight of cotton sliver, which however, cannot be considered as a direct measure of its activity, as the authors themselves indicate. They assume C1 to act in a way to permit an increased moisture uptake, hydrating the cellulose and pushing apart the closely packed chains,” to make the linkages accessible for the action of the hydrolytic ,&1,4-endoglucanse (Cx, carboxymethyl cellulose being used as substrate). C1 activity is enhanced when the organism is grown on cellulose. Different sources of cellulose affect C1 activity to different degrees, due to differences in its production or in its adsorption to the substrate. However, it is found in measurable amounts even in glucose or cellobiose media in the case of T. viride. King (1965) and Li et al. (1965)maintain that the key property of the C1 component is its capacity to attack highly crystalline cellulose. Consequently, they used crystalline aggregates of hydrocellulose, or the commercially available Avicel which constitutes an easily handled “eucellulosic” substrate, for testing a cellulase system. The starting enzyme was obtained from wheat bran-sawdust cultures of T . viride, produced according to Toyama (1963). They have been able to separate this crude preparation into different components with distinct enzymic properties. Although none of the components alone could account for the enzymic overall process of the crude preparation, appropriate combinations did. The components are hydrocellulase, endoglucanase, and exoglucanase. Hydrocellulase corresponds to the C1 component, being the only “

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BIRGITTA NORKRANS

one having the capacity to attack crystalline cellulose at any appreciable rate. The primary product of attack is cellobiose. At the first step of purification, on an Avicel column, irrigated by sodium citrate buffer of pH 4.8,the hydrocellulase was completely retained, contrary to all the other components. Its further purification and properties were not given in detail in this publication; an approximate molecular weight of 60,000 has been given b y King (1965). Endoglucanase has a minimum molecular weight of 26,000 (based on amino acid composition, one methionine per molecule) and a most probable molecular weight of 52,000 according to sedimentation data. It was relatively thermostable and it behaved just as a typical C,enzyme. The optimum length of its substrate chain was at least 6 glucosyl units according to K , values derived from hydrolysis of glucomers varying from cellobiose through cellohexose. By using terminally substituted sugars (cellotetrosyl- and cellopentosyl sorbitol), higher rates of hydrolysis for interior linkages could be shown (cf. Whitaker, 1954, the “end effect”). Exoglucanase has a molecular weight of 76,000, approximated from sedimentation data. The specificity appeared less closely related to the linkage split or length of chain than expected even for an exopolysaccharase. All of 13 P-glucosides tested were hydrolyzed, incuding six aryl-P-glucosides - even one with such a bulky aglycone as the P-bromonaphtylglucoside -P-1,2, /3-1,3, P-1,4, and /3-1,6-glucobioses, and one cellohexose and only small differences in activity against the various substrates used were observed; p-1,3-glucan and carboxymethyl cellulose were attacked by removal of successive glucosyl moieties from the nonreducing end. P-1,2 and /3-1,6-glucans were nonsubstrates. Thus initial attack on crystalline cellulose must be attributed solely to C1. Random attack on relatively long chains of amorphous” cellulose must be assigned primarly to the endoglucanase component, since it apparently has greater affinity for an “amorphous substrate” than has C1 and a greater disposition for long chains than the exoglucanse fraction has. The exoglucanse seems to complete the hydrolysis to glucose of the products obtained both from the action of C1 and the endoglucanase. A certain overlapping action of the endoglucanase seemed to be present, which might be due to a not absolutely complete separation. Li et al. mention that their endoglucanse can be coded according to the Commission on Enzymes of the International Union of Biochemistry (Report, 1961) as a ~-1,4-~-glucan-4-glucanohydrolase (E.C.3.2.1.4). It indicates the substrate as a p-1,4 polymer of Dglucose, which is split by hydrolysis of p-1,4-glucosidic linkages at “

CELLULOSE AND CELLULOLYSIS

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random attack, not confined to the terminal ends. Hydrolases which remove glucose or cellobiose units exclusively, by endwise attack, would be designated “glucohydrolases” and “cellobiohydrolases,” respectively and are both exoglucanases. The exoglucanse of Li et al. is supposed to correspond to a glycohydrolase. The endwise attack seems not to be absolutely pure. This endo-exo-glucanase system seems to correspond to the “po1yase”-“oligase” system proposed by Stone (1958). Reese mentions a P-glucosidase component in his cellulolytic system. This component is given as mainly intracellular, otherwise the differences between Li et al.’s exoglucanase, Stone’s oligase,” aiid Reese’s P-glucosidase do not seem quite distinct now. In 1963, Selby et al. suggested that the cellulase system of Myrothecium verrucaria contained at least two enzyme types that differed in the rate and extent of their attack on fibrous cellulose such as cotton yarn. It was suggested that the A enzyme necessary for extensive degradation of cotton was present in small amounts only in the culture filtrate as normally prepared, and that it was “exhausted” by exposure to cotton yarn. Another cellulase, the B enzyme, was not so deactivated, but was able to weaken cotton to a limited extent only. The B activity was measured as carboxymethyl cellulase activity, the A activity as loss of tensile strength in cotton yarn. Even these Myrothecium verrucaria filtrates have now been fractionated by gel filtration on Sephadex G-75 (Selby and Maitland, 1965).Three major cellulolytic components were obtained. The middle component (11)with a molecular weight of about 30,000, represented 90% of the total carboxymethyl cellulase activity and thus seemed to contain the B enzyme. Reruns after exposure to cotton showed that it only slightly affected this activity. The other two components (I and 111), having molecular weights of about 55,000 and 5300, respectively, were mainly responsible for the activity of the filtrate toward cotton, and were removed or deactivated by exposure to it, behaving as an A enzyme in these respects. These observations accord with the previously reported behavior of the whole culture filtrate. In the previous publication (Selby et al., 1963),it was suggested that the loss of A could result in the genesis of B, possibly by the combination of A with the products of cellulolysis. No evidence of such interconversion was found. Thus if A activity is lost by the formation of a soluble enzyme-carbohydrate complex, the change in molecular weight was too small to be detectable by gel filtration on Sephadex G-75. Maitland (1965) mentions in a brief report that h e has also been able to prepare a component, presumably corresponding to Reese’s CI, from T . uiride. It is essential for the extensive degradation of cotton, but is without significant “

120

BIRGITTA NORKRANS

action of its own, either on cotton, carboxymethyl cellulose, or cellobiose. It acts synergistically with C, as previously found b y Reese. The small solubilizing power of C, acting alone is enhanced when the incubation with cotton takes place on a dialysis membrane. The following interpretation has been given: an inhibitory product d u e to C, action, which C, should have removed, diffuses away on the membrane. As long as only C1 and enzyme A had been found, a “cottonase” acting on specific noncellulosic linkages in the primary wall on cotton would have been suggested. The presence of “hydrocellulase” in the group, however, seems to indicate quite clearly that the activity is directed against highly crystalline cellulose. In an article entitled “Catalytic decomposition of cellulose under biological conditions,” Halliwell (1965a) called attention to the fact that cotton fiber treated with hydrogen peroxide at 0.4% in the presence of 0.2 millimoles ferrous sulfate at pH 4.2 to 4.3 under sterile conditions becomes fragmented in very short fibers within a few days; complete solubilization of 5 mg. of cotton fibers occurred in about 7 days. Thus the course of cotton solubilization in this ferrous sulfate-hydrogen peroxide system was very similar to that obtained by Halliwell (1965b) with Trichoderrna filtrate, except that only very small amounts of soluble degradation products were accumulated in the former. If the concentrations of the reagents were decreased, extensive breakdown occurred, but much more slowly; the ratio of peroxide to ferrous salt was critical. Other ferrous and ferric salts, though none of the other 20 metal salts tested, gave comparable results. Grass, straw, hay, and sawdust were solubilized in the same way. Nothing is said about sterility tests after the treatment periods. Halliwell indicates the peroxide-ferrous sulfate reaction as an alternative mechanism to the hydrolytic action of cellulase in nature, possibly comparable to photolytic degradationiknown for textiles. Halliwell suggests that “microorganisms possessing hydrogenperoxide forming mechanisms, such as glucose oxidase, would be able to provide hydrogen peroxide in amounts suitable for the slower reaction between hydrogen peroxide and ferrous sulphate. Localized attack on cellulose in close proximity to the invading organism is a characteristic feature of the microbial breakdown of fibrous cotton.” It cannot be concluded that Halliwell directly suggests a biogen hydrogen peroxide as a C1 factor, but it does seem that we are on the verge of such a suggestion. The difference between “true cellulolytic” organisms and C, organisms would then be dependent on peroxide formation. This finding brings new material to the discussion aboiit the different behavior of white- and brown-rotters.

CELLULOSE AND CELLULOLYSIS

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Lyr (1956) has found extracellular peroxidase from the white-rotters. Johansson (1966) tested enzyme preparations against the sol of precipitated cellulose from about 50 wood-rotting fungi, about equally divided between white- and brown-rotters. He found the preparations from all the white-rotting fungi active against cellulose sol, whereas those from the brown-rotting fungi were always inactive. As whiterotting fungi, contrary to brown-rotting ones, produce extracellular enzymes, mainly laccase, capable of oxidizing phenolic compounds (e.g., Lindeberg, 1955; Fihraeus and Lindeberg, 1953; Lyr, 1958), Johansson suggested a relation between the presence of active phenol oxidase and cellulase. Since Reese and Levinson (1952) as well as Lyr (1959a,b) had found carboxymethyl cellulase from brown-rotting fungi, Johansson concluded that C1 may either not be present or not active in his preparation from brown-rotters, whereas C, was present, which h e has also confirmed in later experiments with carboxymethyl cellulose as substrate. The cellulose sol does not offer an exclusive C1 substrate, even if it is partly crystalline. Cowling (1961), studying wood decay, makes the following statement about the white-rotting Polyporus versicolor: “the organisms degraded the crystalline and amorphus cellulose simultaneously.” This is contrary to the case of Poria monticola, a brown-rotting fungi which leaves cellulose entities of about crystallite size unattacked. After these findings, further advances may be expected in this field. Extensive purification of Myrothecium verrucaria cellulase enabled Whitaker et al. in 1954 to state that the cellulase was an ellipsoidshaped protein, approximately 50 x 200 in size with a molecular weight of 63,000; by later improved procedures this was amended to 49,000 (Datta et al., 1963).It has been debated whether molecules of that size could really act within the cell wall or the enzyme should be restricted to an action from cell lumen or cell surface and some gross capillaries in connection with these areas. Molecular weights as low as about 10,000 now have been found. Selby and Maitland (1965) even mentioned 5300 for component 111, an estimation which, however, might be quite preliminary as it is only deduced from a Sephadex column, calibrated with proteins and substances of known molecular weights in a range from 670,000 to 255. Even a molecular weight of about 5000, presumably corresponding to protein with a molecular diameter not smaller than 15 A in diameter, seems to be too large at least for action within crystalline cellulose. Pores of about 100 A in diameter, communicating with have been demonstrated in native cellulose (Freypores of 10 Wyssling, 1959) and, in accordance with the fringe micelle, interpreted as intermicrofibrillar and intermicellar spaces, respectively.

A

A,

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BIRGITTA NORKRANS

These sizes of a pore system must still be valid even if the pores -in accordance with the helix model by Manley - may be called intermolecular and intramolecular spaces, respectively. Manley (1965b)says: “If it is assumed that within a single cellulose chain all hydroxyl groups must be bonded to a corresponding receptor oxygen, it is reasonable to suppose that there may be a high degree of specificity in the pattern of intrachain hydrogen bond formation which determines the spatial arrangement prerequisite to the formation of microfibrils.” If this assumption is correct, it is evident that a break of hydrogen bonds would extensively loosen the crystalline structure. The “hydrogen bondase,” suggested some years ago by Siu (1951)and which has ever since been what King calls “something of an intellectual football,” would solve the problem of the C1-hydrocellulase-enzyme A action. The initial attack on the crystalline cellulose seems to open the way for a hydrolytic attack without actually hydrolyzing /3-1,4linkages themselves. The attack might involve an electron transfer to the site of attack due to a side branching of the respiratory chain, Differences in cellulolytic activity of culture filtrates from one and the same hymenomycete seem to be correlated to differences in oxygen supply during growth. Electron transport is mediated by flavoproteins and cytochromes as well as other substances. Trichoderma viride, the outstanding C1 source, exudes in its most cellulolytic phase, at low pH values, strikingly large amounts of a yellowish substance whose absorption spectrum coincides with that of riboflavin (Norkrans, 196313). At the previously mentioned cellulase symposium (Maitland, 1965),phenylisocyanate was reported to modify cotton under nonswelling conditions in a way which protected it against attack of cellulase. The inhibition of cytochromes by cyanic compounds is well known. Hydrogen peroxide -F++ -Fe+++represents a sensitive system for electron exchange; hydrogen peroxide and ferrous-ferric-salts fragmented cotton fiber in the studies by Halliwell mentioned earlier. These facts might possibly fit into the pattern for C1 attack on crystalline cellulose and bring to mind the luciferinluciferase system. Manley (1965a) suggested that the attack of cellulase preferentially takes place in the fold of the chain. D. INHIBITIONOF CELLULASES From the data given above, it is obvious that our knowledge abo,ut the initial attack of native crystalline cellulose still is incomplete. In the past most information about cellulolysis was obtained from studies of crude enzyme preparations. These two facts alone, even if other complicating factors are involved, may explain why inhibition studies

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of cellulases even in in vitro systems offer a lot of seemingly conflicting data. Some very recent works by Mandels and Reese (1963, 1965)-the latter one, though a review also includes some previously unpublished data - cover the problem completely. Thus, this author has confined herself to making a few remarks and additions. Breakdown products often inhibit the enzyme involved in the breakdown, which is true also for cellulase and cellobiose. Enzyme-substrate interactions can be prevented by compounds of similar configuration; methyl cellulose (DS 1.2) may fall into this class (Reese and Mandels, 1957) as well as gluconolactones. These latter strongly inhibit oligoP-glucosidases, thereby inhibiting the complete breakdown of cellulose, although cellulases may be affected only exceptionally. This is known from work, e.g., by Conchie, Festenstein, and others (Conchie and Levvy, 1957; Festenstein, 1959; Norkrans and Wahlstrom, 1961). Keleman and Whelan (1966) have shown a n inhibitory effect by polyols in general and erythritol especially, on glucosidases and galactosidases. Erythritol was supposed to act by combining with the enzyme at the same points as are occupied by the C3 to Cs region of D-glucose. Many common, unspecific protein-denaturing inhibitors are active also against cellulases. Inhibitors which act on free SH-groups also inhibit cellulases. Eriksson and Pettersson (1967) studied an electrophoretically homogeneous P-endoglucanase from Penicillium notatum, having no activity against native cellulose. The molecular weight is 78,000 and the amino acid composition is known, giving evidence for the presence of two half cystines. The enzyme is composed of a single peptide chain, internally cross linked by two cysteine residues in a disulfide bridge. The enzyme was strongly inhabited by mercuric ions. The activity could be restored by cysteine as well as by chloride ions. By electrolytic reduction the disulfide bridge, present in the enzyme, was shown to be essential for the activity. The P-endoglucanase molecule was not attacked by exopeptidases under nondenaturing conditions, indicating a solid structure; on the whole, cellulases are so strikingly stable to, e.g., pH and temperature, that it surprises veteran protein chemists, although the susceptibility of the enzyme increases, naturally enough, with purification. Treatment with endopeptidases caused a pronounced decrease in activity. Active fragments could not be obtained with endopeptidases. An electrophoretically and ultracentrifugally homogeneous cellulase from Myrothecium verrucaria, active with respect to ground or swollen cellulose, carboxyrnethyl cellulose, and a series of oligoglucosides (Whitaker, 1960; Whitaker et al., 1963) was found to have 14 cysteine residues.

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BIRGITTA NORKRANS

Even working with pure enzymes we will also surely find differences in the future between cellulases from different organisms due to inherent variation. Cellulase activity seems to be inherited in a polygenic manner. It is inherited independently of mating type in the brown-rotting Polyporus betdinus (Bell and Burnett, 1966). Monokaryons, derived from the same dikaryon, are known to show a range of cellulase activity in the white-rotting Collybia velutipes (Aschan and Norkrans, 1953; Norkrans and Aschan, 1953; Norkrans, 1957b). A number of related enzymes, possibly isoenzymes, may be produced and different monokaryons produce different combinations of these enzymes. The frequent occurrence of isoenzymes of carbohydrases is known through the work of, among others, Nisizawa et a1. (1962; for isoenzymes see Kaplan, 1963; Wilkinson, 1965). In the aforementioned works by Mandels and Reese, the authors deal extensively with the very interesting natural inhibitors of cellulase, chiefly belonging to phenolics, tannins, and types of polymeric leucoanthocyanins. They are probably widespread in plant tissues. About one-fifth of the plants screened contained reasonable amounts of inhibitors, and unripe fruit of persimmon (Diospyros uirginiana) more than 12 times the amount found in wood of Eucdyptus rostrata, the first localized source of natural cellulase inhibitors (see Youatt, 1961). Cellulases from different sources vary markedly in their resistance to these inhibitors. Mandels and Reese discuss the significance of these natural inhibitors as a factor involved in the resistance of plants, concluding: “Resistance must certainly be a summation of factors, one of which may be the presence of natural enzyme inhibitors.” VI. Applications for Cellulases Disintegration of plant tissue has been experimentally provoked with cellulase preparations. Cocking (1960) succeeded in preparing protoplasts b y removal of cell walls from root tips of tomato seedlings b y means of a Myrotheciurn cellulase. Ruesink and Thimann (1965, 1966) improved the method partly by concentrating the cellulase and obtained protoplasts showing vigorous cytoplasmic streaming, and forming a good experimental material for attacking a number of plant physiological problems such as wall formation, ion transport, and water balance. Cellulases are used as cell wall disintegrators in order to increase the digestibility of vegetable foods (Toyama, 1962, 1963, 1965) or extractibility of proteins, fruit juices, essentia1 oils (Nakayama et al. 1965), agar-agar from seaweeds, etc. (Toyama, 1961; Hachiga and Hayashi, 1965). For a long time, cellulases have found application in the pharmaceutical industry as a digestive aid.

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Microbiological Aspects of the Formation and Degradation of Cellulosic Fibers L. JuRASEK', J.

ROSS COLVIN, AND

D. R. WHITAKER

Division of Biosciences, National Research Council, Ottawu, Canada

I. Introduction ............................................................... 11. Formation of Cellulosic Fibers .............................. A. Biosynthesis of Cellulose Microfibrils ..... B. Orientation of Cellulose Microfibrils .......................... C. Mechanism of Formation of Whole Fibers .................. D. Summary ........... .......... 111. Degradation of Fibers by Microorganisms ....................... A. Microstructure of Cellulosic Fibers ........................... B. Degradation by Fungi ............................................. C. Degradation by Bacteria IV. Cellulases and Related Enzymes .................................... A. Scope and Terminology

131 131 132 139 140 141 14 1 141 145 154 155 155 157 C. Heterogeneity, Homogeneity, and Purification ........... 158 165 D. The Course of Degradation .................... 166 166

1. Introduction This review consists of three separate contributions: on the formation of fibrous cellulose (by J.R.C.), on the decay of fibers, particularly wood fibers, by microorganisms (by L.J.), and on cellulases and related enzymes (by D.R.W.). We have kept them separate for two reasons. The first is a matter of ignorance: the second essay considers processes of ultimate complexity and there are simply too many gaps in present knowledge for us to attempt to give a unified account of fiber formation and decay u p to this level. The second reason is a matter of emphasis which, if it is to be in phase with current research, must shift with each topic. Ultimately these fields will illuminate one another but, at present, each has its own particular set of spotlights. II. Formation of Cellulosic Fibers

Three main subdivisions of the process of formation of any cellulosic fiber of biological origin may be distinguished; (a) biosynthesis National Research Council Postdoctoral Fellow, 1964-66. Present address: h t n y drevarsky vyskumny Gstav, LamaEska 5, Bratislava IX, Czechnslovakia.

131

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L.

JUdSEK,

J. R. COLVIN, AND D. R. WHITAKER

of the component chains of the cellulose microfibrils (or parts of these chains) ( b ) mechanism of orientation of groups of cellulose microfibrils to form bundles, sheets, rings, helices, or other shapes often with remarkable regularity (Roelofsen, 1959) ( c ) ordered association of these aggregates in cells or groups of cells. Frequently, this association is strongly periodic or rhythmic (Roelofsen, 1959). Naturally, the factors involved in each of these three subdivisions of the main process overlap and interact with elements in the others but this classification helps to keep confusion to a minimum. A. BIOSYNTHESIS OF CELLULOSE MICROFIBRILS It is not possible to discuss the synthesis of cellulose microfibrils without reference to their structure. Although the fine structure, size, and morphology of cellulose microfibrils has been and is the subject of controversy (Ohad et al., 1962; Frey-Wyssling and Muhlethaler, 1963; Colvin, 1963; Manley, 1964; Heyn, 1966) the description preferred by Roelofsen (1965) is the one used here for reasons which have been published (Colvin, 1963, 1964, 196613). 1 . B y Bacteria Up to the present only two species of bacteria, Acetobacter acetigenum and Acetobacter xylinum have contributed much to our knowledge of cellulose synthesis. For brevity, we will make no distinction between these two species because there is no evidence as yet of important differences between them. Nearly 20 years ago, Muhlethaler (1949) demonstrated clearly for the first time that some of the strands of bacterial cellulose (microfibrils) were outside the wall of the cell and often at a considerable distance from the wall. Later, Hestrin and Schramm (1954) provided convincing evidence, by chemical analysis of washed cells, that little, if any, of the cellulose was present in the bacterial cell wall. At that time, there was a possibility that these strands were either extruded from the cells or sloughed off from their surfaces, but a number of studies have now produced considerable evidence against this view. If the microfibrils were extruded by the cells, it should be possible to observe the attachment of cells to microfibrils by the electron microscope. All such attempts have failed (Colvin and Beer, 1960; Millman and Colvin, 1961; Ohad et d., 1962). If microfibrils were extruded by cells into the medium, the formation of cellulose microfibrils in a stiff gel might be expected to be hindered or prevented due to the viscous drag of the long strands. This is not observed (Colvin and Beer, 1960). Finally, if cellulose microfibrils were extruded by the bacterial cells, elongation of these microfibrils

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would have to be at one end only and separated from other microfibrils. A recent analysis (Colvin, 1966b) shows, on the contrary, that bacterial cellulose microfibrils elongate at both ends at the same rate and that the ends of microfibrils in bundles grow in close association at approximately the same rate. Likewise, if microfibrils were sloughed off the surface of the cells in liquid media, then one would expect to see some evidence of the process on agar surfaces where both cells and microfibrils are held immobile. As mentioned previously, no such association is observed (Millman and Colvin, 1961). At present, all evidence is consistent with the conclusion that bacterial cellulose microfibrils are formed extracellularly in the culture medium and remote from the cell surface. This conclusion implies that an intermediate(s) must pass from the cells into the medium and the nature of this intermediate(s) has been the subject of much study. The extensive body of knowledge on the biosynthesis of other polysaccharides (Hassid et aZ., 1959) suggested quite early that nucleotide sugar phosphates might play a similar role in bacterial cellulose synthesis. Glaser was the first to provide definite evidence (Glaser, 1958). Working with a particulate fraction from a homogenate of bacterial cells, he demonstrated the formation of bacterial cellulose from uridine diphosphoglucose (UDPG). His observations have been confirmed in a number of laboratories and are supported by the work of Kliingsoyr (1960) who reported that the transfer of glucose residues from cellodextrins to an insoluble cellulose-like material was stimulated b y uridine diphosphate. The assumption of a central role for UDPG was strengthened by Enevoldsen’s isolation of the compound from active cells (Enevoldsen, 1964). At the present time there is no reason to doubt that UDPG plays an essential part in bacterial cellulose biosynthesis but it is equally clear that this role is wholly intracellular. All attempts to find evidence for stimulation of cellulose synthesis b y providing extracellular UDPG or related compounds have failed (Dennis and Colvin, 1965). The source of the energy required to produce the P-glucosidic bond in cellulose has been the subject of some speculation but not much study as yet. If adenosine triphosphate (ATP) is the energy donor via uridine triphosphate (UTP), then ultimately the energy is supplied by oxidation of a portion of the glucose either by the pentose phosphate path (Gromet et al., 1957) or by the glycolytic cycle (Benziman and Burger-Rachamimov, 1962). Some years ago, Schramm and Racker (1957) made the interesting suggestion that at least a portion of the energy required for cellulose synthesis might come from acetyl phosphate produced by a phosphorolytic cleavage of fructose 6-

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phosphate. Quite recently, Benzinian (1966) has presented further evidence that the formation of phosphoenolpyruvate from pyruvate is essential for the synthesis of cellulose. Because glucose is a necessary building block of bacterial cellulose and often a carbon source for the cells, a recurring question is whether the glucose which is incorporated into the microfibril via UDPG has undergone extensive rearrangement. Until recently, the bulk of evidence on this point indicated that there was little, if any, skeletal rearrangement of the glucose supplied (Setterfield and Bayley, 1961). Recently, however, several studies have suggested that a re-examination of the question was worthwhile (Benziman and Burger-Rachamimov, 1962; White and Wang, 1964). This re-examination (Everson and Colvin, 1966) has shown that, at least under certain circumstances, roughly one quarter of the glucose molecules incorporated into bacterial cellulose may be rearranged, probably through sc triose fragments followed by recombination. This result is consistent with the observations of Benziman and Burger-Rachamimov (1962), who found that bacterial cellulose may be formed from pyruvate by triose recombination. In summary, therefore, UDPG is very probably an intermediate in bacterial cellulose synthesis, the energy may come from several sources, and the glucose portion of the UDPG inay have been incorporated without change or built up from smaller fragments, especially trioses. However, the role of UDPG must be completely confined to within the cell (Dennis and Colvin, 1965).How UDPG within the cell may be linked to the formation of an insoluble external product like cellulose has challenged bacterial biochemists for a decade (Groniet et al., 1957). However, two recent trends have closed the gap somewhat. First, there is evidence that the formation of another necessary intermediate may be in or on the cytoplasmic membrane of A. xylinum, riot in the cytoplasm (Dennis and Colvin, 1965). This evidence is supported indirectly by work on cellulose synthesis in cells of Sarcina uentriculi (Canale-Parola and Wolfe, 1964) where no incorlxiration of glucose was observed in a system capable of making UDPG but which contained only the supernatant of crushed cells centrifuged at 10,800 g. for 20 minutes. Incorporat’ion was observed when this supernatant was replaced by suspensions of washed cells. Although the authors state that “centrifugation of the crushed cells at slower velocities did not result in incorporation,” unfortunately, they give no details and their data are still open to the interpretation that the “slower velocities” were still sufficiently high to remove all the wall and membrane fragments. At any rate, for A.

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xylinum these observations have removed the paradox whereby, although an inhibitor of bacterial cellulose could not be demonstrated in the contents of the cells, they did not fill u p with insoluble cellulose. If an intermediate is formed in or on the surface of the membrane and exported into the medium directly, no internal inhibitor of cellulose formation is necessary and the paradox vanishes. Second, there is also evidence that this second intermediate is a lipid glucoside (Colvin, 1964, Dennis and Colvin, 1965) preferentially soluble in water, which acts as a carrier of glucose from the cytoplasmic membrane, across the bacterial cell wall and into the medium. Unfortunately, because this intermediate is transient and present in low concentration at any instant (Colvin and Beer, 1960), it has not been possible thus far to obtain enough of the pure compound for an unequivocal chemical description. Efforts toward this goal are still under way. However, knowledge of the presence of this transitory intermediate outside the cell has led to a satisfactory working hypothesis about the formation of the microfibrils in the medium. This working hypothesis has been outlined recently (Colvin, 1964) and will be extended and brought up to date here. Of necessity, some aspects are speculative and controversial but the reader is warned when substantial evidence ends and speculation begins. The hypothesis is as follows: A watersoluble lipid glucoside is released by the cells to the medium, which also contains an extracellular enzyme capable of transferring the glucose from the glucoside to the tip of an insoluble cellulose microfibril. This enzyme is unusual but not unique in that it is soluble in 80% ethanol (Colvin, 1959; Brown and Gascoigne, 1960) and also has moderate heat resistance (Webb and Colvin, 1967). It seems to be strongly but reversibly adsorbed to the surface of the bacterial cell (Webb and Colvin, 1967). Nothing is known about the way in which it is synthesized by the cells or released initially into the medium. HOWever, it can be shown to be present in the supernatants of celluloseless mutants of the bacteria (Webb and Colvin, 1967),which indicates that the metabolic block to cellulose synthesis in these mutants is in the formation of the biochemical intermediate(s), not in formation of the enzyme. Nothing is known experimentally about the way in which the glucose is transferred from the enzyme-glucoside complex to the tip of the insoluble microfibril, but it is known that the tip is blunt, not tapered or frayed (Colvin and Dennis, 1964). This observation confirms earlier conclusions that there is no amorphous polymeric intermediate in bacterial cellulose synthesis (Colvin, 1964) and therefore that the microfibril must grow by addition of single glucose residues to the ends of the 1,4 p-glucosan chains which extend from

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the microfibril. Under ordinary conditions, this rate of addition is approximately 0.1 p per bacterial cell per minute (Colvin and Beer, 1960; Millman and Colvin, 1961). Furthermore, this mechanism of addition implies that the polymerization of a new residue into the chain and its crystallization into the new phase are nearly simultaneous events. During elongation the microfibrils often twist in the manner of a left-handed screw (Colvin, 196lb) either singly or about one another, and this twisting may reflect the molecular mechanism of extension. If the microfibril grows by a screw dislocation mechanism at its tip (similar to a great many inorganic or metal fibers), then the observed twist may be interpreted as an example of an Eshelby twist (Eshelby, 1953; Gomer, 1958). Furthermore, the pitch of the expected twist x' t'ion may be calculated from the simple rel*

where p is the pitch of the helix, r is the radius of the fiber, and h is the height of the Burgers vector of the dislocation. For a cellulose microfibril, reasonable estimates of b and r are 5 A and 50 A respectively. The predicted pitch of the twist is then about lo4 A (lp) in reasonable agreement with the observed distance (Colvin, 1961b). Unfortunately, it is not yet possible to observe any such dislocation directly in cellulose microfibrils. This mechanism of tip elongation is also consistent with the growth of individual microfibrils at both ends at the same rate (Colvin, 196613). How long an individual microfibril may continue to grow at the observed rates (about 1000 glucose residues per bacterial cell per second) (Colvin and Beer, 1960) is not known. Brown (1962) has shown that individual P-glucosan chains may be terminated (and then perhaps renewed) at the end of about 40 minutes but this gives no definite information about the complete microfibril. We do know that new microfibrils (and therefore new chains) are nucleated in the culture (Brown, 1962; Colvin and Beer, 1960)but we have no information on the molecular mechanism of nucleation or of termination of growth of a microfibril (beyond the trivial case of exhaustion of nutrients in the medium). Although new microfibrils are nucleated at the rate of about 40/103 bacteria per minute, and therefore their numbers may quickly become very large per unit area, they seem to extend without any apparent joining or anastomoses. We are also coin-

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pletely in the dark as to why the microfibrils have such a narrow distribution of diameters about 100 A.2 2. B y Green Plants The reader is referred to the following reviews (Setterfield and Bayley, 1961; Colvin, 1964; Roelofsen, 1965) for discussions of the earlier literature. In general, as indicated by Roelofsen (1965), the weight of evidence at the present time is that synthesis of cellulose in green plants is similar to (but not necessarily identical with) that in bacteria. Nucleotide sugar phosphates play a central role as intermediates within the cell (Barber and Hassid, 1965; Brummond and Gibbons, 1965) but if the cytoplasmic membrane is regarded as the boundary of the cell, the actual synthesis of the microfibril is extracellular (Roelofsen, 1965), often remote from the membrane, and deep in the cell wall (Setterfield and Bayley, 1959). There is some evidence that the transfer of intermediates from within the membrane to the exterior may be by the same general mechanism as in bacteria (Colvin, 1961a) but beyond that very little is known about the molecular mechanism of cellulose microfibril formation in green plants. There has, of course, been considerable speculation (for refs., see Roelofsen, 1965),but all suggestions need much more experimental investigation. Recent work on the intracellular intermediates for cellulose synthesis has disclosed some interesting differences in detail between green plants and bacteria and also, apparently, between different species of green plants. In contrast to bacteria, Elbien et al. (1964) have shown that in mung beans only guanosine diphosphate glucose (GDPG) is an active glucosyl donor for cellulose synthesis. This is true also for synthesis of cellulose in cotton (Barber and Hassid, 1965),but the picture is complicated by the stimulation of incorporation by guanosine diphosphate mannose (GDPM). However, a recent study by Brummond and Gibbons (1965) using Lupins albus, has demonstrated that while GDPG will serve as a donor of glucose for cellulose synthesis in a system from this plant, UDPG is a much better donor. Hence the authors conclude that there may be two enzymes and therefore two enzymic pathways to cellulose. A related *Note: A word about terminology is necessary here. Ohad et al. (1962) have suggested that the true diameter of microfibrils has been grossly overestimated by metal shadowing. One of us is of the opinion that this suggestion is not correct (Colvin, 1963).We stress that, in this article, the term microfibril means a separate independent thread about 100 A in diameter and indefinitely long. Subfilaments may exist within these microfibrils and may be resolved by suitable techniques with the electron microscope but for the sake of clarity, they should not be called microfibrils.

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but different suggestion has been made for cotton by Marx-Figini and co-workers. They have shown (Marx-Figini and Schulz, 1966) kinetically that there are two stages in the incorporation of glucose into the cellulose of cotton which they identify as the formation of the cellulose of the primary and secondary layers. The formation of the cellulose of the secondary layer is consistent with the assumption of a template mechanism whereas this mechanism is not applicable to the formation of the cellulose of the primary layer. Hence, two separate enzymic routes are postulated (Marx-Figini, 1966). Clearly, much more work is required before a clear, unambiguous picture will emerge.

3. By Fungi The walls of several species of fungi are known to contain cellulose quite definitely and there is reasonable basis for assuming that this is true for many others also. However, there is presently very little detailed information on the mechanisms of formation of fungal cellulose (Aronson, 1965). We can only assume that it may be similar in general to the process in bacteria or green plants. 4. By Animals

Cellulose from tunicates has been studied for many years and there is no doubt that this material from animals is very similar to, if not identical with, the product from plant sources (Whistler and Smart, 1953). However, nothing is known about the biosynthesis of cellulose from animal sources and there are no microbiological aspects to the subject except in the following indirect way. Hall and co-workers have detected cellulose in the dermis of ox and humans and in the aorta of humans (Hall et al., 1960; Hall and Saxl, 1961).They reported that, although the concentrations were low in all tissues, this material was particularly evident in the dermis from patients with pathological conditions of the skin. This evidence for “mammalian cellulose” has been attributed to contaminating cotton fibers (Roelofsen, 1959). Even if it is true that all outside sources of vegetable fibers were excluded (Hall and Saxl, 1961), there remain two other possibilities. First, parasitism of mammalian hide or skin by fungi is well known, extremely common, and often associated with visible pathological conditions. The fibers isolated from these samples may simply have been part of the mycelia of a cellulose-producing, parasitic fungus. Whether the cellulose is then still “mammalian” in origin is a question of semantics. Second, the procedures for separation of the

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fibers necessarily required long periods of enzymic digestion under conditions favorable for the development of both bacteria and fungi. The growth of bacteria was suppressed by penicillin and streptomycin but it is possible (indeed probable) that some fungal growth would take place in the specimens during these periods. Once again, it cannot be excluded that the glucose-yielding, anisotropic, tubular fibers observed were not the remains of hyphae from a celluloseproducing fungus. B. ORIENTATIONOF CELLULOSEMICROFIBRILS The biochemistry of cellulose is only a part, although an essential one, of the process of formation of a cellulosic fiber. The orientation of the microfibrils, either during or after actual synthesis, to form larger structures is the next essential step and one about which we know almost nothing. Mechanical forces certainly play a role but it is easy to demonstrate that they are not the only influence or even the most important in many systems (Roleofsen, 1959,1965).

1 . In Bacterial Cellulose Until quite recently, there was little, if any, evidence that microfibrils of bacterial cellulose were oriented to any appreciable extent in undisturbed pellicles at the surface of the culture. Some slight birefringence was evident occasionally when the films were examined between Polaroid films, but this was attributed to slight stretching or compression. In addition, when microfibrils were grown on moist agar surfaces, characteristic sheaves of relatively short oriented microfibrils were observed (Millman and Colvin, 1961). Recently, however, two independent series of observations have indicated that bacterial cellulose microfibrils may be oriented by nonmechanical means during static development of the pellicles. Ben-Hayyim and Ohad (1965) have shown that the introduction of carboxy methyl cellulose or phosphomannans (but not levans) into the medium will cause partial orientation of the microfibrils in the pellicle. They speculate that the orientation may be a result of coulombic interaction between charged microfibrils but the analysis was not pursued. Their observations are extremely interesting as well as suggestive and ought to be continued. In addition, typical two-dimensional analogs of spherulites have been studied in bacterial cellulose which are formed by regular, long-sustained, radial nucleation and growth of microfibrils (Colvin, 1965).These structures may be more than 1cm. in diameter and represent a definite, although

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simple, type of orientation of the component microfibrils. Somewhat later, a nonspherulitic type of birefringence was observed which is due to alignment of bundles of microfibrils in a particular direction throughout the thickness of the pellicles (Colvin, 1966a). Occasionally, these bundles will branch or will induce the formation of parallel structures in their vicinity. No way has yet been found to change noticeably the frequency, the course of development, or the form of both the above types of orientation. Nonetheless, within the last year, a number of chemical compounds have been shown to be able to induce a third type of birefringence (orientation) within pellicles of bacterial cellulose (Carson et al., 1967).These compounds include the diglycerides, deoxycholic acids, and many (but not all) sterols. How any of the above types of orientation are induced at the molecular level is not known definitely yet. In particular, there is as yet no clue as to how the above chemical compounds may induce alignment of very much larger (molecularly) cellulose microfibrils. It is as if the introduction of a barrel of fuel oil into a mill pond were to induce orientation of logs floating in the pond. Nonetheless, the phenomenon occurs, and because these seem to be the first example of the orientation of cellulose microfibrils by other than mechanical means, they may offer a useful key to an old problem.

2. In Fungal and Green Plant Cellulose For a general review of our knowledge in this field the reader is referred to Roelofsen’s excellent recent summary (1965). All the means of orientation which have been studied to date for these organisms have been mechanical in one or more senses, As indicated earlier, other means must exist but their mode of operation is unknown.

c. MECHANISMOF

FORMATION OF WHOLE FIBERS

The mechanism of formation of whole fibers means the process or series of processes whereby the oriented cellulosic structures touched upon previously (bundles, sheets, rings, helices, or other simple shapes) are incorporated with noncellulosic material to form the complete cell or associated cells. Although a great deal is known now about some of the final products (see Section TII,A), very IittIe is known at present about the molecular or supramolecular processes despite an abundance of speculations. In particular, this subject has very few microbiological aspects which have not been summarized

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before (Aronson, 1965), and therefore, will be omitted here. For a general discussion of the present situation with nonmicrobiological cells, the reader ought to consult Roelofsen’s excellent review

(1965).

D. SUMMARY If one makes the basic assumption that the process is similar in general in all organisms, then the formation of cellulosic fibers involves three interrelated aspects which may be termed the biochemical, physical, and biological. In the biochemical phase, a nucleotide sugar phosphate is an intermediate within the cell for external cellulose microfibrils. A subsequent intermediate is a lipid glucoside which acts as a carrier to the space outside the cytoplasmic membrane. Outside the membrane the glucose is transferred to the tips of insoluble cellulose microfibrils by an extracellular enzyme, one residue at a time. The rate of elongation of the microfibrils is comparable to that of other biological fibers but much about its mechanism is obscure. In the physical phase, although it is established that the stiff, strong microfibrils (Sakurada et al., 1964) may associate to form bundles, sheets, or rings, only scraps of information are available at present about the process by which this is accomplished. In addition, in the biological phase, even less is known about the mechanisms of association of the above structures with other substances to form whole cells. Ill. Degradation of Fibers by Microorganisms

A. MICROSTRUCTURE OF 1 . The Cellulose Framework

CELLULOSIC

FIBERS

Cellulosic fibers may be obtained from many different parts of plants: e.g., cotton from seed hairs, flax, jute and ramie from bast, pineapple fibers from leaves and wood fibers from secondary xylem. Except for cotton, the fibers occur in lignified tissues and their main function is to provide mechanical support for plant organs. This support comes from their thick cell walls which persist, and continue to provide support, after the death of the cell. Despite their many shapes, fibers have common structural features. These are best illustrated by the structure of wood cell walls. The following will be only a brief description; detailed accounts are given in the publications edited by Zimmermann (1964) and by Cbte (1965).

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During cell division, the first indication of the cell wall is the amorphous cell plate. This is formed froin the Golgi apparatus (Frey-Wyssling et d., 1964) and develops later into an intercellular layer consisting of hemicelluloses and polyuronides. The cellulose components of the wall are deposited as microfibrils in characteristic arrangements (Wardrop and Harada, 1965; Fig. 1).The primary wall

.

Outer surface

Inner surface I

Primary wall

Inner loyer of secondary wall

Outer loyer of secondary wall

6,) (0-6 Larnellae)

t

’Middle layer of secondary

Ei 1 3 1 wall (S,)

i

(Several Larnelloe iniermidiaie between thoi of the S,ond S, loyersi

.

(4-6 Larnellae alternately oi S or Z rnicrofibrillar orientat ion i

I

( C A 30-150 Lornellaei

(Several Larnellae of orientation interrnedioie between thai of the S,and S, layers)

FIG. 1. A diagrammatic representation of the microfibril orientation in the cell wall of fibers and tracheids (from Wardrop and Haracia, 1965).

(P) is deposited during the phase of surface growth. As the cell grows, the primary wall stretches, becomes weakened, and its microfibrils rearrange; newly formed microfibrils are continually deposited over the extending surface and randomly interwoven microfibrils are characteristic of the primary wall, particularly of its outer surface (Fig. 2). Further growth of the cell wall ceases in areas in which plasmodesmata pass through the cell wall. These areas appear later as thinner regions of the wall and form pits of different shapes. The pit membrane may retain the structure of the primary wall or undergo a rearrangement of its microfibrillar structure. In conifer tracheids, transverse bars of microfibrils (crassulae) cause thickenings of the primary wall between bordered pits. The formation of the secondary wall takes place when surface growth has ceased and the primary cell wall has attained its maximum extent. It starts in the central part of the cell wall and proceeds toward both tips of the cell. The secondary wall is characterized by

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FIG. 2. The outer surface of the primary wall (P) of a spruce tracheid after removal of matrix and encrusting substances. Electron micrographby Dr. V. NeCesanL.

parallel arrangements of microfibrils (Fig. 3). The outer layer (S1) of the secondary wall consists of several lamellae of microfibrils with alternating helical dispositions. The intercellular layer with adhering P and S1 from adjacent cells appears homogeneous under the light microscope and is often referred to as the “compound middle lamella.” The middle layer (Sz) of the secondary wall is the bulkiest component of the wall. The microfibrils composing the numerous thin lamellae of the middle layer are arranged in helices which form steeper angles than those of the outer layer. NeEesany et al. (1965) present evidence that these lamellae reflect day and night periodicity in cell growth. The thin inner layer ( S3) of the secondary wall differs from S z mainly in its less steeply angled helices. Its original designation “tertiary wall” is still in use (Liese, 1963). The inner surface of S3 is covered by an ultramicroscopic layer of isotropic structure. This layer often spreads over small granular particles on the surface of S3. Its appearance (Fig. 4) gives it the name “warty layer” (Liese, 1963).

2. The Matrix and Encrusting Substances In addition to the cellulose framework, the mature cell wall contains

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other materials in the spaces between the fibrils. Those deposited at the same time as the cellulose microfibrils are called “matrix substances’’ (hemicelluloses and polyuronides); those deposited later

FIG. 3. Inner surface of the middle layer of a secondary wall ( S p ) partially covered by the inner layer (SJ of a spruce tracheid. Electron micrograph by Dr. V. Neeesan?.

are called “encrusting substances” (lignin). Meier (1961) studied the distribution of matrix substances by an indirect method. His analyses of differentiating cells at various stages of development indicated that the content of hemicelluloses is low in the inner part of Sz and S3 but high in the outer part and in the compound middle lamella. A high content of hemicelluloses was also found in S3 of summer tracheids of pine. Lange (1954), using a microspectrographic procedure, found

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roughly similar patterns for the distribution of lignin. Electron microscopic observation of the distribution of lignin (Sachs et al., 1963), determined after removal of polysaccharide by chemical treat-

FIG. 4. Warty layer inside a tracheid of Abies ulba (from Liese, 1965).

ment, confirmed the above findings. The compact structure of S3 after the treatment is an indication of high lignin content in this part of the cell wall.

B. DEGRADATION BY FUNGI 1. Growth of Microorganisms in Fibrous Materials Microorganisms require suitable physiological conditions to grow on cellulosic materials. RypaEek (1966) gives a detailed discussion

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of the requirements of wood-rotting fungi. Moisture is frequently the decisive factor. The wood-rotting Basidiomycetes require a comparatively narrow moisture range; bacteria and the soft rot fungi (Ascomycetes and Fungi Imperfecti) can withstand higher moistures and higher temperatures and decompose cellulose under conditions which Basidiomycetes cannot tolerate.

FIG. 5. Hypha of Ophiostoma coeruleum penetrating the cell wall of a pine tracheid (from Liese and Schmid, 1961).

From a morphological point of view, fungi are better equipped to attack cellulosic tissues than bacteria because their hyphae enable them to penetrate deeply into the tissue. However, usually their growth is restricted to the cell lumen. This is the reason why the attack of fungi on wood proceeds faster in an axial direction than in a transverse direction. In beech wood, the relative growth rates of hyphae in axial, radial, and tangential directions were 12.2 : 1.7 : 1.0 respectively (JurASek, 1960). In hardwoods, the hyphae take advantage of long vessels which enable them to grow fast axially. An axial growth rate of up to 0.3 mm./hour was measured in beechwood attacked by SchixophyZlum commune (Navratilova, 1964). The attack on wood by this fungus begins with a rapid occupation of the substrate by a low density of mycelium (100 m. of h y p h a e / ~ mof .~

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wood) without appreciable decomposition. In the occupied areas, the density of the mycelium then increases gradually and reaches values above 1000 m . / ~ min . ~wood which shows signs of only slight decay. As they infest the tissue, the hyphae penetrate cell walls, both through the pits in the walls and at other places, choosing the shortest way across the wall. Observations of Nutman, Cartwright, and Proctor (reviewed by Cartwright and Findlay, 1946) suggest that local enzyme action is responsible for the formation of boreholes but the penetration of cell walls by blue-stain fungi (Fig. 5) was shown to be mechanical (Liese and Schmid, 1961). Liese and Schmid (1962) have described ultramicroscopical microhyphae and mycofibrils as being frequently attached to the hyphae of white rot fungi; these are suspected of participating in the penetration process. The growth of soft rot fungi and bacteria in cellulosic fibers differs from the above scheme and is discussed later.

2. Changes in Microstructure Judging by the comparatively slow rate of weight loss, cellulosic fibers in general seem to be a rather difficult substrate for decomposition b y organisms. Probably the main reason lies in the compact structure of cellulose which hinders free diffusion of the enzymes which degrade it. This problem is more pronounced when the ceIlulosic framework and matrix are encrusted with lignin. In addition, the heterogeneous chemical composition of the cell wall requires the organisms to produce a complex and highly specialized spectrum of enzymes. Peculiarities of enzyme production and/or the morphology of microorganisms appear to be the main factors influencing the mode of decomposition. Each species of fungus tends to have its own characteristic decomposition pattern but they fall into fairly definite categories. For our purpose, we accept Meier’s (1955)four categories: brown rot, simultaneous rot, white rot, and soft rot.

a. Brown Rot The action of brown rot fungi is usually limited to the polysaccharide moiety of the wood. Lignin remains mostly undigested, but not unchanged. The loss of lignin methoxyl, reported by Kiirschner (1927), has been confirmed by many others (e.g., Grohn and Deters, 1959). Hemicelluloses may (RypaEek, 1952) or may not (Cowling, 1961) be digested preferentially. Destruction of pit membranes is one of the early structural changes in pine wood attacked by Coniophora cerebella and Poria vaporaria. The selective breakdown of

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crassulae, leaving holes in the cell wall, suggest that crassulae consist mainly of polysaccharides and are not lignified (Juras’ek, 1958). Although the enzymic attack of the cell wall clearly starts from the inner surface, because the hyphae are located in the cell lumen, no signs of preferential breakdown of the inner layers could be observed in any stage of the decomposition. Liese and Schmid (1962) observed local changes in the Sz layer of pine wood attacked by Lentinus squammosus. The inner surface of the cell wall remains unattacked, even in the immediate vicinity of the hyphae, but enzymes diffusing through S3 cause local lysis of Sz with formation of rhomboid cavities resembling those formed by soft rot fungi.

FIG. 6. Transverse section of spruce tracheids decomposed by. the fungus Merulius lacrymans (from Meier, 1955).

Staining techniques (Juras’ek, 1964a) reveal changes in the compound middle lamella during early stages of decomposition of Abies alba wood by Merulius lacrymans. Possibly diffusion of enzymes is not the rate-limiting step here and the breakdown of polysaccharides proceeds simultaneously in the whole cell wall. As decomposition proceeds, the cell wall gradually loses its birefringence and the compound middle lamella cannot be distinguished by the optical

FORMATION AND DEGRADATION OF CELLULOSIC FIBERS

149

microscope. The cell wall shrinks, becomes brittle, and shows numerous fissures. At this stage, chemical analysis demonstrates that the cell wall consists of almost pure lignin. The porous structure of S p in spruce wood decomposed by Merulius lacrymans is shown by electron micrographs of ultrathin sections (Meier, 1955; Fig. 6); the pores mark the original location of the digested framework and matrix substances. The survival of the compound middle lamella and Ss is in accordance with previous conclusions about high lignin content. In fibers from beech reactionwood, the unlignified inner part of S p (gelatinous layer) completely disappears under the attack of Merulius lacrymans (JuraSek, 1964a).

b. Simultaneous Rot Fungi causing this rot are endowed with the widest spectrum of enzymes and under suitable conditions they are capable of almost complete decomposition of wood. Cowling (1961) studied the action of Polyporus versicolor on sweetgum sapwood and found a striking

FIG. 7. Transverse section of beech wood fibers decomposed by fungus Trametes gibbosa.

parallelism in the removal of all components of the wood (cellulose, hemicelluloses, lignin) during decay. Similar results were obtained

150

L.

JURASEK,

J. R. COLVIN, AND D. R. WHITAKER

with Trametes gibbosa on beech and fir reaction and normal wood Juras’ek, 1964b).

FIG. 8. Hypha of the fungus Polyporus versicolor growing on the inner surface of a beech wood vessel. Lysis zones are visible on both sides of the hypha (from Liese and Schmid, 1961).

The decomposition of wood by simultaneous rot is manifested by gradual loss of thickness of the cell wall (Juras’ek, 1960). The warty structure of the inner surface of the cell wall soon disappears and the parallel fibrillar arrangement of the secondary wall becomes clearly visible. The thinning, however, cannot be explained as a simple gnawing away” of the cell wall. Histochemical analyses show that partial delignification precedes the total decomposition of the inner parts of the cell wall. Advanced stages in the decay of birch wood by “

FORMATION AND DEGRADATION OF CELLULOSIC FIBERS

151

Polyporus versicolor were studied by Meier (1955) with the electron microscope and the intercellular layer and primary wall were shown to be the last survivors of the original structure (Fig. 7 ) .Liese and Schmid (1962) observed sharply defined local zones of lysis of the warty layer of beech wood when hyphae of Polyporus versicolor were grown on the inner surface of the cell wall (Fig. 8). The decomposition of the cell wall by simultaneous rot fungi proceeds gradually, starting from the lumen. This suggests that diffusion of enzymes is the rate-limiting step for decomposition of the cell wall by simultaneous rot fungi (cf. brown rot). The decomposition of polysaccharides is probably influenced by the extent of delignification and vice versa. If not, the proportional loss of all components of the cell wall is hard to explain.

c . White Rot White rot fungi (in the narrow sense used here) represent a small group of fungi which preferentially decompose lignin and hemicelluloses of wood, leaving cellulose nearly undigested.

FIG. 9. Transverse section of birch wood fibers decomposed by Trarnetes pini (from Meier, 1955).

The decomposition of birch and spruce wood by Trametes pini was studied with the electron microscope by Meier (1955). In an

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L. J U d S E K , J. R. COLVIN, AND D. R. WHITAKER

advanced stage of decay, delignification of the cell wall leads to complete dissolution of the intercellular layer and primary wall (Fig. 9). The cellulosic framework of the secondary wall remains and retains its shape. The loss of interfibrillar substance is apparent from the porous structure of the secondary wall. Examination of the inner surface of pine wood attacked by the same fungus (Liese and Schmid, 1966) shows evidence of the local action of the fungal hyphae: e.g., lysis of S, in the close vicinity of hyphae, rhomboid-shaped cavities in S p , and microhyphae penetrating the cell wall. The gradual decomposition of fir or beech wood b y Trametes pini (Juras’ek, 1964a) can be easily followed by histochemical tests for lignification. The delignification starts from the inner surface of the cell wall, with gradual widening of the delignified area. No thinning of the cell wall takes place; in fact, because of the swelling of delignified cellulose, the remaining cell wall becomes even thicker. As soon as delignification extends over the whole Sz, the highly lignified compound middle lamella starts to decompose completely. The tissue falls apart when the compound middle lamella has been dissolved and isolated; completely delignified fibers remain as the residue from the decomposition. The gradual delignification, proceeding from the inner surface, presents convincing evidence of the very slow diffusion of the enzymes which are responsible for delignification. Since the boundary between delignified and native parts of the cell wall is always sharp, and since no signs of decomposition can be found in front of the delignified area, it seems likely that the penetration of enzymes into the cell wall depends on delignification. The slow diffusion of enzymes into the cell wall probably accounts for the slow decomposition of wood by white rot fungi.

d. Soft Rot Soft rot resembles brown rot in its effects upon the chemical composition of wood. Wood polysaccharides are the principal targets and lignin remains mostly undigested, but with a loss of part of its methoxyl content (Levi and Preston, 1965). However, the mode of decomposition by these fungi is so different that the separation of this group is fully justified. The hyphae of soft rot fungi grow both in the cell lumen and in the depths of S2 where they follow the direction of the cellulose microfibrils. Levi and Preston (1965) have shown that hyphae of Chaetomium globosum which penetrate the cell wall transversely often change direction of qrowth and proceed further longitudinally

FORMATION AND DEGRADATION OF CELLULOSIC FIBERS

153

through S p . Hyphae of some soft rot fungi (not Chaetomium) have been shown to cause localized zones of lysis in warty layers or S3 when growing on the inner surface of the cell wall (Liese, 1964). However, hyphae submerged in S z are mainly responsible for decomposition of the cell wall. Their boreholes are narrow at first, about the size of hyphae, and form helical structures clearly visible on longitudinal sections.

FIG. 10. Tracheids of spruce wood from a cooling tower decomposed by soft rot fungi. Longitudinal section.

After further growth, the boreholes start expanding to form wider cavities with characteristic conic ends. Several authors have sought to explain the striking regularity in shape of these cavities (Fig. 10).

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L. JURASEK, J. R. COLVIN, AND D. R. WHITAKER

They have been discussed by Liese (1964), Levi and Preston (1965), and Liese and Schmid (1966).Basically, the phenomenon is attributed to restricted diffusion of enzymes secreted by regions of hyphae growing along the microfibrils in Sz. The restriction of diffusion appears to be determined by the anisotropic structure of Sz. Fungal degradation proceeds finally throughout the whole of the Sz (Fig. 11). The compound middle lamella and often Ss (depending 011 the species of wood and fungus) resist the attack and retain their shape in the last stages of decay (Meier, 1955). The resistance of these parts of the cell wall is probably due to their high lignin content.

FIG. 11. Transverse section of birch wood fibers decomposed by Chaetomium globosum (from Meier, 1955).

It seems that, unlike the easily diffising hydrolases of brown rot fungi, the hydrolases of soft rot fungi diffise very slowly through the cell wall and close contact in Sz between hyphae and substrate appears to be essential.

c. DEGRADATION BY BACTERIA Bacterial attack on pine and spruce wood has been described by Harmsen and Nissen (1965). Bacteria spread comparatively slowly in the tissue. The penetration from one cell to the other occurs only

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155

through pits, whose thin membranes are dissolved by the bacterial enzymes. The infection spreads preferentially along medullary rays which contain numerous pits and which are comparatively rich in nitrogeneous substances. The local action of bacteria on the inner surface etches depressions which reach to the compound middle lamella. Advanced decay is characterized b y complete destruction of the secondary wall which becomes brown and amorphous. At this stage, the bacterial decomposition resembles certain types of soft rot and apparently cell wall polysaccharides are preferentially digested. The compound middle lamella is resistant. The formation of rhomboid-like cavities, characteristic of soft rot, was not observed. The decomposition of isolated cotton fibers by bacteria usually starts from the outer surface (Siu, 1950) in contrast to fungal attack which begins from the lumen of the fiber. Bacteria adhering to the outer surface dissolve the cuticle (the outer layer of the fiber, including the primary layer) and later cause deep erosions of the secondary wall (Fig. 12). Basu and Ghose (1962) found bacteria growing

FIG. 12. Cotton fiber partially digested by bacterium Sporocytophaga myxococcoides (from Siu, 1950).

in the lumen of the cells of jute fibers. The cut ends of the fibers apparently provided the port of entry. The bacteria grew rapidly inside, probably due to the supply of organic nitrogen. IV. Cellulases and Related Enzymes

A. SCOPE AND TERMINOLOGY The earlier literature on cellulases is discussed in the monographs of Siu (1951) and of Gascoigne and Gascoigne (1960). Reese (1963) has edited the proceedings of a symposium held under the auspices

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L. J U A S E K , J. R. COLVIN, AND D. R. WHITAKER

of the American Chemical Society in March, 1962, and Norkrans (19634 has given an excellent review of the literature up to the same period. The literature considered in this review is mostly post-1962. The Report of the Commission on Enzymes of the International Union of Biochemistry (1961) lists “cellulase” as the trivial name for P- 1,4-glucan 4-glucanohydrolases, i.e,, for enzymes which hydrolyse P-1,4-glucans at linkages which are not restricted to terminal linkages (enzymes which degraded P-1,4-glucans by splitting off glucose or cellobiose residues would be designated “P-1,4-glucan glucohydrolases” and “P-1,4-glucan cellobiohydrolases” respectively). T h e report lists “P-glucosidase” as the trivial name for P-D-glucoside glucohydrolases; these are clearly a very heterogeneous group of enzymes. It should be noted that the systematic name specifies one operation, but not necessarily all the operations, of an enzyme. For example, the specification of a cellulose or a glucosidase as a hydrolase does not imply that it is without transferase activity and, if the specificity of a particular cellulase is determined by its ability to form an enzyme-substrate complex with @-1,4-linkedresidues rather than by the cleavage process itself, then the way is open for cellulases to split other types of linkages when the substrate is a heteropolysaccharide such as those studied by Perlin and his associates (see Reese, 1963). Cellulases are defined above in the broadest terms -with no restrictions on the physical state of the glucan or on its occurrence in a natural fiber. Narrower definitions have been suggested at times. Halliwell (see Reese, 1963), for example, favors ability to degrade native cotton fibers as the operative test for “cellulases” (as opposed to “P-polyglucosidases”). The objection to such a narrow definition for enzymes which operate at surfaces is that it attaches no significance to the noncellulose components of the fiber and possibly attaches too much significance to the particular physical structure of the cellulose component. Reese and his co-workers introduced another terminology based on their “CI, CS” hypothesis. “CI” was postulated to be a nonhydrolytic enzyme which, by converting cellulose to linear chains, enabled a hydrolase, Cr, to convert the linear chains to soluble sugars. This hypothesis was later modified to a “multiple C,” hypothesis which assigned the initial attack to the more enterprising hydrolases. Recently there has been some revival of the original C1 notion. Siu (see Reese, 1963) attaches importance to a nonhydrolytic step and, as mentioned later, Mandels and Reese (1964) and King (1965)have dis-

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cussed some of their findings for enzymes from Trichoderma oiride as being possibly indicative of the action of a nonhydrolytic enzyme. The terms “C, activity” and “Cl activity” are often encountered in the literature with a different meaning than the above-“C, activity” usually denoting activity toward a soluble derivative of cellulose and “Cl activity” usually denoting hydrolase activity toward a substrate with a high a-cellulose content or high “crystallinity.” Carboxymethyl cellulose is tending to be supplanted by hydroxyethyl cellulose as the preferred soluble derivative. As shown by Datta et al. (1961), carboxymethyl cellulose is not only a potential substrate but a potential inhibitor of cellulases. The term cellulase is used in this review in the sense of the Commission on Enzymes but without any restriction on the method of degradation as, in some instances, the method of degradation has not been determined.

B. SOURCES

AND

PRODUCTION O F CELLULASES

Recent papers on the production of cellulases cover a broad range of microorganisms: bacteria: Sporocytophaga myxococcoides (Charpentier, 1963, 1965), Ruminococcus albus (Leatherwood, 1965), and a thermophilic bacterium (Verkhovtseva, 1965); a streptomycete: Streptomyces antibioticus (Enger and Sleeper, 1965); molds: Rhizopus sp. (Imada et al., 1962), various species of Penicillium, Trichoderma, and Aspergillus (Konno et al., 1962), Myrothecium verrucuria (Whitaker and Thomas, 1963), Aspergillus terreus and Penicillium variabile (Pal and Ghosh, 1965), a thermophilic strain of Aspergillus fumigatus (Loginova and Tashpulatov, 1965), Pyrenochaeta terrestris (Horton and Keen, 1966); Mycorrhizal fungi (Ritter, 1964) and higher fungi: Fomes annosus (Lyr and Schand, 1964). The cellulase in the digestive juice of the edible snail, Helix pomatia, is prominent in the early literature on cellulases but its origin has been a matter of some controversy. The data of Strasdine and Whitaker (1963) suggest that it is produced by the snail itself, not by its microflora. Myers and Eberhart (1966) have obtained mutants of Neurospora crassa which involve a regulatory gene for activity toward cellobiose and carboxymethyl cellulose. This gene is distinct from a regulatory gene for P-aryl glucosidase activity although cellobiose induces all three activities in wild-type mutants. This promises to be a most significant line of investigation. Four culture methods are in common use for the production of

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cellulases: ( a ) submerged culture in a liquid medium which is aerated from spargers, ( b ) shake cultures in a liquid medium, (c) stationary cultures in a liquid medium and ( d )Koji-type processes in which the organism is grown on a moist solid medium (e.g., wheat bran) from which enzymes are subsequently extracted. Foam control is a major problem in method ( a ) as extracellular enzymes can be completely or partially denatured by foam and by antifoaming agents. Foam can also be a problem in method ( b ) and some organisms, e.g., Collybia velutipes and Rhixina undulata (Norkrans, 196313) and Fomes annosus (Lyr and Schani.1, 1964) have been reported to give satisfactory yields of enzyme in stationary cultures only. Method (d) avoids dilution of the enzyme on the culture medium and is free of foam problems but open trays entail more risk of contamination by other organisms and some of the supporting media used for Koji-processes (and as carbon sources for submerged culture) - wheat bran, for example - favor concomitant production of other types of enzymes (proteases represent the most serious problem) and may yield products which complex with proteins. These, of course, are considerations which matter only if purification of enzymes is in view. Japan has become the major center for production of cellulases on an industrial scale and a great variety of enzyme preparations from submerged culture and Koji-culture of Rhizopus, Trichoderma, Penicillium, Aspergillus, etc. are now available commercially. Toyama (see Reese, 1963) gives a valuable comparison of some of the earlier products.

c. HETEROGENEITY, HOMOGENEITY,AND

PURIFICATION

Conflicting reports on the homogeneity and heterogeneity of cellulases continue in the literature. The extreme example - the early literature on Myrothecium cellulase - has been reviewed before (Whitaker, 1960; also see Reese, 1963). In brief, much of the extreme electrophoretic heterogeneity reported by some laboratories (e.g., 16 components with activity toward carboxymethyl cellulose) appears to have been a matter of dissociable complexes between enzyme and polysaccharide. Jermyn (1962) came to the same conclusion with regard to the apparent heterogeneity of the p-glucosidase of Stachybotrys atra. Some individual examples of reported heterogeneity and homogeneity will be mentioned first. Amberlite XE-64 fractionated an enzyme preparation from Penicillium variabile into a fraction with activity toward carboxymethyl cellulose and a fraction with activity toward carboxymethyl cellulose and a-cellulose; the latter did not

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159

give cellobiose as a hydrolysis product (Konno et al., 1963). A fraction with activity toward carboxymethyl cellulose but not toward swollen cellulose or powdered cellulose has also been obtained from Aspergillus saitoi (Matsurama and Maejima, 1963). Sephadex G-100 separated four components with activity toward carboxymethyl cellulose from an enzyme preparation from Aspergillus niger and three from an enzyme preparation of Penicillium notatum (Pettersson, 1963). Chromatography on calcium phosphate separated two components with activity toward carboxymethyl cellulose from a commercial Aspergillus niger enzyme preparation; treatment with laminarin removed the trace (1+3)-glucan activity (Clarke and Stone, 1965a). Four electrophoretically distinct fractions from Cellvibrio gilvus cleaved cellohexaose, cellohexitol, and cellopentitol at the second and third linkage from the nonreducing end (Cole and King, 1964). Electrophoresis in a block of starch grains separated five components with activity toward carboxymethyl cellulose from an enzyme preparation from Streptomyces gilvus; according to immunodiffusion tests, three of them were immunologically identical (Enger and Sleeper, 1965). The cellulase of a thermophilic bacterium was homogeneous on Sephadex G-100 (Verkhovtseva, 1965) and the cellulase of Aspergillus terreus showed no heterogeneity on Sephadex G-100 or on ion-exchange chromatography; the purified enzyme hydrolyzed carboxymethyl cellulose and a-cellulose (Pal and Ghosh, 1965). Reports on Trichoderma viride cellulase are numerous and varied. 1. King and his co-workers (Li et al., 1963, 1965; King, 1965) have reported fractionation of a commercial enzyme preparation into (a) a “hydrocellulase” fraction with high activity toward Avicel (a commercial microcrystalline cellulose), ( b ) an “endoglucanase” fraction with high activity toward carboxymethyl cellulose (and the soluble oligoglucosides, including cellobiose) and (c) an exoglucanase” fraction which, unlike the endoglucanse, degraded the soluble oligoglucosides b y cleaving glucose units from the nonreducing end. The separations depended on (1)adsorption of the “hydrocellulase” on Avicel in the presence of citrate buffer (and desorption by water) and (2) adsorption of the “endoglucanse” on alkali-swollen cellulose in the presence of phosphate buffer (and desorption by water). The molecular weight of the “hydrocellulase” was estimated from its sedimentation rate to be about 60,000, that of the “endoglucanase” to be about 50,000, and that of the “exoglucanase” to be about 76,000. The endoglucanase was free of carbohydrate. 2. Mandels and Reese (1964) reported a partial separation of “

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activity toward cotton linters and activity toward carboxymethyl cellulose by a fractionation on DEAE-Sephadex (A-50). 3. Ogawa and Toyama (1964, 1965) partially separated activities toward cotton fibers, filter paper, and carboxymethyl cellulose by chromatography on powdered cellulose; chromatography on a gauze column separated the fraction with high activity toward cotton into two fractions with different activities toward vegetable cell walls. 4. Niwa et al. (1964, 1965), using enzyme from the same commercial source as King et al. obtained three to four fractions with varying activities toward filter paper and carboxymethyl cellulose by electrophoresis in a block of starch grains. Chromatography of the crude enzyme on Amberlite XE-64 gave three fractions with activity toward cotton, filter paper, and carboxymethyl cellulose; the major fraction on refractionation on DEAE-Sephadex (A-50)-gave five fractions with activity toward the above substrates and toward soluble oligoglucosides, including cellobiose. The activity of his “hydrocellulase” fraction toward carboxymethyl cellulose and cellotriose is attributed to impurities by King (1965). Thus the findings of King et aZ. and of Mandels and Reese on the one hand and of Niwa et al. on the other can lead to two quite different interpretations of the properties of the T . viride enzyme system. 1. According to one interpretation, the filtrate contains a “C1--CI” system where C1 is responsible for the initial attack on native or crystalline cellulose. King and Mandels and Reese discuss the possibility that C1 is a nonhydrolytic enzyme but there seems to be no significant evidence bearing on this point. 2. According to the other interpretation, the filtrate contains enzymes with a broad spectrum of activities relative to the degree of polymerization and physical state of the substrate. Limited proteolysis may be partly responsible for the apparent multiplicity of such enzymes for Niwa et al. (1964) have noted that the crude enzyme has appreciable protease activity. A recent paper of Selby and Maitland is of interest with respect to the above findings. They report the isolation from T . viride culture filtrates of (a) a cellobiase component with virtually no activity toward carboxymethyl cellulose or cotton, ( b )a “Cz”-component with activity toward carboxymethyl cellulose but with virtually no activity toward cellobiose or cotton, and ( c ) a glycoprotein with virtually no activity toward any of the above substrates. The combination (a) ( c ) accounted for 20% of the filtrate’s activity toward cotton; the combination ( b ) ( c ) accounted for 35% of the

+

+

FORMATION AND DEGRADATION OF CELLULOSIC FIBERS

161

+ +

activity; the combination ( a ) ( b ) (c) accounted for all the activity toward cotton. They designate the glycoprotein ( c ) as a “C ”-cornponent, but although it may, as they suggest, be an enzyme of undetermined function, it may also simply be a nonenzymic glycoprotein which protects a conventional cellulase against inactivation or irreversible adsorption. As discussed below, there are suggestions that Trichoderma koningi cellulase is stabilized by more firmly bound carbohydrates. Data are beginning to accumulate which allow a few purified cellulases to be compared on a fairly broad basis. The following make an interesting comparison: 1. Two cellulases from a commercial preparation of enzymes from Trichoderma koningi (Iwazaki et al., 1965a,b). [These findings are supported by the earlier data of Koaze et al. (1964) who fractionated the enzyme preparation by gel filtration; Wakazawa et at. (1963) give data on its action on wood pulp and cotton linters.] 2. Two cellulases from culture filtrates of Polyporus versicolor (Pettersson et al., 1963; Pettersson and Porath, 1963). 3. A cellulase from culture filtrates of Myrothecium verrucaria (Whitaker et al., 1963; Datta et al., 1963; Hanstein and Whitaker,

1963). The purification methods were briefly as follows: 1. Trichoderma koningi cellulase: enzyme was precipitated by ammonium sulfate, freed of noncellulase impurities by DEAESephadex and Amberlite CG-50, and fractionated on hydroxylapatite into cellulase I (eluted with 0.001 M phosphate) and cellulase I1 (eluted with 0.1 M phosphate buffer). 2. Polyporus versicolor cellulase: enzyme was concentrated by dry Sephadex G-25 and fractionated on Sephadex G-75; one fraction (D), when refractionated on Sephadex G-75, gave an apparently homogeneous enzyme (enzyme D); another fraction (B) on fractionation b y preparative zone electrophoresis in Pevikon powder gave an apparently homogeneous enzyme ( B l ) . 3. Myrothecium cellulase: Method I: Enzyme was precipitated by ammonium sulfate and freed of noncellulase impurities by elution from Sephadex G-75, precipitation with polymethacrylic acid, and displacement from Amberlite CG-50 with a gradient of urea in citrate buffer. Method 11: Enzyme was precipitated with ammonium sulfate and freed of noncellulase impurities by precipitation with polymethacrylic acid, elution from DEAE cellulose with 7 M ureaphosphate buffer and elution from Sephadex G-75. The two methods

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gave products with identical specific activities toward carboxymethyl cellulose. The use of buffered 7 M urea for the elution from DEAE cellulose requires comment. It was used to prevent enzymic attack on the resin; if this was not prevented, the enzyme became complexed with DEAE-substituted oligoglucosides and thereupon became subject to proteolysis; the consequent fall in molecular weight (e.g., from 50,000 to 7,000) was accompanied by loss of activity toward carboxymethyl cellulose. The comparison in Table I, notably the comparison of molecular weights, again raises the question as to whether limited proteolysis is a significant factor in the formation of multiple enzymes. The carbohydrate components raise other questions. For example, it would be of interest to know whether they are covalently bound to protein or whether, like the carbohydrate of Stachybotrys atra P-glucosidase (Jermyn, 1962), they are dissociable. In either event, they might be expected to stabilize the enzyme but possibly a more specific stabilization is at issue here which enables an enzyme to withstand forces at cellulose surfaces which otherwise would disrupt its conformation. The T. koningi cellulase I1 is of particular interest in this respect as it appears to resemble the previously discussed 2‘. viride enzymes of Niwa et al. Purified Myrothecium cellulase is listed in Table I as having weak activity toward ground cotton and the same could be said of the culture filtrate from which it was prepared. However, this assessment is based on reducing sugar formation during a comparatively brief (17 hours) incubation period. Some recent work on Myrothecium filtrates and enzymes has been concerned with the effects of prolonged treatments. Halliwell(l966) used untreated or dewaxed cotton as substrate and measured losses in weight and “fine fiber” formation over periods of up to 5 months. His culture filtrates proved to be extremely variable in their activity but, despite the low load of substrate (2 mg. in 9 ml. of a 1:3 dilution of filtrate at 37“C), none gave more than an 18% conversion to soluble sugar. Some filtrates gave up to an 83% conversion to “fine fiber,” i.e., fiber which remained in suspension when shaken in water. Under the same conditions, Trichoderma koningi filtrates gave complete solubilization in 24 days. Selby et al. (1963) used alkali-treated cotton yarn as substrate and measured the changes in breaking strength. Under their test conditions, the culture filtrate ceased to weaken the fiber after about 5 days. However, it still retained the ability to weaken fresh samples of cotton

TABLE I COMPARISON OF Trichodema, Polyporus,

AND

Myrothecium CELLULASES

Trichodema koningi Enzyme

Molecular weight (Archibald method) Carbohydrate content Sulfhydryl groups (Asx Glx)/ (His + Lys Arg)b N-terminal amino acidC C-terminal amino acid Enzymic activity

+

a

+

Cellulase I1

Cellulase I

Polyporus oersicolor

Myrothecium oerrucaria

Cellulase B1 Cellulase D

Cellulase

50,000

26,000

50,000

11,400

49,000

12%" None detected 4.6

2%

1% 2.7

33%

None detected 6.3

None detected None detected 2.0

None detected Substantially lower a c tivity toward cellobiose and carboxymethyl cellulose than cellulase I. High activity toward powdered cellulose and cotton fiber.

None detected High activity toward cellobiose and carboxymethyl cellulose. Low activity toward powdered cellulose. No appreciable activity on cotton fiber.

-

3.5

Both active against carboxymethyl cellulose and a cellulose sol.

None detected Clycine Very low activity toward cellobiose, activity toward higher oligoglucosides and their p-methyl derivatives increases with D.P. of substrate. High activity toward carboxymethyl cellulose and swollen cellulose. Weak activity toward ground cotton.

This carbohydrate gave mannose and a small amount of hexosamine on hydrolysis.

* Asx denotes (aspartic acid 9 asparagine) residues; Glx denotes (glutamic acid + glutamine) residues. The particular

ratio listed in the table is chosen merely because it indicates one of the most marked differences in amino acid composition between the enzymes. The amino acid analyses of the Trichoderma hydrolyzates show a most unusual dependence on the time of hydrolysis; this may be an effect of carbohydrate. As determined by the DNP and/or Edman method. A negative result is often an indication that the N-terminal residue is acylated.

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to the extent of about 15% and it still retained much of its activity toward carboxymethyl cellulose. If the filtrate was replaced daily by fresh filtrate, the cotton lost virtually all its strength and about 25% of its weight within 30 days. When cotton was exposed to filtrate and then eluted with alkaline borate or with solutions of carboxymethyl cellulose or methyl cellulose, the eluate showed enhanced breaking strength activity. On this and other evidence, Selby et al. (1963) postulated that two types of cellulase were present in the filtrate: (a) a labile “A-enzyme,” readily adsorbed by cellulose, with enhanced activity toward native cellulose and low activity toward carboxymethyl cellulose; and ( b )a stable “B-enzyme,” not readily adsorbed by cellulose, with low activity toward native cellulose and with high activity toward carboxymethyl cellulose. Enzyme B, it was suggested, might be formed from enzyme A by some deactivation process. Selby and Maitland (1965) subsequently found that Sephadex C;-75 fractionated these activities to give two fractions (I and 111) having activities similar to those deduced for “enzyme A” and one fraction (11)having activities similar to those deduced for “enzyme B.” The elution volumes from Sephadex G-75 were taken to indicate molecular weights of 50,000 for I, 30,000 for 11, and 5300 for 111. Apart from their intrinsic interest, these findings illustrate a general problem which runs through the whole literature on cellulases -the problem of comparing data. The variable features of culture filtrates and of most substrates are well known (Whitaker, in Reese, 1963); the problem here stems from differences in test methods. Breaking strength can be a very sensitive measure of attack on fibers and a filtrate with high breaking strength activity might still be rated as having low activity toward native cellulose by measurements of weight loss or reducing sugar formation. According to Selby and Maitland’s estimates of molecular weights, the enzyme in Fraction I matches the Myrothecium enzyme listed in Table I, but the latter was complexed with polypeptides in the culture filtrate and, at a corresponding stage of purification, would have been rated as having a molecular weight closer to 60,000. The original data on elution volumes do not resolve the problem for, apart from differences in load and temperature, the Sephadex preparations are not comparable (that used by Whitaker et al. was one of the first batches released for commercial use). The absolute values of the molecular weights are also not necessarily comparable, for Selby and Maitland’s estimates rest on assumptions with regard to shape, composition, and binding properties which are not always valid [The data of JuraSek

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and Whitaker (1965) and Whitaker et al. (1966) for a bacteriolytic enzyme provide a recent example]. In short, two sets of data cannot be compared with certainty and, unfortunately, such is the normal situation in this field. D. THE COURSE OF DEGRADATION By ordinary standards, the degradation of native cellulose by enzymes or microorganisms is a slow process as only a small fraction of the linkages are accessible to an enzyme at any given time. The kinetics of the process have not received any serious attention as yet -and probably with good reason - for an adequate treatment would be an extremely complicated treatment. McLaren (1963), in a paper dealing with insoluble substrates in general, has derived an equation for the cases in which the rate of hydrolysis is proportional to the amount of enzyme adsorbed on a surface in accordance with a Gyani-Freundlich isotherm. He has shown that this equation has some applicability to data on cellulases but a more searching test would require better data on initial rates than those available at present. Random cleavage of accessible linkages appears to be the general method of degradation for unsubstituted celluloses but it is difficult to check the method rigorously unless the cellulose is highly swollen. Selby (1961) has reported a substantial deviation from random cleavage in the degradation of mercerized cotton by Myrothecium cellulase but he did not establish whether anything more than accessibility factors were responsible for the deviation. Soluble derivatives of cellulose are never cleaved in a strictly random manner as a linkage's susceptibility to cleavage depends on the proximity of the linkage to a substituted glucose residue. Klop and Kooiman (1965) have made a detailed study of the action pattern of Myrothecium cellulase and Luizym on many soluble derivatives of cellulose. Their findings suggest that a linkage - GI - o - 4G1- is susceptible to hydrolysis by these enzymes if it meets two conditions: (a) G must be an unsubstituted glucosyl residue and (b) G' must be either an unsubstituted or a 6-substituted glucosyl residue. The soluble oligoglucosides are also never cleaved in a strictly random manner as the rate of hydrolysis of a linkage in a short chain depends on its proximity to the ends of the chain. Hanstein and Whitaker (1963)have extended the earlier data on the rates and action patterns of Myrothecium cellulase with more comprehensive data on the cleavage of @-methyl oligoglucosides. Action patterns of Cellvibrio gilvus (Cole and King,

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1964), Aspergillus niger (Clarke and Stone, 1965b), and Trichoderma viride (Li et al., 1965) enzymes on the oligoglucosides and their reduction products have also been determined. With the exception of the exoglucanase of T. viride, all these enzymes tend to cleave interior linkages more rapidly than terminal linkages, The exoglucanase of 7‘. viride cleaves terminal linkages only and thus resembles the P-glucosidase of Stachybotrys a t m (Youatt, 1958).

E. APPLICATIONS Most of the cellulase produced in Japan goes to the pharmaceutical industry, e.g., for use in digestive tablets. This is an old application; Luizym, the Aspergillus enzyme preparation which figures so prominently in the older literature, was and still is produced for that purpose at the Luitpoldwerke in Munich. Analytical procedures for determining cellulases in pharmaceutical preparations are discussed in a review by Courtois and Bui-Khac-Diep (1965). A rough rule of the fermentation industry states that the broth in a fermenter must be worth 25 cents per liter if its production is to be profitable (Aiba et al., 1965). This gives an indication of the economic limitation on other applications of cellulases (as distinct from applications of cellulolytic organisms). At present, these applications tend to exploit the ability of cellulases to weaken or eliminate unwanted fiber (e.g., fiber which interferes with a processing operation) rather than their potential ability to convert waste cellulose to edible sugar (Underkofler and Toyama, in Reese, 1963). Inhibitors of cellulases have possible applications as agents for protecting cellulosic materials from microbial attack but, apart from the implications of their being large molecules, cellulases appear to be less vulnerable targets for practical inhibitors than their parent organisms. ACKNOWLEDGMENT We wish to thank Dr. V. NeEesany for the electron micrographs shown in Figs. 2 and 3 and the following publishers for permission to reproduce published figures: Clarendon Press, Oxford (Fig. I), Syracuse University Press (Fig. 4), Vereinigung f i r Angewandte Botanik, Berlin (Figs. 5 and 8), Springer-Verlag,Berlin (Figs. 6,9 and 11) and the Textile Research Institute, Princeton (Fig. 12).

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The Biotransformation of Lignin to HumusFacts a n d Postulates

R. T. OGLESBY,R. F. CHRISTMAN,AND C. H. DRIVER Departments of Civil Engineering and College of Forestry, University of Washington, Seattle, Washington

I. Introduction ............................................................... 11. Mechanisms of Lignin Biodegradation ........................... 111. Humification ............... IV. The White-Ro A. Taxonomy ............................................................ B. Natural Occurrence ............................................... C. Factors of Growth ...... .......... V. Other Organisms Responsible for Lignolysis and Humification .............................................. A. T h e Ultimate Decomposition of Lignin .................. B. Organisms Involved in Humification ....................... References .................................................................

171 173 175 178 178 178 181 182 182 182 183

1. Introduction Sarkanen (1963) has stated that between 18 and 30% of the tissue in mature wood is composed of lignin which forms, together with the cellulose and other carbohydrates of the cell walls, a material of excellent strength and durability. Although lignin is undoubtedly durable, it is equally obvious that mechanisms exist in nature for its eventual aerobic decomposition. For some 30 years it has been generally recognized that lignolysis under natural conditions is primarily a biodegradative process and that the principal lignolytic organisms were a group of basidiomycetes known as the white-rot fungi. Much of the early work on this subject was devoted to ascertaining what organisms were capable of degrading intact lignin and the general rates at which such attacks proceeded. Cook (1957) has reviewed this research and one may conclude from the data which he presents that in approximately 6 to 8 months 60 to 75% of the lignin content of natural substrates is destroyed by white-rot fungi. This figure is consistent with the more detailed findings of Ichihara et al. (1956). In a forest a second process occurs in close physical proximity to the site of lignin degradation. This is the formation of humus on the forest floor. Humus performs several important functions in nature. In the soil it influences structure and texture, thus exerting an effect on aeration and moisture-holding capacity. Humus has a pronounced 171

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base exchange capacity which enables it to store and release nutrients to the surrounding soil and it serves as a source of carbon dioxide which may subsequently be used by green plants. Kononova and Aleksandrova (1959) indicate that humus may also directly participate in plant metabolism through stimulation of enzyme systems. When considering lignin and humus the question naturally occurs as to what, if any, connection exists between the processes of lignolysis and humus formation. Umbreit (1962)has presented a general scheme (Fig. 1) illustrating a sequence of biotransformations beginning with

Start

After a week

After a month

After o year

FIG. 1. The formation of humus resulting from the biodegradation of plants. Figures are given in grams of carbon. After Umbreit (1962).

lignin and other compounds of plants and generating soil humus as an end product. To completely delineate such a sequence, one would first of all need to know the molecular structures of both materials and then trace the metabolic pathways between them. To date the chemical structures of neither lignin nor humus have been fully elucidated but enough it known about the chemical characteristics of each to indicate that not only are these highly complex macromolecules but also highly variable as well. The latter is particularly true of humus, which is not really a discrete compound but probably a mixture of fractions classically referred to as humin (ulvin), fulvic acid, hymatomelanic acid, and humic acid. The latter fraction usually predominates but the ratios between the different fractions of humus may vary. Humic acid is generally regarded as being the most chemically

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complex of the four and it is often inferred that the other fractions of humus are precursors of humic acid. During the last 15 years or so a number of papers, principally reporting the research of Japanese workers, have indicated the probable mode of initial attack by white-rot fungi on the lignin macromolecule and the nature of some of the early degradative intermediates. During the same period, researchers in Europe and North America have elucidated some of the probable steps in the formation of humic acid. Despite the many gaps in available information, Christman and Oglesby (1967) have postulated metabolic sequences leading from lignolysis to humic acid formation. Their work is summarized below. Except for the white-rot fungi, which are generally conceded to be the principal agents of biological attack on raw lignin, the nature of the organisms which participate in the intermediate reactions, and ultimately the formation of humus, is mainly speculative. For this reason the authors have focused their attention principally on the higher fungi but will briefly hypothesize on the possible nature and role of some of the other forms of life relating to the sequence of chemical events. II. Mechanisms of l i g n i n Biodegradation

The initial attack by white-rot fungi on the lignin macromolecule is almost certainly dependent upon their extracellular enzyme activity. Most lignolytic fungi contain the phenoloxidase-type enzymes laccase and tyrosinase (phenolase) and a few species have been found to also produce a peroxidase similar to that found in the horseradish. Schubert (1965) has discussed lignolytic enzymes in detail and Lyr (1962) has proposed a possible second function for such enzymes, namely the detoxification of a number of materials in heartwood which would otherwise cause uncoupling of oxidative phosphorylation. He has also suggested that peroxidases would be particularly appropriate enzymes for fungi living in heartwood because they do not compete with intracellular substrate oxidations for oxygen. Work by Higuchi et al. (1955), Fukuzumi (1960), Ishikawa et al. (1963a,b), and Ishikawa and Oki (1964) indicates that the first step in lignin biodegradation is an attack on the guaiacyl-type monomeric units, resulting in the liberation of guaiacylglycerol-P-coniferylether. These workers have also identified a number of phenylpropanoid compounds presumably produced from guaiacylglycerol-0-coniferyl ether and existing as intermediates in the subsequent degradation to

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benzyl derivatives which are ultimately oxidized to COz and/or compounds in the citric acid cycle. These investigations resulted in the development of two different concepts of the pathways employed by microorganisms in converting

H-c-0

OCH, OH Guaiacylglycerol-0coniferyl ether units

1

COOH

Q

OCH,

OH

3-Methoxy-4-hydroxyphenylpyruvic acid

/ \ @CH1CWH CH,O

+ cq

OH

6 -6 OH

OCH,

OH

OCH,

1

Demethoxylation, h ydroxylation

Demethoxylation

COOH

9 I

OH

OH

OH

Ring f i s s u r e

OH

Ring f i s s u r e

FIG. 2. Possible pathways of aromatic ring metabolism by white-rot fungi. After Christman and Oglesby (1967).

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175

guaiacylglycerol-P-coniferyl ether units to simple benzyl derivatives. One view, proposed by Ishikawa and Oki (1964), consists of conversion to vanillic acid through coniferaldehyde and vanillin. Fukuzumi (1962),however, does not consider coniferaldehyde and vanillin as degradation products produced by Poria and has hypothesized conversion of guaiacylglycerol-P-coniferyl ether units to methoxy gentisic acid via guaiacylpyruvic acid, P-oxyconiferyl alcohol. and coniferyl alcohol. This concept, although intriguing, is supported by less evidence than the former. At any rate, it is apparent from both the low yields of degradation products obtained by Ishikawa and oxygen uptake data presented by Fukuzumi that lignolytic organisms do not terminate their degradation of the substrate with the formation of guaiacylpyruvic or vanillic acids. The metabolic pathways employed in further degrading these benzyl derivatives are, at present, entirely speculative. However, a consideration of known methods of microbial attack on aromatic nuclei would indicate that such substrates should be readily degradable and that the course of degradation might well follow a number of different pathways. Christman and Oglesby (1967)have indicated how protocatechuic acid and/or gentisic acid could be formed from 3methoxy-4-hydroxyphenylpyruvic acid and then subsequently degraded along pathways consistent with theories of aromatic ring metabolism (Fig. 2). I I I. Hum ification Present knowledge indicates that at least three general possibilities exist for the conversion of lignin to humus. First, the solid material left after fragmentation of the lignin macromolecule by microorganisms could form the basic material for humification. Second, reactive phenylpropanoid or benzyl degradation products may undergo either direct or enzymic extracellular polymerization with the formation of new humus-like macromolecules. Finally, humus may be formed as a direct metabolic by-product of organisms involving cellular resynthesis of reactive compounds followed by excretion and subsequent repolymerization. The overall process of converting lignin to humus would have to be oxidative in nature. An increase in carboxyl acidity and a decrease in alcoholic hydroxyl content, probably due to side chain oxidation, would have to take place. Christman and Oglesby (1967), in comparing functional group analyses for a hypothetical gymnosperm lignin and a humic acid concluded that the increased carboxyl content could

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not arise solely from side chain oxidation but also probably involved ring cleavage of some of the aromatic configurations in the original lignin molecule. Ring oxidation is also indicated by the relatively large carbonyl content of humic acid. In addition to oxidation, the conversion of lignin to humus involves the addition of nitrogen. A general scheme which might account for these conversions is shown in Fig. 3. Lignin

1

Microbial degradation

Guaiacylglycerol0-aryl ether

+

Residue

I t

Phenolic and quinonoid intermediates

k

Amino acids and other polyphenols

Humus

FIG.3. Hypothesized general pathway for the conversion of residual decayed lignin to humus. After Christman and Oglesby, (1967).

Polymerization of reactive intermediates produced during lignin degradation is also an essential feature of humus formation. Flaig (1960) and Murphy and Moore (1960) have studied the nonenzymic polymerization of simple phenolic compounds leading to humiclike compounds and Flaig (1960) also studied polymerization reactions involving quinones. He showed that a -hydroxy-o-benzoquinones are important intermediates in the oxidative polymerization of lignin-like phenols. From the experimental results of these workers Christman and Oglesby (1967) concluded that the fractions of the vanillic acid-like degradation products of lignin not metabolized by microorganisms are oxidized to a series of quinonoid intermediates, probably of the a-hydroxy-o-benzoquinonetype prior to humus formation. Polymerization must be preceded by demethoxylation to account for the low methoxyl content of humic acid. Such reactions are theoretically required for metabolic utilization involving ring fissure

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177

and it is therefore conceivable that benzyl derivatives are demethoxylated prior to either metabolic utilization reactions or to polymerizations leading to humification. Possible pathways are summarized in Fig. 4. Intermediate methoxyphenols De methoxylation

/

t

Intermediate phenols I

(Y

-Hydroxy-o-benzoquinoiies

Ring fis\

Hydroxylation

Intermediate phenols I1

g/

fissure

Cell t i s s u e + C$

t

Humus

FIG.4. Hypothesized conversion of lignin degradation products to humus or cell tissue. After Christman and Oglesby (1967).

The above discussion was solely concerned with lignin humification as an extracellular process. Kononova and Aleksandrova (1959) have reported on experiments which indicate that two common soil fungi, Asperigillus niger and Penicillium spp., are apparently capable of synthesizing humic precursors within the cell. They grew these organisms on media containing glucose as the sole carbon source and subsequently isolated a brown residue closely resembling humic acid from the culture medium. Both fungi were able to incorporate inorganic nitrogen into the resulting humic-like residue. Emanual (1964) and Craigie and McLachlan (1964) have also noted the production of humic substances by microorganisms growing on simple carbon sources. Considering the size of humic acid molecules, it seems certain that only humic precursors, probably phenolic or quinonoid in nature, are formed inside the cell. Following excretion these intermediates could be oxidatively polymerized, as indicated above, with the resultant formation of humic acid. The reader should not infer from the foregoing discussion that lignin degradation is the sole process by which humus is formed. Many other substances are subject to humification and, even in cases

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in which lignin appears to be the primary precursor, our knowledge of the chemical structure of humus would indicate the probability that other chemical moities must be present to react with lignin degradation products before a true humus can be formed.

IV. The White-Rot Fungi Despite the economic importance and ecological significance of

the white-rot fungi, surprisingly little is known about their genetics, the physical and chemical environments needed for their growth, or their interactions with other organisms. At present our store of knowledge concerning these fungi seems to be reasonably complete only in regard to which forms possess lignolytic capabilities and in regard to techniques for growing them in laboratory culture. From the latter, certain general inferences may be made as to their growth requirements.

A. TAXONOMY Lawson and Still (1956) have attempted to compile a comprehensive listing of the organisms responsible for white-rot decay. And Nobles (1965) has developed a key for identifying cultures of woodinhabiting hymenomycetes. Cowling (1961) has summarized the taxa responsible for white-rot decay and has indicated that a total of 250 species of fungi fall into this category. Of these, seven species belonged to the Ascomycetes and Fungi Imperfecti and the remainder to the Basidiomycetes. The family Polyporaceae includes 184 species of white-rot fungi, 41 species are found in the Thelephoraceae, 11in the Agaricaceae, and 7 species are members of the Hydnaceae. All of the basidiomycetes are capable of spreading by either vegetative growth of the mycelia or through the formation of airborne basidiospores. Identification of the white-rot basidiomycetes is complicated by the great variety of growth forms which a single species may take. This is illustrated in Fig. 5 which shows photographs of Fomes annosus, one of the most ubiquitous as well as one of the more morphologically variable white-rots.

B. NATURALOCCURRENCE White-rot fungi attack the lignin of both softwood (gymnosperm or coniferous) and hardwood (angiosperm) trees, and Cowling (1961) has stated that 75% of all those reported in the United States are associated with the latter group. However, many of those known to

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occur on coniferous trees are pathogenic and produce patterns of decay more extensive than the heartwood type characteristic of whiterots associated with the angiosperms. Fomes annosus, for example, is reported by Rishbeth (1950) to be the most limiting factor in the management of conifer forests in Europe, and Driver and Ginns (1964) indicates a similar condition for the intensively managed forests of the southeastern United States. Wilcox (1965) and Cowling (1961) have both indicated that lignin in the wood of angiosperms is degraded more rapidly than is that of the softwoods. Although the latter have a higher lignin content than do the angiosperms, there is a chemical difference between the lignins of these two groups of trees as well as a difference in the location of the lignin in relation to the cells. Sarkanen (1963) has stated that softwood lignin appears to contain primarily guaiacylpropane units while that of the hardwoods contains both guaiacyl-

FIG.5. A. The white stringy root-rot of slash pine induced by Fomes unnosus (Fr.) Cke. The white irregular structures on the roots are resupinate fruiting bodies of the causative fungus. B. A typical perennial bracket fruiting body of F. annosus at the base of a young slash pine killed by the actions of the fungus. C. Annual fruiting bodies of F . annosus on a slash pine stump. The scale in the figure is in inches.

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and syringylpropane units. Whether or not these chemical differences are related to a difference in the rate of white-rot fungal attack is not known. Wilcox (1965) and Cowling (1961) postulate that angiosperm wood is more susceptible to attack due to the location of lignin in the middle lamellae of the tissue rather than concentrated in the cell walls as is the case with the lignin of conifers. Lignin would thus be more available to the exoenzymes of the fungi in the former case.

c. FACTORS OF GROWTH In considering the chemical factors which affect the growth of white-rot fungi, little can be said beyond citing the fact that synthetic media rich in organics, such as the malt extract agars used by Nobles (19651, K2iarik (1965), and others, produce excellent growth and high enzyme activity. Cook (1957) cites several works on submerged culture media, using lignin as a carbon source, including the vitamin requirements of one species and the nitrogen requirements of several others. The hydrogen ion concentration as it affects fungal enzyme activity has been reported on by Law (1950),Boiden (1951),Lyndeberg and Holm (1952),Kaarik (1965),and Fukuzumi (1959).In general they all found a maximum phenoloxidase activity in the acid range but their values for p H optima varied widely from a minimum of about pH 2 to a maximum of around pH 5.5, perhaps reflecting differences in the phenoloxidase systems studied. In the laboratory most studies of lignolytic activity by the whiterot fungi have been conducted within the temperature range of 20" to 28°C. However, two efforts at research to demonstrate the temperature-growth relationship of these organisms gave rather surprising results. Cowling and Kelman (1964) in studying the growth rates of numerous strains of Fomes annosus found little difference between any of the cultures grown over a temperature range of 5" to 28°C. Kaarik (1965) reported little difference in phenoloxidase activity for a group of white-rot fungi when tests were conducted over a temperature range of 15" to 30°C. Biological factors of growth which have received some attention in laboratory studies include variations in enzyme production by different isolates of a single species and the effects of culture age on phenoloxidase concentration. Kaarik (1965) has shown that some species exhibit little variation between strains in terms of the enzymes which they secrete but a few, such as Peniophora gigantea and Stereum sanguinolentum, are quite variable in this regard. He also

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demonstrated that older cultures exhibit greater enzyme activity. Whether this aging effect is due to change(s) in the medium, such as a lowering of pH, caused by metabolic activity of the fungi, or whether it is due to cell autolysis was not investigated. V. Other Organisms Responsible for Lignolysis a n d Humification

A. THEULTIMATEDECOMPOSITION OF LIGNIN The role of microorganisms other than white-rot fungi in the decomposition of lignin has been discussed by many but so far has not been demonstrated in natural ecosystems. However, since the initial attack on the lignin macromolecule most probably involves extracellular reactions, a variety of products could well be made available for uptake not only by white-rot fungi but also by any other microorganisms present in the vicinity. Certainly a wide variety of soil bacteria and fungi would be capable of degrading such fragments. For example, Evans (1963) in reviewing the nature of microbial attacks on aromatic nuclei cites the extensive capabilities of the soil bacterium Pseudomonas in this regard and Henderson (1957) has demonstrated that the soil fungi Haplographium spp., Hormodendrum spp., and Penicilliurn spp. are capable of converting ortho-, meta-, and paramethoxy benzoic acids to the corresponding monohydroxybenzoic acid and that Hormodendrum and Penicillium further metabolized p-hydroxybenzoic acid via protocatechuic acid. Pathways such as these are not only important in obtaining a full understanding of how lignin is degraded but also may be used to help explain some of the differences which exist in aromatic functional groups between humic acid and lignin.

B.

ORGANISMS INVOLVED IN

HUMIFICATION

As cited above, laboratory studies have indicated the probability that soil fungi are directly responsible for producing at least a portion of the soil humus from more highly oxidized precursors. Such work lends credence to the hypothesis that humic acid may be, at least in part, an excreted metabolic end product and hence raises the question of whether or not soil protozoans and nematode worms may not also play a role in the formation of soil humus. Of all organisms in the soil, bacteria are by far the most numerous and have b y far the greatest surface area. Since this latter parameter reflects biochemical activity, it is logical to hypothesize a significant role for bacteria in the lignin-to-humus pathway. Sorensen (1962) has

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provided the only direct piece of evidence so far which shows a bacterial degradation of a prepared lignin and the subsequent formation of a humic acid-like compound. He used an isolated Braun’s native lignin as the carbon source for cultures of Pseudomonas-14 and mixed cultures of soil bacteria which incIuded Flauobacterium. Both cultures decomposed about 30% of the substrate in 3 to 4 weeks. Following biological utilization, nonbiological changes occurred which resulted in the residual lignin being converted into a colored, water-soluble substance which reacted with organic nitrogen compounds to form the humic acid-like residue. REFERENCES Boiden, J. (1951).Reo. Mycol. 16, 173. Christman, R. F., and Oglesby, R. T. (1967). In “Lignins-Chemistry and Utilization” (K. V. Sarkanen, ed.). Wiley (Interscience), New York. In press. Cook. W. B. (1957). Tappi 40,301. Cowling, E. B. (1961). U.S.Dept. Agr. Tech. Bull. 1258, 79 pp. Cowling, E. B., and Kelman, A. (1964). Phytopathology 54, 373. Craigie, J. S., and McLachlan, J. (1964).Can.]. Botany 42,23. Driver, C . H., and Ginns, J. H. (1964). Plant Disease Reptr. 48, 803. Emanual, C. F. (1964).J. Water Pollution Control Federation 36, 1229. Evans, W. C. (1963).J . Gen. Microbiol. 32, 177. Flaig, W. (1960). Sci. Proc. Roy. Dublin S O C . Ser. A 1, 149. Fukuzumi, T. (1959). Nippon Mokuzai Gakkaishi 5,222. Fukuzumi, T. (1960). Bull. Agr. Chem. SOC. Japan 24,728. Fukuzumi, T. (1962).Agr. Biol. Chem. (Tokyo) 26,447. Henderson, M. E. K. (1957). J . Gen. Microbiol. 16, 686. Higuchi, T., Kawamura, I., and Kawamura, H. (1955). Nippon Ringaku Kaishi 37,298. Ichihara, K., Ikeda, S., and Sakamoto, Y. (1956).J. Biochem. 43, 129. Ishikawa, H., and Oki, T. (1964). Nippon Mokuzai Gakaishi 10,207. Ishikawa, H., Schubert, W. J., and Nord, F. F. (1963a).Arch. Biochem. Biophys. 100, 131. Ishikawa, H., Schubert, W. J., and Nord, F. F. (1963b).Arch. Biochem. Biophys. 100, 140. Kaarik, A. (1965). Studia Forest. Suecica 31, 80 pp. Kononova, M. M., and Aleksandrova, I. V. (1959). Soils Fertilizers 22, 77. Transl. from Izu. Akad. Nauk SSR Ser. Biol. 1, 79 (1958). Law, K. (1950). Ann. Botany (London) 14,69. Lawson, L. R., and Still, C. N. (1956). Inform. Serv. Center. 79 pp., 272 refs. West V. Pulp Paper Co., Charleston, S.C. Published for private use. Lyndeberg, and Holm (1952).Physaol. Plantarum 5, 100. Lyr, H. (1962).Nature 195,289. Murphy, D., and Moore, A. W. (1960). Sci. Proc. Roy. Dublin S O C . Ser. A 1, 191. Nobles, M. K. (1965). Can. J . Botany 43, 1097. Rishbeth, J. (1950).Ann. Botany (London) 14, 365. Sarkanen, K. V. (1963). In “The Chemistry of Wood” (B. L. Browning, ed.), Chapt. 6. Wiley, New York.

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Schubert, W. J. (1965). “Lignin Biochemistry,” pp. 77-119. Academic Press, New York. Sorensen, H. (1962).J . Gen. Microbiol. 27,21. Umbreit, W. W. (1962). “Modern Microbiology,” p. 331. Freeman, San Francisco, California. Wilcox, W. W. (1965). Forest Prod. J. 15, 255.

Bulking of Activated Sludge WESLEY0. PIPES Department of Civil Engineering Northwestern University, Evanston, Illinois

I. Introduction ... A. Applied Researc B. Operating Problems ............................................... 11. Types of Settli A. Floc Formation Problems B. Density Problems .................................................. C. Compaction Problems ............................................ 111. Superficial Aspects of Bulking ......... .................... A. Plant Operation ...... B. Waste Composition ............... C. Aeration Tank Environment .. IV. Fundamental Aspects of Filmentous Bulking.. ................ A. Hypotheses About the Mechanisms of Filamentous B. Filamentous Bulking Organisms

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185 185 187 189 191 196 20 1 204 204 209 214 217 218 225 229 232

I. Introduction Last year in this series I tried to point out that the activated sludge process provides some intellectually challenging and practicalIy significant research problems in applied microbiology (Pipes, 1966). Activated sludge contains a range of different types of microorganisms comparable with that of soils and the ecological relationships between the various components of the population are undoubtedly quite complex. In addition, the activated sludge milieu provides an environment which is more readily definable in quantitative terms than the soil environment and it is amenable to controlled experimentation in the field or laboratory. However, by and large, applied microbiologists who readily recognize the significant problems of soil microbiology have left research on the activated sludge process to engineers and chemists. These research endeavors have had some signal successes but, not surprisingly, those problems which should yield most readily to biological investigations remain unsolved. A. APPLIED RESEARCHON ACTIVATED SLUDGE There is no objection to using activated sludge for pure research. However, since the process is used in an attempt to solve one of the 185

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significant practical problems of modern civilization, it is quite likely that any advances in our knowledge of sludge organisms will ultimately be applied to process improvements. It is reasonable for the investigator to be aware of the practical significance of his studies and the possible application of his results. Thus, all studies of activated sludge organisms become applied research. Applied research may be superficial, dealing with description of phenomena which are empirically demonstrable in practical situations, or fundamental, attempting to elucidate relationships between various phenomena. The obvious place to start any research problem is at the superficial level and, to be successful, applied research must ultimately return to the superficial level in order to demonstrate its usefulness. The various levels of abstraction of fundamental research are expediencies in that they greatly reduce the amount of superficial research which has to be carried out and provide an understanding of a much wider range of phenomena. Unfortunately, many of the research studies of activated sludge have jumped to what was presumed to be a fundamental level without an adequate investigation at the superficial level. Applied research, in this instance, must be related to the solution of either design problems or operating problems. In the past most of the successful research on activated sludge has been related to design problems. It is now possible to design an activated sludge process which will provide a satisfactory degree of waste treatment for many types of wastes most of the time. Many activated sludge processes, perhaps the majority of them, are left to operate themselves with only routine maintenance of the mechanical appurtenances. When these processes fail to provide a satisfactory degree of treatment, either no one notices or no one in the area is sufficiently concerned with water pollution to do anything about it. There are waste treatment plants in which an attempt is made to control the activated sludge process so that it does provide an adequate level of treatment all of the time. In a few of these plants, the attempts at control are successful all of the time but, in the great majority of them, there are failures from time to time. In some cases, the solution to the process failure is a change in operation and in other cases it may be a modification of the design. Studies of operating problems are likely to provide not only solutions to operating problems but also identification of those problems which can be solved by further improvements in design.

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B. OPERATING PROBLEMS The types of wastes amenable to treatment by the activated sludge process have been defined to some extent by studies of the concentrations of nitrogen and phosphorus required in relation to the concentration of organic matter (Helmers et al., 1952) and studies of toxicity problems (Barth et al., 1965). In any case, the biological treatability of a particular waste can be determined in the laboratory in a relatively short period of time (Symons et al., 1960). However, the proper method of operating an activated sludge process in all situations is not known. There are properly designed, properly maintained activated sludge plants treating biologically treatable wastes which produce very poor results from time to time even with careful application of the best operating procedures known. Obtaining good results from an activated sludge process is accomplished by producing a sludge which will meet the requirements of the process and maintaining the proper environment so that the sludge can do its work. Activated sludge itself is a mass of microorganisms plus some inert solid material which is either introduced with the waste or produced by the microorganisms themselves as byproducts. The requirements of the process are: (a) the sludge must be able to separate organic matter from the waste in a relatively short period of time, and (b) the sludge must separate from the clarified waste by sedimentation. The first of these two requirements corresponds to a part of the question of the biological treatability of the waste. If the major components of the organic matter in the waste are compounds which can be assimilated and oxidized by some microorganisms, if adequate amounts of nitrogen, phosphorus, and other nutrients are present so that the organic matter can be metabolized, and if no toxicity problems occur, then some microorganisms will be able to grow in the waste. If microorganisms can grow in the waste, they separate the organic matter from the waste and, in most instances, separate it rapidly enough that the activated sludge process is a feasible method of treating the waste. After the organic matter is separated from the waste, it is either oxidized or synthesized into fairly stable cellular components. In some cases microorganisms will grow in the waste, but they grow in a form such that they do not separate from the waste by sedimentation. All of the operating problems which are the product of deficiencies in the design, maintenance, or operation of the activated sludge

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process are manifest as sedimentation problems; i.e., activated sludge appears in the effluent from the process. There are no recorded occurrences of an activated sludge which would settle but would not remove organic matter from the waste, but there are a great many instances in which an activated sludge could separate organic matter from the waste but would not settle properly. Except for the question of the biological treatability of the waste, all problems with operation of an activated sludge process reduce to the question of whether or not the sludge will settle. The biological treatability of domestic sewage and many industrial wastes has already been established and simple laboratory techniques to resolve the question of biological treatability of any waste are available; the obvious solution when a waste is not biologically treatable is to exclude it from the activated sludge process, The questions which are as yet unanswered, the problems for which no solution is now available, and many problems which can be solved with present knowledge but have not been solved, focus upon the question of settleability of the sludge. Thus, it is apparent that the major effort of applied research on activated sludge should be to resolve this question of settleability. There are many different phenomena which can prevent activated sludge from settling properly. All of these have been called “bulking” at one time or another; i.e., some people define bulking as the condition of activated sludge passing out into the effluent. On the other hand, bulking has been defined by some authorities in a narrow way so that it includes only a limited number of these phenomena. It is the more limited definition of bulking which will be used herein. In a publication of this type, it would be intellectually more satisfying to produce a defiriition of bulking and the proceed to a discussion of the more fundamental aspects of the problem. However, in the past there has been a major confusion because the superficial aspects of the penomena of bulking have not been treated adequately and much of what was intended to be fundamental research on problems of the settling characteristics of activated sludge is of questionable value because of the lack of a concise description of the phenomenon being investigated. Therefore, a large section of this paper will be devoted to a description of bulking and of other phenomena which can result in sludge with poor settling characteristics so that they can be differentiated from bulking. The discussion of the fundamental aspects of bulking will be limited to those investigations which can definitely be related to the description of phenomena occurring in actual activated sludge processes.

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II. Types of Settling Problems When a biologically treatable waste is introduced into an activated sludge process, any one of many different types of sludge may form. Presumably, there is an optimum type of activated sludge to treat each type of waste and an activated sludge process could be designed and operated to produce the optimum type of sludge. However, before it can be shown that this presumption is true, it will be necessary to be able to measure several characteristics of activated sludge, classify the types of activated sludge according to these measurable characteristics, and relate the types of sludge to the systems in which they are produced. The characteristics which should be measured have not yet been defined. In some research studies and a few operating plants, microscopic examination of activated sludge is used for qualitative estimation of sludge condition. The bacteria and fungi in activated sludge can be present as isolated cells, as clumps of cells, or as filaments. Microscopic examination can be used to determine the relative amounts of each form of growth present and to differentiate between bacterial filaments and fungal hyphae. The types and relative numbers of the protozoa present can be estimated by microscopic examination, as can the types and numbers of invertebrates present. All of this information may be of value to a particular operator who has studied his process for a number of years but quantitative relationships between the information which can be obtained by microscopic examination of the sludge and the results produced by the process have not been formulated into generalizations which apply to a large number of plants. The only characteristics of the activated sludge itself which are measured in operating practice are the concentration of solids in the aerating mixture of sludge and waste and the volume occupied by the sludge after it has settled for a specified length of time. The aerating mixture of waste and sludge is called “mixed liquor” and the concentration of suspended solids in the mixed liquor is abbreviated as MLSS. The volume occupied by the sludge after settling is called settled volume (SV) and usually the 30-minute SV is determined. A few other characteristics of activated sludge have been measured during investigations of one aspect or another of the process but these measurements have not been adopted for operational control and very little progress has been made toward using any of them for describing the different types of activated sludge.

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Quantitative evaluation of the settling characteristics of activated sludge for purposes of operational control has so far been limited to determination of the 30-minute settled volume. This determination is performed by placing a liter of mixed liquor in a graduated cylinder, allowing it to sit for 30 minutes, and then reading the volume of sludge from the graduations on the cylinder. This gives the SV in units of milliliters of sludge per liter of mixed liquor. Sometimes there is a distinct demarcation between the sludge and the supernatant and sometimes the sludge line is quite hard to locate. The supernatant may have varying amounts of turbidity and it may contain suspended solids large enough to be seen as individual particles. An experienced operator can tell a great deal about the condition of his process by observing the color and appearance of the sludge and supernatant, but no quantitative measurements of these qualities have been used. The 30-minute settling time was selected arbitrarily because many activated sludge processes have a sedimentation tank with a detention period of approximately 30 minutes. More information could be obtained from this determination by recording the sludge volume at several different time intervals and calculating the subsidence rate as well as the 30-minute SV. The concentration of MLSS is determined by filtering a measured volume of mixed liquor through a tared filtering crucible, drying at 103”C.,and weighing. MLSS is most conveniently expressed in units of grams per liter. The MLSS determination measures not only the mass of microorganisms which can be separated from the mixed liquor by sedimentation but also some varying quantity of inert suspended solids. Several attempts have been made to develop a method of measuring the biological solids or “active mass” as a fraction of the MLSS but none of these have been very successful. The most widely used refinement on the MLSS test is to determine the solids which volatilize in 20 minutes at 600°C. (called MLVSS) and the solids which remain after ignition (called “ash”). The MLVSS as a percentage of the MLSS can be a useful parameter for operational control of activated sludge processes (Ruchhoft and Smith, 1939). The most widely used parameter describing the settling characteristics of activated sludge is the sludge volume index (SVI) described by Mohlman (1934). The SVI is calculated by dividing the 30-minute SV by the MLSS, giving it the units of milliliters per gram. Other parameters of activated sludge settling characteristics have been proposed but have fallen into disuse in recent years. The most frequently encountered of these parameters in the older literature is

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the sludge density index (Donaldson, 1932) which is numerically equivalent to 100 divided by the SVI. The SVI is a very useful parameter for making quantitative measurements of bulking or of the approach of activated sludge to the bulking condition because bulking sludge will settle to some extent. However, in many cases, the settling problem is that some of the sludge does not settle at all or it settles and then rises. The assumptions made in using the SVI as the sole measure of settling characteristics are that the sludge settles as a mass, not as individual particles, that there is a distinct differentiation between the sludge and the supernatant, and that the sludge remains settled for several hours. When these assumptions hold, the SVI is an excellent parameter; when they do not hold, it is worthless. The types of problems which occur during settling of activated sludge can be classified into three large groups according to the observations which can be made on the settled volume test. One type includes those cases in which some of the sludge settles rapidly but a portion of it settles very slowly and, after 30 minutes, remains SUSpended in the supernatant. These types of problems are discussed under the heading of floc-formation problems. The second category of problems is called sludge density problems and includes those phenomena in which the sludge floats instead of settling or settles and then rises to the top of the cylinder. The third category is bulking.

A. FLOC FORMATION PROBLEMS

The microorganisms in activated sludge usually agglomerate with each other and with inert suspended solids into clumps. T h e process of agglomeration is called “flocculation” and the individual clumps are “floc particles” or just “floc.” The phenomenon of floc formation may be due to bacteria which normally grow as cells embedded in a gelatinous matrix (Butterfield, 1935), bacteria which normally grow as isolated cells but flocculate under some conditions (McKinney and Weichlein, 1953), bacteria sticking to extracellular material produced by fungi (Lackey, 1949), bacteria being flocculated by protozoa (Curds, 1963), or possibly all of these plus some other mechanisms. There is ample room for further research on flocculation phenomena. Problems with the flocculation of activated sludge can best be evaluated b y the type and amount of suspended material remaining in the supernatant after the sludge has settled. This suspended material may be microscopic, producing a uniform turbidity. In other cases it may be in large enough pieces to be identified as individual

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particles by the unaided eye. Unfortunately, poor flocculation has often been confused with bulking. In actuality, when bulking occurs the flocculation of the sludge is excellent. The supernatant produced by a sludge with a high SVI is sparkling clear but there is not very much of it. In the case of flocculation problems, the sludge which will settle settles well but the problem is caused by the sludge which does not settle.

1. Normal Sludge Activated sludge in good condition will settle rapidly in a 1-liter cylinder, as pictured in Fig. 1. The sludge itself is golden brown in

FIG.1. Normal activated sludge after 30 minutes’ settling.

color and has a slightly musty odor. The supernatant above the sludge is somewhat turbid and has a light brown or yellow color. When first poured into the cylinder, the sludge looks like a uniform suspension but as it settles it appears to pull together into clumps which are quite large. The SVI is in the range of 50 to 200 ml./gm.

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As shown in Fig. 2, microscopically normal activated sludge appears to be composed of irregular particles which are 50 to 500 p in diameter. The particles are composed of bacterial cells embedded in some kind of a matrix along with some larger spherical bodies which could be yeast cells or possibly protozoan cysts. A few short filaments project out from each particle. The protozoa present are mostly freeswimming ciliates and stalked ciliates. Nematodes, rotifers, crustaceans, and insects may sometimes be present. The supernatant above the sludge contains a few individual bacterial cells, short filaments, some flagellated protozoans, and some small particles which are not identifiable.

FIG.2. Microscopic appearance of normal activated sludge.

2. Dispersed Growth As shown in Fig. 3, dispersed growth does not form any identifiable sludge after settling for 30 minutes in a cylinder. The top of the suspension may be somewhat more translucent than the bottom but there is no distinction between the sludge and the supernatant. The color of the suspension may be white, brown, gray, or black, depending upon the nature of the waste. Under the microscope, dispersed growth is composed of bacterial cells, short bacterial filaments, yeast cells, and flagellated protozoans (Fig. 4).

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FIG.3. Dispersed growth after 30 minutes’ settling.

FIG. 4. Microscopic appearance of dispersed growth.

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Dispersed growth is considered to be something other than activated sludge (Heukelekian, 1949). It is included here because it is what is produced in an activated sludge process when the concentration of soluble organic matter in the waste is extremely high and microscopically it Iooks very much like what can be found in the supernatant above normal activated sludge. If the types of wastes which produce dispersed growth were diluted before being introduced into the activated sludge process, they would produce a settleable biological sludge.

3. Dejlocculation At times normal activated sludge appears to break up into dispersed growth. Some settleable sludge remains but the supernatant becomes extremely turbid and contains large numbers of bacterial cells (Fig. 5). Deflocculation is usually a temporary phenomenon and normal sludge will re-form after the organisms have acclimated to the new conditions or the conditions change back to what they were before the shock occurred.

FIG.5. Deflocculated sludge.

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There are three possible explanations of deflocculation. The sludge may simply cease reacting with the waste, allowing the suspended material coming into the process to pass through untouched. The protozoa in the sludge may cease their activities, allowing the singlecell bacteria and yeasts upon which they normally feed to multiply rapidly. The bacteria in the floc may die and the floc particles actually break up. Possibly all three of these mechanisms are involved, either simultaneously or individually, in different cases of deflocculation.

4 . Pin Point Floc Pin point floc are small distinct particles which remain suspended in the supernatant above the sludge after it settles. I n the process, the pin point floc passes out in the effluent and can cause pollution problems. Most activated sludge plants produce pin point floc from time to time but there has been no definitive investigation of it as yet. There are at least two types of pin point floc. One type is a grayish material which looks like fly-ash, is inert, and has no biochemical oxygen demand (BOD). Under the microscope it is an unidentifiable, amorphous mass. The other type of pin point floc looks like normal floc particles but it has no tendency to either settle or rise. It does have a high BOD, probably due to the respiratory activity of the bacteria of which the particles are composed.

5. Billowing Sludge Billowing sludge is actually normal activated sludge which settles well in a cylinder. The phenomenon of billowing is caused by hydraulic overloading of the sedimentation tank or b y flow imbalance or by stirring of the sludge by the sludge collection mechanisms. Stirring activated sludge gently causes it to billow up like clouds in the sky. I got the name “billowing sludge” from a plant operator who explained to me exactly what it is. However, I have also had several plant operators tell me that they had bulking sludge and show me billowing sludge. This definitely is an engineering problem and not a microbial problem.

B. DENSITYPROBLEMS In the cases of poor flocculation, the material in the supernatant above the sludge has very little tendency to settle and just remains suspended. There are several conditions which cause the production of an activated sludge which has a density less than that of water and floats instead of settling. In this category there are three types of sludge composed of particles which individually are heavy enough

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to settle but when compacted into a mass containing sufficient number of entrapped gas bubbles form a sludge which floats. There are also several conditions which lead to the formation of sludge particles which individually have a density less than that of water and always float. All of these conditions have been, and still are, frequently confused with bulking.

1 . Rising Sludge Rising sludge initially settles quite well when placed in a graduated cylinder and at the end of 30 minutes has compacted into a small volume. Thus it has a low SVI and if the cylinder is cleaned out at this time the rising sludge problem is not noticed in the laboratory. However, if the sludge is allowed to sit for a few more minutes, it slowly rises to the top of the cylinder. Figure 6 shows an example of

FIG. 6. Demonstration of rising sludge. The sludge in the cylinder on the left has settled for 20 minutes but that in the right-hand cylinder has settled for 40 minutes.

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rising sludge. Microscopically, rising sludge looks just like normal sludge and probably is composed of much the same group of organisms. Rising sludge is still sometimes confused with bulking sludge despite the fact that many investigators have recognized it as a separate phenomenon and the mechanisms causing rising sludge have been thoroughly worked out. The most comprehesive study of rising sludge in actual plant operation was reported by Sawyer and Bradney (1945). Rising sludge occurs when nitrification is taking place in the aeration tank and denitrification is taking place in the sedimentation tank. T h e nitrogen gas produced by denitrification accumulates in small bubbles in the sludge and produces a density less than that of water in a mass of sludge which has compacted together.

2. Anaerobic Sludge Any biological sludge which compacts well will eventually become anaerobic and rise if it is allowed to sit around long enough. The distinctions of rising sludge are that the transition from settling to floating occurs in a short period of time and that it is caused by denitrification. If nitrates are not present, a sludge will compact for several hours and sometimes several days before it produces enough gas to rise. The gases produced in the absence of denitrification are COZ, and HZ, and the other gaseous fermentation products. Usually HZS is also produced and the sludge becomes black and odorous. The only sludges which will not rise eventually are those which do not compact well enough to entrap small gas bubbles, i.e., the bulking sludges. Anaerobic sludge can be demonstrated in the laboratory by allowing any normal sludge to sit in a graduated cylinder until it produces enough gas to float. It only occurs in actual plants which have a settling tank so poorly designed that some portion of the settled sludge is allowed to compact for hours without being swept out by the sludge collection mechanism and returned to the aeration tank. Unfortunately, many plants are that poorly designed.

3. Overaerated Sludge Most sludges can be aerated and mixed violently enough that the air is broken up into very small bubbles which attach to sludge particles. The overaerated sludge will then immediately rise to the top of a graduated cylinder. However, this sludge is poorly compacted and over a period of an hour or so the air bubbles will coalesce and

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separate from the sludge particles, allowing them to settle. This phenomenon is quite rare in actual practice and probably only occurs when some organic material (such as grease) which greatly lowers the surface tension of water is present.

4 . Floating Sludge Some activated sludges are composed partially of particles which individually have a density less than that of water and naturally float instead of settling. I t is usual to find a few floating particles in any sample of activated sludge, but normally such a tiny fraction of the sludge floats that its effect on the overall process performance is negligible. However, as pictured in Fig. 7 , there are cases in which

FIG.7. Floating sludge.

a sizable fraction of the sludge floats. This results in the loss of much of the sludge into the effluent. In a well-operated activated sludge process, floating sludge is only a transitory phenomenon. If the plant

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operator is aware that he has lost most of his sludge, he can operate the process on a start-up basis until enough normal sludge is produced so that he can revert to normal operation. If the operator is not alert,

FIG.8. Floating sludge caused by extensive growth of predatory fungi.

FIG.9. Floating sludge caused by extensive growth of a saprophytic fungus.

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20 1

the loss of large amounts of floating sludge can lead to conditions which produce deflocculation or conditions which produce bulking. Floating sludge is quite filamentous and has been confused with bulking by a number of people, including myself. There are at least three different phenomena which produce floating sludge. Dead stalked ciliates float and carry sludge particles out in the effluent; this has been described as bulking by Lackey and Wattie (1940) and Pillai and Subrahmanyan (1943). Rotifers and nematodes killed by predatory fungi also float, carrying along sludge particles which adhere to the fungus mycelium; this has been described as bulking by Cooke and Ludzack (1958) and by Pipes (1965). Figure 8 is a photomicrograph of activated sludge containing both rotifer- and nematodetrapping fungi. Sometimes saprophytic fungi having large-diameter hyphae and vacuoles apparently containing lipids will grow out of sludge particles, causing them to float; this has not been previously described in the literature but is pictured in Fig. 9.

C . COMPACTION PROBLEMS Sludge which settles slowly and compacts poorly, leaving a small amount of clear supernatant, is properly called bulking sludge. Two types of bulking sludge were described by a committee of the American Public Health Association (Pearse et al., 1937) as (a) a flocculated sludge with a high SVI and a low settling rate, and ( b )a filamentous sludge with an extremely low settling rate. The two types of bulking have come to be known as “zoogleal bulking” and “sphaerotilus bulking” b y misapplication of two generic names. (To many sanitary engineers any bacterium or fungus which flocculates is “zooglea” and any filamentous bacterium or fungus is “sphaerotilus.”) The real problem with bulking is that the sludge starts to compact at a very low solids concentration instead of settling rapidly to a solids concentration of greater than 5 gm/liter before reaching the compaction stage. An activated sludge process can be operated with bulking sludge and still produce excellent results if the plant is designed with adequate capacity and flexibility and the operator knows what he is doing. Unfortunately, most plants do not have the required capacity to operate well with bulking sludge and many operators would not know how to use the capacity if they had it. 1 . Zoogleal Bulking An excellent description of nonfilamentous bulking in an activated sludge process plus some interesting laboratory experiments on bulking sludges was reported by Heukelekian and Weisberg (1956).They

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ascribe the phenomenon of nonfilamentous bulking to the formation of floc particles which contain excessive amounts of bound water. The sludge settles some, but the 30-minute SVI is a function of the nature of the sludge instead of the MLSS and the SVI test is of little value in describing the degree of bulking. Under the microscope the sludge particles are described as being ragged and diffuse with relatively large surface area (Smith and Purdy, 1936). It is possible that nonfilamentous bulking is more closely related to one of the deflocculation phenomena than to filamentous bulking.

2 . Filamentous Bulking A filamentous bulking sludge settles very slowly in a graduated cylinder giving an SVI of greater than 100 and sometimes as great as 2000 or more (Kraus, 1949). The supernatant above the sludge is very clear, a fact that has been noted by several investigators (Haseltine, 1932; Heukelekian, 1941; Keefer, 1963). Bulking sludge is light brown, grey, or white in color and sometimes has a sweetish or fruitlike odor. A typical bulking sludge is shown in Fig. 10.

FIG.10. Filamentous bulking sludge after 30 minutes’ settling.

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The sludge particles of a bulking sludge appear to be similar to normal sludge except that the filaments extending from the clumps are much more numerous and much longer. In certain instances, the

FIG.11. Filamentous bulking caused by a filamentous bacterium.

FIG. 12. Filamentous bulking caused by a fungus.

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sludge may be composed almost entirely of filaments with very few bacterial flocs present. The filaments may have a diameter of about 1 p and be unbranched, as pictured in Fig. 11, or they may be definitely fungal, having a much larger diameter and distinct branching, as shown in Fig. 12. Ill. Superficial Aspects of Bulking

Three of the ten different types of sludge which do not settle properly - namely, billowing sludge, anaerobic sludge, and overaerated sludge-are directly due to deficiences in the design and/or operation of the process and are independent of the microbial population of the sludge. The other seven types are the result of some unusual feature of the microbial population. The organisms which cause rising sludge and the mechanisms by which they produce it are well known and this problem can be cured readily. However, there are a large number of questions about the organisms present in the other six types of poorly settling sludge and the conditions under which these organisms flourish. In each case there are the questions of the number of different mechanisms which can cause the problem, of the identity of the organisms involved, and of proper methods of controlling the growth of the organisms. This particular essay is concerned with the problem of filamentous bulking and the attempts to discover what this problem is and how it may be controlled. The literature describing bulking is voluminous but rather difficult to interpret because many authors have not provided evidence that they were studying filamentous bulking and not one of the other types of activated sludge which settles poorly. An attempt is made in the material which follows to deal only with welldefined classes of filamentous bulking and to separate this from the other problems. The conditions which lead to filamentous bulking in process operation will be described. The objective is to provide a firm basis for a discussion of the microbial ecology of bulking sludge on a more basic level. A. PLANT OPERATION

A normal activated sludge with good settling characteristics can be produced in an activated sludge process treating domestic sewage if the proper operating conditions are maintained. However, many plants treating a waste which is mostly domestic sewage have problems with filamentous bulking and have had to experiment with various operating conditions. Figure 13 is a flow diagram of the activated

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sludge process which is used as a framework for the following discussion.

II wnctr ..-1.-

By - P O S S

Untreated Woste

rChernicols Addltlon

1

Purified

w\/ Tonk

\

/

Reoerotion Sludge

Chemical Addition

Woste Sludge

FIG. 13. Flow diagram of the activated sludge process.

The plant operator does have control of the amount of waste treated and sometimes the waste composition by means of a by-pass arrangement preceding the process. However, use of this method of control is frowned upon by regulatory agencies concerned with stream pollution and it is an expediency to which an operator should not have to resort. In order to control the process properly, the operator should be able to vary the return sludge rate between 10% and 100% of the influent flow rate, vary the waste sludge rate u p to 10%of the influent rate, and vary the air supply between 0.5 and 2.0 cubic feet per gallon of waste treated. H e should also be able to reaerate the return sludge or hold it under anaerobic conditions and to add chemicals to the influent waste, the mixed liquor, or the return sludge. Unfortunately, very few plants are designed to give the operator this much flexibility of operation. 1. Operating with Filamentous Bulking A process with sufficient capacity in the sedimentation tanks and the return sludge pumps can be operated with a high SVI without losing sludge into the effluent. Since a filamentous bulking sludge produces the best quality effluent, if the sludge can be kept out of the effluent, a few processes are operated with the objective of maintaining a high SVI. I n most cases, however, the operator attempts to maintain an SVI well below 100 and operates at higher SVI values

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only when the sludge microorganisms are not cooperative. A problem often arises because the steps which must be taken to operate the process with a high SVI are not compatible with the steps which should be taken to reduce the SVI. The means of controlling an activated sludge process with a filamentous bulking sludge have been clearly described in articles by Bloodgood (1947) and Kraus (1949). A filamentous bulking sludge compacts very poorly, giving a lower concentration of suspended solids in the return sludge; thus, the return sludge flow rate must be increased in order to return the same weight of solids to the aeration tank. Sometimes when bulking occurs it is necessary to increase the return sludge rate from the normal 25% of influent flow to 100% of influent flow. Increasing the return sludge rate does not change the average detention time of the waste in the aeration tank; however, it does increase the surface loading on the sedimentation tank directly. An activated sludge process should have a sedimentation tank designed to handle at least twice the influent flow in order to control a bulking sludge. In many activated sludge processes, the return sludge pump capacity is only 30 or 40% of the influent flow rate. If bulking occurs and the 30-minute SV of the mixed liquor rises to 500 ml./liter (50%), then the extra volume of sludge accumulates in the sedimentation tank. In this case the only thing which can be done to prevent the sludge from filling up the sedimentation tank and going out in the effluent is to increase the waste sludge rate. Increasing sludge wasting decreases the MLSS and this usually results in pushing the SVI up higher. Increased sludge wasting is a temporary expedient to prevent sludge from being lost into the effluent; it is not a method of controlling bulking. Some plants have no provision for handling any increase in the amount of waste activated sludge. In these plants, the operator pumps the sludge around from one part of the plant to another until it finally gets back into the activated sludge process and from there into the effluent. It is not too surprising that in the face of this condition some operators give up any attempt to control the activated sludge process at all. Many engineers and operators have tried weighting the sludge with inert solids or adding coagulants which cause the sludge particles to clump together. The weighting agents employed successfully include clay (Ely, 1938), activated carbon (Haywood, 1937), hydrated lime (Haseltine, 1938),digested sewage solids (Kraus, 1945), and raw sewage solids (Palmer, 1949). Coagulants which can be used to reduce

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SVI are alum and ferric salts (Andersen, 1936) and synthetic polyelectrolyte coagulants (Singer et al., 1968). With the possible exception of lime, none of these chemicals would b e expected to change the microbial population of activated sludge but they do improve the settling characteristics of bulking sludge enough so that the process can be operated.

2. Operating to Prevent Filamentous Bulking In routine operation of an activated sludge process, the operator usually wants to avoid addition of chemicals because of the expense involved. H e also wants to maintain a stable sludge, i.e., one which has good settling characteristics and which does not change its settling characteristics rapidly. If the air supply is adequate, the two main points of control for routine operation are the concentration of suspended solids in the aeration tank and the manner in which the return sludge is handled.

a. Maintaining Proper M L S S When a waste is mixed with activated sludge, aerated for a short time, and then separated from the sludge, the BOD of the waste is reduced a great deal more than the amount of oxygen consumed by the sludge. However, the sludge now consumes oxygen at a higher rate than it did before it was in contact with the waste and it requires several hours of aeration before its rate of oxygen consumption tapers off again. This simple experiment, which has been performed many, many times, led to the development of the concept of two phases of purification of waste by the activated sludge process. The first phase is the transfer of organic matter from the waste to the sludge and has variously been called clarification, adsorption, biosorption, and the contact phase. The second phase is the conversion of the organic matter accumulated in the sludge into stable sludge components and is usually called either oxidation or the endogenous phase. Haseltine (1932) expressed rather well the concept that, in order to avoid bulking, enough sludge must be provided to take up the organic matter from the waste and enough aeration time must be provided for the sludge to oxidize the organic matter. This concept has been used by many authors since. It was formulated into a quantitative expression b y a National Research Council Committee (Mohlman et al., 1946) as a constant times the ratio of the product of the influent BOD and influent flow rate to the product of the MLSS, the aeration tank volume, and the average detention time in the aeration tank. This ratio has been called a loading parameter, a loading velocity, and a

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food-to-microorganism (F/M ) ratio by various authors. Sometimes the reciprocal of this ratio is called “sludge age” but there are other expressions which are also called sludge age (Torpey, 1948). Logan and Budd (1956) reported that in pilot plant experiments the lowest SVI was produced at a loading of 0.22 to 0.38 and that the SVI increased at higher or lower loadings. In a series of experiments with laboratory activated sludge processes, Orford et al. (1960) found that the SVI was a minimum at a loading of 0.17 but increased at higher or lower loadings. Stewart (1964) presented the opinion that SVI is very high at loadings in the range of 0.35 to 0.8 and decreases at higher or lower loadings. Despite the different opinions about the form of the relationship, it is apparent that there is some relationship between the loading and the SVI, that filamentous bulking is quite likely to occur at loadings above 0.35, and that most activated sludge processes will produce a sludge with good settling characteristics when the loading is in the range of 0.2 to 0.3. A sludge produced in a process with a loading greater than 0.3 can be considered to be an underoxidized sludge and one produced in a process with a loading less than 0.2 is an overoxidized sludge. If an attempt is made to treat all of the waste received at the plant, the only method of controlling the loading on the activated sludge process available to the operator is selection of the MLSS by controlled sludge wasting. In most instances, filamentous bulking is prevented by keeping the MLSS high enough that the process loading is less than 0.3. If the SVI starts to increase because the loading of the process is too great, the proper corrective measure is to increase the MLSS. Reducing the MLSS is a temporary expedient to prevent loss of sludge into the effluent when the SVI is already high; it causes an increase in loading and thus is likely to produce an even higher SVI.

b. Control of Sludge Return Harris et al. (1926) made the observation that holding activated sludge in the sedimentation tank and the return sludge lines for a long time had a tendency to produce bulking. Since then many authors have recommended that the return sludge should be pumped back to the aeration tank as quickly as possible. This requires operating at a rather high sludge return rate. Many investigators have observed that reaerating the return sludge before it is mixed with more waste improves the settling characteristics of the sludge and makes the process much more stable. The first full-scale activated sludge process used in this country employed re-

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turn sludge reaeration but, despite this, every few years someone invents a new activated sludge process based on return sludge reaeration and makes extravagant claims about its superiority over the conventional activated sludge process. One of the best summaries of operating experiences with return sludge reaeration was published by Haseltine (1961). In an activated sludge process with a fixed aeration tank capacity but some flexibility for use of this capacity for either reaeration of return sludge or aeration of mixed liquor, increasing the volume used for sludge reaeration greatly increases the weight of sludge being aerated, because the return sludge concentration is usually 4 or 5 times greater than the MLSS, but it has no effect on the average detention time of the waste in the aeration tank. Thus, using some of the aeration tank capacity for return sludge reaeration increases the total amount of organic matter which can be handled by the process without increasing the loading on the process. Most processes in which return sludge reaeration is practiced use between 113 and 314 of the aeration capacity for reaeration. The effect of return sludge reaeration upon the settling characteristics of the sludge produced appears to be greater than can be explained by the effect of lowering the process loading although this observation has not been established by careful experimentation. Reaeration of return sludge seems to produce an activated sludge which does not change its settling characteristics rapidly and is more resistant to sudden changes in the environment of the aeration tank than sludge produced in a conventional activated sludge process with the same loading. Kraus (1946) found that an even more stable sludge could be produced if digester supernatantwere added to the reaeration tank. Supernatant from an anaerobic sludge digester upsets a conventional activated sludge process but improves the settling characteristics of sludge in a reaeration system (Ullrich and Smith, 1957).

B. WASTE COMPOSITION Although it has been observed that activated sludge processes treating domestic sewage can produce filamentous bulking if the plant is poorly operated or poorly designed so that the process cannot be operated properly, it has also been observed that activated sludge processes treating industrial wastes or a combination of domestic sewage and industrial wastes are much more IikeIy to produce filamentous bulking even when properly designed and operated by present standards. This has led to many studies which attempted to define the types of compounds which might occur in industrial wastes

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and would have some effect upon bulking. These types of compounds include organic compounds which are readily assimilated by some activated sludge organisms, inorganic compounds which can serve as nutrients, and toxic compounds both organic and inorganic.

1. Organic Content Many of the industrial wastes which cause filamentous bulking problems are those containing high concentrations of carbohydrates (Smit, 1932). One of the classic investigations on filamentous bulking was that of Morgan and Beck (1928) in which they definitely traced their bulking problem to sugar in the waste from an illegal distillery and solved the problem by having revenue agents knock over the still. Several authors have suggested grease or oil as materials which produce bulking (Greeley, 1945) but this apparently is a case of floating sludge confused with bulking. A number of investigators have tried developing an activated sludge culture in the laboratory using different organic components in the medium fed to the culture to see what type of sludge would be produced. Scott (1928) tried adding brewery waste, tannery waste, milk, starch, yeast, and dextrose to sewage fed to fill-and-draw activated sludge cultures and found that, except for tannery waste, they all produced a very filamentous sludge. Smit (1932) tried glucose, sucrose, lactose, and,starch and was able to produce filamentous bulking with all except starch. Ingols and Heukelekian (1939) developed activated sludge cultures on starch, glucose, peptone, and calcium butyrate and found that although starch and glucose produced filamentous sludges, peptone and calcium butyrate did not. Sanitary engineers now are still doing the same laboratory experiments that sanitary engineers were doing 30 years ago but at least a few refinements have been added. Genetelli and Heukelekian (1964) used continuous culture technique for their laboratory activated sludge cultures and found that egg albumin produced good settling sludge at a loading of 0.3 but a filamentous sludge at loadings of 0.5 and 0.7 while hydrolyzed casein and glucose both produced filamentous bulking at loadings of 0.3, 0.5, and 0.7. Although it is somewhat difficult to reconcile some of the differences in the results reported by the different investigators, it appears that there are at least two common factors in the various studies of filamentous bulking in laboratory activated sludge cultures. It appears that simple, soluble organic compounds which are readily metabolized by the majority of microorganisms favor the growth of filamentous

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organisms while complex, insoluble compounds which have to be hydrolyzed before being metabolized favor the growth of organisms which form sludge with good settling properties. It also appears that filamentous bulking can be produced with any substrate if the loading is high enough. Domestic sewage which has remained in the sewers long enough to become anaerobic is called stale sewage and if it has a distinct sulfide odor it is called septic sewage. Stale or septic sewage is considered to be much more likely to produce filamentous bulking than fresh sewage and sometimes sulfide itself is suggested as the direct cause of bulking (Greeley, 1945). Morgan et al. (1936) found that filamentous bulking occurred during periods of low flow in hot weather but disappeared when the weather cooled off or the sewage flow increased. They solved their bulking problem by flushing the sewers during periods of low flow. Andersen (1936) found that chlorination did not cure a case of filamentous bulking, thought to be caused by septic sewage. Chlorination, of course, would convert sulfide to free sulfur. Septic sewage is somewhat similar to digester supernatant which has been found to cause bulking if introduced directly into the aeration tank (Ullrich and Smith, 1957) but cure bulking when aerated with the return sludge (Kraus, 1946). In addition to sulfide, both septic sewage and digester supernatant contain simple, soluble organic compounds (fermentation products) which are readily assimilated by many microorganisms.

2 . lnorganic Content In addition to organic substrates, activated sludge microorganisms require readily available sources of nitrogen, phosphorus, potassium, magnesium, iron, and the trace elements. All of these elements except nitrogen and phosphorus should be supplied in adequate amounts by the carrage water. Inadequate amounts of both nitrogen and phosphorus have been suggested as conditions leading to the occurrence of filamentous bulking. Ingols and Heukelekian (1939)found that bulking in their laboratory cultures caused by glucose could be prevented if enough urea were added to their medium so that the carbon-to-nitrogen ratio was less than 10 and the loading was kept low enough. Unfortunately, the investigators who carried out the most extensive study of the amounts of nitrogen and phosphorus in relation to the amount of oxidizable organic matter required for proper operation of an activated sludge process did not report if the failure of the process at high C :N or C :P

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ratios was due to filamentous bulking or to some other problem (Helmers et d., 1952). Greenberg et a2. (1955) studied the effect of low phosphorus concentrations in a synthetic sewage composed of 50 mg./liter of soap and 200 mg-/liter of starch on laboratory, fill-and-draw, activated sludge cultures. They found that when the phosphorus concentration was greater than 2 mg./liter, the sludge produced had good settling characteristics, but when it was less than 2 mg./liter, a very filamentous sludge with poor settling characteristics was produced. Fortunately, they also found that when no available phosphorus was supplied in the medium, no activated sludge was produced. Domestic sewage contains concentrations of nitrogen and phosphorus far in excess of the amounts required for the microorganisms in activated sludge to assimilate all of the organic matter present. Low nitrogen or phosphorus content can be a problem with some industrial wastes or when an attempt is made to treat industrial wastes along with domestic sewage.

3. Toxic Compounds Toxic materials in the influent waste definitely cause problems in the activated sludge process but, from the literature, it is somewhat difficult to determine what types of toxic materials are likely to cause which problems. The toxic material may be present in the waste continuously at low concentrations or it may be received at the plant in a slug or shock load and the response of the process will depend upon the manner in which the material is received at the plant. A toxic material continuously present in the waste may prevent the formation of activated sludge, it may be selectively toxic to filamentous organisms promoting the development of a low SVI sludge, or it may be toxic to normal sludge organisms but not to filamentous organisms. Many investigators have not differentiated between all of these possibilities. Many toxic organic compounds such as phenols and cyanides which were at one time believed to prevent operation of the activated sludge process (Haseltine, 1941) have since been shown to be treatable if a proper population of microorganisms is developed in the sludge. For instance, Ludzack, et u2. (1961a), working with laboratory activated sludge cultures, found that sludges which could oxidize a number of different organic cyanides could be produced if several weeks' acclimation was allowed for the sludge to build up. They did report that several of the sludges they developed were filamentous. However,

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Ludzack and Schaffer (1962) did not report excessive numbers of filamentous organisms in activated sludges developed on cyanide-, cyanate-, and thiocyanate-containing media. I n all probability, any organic compound which can be metabolized by some microorganism can be oxidized in the activated sludge process if the proper operating conditions can be found. A number of heavy metals are toxic to all organisms in high concentrations and definitely interfere with the activated sludge process. However, many of them can be tolerated in low concentrations. Ingols and Fetner (1961) reported that low concentrations of chromium in the waste would cause the conversion of a normal activated sludge to a filamentous sludge. Barth et aE. (1965) reported that copper at 1 mg./ liter, nickel at 2 mg./liter, zinc at 5 mg./liter, and chromium at 10 mg./liter could be tolerated by activated sludge without reducing efficiency and that when low concentrations of one of these metals were present, the sludge contained no filamentous organisms. They even suggested use of low concentrations of heavy metals as a method of curing a filamentous bulking problem. There are no reports of attempts to solve filamentous bulking problems by adding heavy metals, but many investigators have tried chlorination of return sludge, hoping that it would be more toxic to the filamentous organisms than to the normal sludge organisms. Both Smith and Purdy (1936) and Tapleshay (1945) reported that chlorination of return sludge was a good method of reducing the SVI. However, Ridenour et al. (1937) reported that sometimes chlorination of return sludge did not reduce the SVI and concluded that some filamentous organisms were more resistant to chlorine than others. Heukelekian and Weisberg (1956) found that chlorination had no effect on filamentous bulking but that it rapidly reduced the SVI in a case of zoogleal bulking. They concluded that the effect of chlorination was a physical change in the form of the sludge particles rather than a biological change in the microbial population.

4. Shock Loading All sudden changes in the concentration, composition, or flow rate of the waste received by an activated sludge process is called a shock load. Guady and Engelbrecht (1961) defined quantitative, qualitative, toxic, and hydraulic shock loads but, unfortunately, in their investigations paid no attention to the settling properties of the sludge. Many authors have suggested that large fluctuations in the waste flow rate or composition are likely to cause bulking (Greeley, 1945).

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A sudden increase in the influent flow rate causes a decrease in the detention time in the aeration tank and an increase in the surface loading on the sedimentation tank. If the increase in flow is great enough, it can wash even sludge with good settling characteristics out of the process and reduce the MLSS to the point at which the process is overloaded. A plant with some extra capacity in the pretreatment and primary treatment units can be operated so as to prevent the effects of a hydraulic shock from reaching the activated sludge process. Although toxic shock b a d s have been given as causes of filamentous bulking, it is likely that these were cases of deflocculation confused with bulking. Edwards and Nussberger (1947) described how a slug of waste containing a very high concentration of chromate ion and a low pH passed through an activated sludge process, causing a great increase in effluent turbidity but no change in the SVI of the sludge which settled. A number of toxic materials have been tested as shock loads on laboratory activated sludge cultures and found to produce a very rapid increase in effluent turbidity (Ludzack et al., 1961a; Ludzack and Schaffer, 1962; Barth et al., 1965). If the toxic shock load occurs for only a short period of time, only a part of the sludge goes out as effluent turbidity and the process can recover in a day or two. If the toxic load continues long enough, all of the sludge breaks up and then the operator is faced with the job of producing enough activated sludge to start up his operation again. A sudden increase in the concentration of the oxidizable organic matter in the waste causes an overload of the activated sludge process. If continued long enough, this can lead to filamentous bulking but on a short-term basis it probably will not adversely affect the settling properties of the sludge. A sudden change in the type of oxidizable organic matter in the waste could conceivably cause a change in the microbial population of the sludge to filamentous organisms but this has not yet been demonstrated. It has been observed a number of times that activated sludge systems which use return sludge reaeration are much more resistant to shock loadings (Haseltine, 1961).

C. AERATION TANKENVIRONMENT The environmental conditions in the activated sludge process are determined b y the nature of the waste being treated, any substances added to the waste to make it treatable, the operation of the process, and the activities of the organisms present. The range of operating conditions is limited by the design of the process. It is assumed that the environmental conditions in the process determine the population

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of microorganisms present and the physical form of their associations. Thus, the environmental conditions determine the type of sludge produced. There are three distinct and different environments in the activated sludge process; namely, the aeration tank environment, the sedimentation tank environment, and the return sludge environment. Any organisms which are active in the process must be able to survive passage through each of these environments and to grow in at least one of them. Which of these three environments has the controlling influence on the sludge population has not yet been determined for even one situation. However, in a properly operated process, the activated sludge spends most of its time in the aeration tank and this should be the controlling environment.

1. pH

The pH in the aeration tank can have a profound influence upon the types of organisms which can grow in the sludge. Keefer and Meisel (1951) tried controlling the pH in pilot plant activated sludge processes treating domestic sewage. They found that the process would operate in the range pH 6 to pH 9 but outside this range normal activated sludge would not form. They also observed that the SVI was lower at pH 6.5 and p H 7.5 than at pH 7.0. It is unlikely that an activated sludge process would have a p H greater than 9 for very long because not very much highly alkaline industrial waste is produced any more and COe produced b y microbial metabolism is quite effective in lowering the pH to less than 9. A p H of less than 5 is possible in an activated sludge process if the operator does not know what he is doing. As a shock load, acid will cause deflocculation. Over an extended period of time, a low p H will cause the production of a sludge composed almost entirely of filamentous organisms and having a very high SVI. 2 . Temperature It is a common observation that filamentous bulking is much more likely to occur in the summer than in the winter (Ruchhoft and Kachmar, 1941). Keefer (1962) reported data from plant operation which indicated that the SVI in his process was, on the average, slightly higher at 26°C. than at 12°C. but that the concentration of suspended solids in the effluent was much lower at the higher temperature. Dougherty and McNary (1958) found that pushing the temperature in a laboratory activatd sludge culture u p to 30°C. and above

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produced a highly filamentous sludge. However, Ludzack et ul. (1961b) found filamentous bulking in a laboratory activated sludge culture at 5" C. while an identical culture at 30" C. produced normal sludge. Logan and Budd (1956) reported that, in their pilot plant studies, the loading had to be reduced when the temperature was decreased from 25" C. to 9" C. in order to prevent the SVI from increasing. It appears that at temperatures above 20" C., filamentous organisms can replace normal sludge organisms quite rapidly if some conditions which favor the growth of filamentous organisms occur. At temperatures below 10"C., a higher MLSS must be maintained in order to have an adequate population of microorganisms to oxidize the organic matter accumulated by the sludge.

3. Dissolved Oxygen Fairly early in the history of the activated sludge process, it was recognized that some dissolved oxygen concentration (DO) in the mixed liquor was required to maintain a population of aerobic microorganisms in the sludge and low DO has been suggested as a cause of filamentous bulking a number of times. Increasing aeration rate has solved bulking problems in actual process operation (Andersen, 1936), but most of the experimental work on the effect of low DO on activated sludge has been done in the laboratory. Heukelekian and Ingols (1940) were strongly convinced that bulking produced in their laboratory activated sludge cultures fed on domestic sewage was due to low mixed liquor DO and later (Ingols and Heukelekian, 1940) reported that filamentous bulking caused by feeding sugar to their cultures could b e overcome by increasing the aeration rate. Okun (1949) tried aerating activated sludge cultures with pure oxygen instead of air and observed that a high DO in the mixed liquor prevented the development of filamentous growths. However, Orford et ul. (1960) analyzed data from a very extensive set of laboratory activated sludge cultures and could find no statistically significant correlation between mixed liquor DO and the settling characteristics of the sludge. The air supply to most activated sludge processes is operated in an attempt to maintain at least 1 mg./liter DO in the mixed liquor. All of the laboratory investigations were considering something less than 0.5 mg./liter DO as low oxygen concentration. Thus, it is not likely that low DO would cause filamentous bulking in an activated sludge process unless the aeration system was not adequately designed or the process was poorly operated.

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The DO in the mixed liquor is the oxygen resource which must carry the sludge organisms through sedimentation and return to the aeration tank. It is quite likely that the need for quick return of the sludge to the aeration tank is due to a need to prevent the sludge from being exposed to anaerobic conditions for too long a time. If this is true, then the higher the DO in the mixed liquor as it enters the sedimentation tank, the longer it takes for the return sludge to become anaerobic. However, Ford and Eckenfelder (1967) recently reported a case of filamentous bulking which was cured by holding the return sludge under anaerobic conditions for several hours, but this is probably an exceptional case. IV. Fundamental Aspects of Filamentous Bulking An analogy can be drawn between normal activated sludge and a healthy person. Normal activated sludge works hard, does a good job, and is resistant to infection. It can come in contact with all types of pathogenic organisms in the environment and still not become sick. However, if it is overfed or underfed or poisoned or smothered or mistreated in other ways, it loses its resistance to infection. The universal symptom of sickness of activated sludge is loss of solids into the effluent-like fever is a universal symptom of illness in people. At one time, most fevers were called ague and little progress was made toward curing human disease until fever was recognized as a symptom rather than an illness itself. As long as any loss of solids in the effluent from an activated sludge process is called bulking and believed to be an illness rather than a symptom, it is unlikely that the pathology of the process will be adequately understood. Approximately 100 years ago, physicians were making rapid progress toward defining and distinguishing between the various human diseases and the complex of symptoms associated with each. At that time, a revolution in applied microbiology provided the principles and techniques which were used to elucidate the etiology of many human diseases and ultimately resulted in the development of cures for many of these diseases. At this time, sanitary engineers are, or at least should be, describing the various illness of activated sludge. The same principles employed in tracing out the etiology and pathogenesis of human disease can be directly applied to the study of the diseases of activated sludge. Since many new and improved techniques are now available to applied microbiologists, the task should proceed quite rapidly if approached properly. A fundamental approach to the study and solution of problems with

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the settling characteristics of activated sludge should consist of the following steps: (1) formulation of hypotheses describing the mechanisms by which the settling characteristics of activated sludge can change; (2) application of van Niel’s postulates (Gibson, 1957) to the determination of which organisms cause a change in settling characteristics; (3) testing of the hypotheses by pure culture experimentation in the laboratory; and (4)use of the validated hypotheses in devising and testing methods of curing settling problems in full-scale activated sludge processes. An attempt will be made in the material which follows to describe the progress which has been made in research on the filamentous bulking problem which can be used in this approach to the solution of the problem. A. HYPOTHESESABOUT THE MECHANISMS O F FILAMENTOUS

BULKING

The science of physiology developed rapidly at the same time that medical microbiology was identifying the pathogenic parasites, and developments in one field stimulated advances in the other through the study of the mechanisms of pathogenesis. Studies of the microorganisms causing settling problems in the activated sludge process should be related to the ecology of normal activated sludge by investigations of the mechanisms by which the settling characteristics of activated sludge can change. Unfortunately, there is a great deal which is not known about the ecology of normal activated sludge (Pipes, 1966).

1 . The Nature of Filamentous Bulking The settling characteristics of activated sludge are the result of physical properties which determine the manner in which individual particles settle through water, individual particles agglomerate with each other, and water flows up through a mass of compacting sludge. The physical properties of the individual particles are determined b y the microorganisms which make up the particle, the form in which they grow, the nature and amount of inert material agglomerated with the microorganisms in the particle, and the chemical composition and physical properties of the solution in which the particles are suspended. The extent to which the settling characteristics of activated sludge are determined either by the microbial population or by other factors has not yet been determined but there have been some guesses about it. Haseltine (1932) guessed that bulking was not a replacement of

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normal sludge organisms by filamentous organisms but some condition which produced both a change in the settling characteristics and an opportunity for the filamentous organisms to grow. Kraus (1949) used a formulation which implied that he believed that bulking was a simple replacement of normal sludge with filamentous sludge. Heukelekian and Weisberg (1956) performed a valuable service when they showed that zoogleal bulking and filamentous bulking were completely different phenomena on the basis of physical measurements and that activated sludge could have a high sludge volume index without being very filamentous. Recently, Finstein and Heukelekian (1965), using a technique adapted from soil microbiology, showed that in some activated sludge processes t h e SVI of the sludge produced was directly proportional to the number and length of filaments projecting from the particles. Thus, the evidence indicates that a profuse growth of filamentous organisms in activated sludge produces an increase in SVI, but other factors can also produce a poorly compacting sludge. Since the bacterial and fungal populations of sludge with different settling characteristics have not yet been determined, it is possible that the filamentous bulking organisms grow only as filaments and are not present in normal activated sludge. It is also possible that they can grow as either floc particles or as filaments and make u p a sizable proportion of the microbial population of many types of activated sludge which settles well. Ingols and Heukelekian (1939) expressed the opinion that zoogleal bulking was a growth of Sphaerotilus in flocculant form and that filamentous bulking would succeed zoogleal bulking if the conditions in the process were not changed to prevent the continued growth of Sphaerotilus. Unfortunately, Heukelekian and Weisberg (1956) did not identify the organisms in zoogleal bulking when they were comparing it with filamentous bulking so this question must remain open at present.

2. The AIV and Related Hypotheses A long filament has a much greater surface area-to-volume ratio than a clump of cells. Thus, filamentous organisms should have a competitive advantage over floc-forming organisms in a continuous culture in which the factor-limiting growth is in solution. Many investigators have used this concept as a hypothesis to explain why filamentous organisms sometimes predominate in activated sludge and sometimes do not. If the identity of the floc-forming organisms and the filamentous organisms which predominate in activated sludge

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under different conditions were known, they could easily be obtained in pure culture and this hypothesis tested in well-controlled laboratory experiments. a. Competition f o r Soluble Substrates Littman (1940) observed that Sphaerotilus natans in pure culture utilizes simple, soluble substrates much more readily than insoluble or complex substrates and then used the A/V hypothesis to explain filamentous bulking induced by sugars or other soluble organic matter in the influent waste. The A/V hypothesis applied to cornpetition for soluble substrates seems a reasonable hypothesis to explain some types of filamentous bulking. Particulate and colloidal organic matter would be coagulated by floc-forming organisms and they would have the advantage when there is not much soluble organic matter in the waste. Sugars and other readily available, soluble substrates would favor the growth of filamentous microorganisms. The A/V hypothesis and competition for substrates can be elaborated to explain filamentous bulking on nonsoluble organic wastes when the process loading is too high. It is further hypothesized that the particulate organic matter coagulated by floc-forming organisms must be hydrolyzed and that the growth rate of the floc-forming organisms is a function of the rate of diffusion of the soluble hydrolysis products into their cells. At high process loading, the organisms must grow more rapidly and the concentration of hydrolysis products (and possibly metabolic intermediates diffusing out from the cells) in solution must be greater. Thus, at high process loading, enough soluble organic matter will be present that the filamentous organisms can grow well even though the organic matter coming into the process is in a particulate form which they cannot readily assimilate. Furthermore, this same concept can be used to explain filamentous bulking due to stale sewage. If the sewage remains in the sewer a long time, the particulate matter can settle out and be decomposed anaerobically before it reaches the treatment plant. The products of this anaerobic decomposition are soluble substrates which can be readily assimilated. This same argument applies to the case of filamentous bulking caused by the addition of digester supernatant to the aeration tank. Sugars, stale sewage, or digester supernatant do not cause filamentous bulking if the process loading is low enough. In order to use the A/V hypothesis and competition for soluble substrates to explain these cases of bulking, it must be assumed that the floc-forming

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organisms can utilize simple, soluble substrates and that when the growth rate in the culture is very low they can actually assimilate them more rapidly than the filamentous organisms can. This necessary assumption does not seem to be very likely to be true and casts a large doubt on the use of the A/V hypothesis and competition for soluble substrate as an explanation for filamentous bulking.

b. Nitrogen or Phosphorus Limitation A case of filamentous bulking when the wastes contain inadequate concentrations of either nitrogen or phosphorus in relation to the amount of oxidizable organic matter present could be explained by the A/V hypothesis if the nitrogen or phosphorus compounds which are available are soluble. Usually when a case of nitrogen or phosphorus limitation does occur, it is in a waste in which most of the nutrients are in soluble form. The A/V hypothesis is an attractive explanation for these cases of filamentous bulking.

c. Competition for Oxygen Heukelekian (1941) used the A/V hypothesis to explain filamentous bulking due to sugars in the waste and overloading by assuming that the primary competition in these instances was for dissolved oxygen rather than for oxidizable substrates. When an activated sludge process is overloaded or when large concentrations of soluble organic matter are present in the waste, the sludge in the aeration tank does have an extremely high rate of oxygen uptake and if the aeration rate is not increased, the DO value falls to a very low level. The use of the A/V hypothesis and competition for DO to explain these cases of filamentous bulking seems quite reasonable and the same argument applies to cases of filamentous bulking caused by stale sewage or by digester supernatant. Okun (1949) went a step further and suggested that high DO would be toxic to filamentous organisms but there are no data to support this contention. Wuhrmann (1964) calculated the rate of diffusion of oxygen into a floc particle by assuming a value for the oxygen transfer coefficient for the extracellular material which binds microorganisms together. His calculations showed that unless the DO in the mixed liquor is 2 mg./liter or greater, the oxygen concentration in the center of a 500 p diameter floe particle will be less than 0.1 mg./liter. It is possible that a sudden drop in DO could cause a breaking up of floc particles while a long-contained low DO would cause filamentous bulking.

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Mulder (1964) actually tried to verify the A/V hypothesis applied to competition for DO in the laboratory. He used Sphaerotilus natans as the filamentous organism and Arthrobacter globiforrnis which was isolated from activated sludge as the floc-forming organism. It was shown that Sphaerotilus grows more rapidly under poorly aerated conditions and Arthrobacter grows more rapidly under well-aerated conditions. However, the poorly aerated conditions were flask cultures sitting on a table top and the well-aerated conditions were flask cultures on a shaker. It would be much more convincing if the hypothesis were tested using continuous culture technique and the actual DO values were reported. The A/V hypothesis and competition for DO can b e used to explain filamentous bulking due to organic shock loads. A rapid increase in the concentration of organic matter in the aeration tank stimulates the respiratory gas exchange of the sludge microorganisms greatly and the DO drops to a low level (Gaudy and Turner, 1964).This low DO could favor the growth of filamentous organisms over the growth of flocforming organisms or it could cause the floc particles to break up if Wuhrmann (1964) is correct in his contention that anaerobic conditions inside the floc particle cause it to break apart.

3. The Fat Sludge Hypothesis In explaining the necessity of maintaining the loading on an activated sludge process in the proper range to plant operators, many engineers use an analogy of feeding corn to pigs. According to this analogy, if the sludge is fed too much (loading too great), it becomes fat and lazy and does not move very rapidly. This analogy provides a clue to a possible hypothesis explaining some cases of filamentous bulking. If it is assumed that the filamentous organisms are capable of storing large quantities of a carbohydrate or lipid food reserve but that the floc-forming organisms are not, then the filamentous organisms would have an advantage under certain conditions. The conditions under which they would store large amounts of food reserve would be when large concentrations of simple, soluble substrates are available or when there are inadequate concentrations of either nitrogen or phosphorus to allow complete assimilation of the organic substrate. It could further be assumed that storage of large quantities of food reserve would decrease the specific gravity of the filamentous organisms. If this is true, then some cases of filamentous bulking should ultimately result in floating sludge.

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4 . The Selective Toxicity Hypothesis It could be postulated that certain substances are more toxic to floc-forming organisms than to filamentous organisms and their presence in the waste would produce filamentous bulking of the activated sludge. Among the substances which could b e more toxic to the flocforming organisms are soluble organic compounds such as phenolic compounds, organic acids, aldehydes, ketones, etc. Hydrogen ion would have to be considered to be more toxic to floc-forming organisms than to some filamentous organisms since at low p H values activated sludge always becomes very filamentous. A slightly reduced pH would be expected to increase the toxicity of phenols and organic acids. A similar but slightly different hypothesis would be that filamentous organisms are more resistent to anaerobic conditions than floc-forming bacteria. According to this hypothesis, holding the return sludge under anaerobic conditions for several hours would kill some of the floc-forming organisms and leave the filamentous organisms to pick up their growth after the sludge is returned to aeration. A greater toxicity of sulfide to floc-forming organisms than to filamentous organisms could also explain filamentous bulking due to anaerobic return sludge.

5. Miscell aneous Hypotheses A number of other hypotheses have been advanced to explain bulking. Some of these have merit as concepts but have not yet been formulated concisely enough that they can be tested easily. Others were obviously formulated on the basis of a false analogy or apply to settling problems other than filamentous bulking.

a. The Protozoan Hypothesis The ciliated protozoa in activated sludge are believed to be predators which feed on individual bacterial cells, allowing the flocforming organisms and filamentous organisms to grow. Single bacterial cells have a much higher A/V ratio than filamentous organisms and could be expected to predominate in any competition for solutes unless there is some other controlling mechanism. It is quite likely that selective predation controls the population of saprophytic organisms in activated sludge under certain conditions. Larsen (1936) elaborated the theory that filamentous bulking occurs after the protozoa have consumed the normal sludge organisms and

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removed them from competition with the filamentous organisms. For this hypothesis to be valid, it would be necessary to demonstrate that some protozoa are able to break up floc particles to get at the organisms inside. This has not yet been demonstrated and it seems likely that if it did happen, some investigator would have published observations on it. Pillai and Subrahmanyan (1943) advanced the hypothesis that low DO causes bulking because the stalked ciliates die under anaerobic conditions, the dead cells are invaded by bacteria, and the decomposition of the protozoa produces gas bubbles which decrease the specific gravity of the sludge. Their observations are undoubtedly correct, but what they have described is one of the types of floating sludge rather than one of the types of filamentous bulking.

b. The Bacterial Equilibrium Hypothesis Some investigators have had the idea that the production of normal activated sludge depends upon the establishment of an equilibrium between the microbial population of the sludge and the environment or among the various components of the microbial population. Ely (1938) attributed bulking to the addition of some foreign substance which destroys the equilibrium between the bacteria. However, recently it has been shown that a laboratory activated sludge culture in which the environmental conditions change much less than they do in a full-scale process never reaches an equilibrium in respect to its microbial population (Cassell et al., 1966). Actually, it is more likely that normal activated sludge depends upon variations in the environmental conditions to maintain a diversified population capable of oxidizing a large variety of organic compounds. c. The Biophysical Strain Hypothesis

Ruchhoft and Kachmar (1941) expounded the theory that bulking was caused b y “biophysical strain” which was defined as any sudden change in the sludge itself, the food supply, the oxygen supply, etc. Sometimes filamentous bulking does appear as a very sudden change in the SVI and sometimes it does appear after a sudden change in the character of the waste being treated. However, this hypothesis is not formulated in useful terms because, as it now stands, there is no clearcut method of testing it.

d. The Laxative Hypothesis Goudey (1933) advanced the hypothesis that bulking was caused b y

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anything that is laxative to people such as large amounts of sugar, fruit pulp, etc. He also believed that the presence of filamentous organisms in bulking sludge was irrelevant and that the way to cure bulking was to add a constipating substance, such as milk of magnesia or cheese. The best I can figure out is that he must have been describing a case of overaerated sludge in which the incorporation of fruit pulp physically in the sludge without assimilation by the microorganisms caused small air bubbles to adhere to the sludge, decreasing its density.

B. FILAMENTOUS BULKINGORGANISMS

The verification of some of the hypotheses about the mechanisms of filamentous bulking can be accomplished only by laboratory studies with pure cultures in which the environment can be carefully controlled. Once a sufficient number of these hypotheses have been verified, it will be possible to formulate a theory of filamentous bulking which can be used to predict when bulking will occur and how it can be cured. This theory can then be tested in practice on operating activated sludge processes. However, the successful completion of this fundamental approach to the filamentous bulking problem depends initially upon knowing the identity of the filamentous bulking organisms and being able to study them in pure culture. In practically all cases described in the literature, the filamentous organism in bulking sludge has been called “spaerotilus” with no real attempt to establish its identity. I am presently in the midst of attempting to establish the identities of some filamentous organisms isolated from samples of activated sludge having high sludge volume indices. My examination of these organisms is quite incomplete so some of this material must be considered as my personal opinion not yet supported by published data.

1 . Sphaerotilus natans

Sphaerotilus natans from the case of bulking described b y Morgan and Beck (1928), was isolated and properly identified by Ruchhoft and Watkins (1928). Smit (1934) isolated a filamentous bacterium resembling Sphaerotilus natans from bulking sludge but h e was not willing to state positively that it was Sphaerotilus. Lackey and Wattie (1940) isolated Sphaerotilus natans from activated sludge by enrichment culture technique and their work had a great deal to do with the acceptance of Sphaerotilus as “the bulking organism” despite the fact that they noted in passing that several other organisms could cause

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bulking. Tkachenko and Droblyanets (1959) isolated what they called Spaerotilus dichotomus (a bastardization of Cladothrix dichotoma which is a growth form of Sphaerotilus natans) from bulking activated sludge. Also, Sphaerotihs natans has been isolated from polluted waters and identified many times by competent bacteriologists. There is no doubt about the fact that Sphaerotilus does cause some cases of bulking. A number of investigators have reported on the types of organic compounds which can serve as growth substrates for Sphaerotilus (Lackey and Wattie, 1940; Stokes, 1954; Hohnl, 1955). It can grow well on a variety of simple, soluble substrates but its growth on complex carbohydrates, fats, or proteins is very slow and could be explained by traces of impurities in the media. It can utilize either ammonia or nitrate as its sole nitrogen source, but requires vitamin BIZ unless methionine is supplied (Mulder and van Veen, 1963). There has been some confusion in the literature about the ability of Sphaerotilus to grow under anaerobic conditions. It is a strict aerobe but does remain viable for a long time under anaerobic conditions (Mulder, 1964). Ruchhoft and Kachmar (1941)found that it could grow well at DO concentration less than 0.1 mg./liter. Both Waitz and Lackey (1959) and Mulder (1964) demonstrated that it can grow as well in a poorly aerated culture as in a well-aerated culture but quantitative data relating its growth rate to the DO concentration are lacking. Sphaerotilus is limited to a p H range of 5.8 to 8.1 (Stokes, 1954) and could not be the cause of filamentous bulking due either to exceptionally high or low p H of the influent waste. It has a temperature optimum near 30°C. but does not grow at less than 15"C. (Stokes, 1954); thus it could not be the cause of low temperature bulking, as described by Ludzack et aE. (1961b). Rouf and Stokes (1962) demonstrated that Sphaerotilus natans could store large quantities of poly-P-hydroxybutyrate, making the fat sludge hypothesis reasonable from this standpoint. Skerman et al. (1957) reported that Sphaerotilus natans could oxidize hydrogen sulfide and deposit elemental sulfur in its cells. Their experiments were repeated successfully by Waitz and Lackey (1959) but neither group demonstrated that Sphaerotilus could obtain energy for growth from the oxidation of HZS and apparently the cells lose their viability after a few hours of exposure to HZS even in an aerobic environment. Several investigators have reported that Sphaerotilus can grow as isolated cells without forming a sheath. Gaudy and Wolfe (1961) investigated the conditions under which it grows as single cells and developed a technique for separating the filaments from the isolated

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cells. Under certain conditions, Sphaerotilus can produce large quantities of extracellular polysaccharide (Gaudy and WoIfe, 1962) and I have observed formation of floc particles in a pure culture of Sphaerotilus.

2. Bacillus I have isolated from three different samples of bulking activated sludge bacteria which grow as filaments on agar media for 2 or 3 days and then break up into single cells, most of which form endospores. Shive and Buswell (1928) reported producing a growth which looked very much like filamentous bulking sludge in a pure culture of Bacillus subtilus. Lackey and Wattie (1940) suggested that Bacillus mycoides could be a filamentous bulking organism and stated that it was difficult to distinguish between Bacillus and Sphaerotilus on the basis of morphology, even in pure culture. Dondero et al. (1961) also commented on the possibility of confusing some members of the Bacillus group with Sphaerotilus. The genus Bacillus contains some filamentous bulking organisms but which species are bulking organisms and which are not has not yet been established. A summary of the nutrition and physiology of the various species of the genus Bacillus was published by Knight and Proom (1951).There is considerable variation from one species to another and this information will not be a great deal of help in evaluating hypotheses about the mechanisms of bulking until the identities of the species which are filamentous bulking organisms have been established. Most members of this genus can grow on either simple, soluble substrates or complex organic molecules. Most of them have requirements for specific amino acids or vitamins but a few species can utilize ammonia as the sole nitrogen source without added growth factors. They cover a very wide temperature range and because of the ability to form endospores they are quite tolerant of many environmental conditions which are toxic to most other microorganisms. Macrae and Wilkinson (1958) reported on the accumulation of poly-P-hydroxybutyrate by Bacillus cereus and bacillus megatherium. It can be stated that the members of this group which are able to grow as filaments are also able to grow as single cells. Shive and Buswell (1928) reported that Bacillus subtilis could form into floc particles under certain conditions but Lackey and Wattie (1940) stated that Bacillus mycoides does not form floc.

3. Beggia toa

I have found one case of filamentous bulking in which the pre-

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dominant organism in the sludge appears to be a species of Beggiatoa. I have not been able to find any reports of Beggiatoa having been isolated from activated sludge previously but it has been reported from trickling filters (Schulze, 1957) and from polluted streams (Harrison and Heukelekian, 1958). In primary isolation cultures, Beggiatoa looks very much like Sphaerotilus (Faust and Wolfe, 1961). Pringsheim (1964) summarized what is known about the nutrition and physiology of Beggiatoa. It grows well on simple, soluble substrates but does not hydrolyze complex substrates. Most strains can utilize ammonia as the sole nitrogen source. It is limited to a narrow p H range near neutrality. It can store large quantities of poly-phydroxybutyrate. It can oxidize HZS and desposit elemental sulfur inside the cell but high concentrations of HZS are toxic to it. In general, it is quite similar to Sphaerotilus in its metabolic capabilities. Beggiatoa does not grow as single cells; however, the trichomes of Beggiatoa do break up into very short filaments under some conditions. Also, it has been reported as forming into a flocculant growth (Pringsheim, 1964).

4 . Geotrichum candidum It has been obvious to some investigators (presumably those who bothered to look at the sludge under a microscope) that some cases of bulking are caused by fungi and not by filamentous bacteria (Genetelli and Heukelekian, 1964) but there have been few attempts to identify the fungi associated with bulking. Smit (1934) identified a bulking fungus as Geotrichoides paludosus which is presumably the same organism as Geotrichum candidum which has been reported as causing bulking in Yardley, England (Hawkes, 1960), and Providence, Rhode Island (Jones, 1964). I am personally on record as having isolated Geotrichum candidum from activated sludge and having confused it with Sphaerotilus natans (Pipes and Jones, 1963). T h e best information on the nutrition and physiology of Geotrichum is in three Ph.D. dissertations (Bobrov, 1951; Carmichael, 1954; Jones, 1964). It grows quite rapidly on simple, soluble substrates but also grows well on complex substrates. It can utilize inorganic nitrogen compounds as its sole nitrogen source and can grow rapidly in media containing very low concentrations of nitrogen or phosphorus. In nitrogen- or phosphorus-deficient media, it accumulates large amounts of a lipid food reserve. It can grow in media having p H values in the range of 3 to 12 and thus could cause bulking under acid conditions. Geotrichum normally grows as a mycelium but reproduces by

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breaking up into arthrospores. In agitated cultures it produces very short hyphae which readily break up into arthrospores. It has not been reported to be a floc-forming organism.

5. Other Fungi Lackey and Wattie (1940) stated that filamentous yeasts sometimes cause bulking. I have isolated some yeasts which have the capability of growing either as single cells or as filaments from several different samples of activated sludge but I have not yet found a particular case of bulking which was definitely due to a profuse growth of yeasts. Many of the yeasts d o have properties similar to the organisms which have been found in bulking sludge. It has been known for many years (Pasteur, 1862) that many yeasts and some other fungi will grow as single cells under anaerobic conditions and as filaments under aerobic conditions. Ford and Eckenfelder (1967) recently reported a case of filamentous bulking in which the solution to the problem was to hold the return sludge under anaerobic conditions for several hours. It is likely that this was a case of yeast bulking and the anaerobic conditions caused the filaments to break u p into single cells. V. Summary

The proper operation of the activated sludge process depends upon finding the solution to two problems; namely, (a) an aerobic microbial population capable of metabolizing the major organic components of the waste must be grown in the aeration tank, and (b) the microbial population must be separated from the purified waste in the sedimentation tank. Previous research studies have delineated the conditions under which the first problem can be solved, how it can be solved, and the conditions under which it cannot be solved. However, previous research has not produced a method of attack which can be counted upon to provide a solution to the second problem in all cases. One of the reasons that the settling problem cannot always be solved is that the different phenomena which cause problems with settling have never been clearly defined. There are at least ten different types of sludge which do not separate properly in the sedimentation tank and all ten of these types have been called “bulking sludge” at one time or another. At least three of these types, deflocculation, floating sludge, and filamentous bulking sludge, are complexes of several different phenomena which can be produced by a number of different causes.

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Research on filamentous bulking has been rather confused due to a failure to identify the organisms involved. With very few exceptions, a filamentous organism found in activated sludge has been called Sphaerotilus (or Cladothrix which is a synonym of Sphaerotilus). However, there are a number of other filamentous bacteria (including Beggiatoa and filamentous members of the genus BaciZZus) and some fungi (particularly Geotrichum and other yeast-like imperfect fungi) which sometimes occur in large numbers in activated sludge and can cause filamentous bulking. There is an urgent need for studies which will define the relationships between specific filamentous organisms in specific occurrences of filamentous bulking in full-scale process operation under well-characterized environmental conditions in the process. There is also a need for the establishment of collections of cultures of filamentous and other sludge organisms so that hyphotheses about the mechanisms of filamentous bulking can be tested in laboratory experiments with pure cultures. The following statements can be considered to be working hypotheses which are useful guidelines when attempting to discover the cause of a particular case of bulking but they need to be either verified or disproved before a useful theory of filamentous bulking can be developed. 1. The condition of filamentous bulking is a function of the ratio of filamentous organisms to floc-forming organisms in the sludge, a high SVI being directly due to a high proportion of filamentous organisms. 2. Many of the filamentous bulking organisms can grow as either single cells or filaments and sometimes form floc particles in pure culture; thus, they may make up a sizable proportion of the population of normal activated sludge. 3. The ratio of the filamentous to floc-forming organisms is sometimes determined by the competition between filamentous and flocforming organisms for some resource in the environment. 4. The higher surface area-to-volume ratio of filamentous organisms gives them an advantage over floc-forming organisms when the primary competition is for soluble material such as dissolved nutrients or oxygen. 5. The predatory activity of protozoa and rotifers feeding upon isolated bacterial or yeast cells eliminates them from competition with filamentous and floc-forming organisms. 6. Filamentous bulking organisms assimilate simple, soluble substrates very rapidly but grow poorly on complex or insoluble substrates. 7 . Floc-forming organisms can utilize either soluble or insoluble

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substrates for growth but must hydrolyze insoluble substrates to simple compounds before assimilating them. 8. At high process loadings, soluble hydrolysis products and metabolic intermediates produced by the floc-forming organisms build up to appreciable concentrations in the mixed liquor. 9. At low dissolved oxygen concentration the filamentous organisms grow more rapidly than the floc-forming organisms. 10. Filamentous bulking organisms have the capacity to store large amounts of food reserve but the floc-forming organisms do not. 11. Some soluble organic compounds are toxic to floc-forming organisms but can be assimilated by filamentous bulking organisms. 12. Some metallic ions are more toxic to filamentous bulking organisms than to floc-forming organisms. 13. Filamentous bulking organisms can remain viable under anaerobic conditions longer than floc-forming organisms can. 14. Some of the yeast-like fungi which are only rarely bulking organisms grow as filaments under aerobic conditions and single cells under anaerobic conditions; thus, when bulking is due to the presence of one of these organisms, the sludge can be made less filamentous by holding it under anaerobic conditions for a few hours. 15. Filamentous bacteria which are found in bulking sludge do not grow when the pH is less 6.0. 16. Several different fungi can grow well at low pH and one of these will make up the bulk of the activated sludge population when the pH is less than 5.0. 17. Some predators in activated sludge can feed on filamentous organisms but not on flocculated organisms while others can feed on flocculated organisms but not on filamentous organisms. Investigations of the validity of these hypotheses not only will provide a firm basis for a solution to the problem of filamentous bulking but also could contribute to the development of microbial ecology. For instance, the determination of the conditions under which protozoa predation determines the bacterial predominance as differentiated from the conditions under which bacterial predominance is the result of competition for some resource would have general applicability in a number of fields of research. There is an opportunity for microbiology to contribute a great deal to the study of activated sludge and vice versa. ACKNOWLEDGMENTS My research on filamentous bulking has been supported in part by Research Grant No. WP-00588, originally from the Division of Water Supply and Polliition Control.

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U.S. Public Health Service and, more recently, from the Federal Water Pollution Control Administration, U.S. Department of the Interior. The photographic illustrations in this paper are the work of Mrs. Caroline L. Wienke and Roger W. Thompson and their assistance is gratefully acknowledged.

REFERENCES Andersen, C. G. (1936).Sewage Works]. 8,785-792. Barth, E. F., Ettinger, M. B., Salotto, B. V., and McDermott, G. M. (1965).J. Water Pollution Control Federation 37,86-96. Bloodgood, D. E. (1947).Sewage WorksJ.19,202-213. Bobrov, R. A. (1951). Ph.D. Thesis, Univ. of Calif., Los Angeles, Calif. Butterfield, C. T. (1935).Public Health Rept. (U.S.) 50,671-684. Carmichael, J. W. (1954).Ph.D. Thesis, Harvard Univ., Cambridge, Massachusetts. Cassell, E. A., Sulzer, F. T., and Lamb, J. C. (1966).J. Water Pollution Control Federation 38,1398-1409. Cooke, W. B., and Ludzack, F. J. (1958).Sewage Ind. Waste 30,1490-1495. Curds, C. R.(1963). J . Gen. Microbiol. 33,359-363. Donaldson, W. (1932). Sewage WorksJ . 4,48-52. Dondero, N. C., Phillips, R. A., and Heukelekian, H. (1961).Appl. Microbiol. 9, 219227. Dougherty, M. H., and McNary, R. R. (1958).Sewage Ind. Waste 30,1263-1265. Edwards, G. P., and Nussberger, F. E. (1947). Sewage WorksJ . 19,598-602. Ely, H.M. (1938).Cali$ Sewage WorksJ.10,17-18. Faust, L., and Wolfe, R. S. (1961).J. Bacteriol. 81,99-110. Finstein, M. S.,and Heukelekian, H. (1965).Purdue Univ., Eng. Bull., E x t . Ser. 49, (4), 175-181 Ford, D. L., and Eckenfelder, W. W. (1967). J . Water Pollution Control Federation 39 (in press). Gaudy, A. F., and Engelbrecht, R. S. (1961).J . Water Pollution Control Federation 33, 800-816. Gaudy, A. F.,and Turner, B. G. (1964).J. Water Pollution Control Federation 36,767781. Gaudy, E., and Wolfe, R. S. (1961).A p p l . Microbiol. 9,580-584. Gaudy, E., and Wolfe, R. S. (1962).A p p l . Microbiol. 10,200-205. Genetelli, E. J., and Heukelekian, H. (1964).J. Water Pollution Control Federation 36, 643-649. Gibson, J. (1957).I n “Microbial Ecology” (R. E. 0. Williams and C. C. Spicer, eds.), pp. 22-41,Cambridge Univ. Press, London and New York. Goudey, R. F. (1933).Cali5 Sewage WorksJ . 5,61-62. Greeley, S. A. (1945). Sewage WorksJ.17,1135-1145. Greenberg, A. E., Klein, G., and Kaufman, W. J. (1955).Sewage Ind. Wastes 27, 277282. Harris, F. W., Cockburn, T., and Anderson, T. (1926).Surueyor70,30-31,53-55. Harrison, M. E., and Heukelekian, H. (1958).Sewage Ind. Wastes 30,1278-1302. Haseltine, T. R.(1932).Sewage Works].4,461-489. Haseltine, T. R. (1938).Sewage WorksJ . 10,1017-1042. Haseltine, T. R. (1941).Water Works Sewerage 88,274-279. Haseltine, T . R. (1961).J. Water Pollution Control Federation 33,946-967. Hawkes, H. A. (1960).In “Waste Treatment” (P. C . C . Isaac, ed.), pp. 52-98,Macmillan (Pergamon), New York.

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Haywood, R. W. (1937).Sewage WorksJ. 9,785-794. Helmers, E. N., Frame, J. D., Greenberg, A. E., and Sawyer, C. N. (1952). Sewage Ind. Wastes 24,496-507. Heukelekian, H. (1941).Sewage Works]. 13,39-42. Heukelekian, H. (1949).Ind. Eng. Chem. 41,743-769. Heukelekian, H., and lngols, R. S. (1940).Sewage Works]. 12,694-714. Heukelekian, H., and Weisberg, E. (1956). Sewage Ind. Wastes 28,558-574. Hohnl, G . (1955).Arch. Mikrobiol. 23,207-250. Ingols, R. S., and Fetner, R. H. (1961).J. Water Pollution Control Federation 33,366370. Ingols, R. S., and Heukelekian, H. (1939).Sewage WorksJ . 12,927-945. Ingols, R. S., and Heukelekian, H. (1940).Sewage Works). 12,849-862. Jones, P. H. (1964).Ph.D. Thesis, Northwestern Univ., Evanston, Illinois. Keefer, C. E. (1962)J. Water Pollution Control Federation 34,1186-1196. Keefer, C . E. (1963).J.Water Pollution Control Federation 35,1166-1173. Keefer, C . E., and Meisel, J. (1951).Sewage Ind. Wastes 23,982-991. Knight, B . C. J. G., and Proom, H. (1950).J . Gen. Microbiol. 4,508-538. Kraus, L. S. (1945).Sewage WorksJ.17,1177-1194. Kraus, L. S. (1946). Sewage Works]. 18,1099-1112. Kraus, L. S. (1949).Sewage WorksJ. 21,613-622. Lackey, J. B. (1949). Sewage WorksJ . 21,659-665. Lackey, J. B., and Wattie, E. (1940).Sewage WorksJ.12,669-684. Larsen, W. A. (1936). Munic. Sanit. 7,52-54. Littman, M. L. (1940).Sewage WorksJ. 12,685-693. Logan, R. P., and Budd, W. E. (1956). In “Biological Treatment of Sewage and Industrial Wastes” (W. W. Eckenfelder and J. McCabe, eds.), Vol. 1, pp. 271-276. Reinhold, New York. Ludzack, F. J., and Schaffer, R. B. (1962). J . Water Pollution Control Federation 34, 320-340. Ludzack, F. J., Schaffer, R. B., and Bloomhuff, R. N. (1961a).J.Water Pollution Control Federation 33,492-505. Ludzack, F. J., Schaffer, R. B., and Ettinger, M. B. (1961b).J . Water Pollution Control Federation 33,141-156. McKinney, R. E., and Weichlein, R. G. (1953).Appl. Microbiol. 1,259-261. Macrae, R. M. and Wilkinson, J. F. (1958).J.Gen. Microbiol. 19,210-222. Mohlman, F . W. (1934).Sewage Works]. 6,119-121. Mohlman, F. W., Thomas, H. A., and NRC Committee (1946). Sewage WorksJ.18,7941028. Morgan, E. H., and Beck, A. J. (1928).Sewage WorksJ. 1,46-51. Morgan, P. F., Durdin, A. C., and Spiegal, M. (1936).Sewage WorksJ . 8,933-942. Mulder, E. G. (1964).J.A p p l . Bacteriol. 27,151-173. Mulder, E. G., and van Veen, W. L. (1963). Antonie wan Leeuwenhoek J . Microbiol. Serol. 29,121-153. Okun, D. A. (1949). Sewage Works]. 21,763-791. Orford, H . E., Heukelekian, H., and Isenberg, E. (1960). Conf. Biol. Waste Treat., 3rd, Manhattan Coll., New York, 1960. Preprints pp. 68-84. Palmer, J. R. (1949).Sewage WorksJ . 13,168-169. Pasteur, L. (1862).Ann.Chim. Phys. Ser. 3 64,5-110. Pearse, L., and APHA Committee (1937).A m . Public Health Assoc. Year Book 27,164178.

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Pillai, S. C., and Subrahmanyan, V. (1943).Sci. Cult. (Calcutta)8,376-378. Pipes, W. 0.(1965).Purdue Uniu., Eng. Bull., E x t . Ser. 49 (4),647-656. Pipes, W. 0.(1966).Adoan. Appl. Microbiol. 8,77-103. Pipes, W. O., and Jones, P. H. (1963).Biotechnol. Bioeng. 5,287-293. Pringsheim, E . G. (1964).Am.]. Botany 51,898-913. Ridenour, G. M., Henderson, C. N., and Schulloff, H. B. (1937). Sewage Works J. 9, 63-69. Rouf, M. A,, and Stokes, J. L. (1962).]. Bacteriol. 83,343-347. Ruchhoft, C. C., and Kachmar, J. F. (1941).Sewage WorksJ . 13,3-32. Ruchhoft, C. C., and Smith, R. S. (1939).Sewageworks]. 11,409-413. Ruchhoft, C . C., and Watkins,-J. H. (1928).Sewage WorksJ.1,52-54. Sawyer, C . N., and Bradney, L. (1945).Sewage Works]. 17,1191-1209. Schulze, K. L. (1957).Sewage Ind. Wastes 29,458-466. Scott, W. (1928).Surveyor 73,345-347. Shive, R. A., and Buswell, A. M. (1928).Illinois State Water Survey, Bull. 25,37-83. Singer, P. C., Pipes, W. O., and Hermann, E. R. (1967).1. Water Pollution Control Federation 39 (in press). Skerman, V. B. D., Dementijeva, G., and Carey, R. J. (1957).]. Bacteriol. 73,504-512. Smit, J. (1932).Sewage Works]. 4,960-972. Smit, J. (1934).Sewage Works]. 6,1041-1053. Smith, R. S., and Purdy, W. C. (1936).Sewage WorksJ. 8,223-230. Stewart, M. J. (1964).Water Sewage Works 111,295-297. Stokes, J. L. (1954).J.Bacteriol. 67,278-291. Symons, J . M., McKinney, R. E., and Harris, H. H. (1960).]. Water Pollution Control Federation 32,841-852. Tapleshay, J. A. (1945).Sewage Works]. 17,210-214. Tkachenko, N., and Droblyanets, E. E. (1959).Mikrobiologiya 28,708-712. Torpey, W. N. (1948).Sewage Works]. 20,781-794. Ullrich, A. H., and Smith, M. W. (1957).Sewage Ind. Wastes 29,400-413. Waitz, S., and Lackey, J. B. (1959).Quart. J . Florida Acad. Sci. 21,335-340. Wuhrmann, K. (1964).Aduan. Appl. Microbial. 6,119-150.

Money, wine and women have good and bud things in them-Randle Cotgrave

Ma lo-lactic Fermentation RALPH E. KUNKEE Department of Viticulture and Enology University of California, Davis, California

I. Introduction ......................................................... ....... ..... 11. Early Observations 111. Occurrence ................................................................. IV. Malo-lactic Bacteria .., .............................................. .... A. Taxonomy. ............................................ C. Habitat .....................

..........

F. End Products .......................... V. VI. A. Mechanism ................ B. Enzyme Induction ............................................... C. Energetics .......................................................... D. Secondary Effects of Deacidification VII. Control of Malo-lactic Fermentation .................... A. Desirability of Control ....................... €3. Stimulation ......................................................... C. Inhibition ........................................................... VIII. Conclusions .............................................................. References

235 236 239 241 24 1 243 246 247 253 257 259 260 260 262 263 269 270 270 271 272 2 73 274

1. Introduction

Acidity and acid taste are pronounced and desirable chemical and sensory characteristics of wine. During storage of new wine a decrease in acidity often occurs. One of the causes of this loss was discovered around the turn of the century when it was found that after the alcoholic fermentation by yeast, certain lactic acid bacteria may carry out a secondary fermentation of wine. During this secondary fermentation, malic acid (a dicarboxylic acid which may constitute as much as half of the acid of grapes) is converted to lactic acid, a monocarboxylic acid. This conversion, now referred to as malo-lactic fermentation, results in a deacidification. This may be desirable in wines of high acidity but is detrimental to wines with acidities that are already low. In addition to a change in acidity brought about by the 235

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bacteria, flavor changes, either favorable or unfavorable, may take place. One can see that control (either stimulation or inhibition) of malo-lactic fermentation is of importance to the wine maker. This review includes information on the biochemistry and metabolism of bacteria causing malo-lactic fermentation, and discusses how this knowledge may be applied to control malo-lactic fermentation. The concentration of malic acid is high in apples’ as well as grapes, and malo-lactic fermentation also occurs in cider. Some of the information about the bacteria reviewed here has been obtained from research on microbiology of cider. However, the discussion of the application of malo-lactic fermentation will be restricted to wine making. Other discussions of malo-lactic fermentation may be found in enologic texts and reference works such as Amerine et al. (1967), Amerine and Singleton (1965), and Ribereau-Gayon and Peynaud (1961), and in articles b y Fell (1961), Fornachon (1963a), Lambion and Meskhi (1957), Peynaud and Domercq (1961a), Radler (1962a, 1963, 1966), Rankine (1963a), Schanderl (1959), Suverkrop and Tchelistcheff (1949), Vaughn (1955), and Vaughn and Tchelistcheff

(1957). II. Early Observations Gross changes in wine can be noticed during malo-lactic fermentation. There is often an increase in cloudiness because of the growth of bacteria, and a deposition of tartrate because of the change in pH (see Section V1,D). Off-odors, which may later disappear, frequently accompany the fermentation. Also noticeable is the effervescence caused b y the evolution of carbon dioxide. In the past the loss of acidity was the most mentioned aspecte2Of course some loss of acid is common in all wine after alcoholic fermentation. The presence of alcohol decreases the solubility of potassium acid tartrate, causing it to precipitate, as does the cool temperature at which wines are stored. The earliest observations of malo-lactic fermentation may have confused this precipitation of tartrate with microbial decomposition of

’Indeed, “malic” is derived from the Latin word for apple, malum. In English, malic acid is sometimes called “apple acid,” and the translation of this term is used in many foreign languages. ‘Even today the most common German translation for “malo-lactic fermentation” is biologischer Saureabbau or Saureabnahme. Cognates of “malo-lactic” are used in other languages. The German term now appears even more ambiguous as it may refer to alcoholic fermentation by some Schizosaccharomyces yeasts which can also ferment malic acid (to alcohol).

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tartrate.3 The first observations of a loss of total acidity that was greater than that caused by loss of tartrate has been attributed to Berthelot and de Fleurieu (1864). In one wine analyzed by them, the total fixed acidity dropped from 10.0 gm./liter to 5.8, but the tartaric acid loss was only 2.5 gm./liter. However, it is not certain that this was a malo-lactic fermentation since the loss of acidity occurred during the alcoholic fermentation. Boussingault (1868) also found a loss of about half the acid in cider, and this may have been a malo-lactic fermentation. In 1891, Ordonneau reported the loss of acid in some aged wine to be the loss of malic acid, and he suggested that malic acid was transformed into another acid. Pasteur (1858) of course proved that lactic acid was produced by bacteria (or “new yeast” as he called them). Although he did not give much special attention to the loss of acidity in wine, characteristically he correctly related the loss to microbial action. In his studies on wine spoilage, he quoted a letter from de Vergnette-Lamotte (Pasteur, 1866, pp. 66-68). The letter described a wine “disease” which, among other things, brought about a flat (fade) taste. Pasteur (1866, pp. 36-37) described the release of carbon dioxide during the pousse or gassy stage of tourne spoilage. Some of this undoubtedly was caused by malo-lactic fermentation. [It must be pointed out here that although loss of acidity in wine may sometimes be considered spoilage, it does not follow that all bacteria that cause malo-lactic fermentation are necessarily spoilage organisms. One should not identify “malo-” of “malo-lactic” with “bad” instead of with “malic.” Balard (1861) very early recognized the formation of lactic acid in sound wine by microorganisms. Many of the “grand wines” apparently require bacterial activity for their quality, and malo-lactic fermentation is a necessity in the wines of high acidity from the coldest viniculture areas.] Pasteur (1873, pp. 270-277) prevented a small loss of acidity in wine stored for nearly a decade by heat treatment before storage. The untreated wine lost about 0.5 gm. acid per liter of wine. However, this loss seems too small to be a malo-lactic fermentation;, Pasteur claimed the loss was tartaric acid. Kulisch (1889) was probably the first to prove the biological nature of what is now called malo-lactic fermentation. H e pasteurized cider (heated to 60°C.) which then lost very little acid, but in 6 months the 31n the early literature one finds reports of decomposition of tartaric acid in wine by bacteria which seem to have been Lactobacillus spp. These reports may be erroneous; only recently have workers been able to isolate bacteria from wine that are capable of metabolizing tartaric acid. See Berry and Vaughn (1952), Vaughn (1955), Krumperman (1964),and Krumperman and Vaughn (1966).

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total fixed acidity of the untreated cider dropped from 0.8% to 0.45%. Kulisch (1889) also claimed h e was able to induce the loss of acidity by addition of what he considered a pure yeast culture of “Saccharomyces ellipsoideus.” Consequently he concluded the acid reduction was caused by yeast. About the same time, Muller-Thurgau (1891a) reported several experiments, probably the first ones, on the control of acid reduction. From his observations he was convinced that bacteria, not yeast, were responsible for the change in acidity. He always found bacteria in wines which had undergone the change, and in “hundreds” of examples h e was never able to induce acid reduction in wine by addition of pure yeast. Unfortunately his attempts (at that time) to induce the acid change by inoculation with the bacteria h e had isolated from wine were unsuccessful. The contention that yeasts were causative agents was supported by many other workers: e.g., Amthor (1889), Wortmann (1894), Schukow (1896),and Mestrezat (1907). Alfred Koch (1900) was the first to isolate malo-lactic bacteria and to induce malo-lactic fermentation by inoculation with these organisms. Furthermore, with the use of salicylic acid he discredited claims that yeast were responsible. Koch (1898) showed that salicylic acid strongly inhibited bacterial growth in wine but had little effect on yeast. Wine with added salicylic acid did not undergo acid reduction, whereas the control wine did (Koch, 1900). H e thus proved Muller-Thurgau’s hypothesis that bacteria were causative agents. Koch also recognized that the loss of acidity involved mainly the loss of malic acid, and he seemed to understand the fastidious nutritional nature of the bacteria and that important bacterial growth factors might arise in commercial winemaking from autolysis of yeast. Seifert (1901, 1903) also isolated bacteria which would induce loss of acidity. Both Seifert and Koch isolated their bacteria from wine sediment. Seifert gave both a description and a microscopic illustration of paired cocci 1p in diameter. H e showed that the loss of acidity involved the conversion of malic acid to lactic acid and h e named the isolate Micrococcus malolacticus. Moslinger (1901) is credited with first presentation of the overall malo-lactic equation (malic acid to lactic acid and carbon dioxide), although Seifert (1901) also published the equation the same year. Moslinger (1901) realized that lactic acid arose both from decomposition of malic acid (malo-lactic fermentation) and by fermentation of other [carbohydrate] materials (lactic acid fermentation). During the next several years, four mare strains of bacteria capable of malo-lactic fermentation were isolated by Muller-Thurgau and by

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him and Osterwalder. A detailed account of their work gave a description of these bacteria (Muller-Thurgau and Ostenvalder, 1913). This report included a taxonomic key for classification of all bacteria (except acetic acid bacteria and bacteria from “ropy” wines) which at that time had been isolated from wine or other fermented beverages. Koch (1900) suggested that the claims of loss of acidity by yeast were the result of aerobic metabolism, and that wine stored anaerobically did not lose acidity (except from bacterial activity). It is tempting today to attribute the controversy (see also Muller-Thurgau, 1891b; Kulisch, 1891) over yeast as the cause of malo-lactic fermentation to primitive microbiological techniques. Some of the so-called “pure yeast cultures” or the “sterilized” musts and wines might have been contaminated with aerobic spoilage yeast or perhaps with fermentative yeasts which decompose malic acid, such as some Schizosaccharomyces. Mestrezat (1908) pointed out that most of the claims of loss of malic acid by yeasts were based on loss of total acidity rather than on determinations of malic acid itself. Nevertheless, the early literature is amazing because of the perspicacity of these researchers. The principles put forward by them are still generally valid. 111. Occurrence Discoveries of malo-lactic fermentation in wines of various regions of the world spread with the application of technology to the practice of winemaking. Following the first proofs of the microbiological nature of the fermentation in German and Austrian wines (see Section 11)came the reports of malo-lactic fermentation in wines of many areas of western Europe. Moreau (1906) noted the deacidification (by “yeasts”) in wines of Anjou. Rosenstiehl (1908) was of the opinion that wines of Gironde were improved by a slow malo-lactic fermentation, and h e also mentioned observing a vigorous secondary fermentation in Alsace. Mestrezat (1908) noted loss of malic acid in wines of southern France (Midi). Although he recognized a difference between a rapid loss of the acid apparently caused by yeast at about the time of alcoholic fermentation and a slower disappearance during aging, he objected to the term “malo-lactic fermentation.” Astruc (1925) also examined wines of southern France and found malo-lactic fermentation. Ferre (1922) and Rousseaux and Ferre (1926) reported malolactic fermentation in both white and red wines of Burgundy. Malolactic fermentation had already been detected in Bordeaux wines (Rosenstiehl, 1908), and it was further studied by Ribereau-Gayon and Peynaud (cf. 1961, p. 434).

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After World War 11, paper chromatography became a routine tool for research biologists, and its use became more and more widespread in enology laboratories. By replacing the involved chemical procedures for the determination of organic acids with chromatographic methods, winemakers throughout the world found it easier to detect and follow the malo-lactic fermentation. In many wine laboratories this determination is now routine. Today malo-lactic fermentation has been reported practically everywhere wine is made. Besides the geographical areas mentioned above, the following countries, at least, can be listed: In Europe: Switzerland (Tonduz, 1920), Spain (Feduchy Marino, 1964), Portugal (Marques Gomes et al., 1956), Italy (Tarantola, 1959), Yugoslavia (Milisavljevic, 1964), and the Soviet Union (Saenko et al., 1965); Africa: Algeria (Bremond, 1937), South Africa (du Plessis, 1964); Australia (Fornachon, 1957); Japan (Nonomura et a,?., 1963); South America: Argentina (Arena, 1936), Uruguay (Poittevin et al., 1963), Chile (Hernandez and Ortega Tello, 1964); and North America: Canada [Adams (1964) reported high concentrations of lactic acid-presumably from malo-lactic fermentation -in Ontario wines]; and the United States: New York (Rihe, 1965) and California (Suverkrop and Tchelistcheff, 1949; Ingraham and Cooke, 1960; Kunkee et al., 1965). Sometimes such sophisticated techniques as paper chromatography are not required for detection of malo-lactic fermentation. Some wines of northern Portugal (and certain other parts of Europe) which, because of viticultural practices are very high in acid, require malolactic fermentation for palatability (cf. Amerine et at., 1967, pp. 37, 457). The fermentation often occurs after bottling, and the carbon dioxide is retained. This results in a vinho frisante or slightly gassy wine which has become a characteristic type. It is generally true that malo-lactic fermentation is more common in red wine than white wine. Part of the reason for this is undoubtedly the inhibitory effect on the bacteria of the greater acidity and the higher concentration of sulfur dioxide usually found in white wire. The presence of material extracted from the skins may also stimulate the fermentation of red wine (cf. Garino-Canina, 1943; Kunkee, 1966). Nevertheless, malo-lactic fermentation is an important occurrence in some white wines of high acidity such as those of Switzerland (cf. Ribereau-Gayon and Peynaud, 1960, pp. 438-439). Malo-lactic fermentation is also inhibited by high alcohol concentration. It has been found only in table wines or wines with alcohol concentration not much higher than 14%.4In California, sherries and *All alcohol concentrations are given as ethanol, percent by volume.

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24 1

other appetizer and dessert wines must be at least 19.5% alcohol. Except for some bacilli (Gini and Vaughn, 1962), Lactobacillus trichodes is the onIy bacterium ever found in these high-alcohol wines, and this species does not attack malic acid (Fornachon et al., 1949). IV. Malo-lactic Bacteria

A. TAXONOMY By definition, malo-lactic bacteria are lactic acid bacteria which can ferment malic acid. They are members of the family Lactobacillaceae, but the ability to ferment malic acid cuts across generic and*possibly specific lines; and these bacteria are not placed in a taxonomic group of their own (Breed et al., 1957).This sometimes has led to difficulties in classification of these organisms. In the first systematic classification which included malo-lactic bacteria, MullerThurgau and Osterwalder (1913) used the ability to ferment malic acid as part of the dichotomous key. They listed four malo-lactic bacteria: Seifert’s Micrococcus malolacticus, their own M . acidovorax and M . varicoccus, and Muller-Thurgau’s Bacterium gracile. By today’s classification, the above micrococci should be renamed because they produce lactic acid and are undoubtedly catalase negative (cf. Arena, 1936).The taxonomy of these organisms has been discussed in detail by Vaughn (1955) who placed them either in the genus Pediococcus or Streptococcus. Since they were isolated from nonanimal sources and do not form chains (cf. Muller-Thurgau and Osterwalder, 1913; Arena, 1936), they should most likely be placed in the genus P e d i o c ~ c c u soptical ;~ activity of the lactic acid produced is required for definite classification (Breed et al., 1957). B . gracile, also, is not an accepted name today. Carr (1952) suggested this organism ought to be renamed Leuconostoc mesenteroides, although the photographs of it (Miiller-Thurgau and Osterwalder, 1918) give the impression of a definite rod rather than an elongated coccus. Ribereau-Gayon and Peynaud (1961, pp. 465-479) gave a resume of names and classifications used for the most important malo-lactic bacteria isolated from wine since the time of Muller-Thurgau and Osterwalder. Using modern classification (Breed et al., 1957), we It is ironic that the first characterized malo-lactic organism does not fit modern classification. Miiller-Thurgau and Osterwalder (1913) described Seifert’s M . malolacticus as producing no lactic acid from glucose and not fermenting fructose. We have to consider these observations as errors if we place the organisms in the family Lactobacillaceae. Bidan (1956) isolated an orgamism similar to Pediococcus cereoisiae which he called identical to M . malolacticus and M . varicoccus.

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find many members of the Lactobacillaceae are malo-lactic bacteria. Aadler (1962a) listed the following lactic acid bacteria isolated from wine which decomposed malic acid: Lactobacillus buchneri, L. casei, L. delbrueckii, L. fermenti, L. hilgardii, L. pastorianus, and L. plantarum; Leuconostoc citrovorum, L. dextranicum, and L. mesenteroides; and Pediococcus cereuisiae. To this list should be added Lactobacillus brevis (Vaughn and Tchelistcheff, 1957) and Lactobacillus leichmannii (du Plessis, 1964). With the exception of L. hilgardii (Vaughn et al., 1949),-each of the above species is described in “Bergey’s Manual” (Breed et ul., 1957). Several species of Streptococcus which are malo-lactic have reportedly been isolated from spoiled wines: S. mucilaginosus var. vini, S. malolacticus, and an organism similar to S . damnosus (Carr, 1962; cf. Vaughn, 1.955).There is a question as to the correct classification of some of these streptococci (Vaughn, 1955). The high concentration of alcohol and acid in wine, plus the presence of sulfur dioxide and the lack of nutrients make wine a fairly hostile environment for microorganisms. Only a few kinds of organisms are found in wine, and part of the classification of malo-lactic bacteria is relatively easy. The family Lactobacillaceae is part of the order Eubacteriales. Members of the family are Gram-positive, asporogenous, nonmotile, anaerobic or microaerophilic, lactic-acid producing bacteria. The absence of catalase6 and a minimum of surface growth in a stab culture separate lactic acid bacteria from other bacteria sometimes found in wine, i.e., from the acetic acid or vinegar bacteria in table wine, and from bacilli in fortified wine. The Lactobacillaceae family is divided on basis of cell morphology into tribes of cocci and rods (Breed et al., 1957). Of the cocci, only the facultative anaerobes are important as malo-lactic bacteria, and of these only the two genera Pediococcus (homofermentative) and Leuconostoc (heterofermentative) are important. Of the rods, again, only the facultative anaerobes are important; and these include only the genus Lactobacillus. Further subdivision of the three genera is dependent on optimal temperature of growth, optical activity of lactic acid produced, and kinds of sugars fermented. The heterofermentative cocci sometimes are hard to classify because they are especially nutritionally fastidious, which makes it difficult to obtain a defined medium for determination of sugar fermentability (cf. Ingraham et al., Some pediococci and lactobacilli have been reported to be weakly catalase positive in presence of low concentrations of energy source (Felton et at., 1953; Dacre and Sharpe, 1956; Whittenbury, 1964).

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1960). For example, the separation of L. citrovorum from L. mesenteroides and L. dextranicum is based on the capability to ferment sucrose (Breed et al., 1957). The response of a leuconostoc isolated by Ingraham et al. (1960) and designated “ML 34” (Webb and Ingraham, 1960) was poor on all synthetic media. In a complex medium with glucose as the only known carbon source, growth was obtained, but none was found when sucrose was the only known carbon source. The organism was classified L. citrovorum (Pilone et al., 1966). Fornachon (1964)found difficulty in classification of a heterofermentative coccus The organism he described did ferment sucrose and was hence classified as L. mesenteroides, but it did not form dextran when sucrose was the carbon source. Bidan (1956) and Radler (1958a) also found heterofermentative cocci which were similar but not identical to L. citrovorum. Classification difficulties have been found with some homofermentative rods which are similar to Lactobacillus plantarum (Bidan, 1956; Radler, 1962a). Lambion and Meskhi (1957) suggested the homofermentative malo-lactic rods be named Lactobacillus plantarum var. gracile and the heterofermentative malolactic cocci, Leuconostoc mesenteroides var. gracile.

B. ISOLATION, CULTIVATION,

AND

IDENTIFICATION

Koch (1900) and Seifert (1901) first isolated malo-lactic bacteria from sediments of wines which had undergone malolactic fermentation. Wine sediment can be a good source of the bacteria; however, one can also use wine itself. Two major difficulties in the isolation of the bacteria are (a) the fastidious nature of the organisms and (b) the presence of yeast in the sample. The first difficulty is solved by use of a culture medium of great complexity. Synthetic media have been described for malo-lactic bacteria (e.g., Peynaud et al., 1965), but these have not proved suitable for the most fastidious malo-lactic bacteria (Kunkee, unpublished results). Media which contains tomato juice has often proved to be excellent for culturing the bacteria (Rogosa et al., 1953). Ingraham (1963) made some modifications of the Rogosa medium to give the following formula: 2%tryptone, 0.5% yeast extract, 0.5% peptone, 0.3% glucose, 0.2% lactose, 0.1% liver extract, and 0.05% Tween 80 in 4.2-fold dilution of tomato juice (canned without preservative) filtered through filter aid. The broth was adjusted to pH 5.5 with hydrochloric acid and 2% agar added for solid medium. In this laboratory, either tryptone or tryptose was found to be satisfactory in this formula, but the shelf age of the tryptone can be important. We have had good results with Difco’s

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Rogosa SL broth prepared in diluted, filtered tomato juice. We have also used fresh grape (Vitis uin$era) juice to which 0.05% yeast extract was added, the mixture being adjusted with sodium hydroxide to pH 4.5. Because of the low pH of the medium, extreme sterilization procedures are often not required (Rogosa and Sharpe, 1959). However, little or no deleterious effects from standard autoclave treatment have been detected. Whiting and Coggins (1963) reported improvement in some media after being autoclaved. The second difficulty, the presence of yeast, has been avoided by use of the fungicide cycloheximide (Acti-dione, Upjohn Co.) at a final concentration from 20 p g h l . (Peynaud and Domercq, 19614 to 100 pg./ml. (Webb and Ingraham, 1960). Sorbic acid has also been used to inhibit yeast (Fornachon, 19641, but Ingraham et al. (1960) found it somewhat inhibitory to malo-lactic bacteria. Peynaud and Domercq (1961a) suggested differential centrifugation of the wine as a preliminary step in removal of the yeast cells. This is undoubtedly also a good practice with old wine in which the proportion of viable bacteria may be small. Initial inoculations have been made both in liquid and on solid media. Inoculated agar plates are often incubated in sealed glass jars in which the oxygen has been exhausted with alkaline pyrogallol (Peynaud and Domercq, 1961a; Meynell and Meynell, 1965) to inhibit aerobic organisms. An external supply of carbon dioxide has been shown to stimulate growth of lactic acid bacteria under some conditions (cf. Snell, 1952). Rogosa and Sharpe (1959) suggested incubation of lactobacilli in an atmosphere of 5% carbon dioxide. The oxygen concentration in the atmosphere of incubation jars can also be lowered and the carbon dioxide increased b y use of a lighted candle (Ingraham et al., 1960). Colony formation on solid media inoculated with 0.1 ml. wine sometimes takes as long as 2 weeks. Because of this relatively long incubation period, mold contamination sometimes develops. Beech and Carr (1955) suggested use of 100 pg./ml. diphenyl to inhibit mold. As with any microbial isolation, one assumes that the predominant kinds of colonies are the important organisms. Microscopic examination should be made of the isolated organisms to be assured that they are bacteria. After purification of the bacteria by restreaking, they can be stored as stab cultures. In this laboratory, cultures are kept in the cold and transferred every 3 months. Fell (1961) successfully stored malo-lactic bacteria by lyophilization of a mixed culture of the bacteria and yeast. It is well to point out that often lactic acid bacteria die off quickly and relatively large inocula should b e used in making transfers (Rogosa and Sharpe, 1959; Davis, 1960).

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For classifications, standard methods for lactic acid bacteria are used, but because of the limited numbers of genera found in wine, the procedure can be somewhat abbreviated. The cultures are first checked for absence of catalase and for minimal surface growth in stab cultures. These positive tests for anaerobes and positive Gram strains of young cultures are presumptive evidence for Lactobacillaceae. The production of lactic acid can be determined by paper chromatography of the culture medium (Section V) to confirm the presumption. Cell morphology is used at the next stage of classification. Some Leuconostoc become elongated when grown in fruit juice medium (Vaughn, 1955; Breed et al., 1957). Very short rods ought to be “keyed-out” both as cocci and rods before final classification. Heterolactic fermentability is determined by gas production from glucose (Hayward, 1957; Pilone et al., 1966) and by mannitol formation from fructose. The latter is determined by the formation of rosette macrocrystals in evaporated medium (cf. Ribereau-Gayon and Peynaud, 1958, pp. 256-258). The distinction between homo- and heterofermentative types is sometimes vague (Davis, 1963). From these tests one is able to identify the organism as Pediococcus, Leuconostoc, or Lactobacillus. For further identification, optimal temperature of growth can be determined from an Arrhenius plot of logarithms of specific growth rates (determined by increase in turbidity with time) versus reciprocal absolute temperatures. Optical activity of lactic acid is most easily determined by use of an enzyme assay for L-lactic acid (Hohorst, 1963; Pfleiderer and Dose, 1955) and a chemical method for total lactic acid (Bruno and Moore, 1962; Amerine, 1965, pp. 64-66); the D-lactic acid is then obtained by difference (Cato and Moore, 1965). Determination of fermentable sugars must be made on media with no energy source other than the test sugar. We have used the modified Rogosa medium listed above but lacking glucose, lactose, and tomato juice. A suitable synthetic medium would be preferred. Malic acid fermentability can be determined by growth of the bacteria in broth which contains 0.3% L-malic acid. Paper chromatography (Section V) of the medium will show whether malic acid was utilized. In the same manner, utilization of citric acid can also be determined. A symposium on isolation, cultivation, and classification of malolactic bacteria was recently held by the Commission 11: Oenologie of the Ofice International de la Vigne et du Vin (Husfeld, 1964; Ulbrich, 1964; Milisavljevic, 1964; Peynaud and Dupuy, 1964; Florenzano and Verona, 1964; Fell, 1964; Feduchy Marino, 1964). It is comforting for the wine lover to learn that no Leuconostoc

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or LactobaciZZi are known to be pathogenic (Niven, 1952), and that no pathogenic bacteria have been found in spoiled or sound wine (Vaughn, 1955). C. HABITAT It is natural to wonder about the source of malo-lactic bacteria in wine. On the one hand, the most obvious source would seem-to be the grapes themselves. Peynaud and Domercq (1961b) and Kunkee et al. (1965) got spontaneous malo-lactic fermentation in wines in which care had been taken to obtain the musts aseptically. Japanese workers have obtained malo-lactic fermentation in grapes stored at 40°C. (Otsuka et uZ., 1964). [We have been unable to confirm this with tests on a dozen varieties of grapes (Kunkee, unpublished experiments).] Radler (1958a) detected malo-lactic bacteria on grape leaves. Garino-Canina (1943) reported an old experiment of Nessler which indicated the presence of bacteria on grape skins. On the other hand, the sources of the bacteria might be the winery. Webb and Ingraham (1960) obtained spontaneous malo-lactic fermentation in grapes vinified in one winery but not when the same grapes were used in another winery. Radler (1958a, 1966) was unable to isolate malo-lactic bacteria from fresh must, but he was able to obtain bacteria from it 2 weeks after the beginning of alcoholic fermentation. I n the results of Peynaud and Domercq (1961b) mentioned above, the spontaneous fermentations occurred only in a few instances. Both sources of the bacteria, in fact, may be important. We might suppose that the bacteria arise originally from a small population on the grapes. Under favorable conditions, especially after alcoholic fermentation has brought about added nutrients from yeast autolysis, the bacteria would multiply to an easily detectable level. Peynaud and Domercu (1959) pointed out that malo-lactic fermentation more likely occurs in large vessels where the chance for significant levels of viable bacteria is higher than in smaller vessels. Perhaps the presence of an occasional grape leaf also contributes to the original natural inoculum. It would seem reasonable that fermentation and storage containers, especially those which are difficult to clean, would retain malo-lactic bacteria after the secondary fermentation. Ribereau-Gayon and Peynaud (1961, pp. 440,468) reported malo-lactic organisms on walls or floors of wine cellars and on wooden barrels. Bacteria also might be associated with pomace. Wineries often dispose of pomace by spreqding it in nearby vineyards. Thus, winery operations might lead to a perpetuation of the bacteria in and around the winery from year to

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year until characteristic malo-lactic microflora were built up in much the same way certain yeast strains become established in vineyard areas (Amerine and Singleton, 1965, p. 54).

D. GROWTHCONDITIONS Descriptions of isolated malo-lactic bacteria usually include some information about the growth requirements for the organisms. Even in the very first reports, Koch (1900) indicated a need for special growth factors for the bacteria he isolated, and Seifert (1903) showed that M. malolacticus was inhibited by high alcohol and high acidity. The requirements for growth of malo-lactic bacteria are generally those of lactic acid bacteria. However, as a class malo-lactic bacteria are more tolerant to alcohol and to acidity than many other lactic acid bacteria. Even though one determines the best conditions for growth of a particular organism, the knowledge of the requirements may not be directly helpful to the enologist, since the information obtained under laboratory conditions is not necessarily applicable to winery operations. The winemaker usually does not have control over the kinds of organisms with which he must be concerned. Furthermore, the most favorable conditions for bacterial growth may not be the same as those for most rapid conversion of malic acid to lactic acid. More information on the latter subject is especially needed. Experiments on malo-lactic fermentation itself can tell us something about growth and metabolism of malo-lactic bacteria, but careful interpretation is required before these kinds of results can be generally applied. In this report, detailed descriptions will not be given of specific bacteria. Rather, a generalized discussion of malo-lactic bacteria as a group will be presented.

1 . Nutrition Lactic acid bacteria require an extensive complement of amino acids, purines, pyrimidines, and vitamins, besides minerals and carbon and nitrogen sources. These bacteria are often used in bioassays for growth factors. Several synthetic media for malo-lactic bacteria have been published (e.g., Luthi and Vetsch, 1959; Peynaud et al., 1965). du Plessis (1963) and Radler (1966) have published lists of amino acids and other growth factors required for strains of malolactic bacteria studied by them. We have found that complex medium containing all of the suggested growth factors gave only minimal

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growth for some bacteria. As mentioned above, L. citrovorurn ML 34 is especially fastidious. It grew poorly in a medium containing, in addition to the components of Difco’s C F [Citrovorum Factor Assay Medium (itself a very complex mixture)]: folinic acid, lipoic acid, cyanocobalamine, inositol, L-asparagine, y-aminobutyric acid, thymidine, deoxycytodine, Tween 80, sodium pyruvate, .xylose, arabinose, galactose, and sucrose (Kunkee, unpublished results). The medium was adjusted to a final pH of 4.5 and sterilized by filtration. It also contained 0.5 pg./ml. pantothenic acid, which is sometimes needed by lactic acid bacteria at a relatively large concentration (Snell, 1952). Addition of L-malic acid, citric acid, acetic acid, or ethanol to the medium did not give a better growth response. y-Aminobutyric acid was added because it was found to be an unexpectedly large component of the amino acids of grapes (Nassar and Kliewer, 1966); Radler (1966) found it ineffective in his tests of it as a nutrient. Not all malo-lactic bacteria are as fastidious as this, of course, and some will grow well on simpler media. All nonsynthetic media for malo-lactic bacteria have yeast extract or yeast autolyzate added. It was discovered very early that the presence of yeast can supply the nutrients for the fastidious bacteria (Koch, 1900). Storage of wine on gross lees for several weeks stimulates malo-lactic fermentation (Fornachon, 1957). Liithi and Vetsch (1959) commented on the ancient practice of adding one wine to another to “start” malo-lactic fermentation. The practice was effective because it supplied bacteria as inoculum and probably because the addition of resting yeast cells supplied nutrients for them. The nutritionally active components of yeast extract have been isolated (Luthi and Vetsch, 1959), and seemed to be mostly simple nitrogen compounds. Discovery of bacterial contamination of tomato ketchup led to the use of tomato juice in nutrient media for bacteria (Mickle, 1924). For all the malo-lactic bacteria we have tested, the addition of tomato juice, or grape juice, was helpful. Stamer et al. (1964) found the ash of tomato juice to be the active fraction. By testing various minerals, they found manganous sulfate addition resulted in bacterial growth equal to that obtained on medium supplemented with tomato juice. Other inorganic components are also important. Zickler (1964) showed that potassium, sodium, magnesium, and manganese ions were required for growth of malo-lactic B . gracile. Malic acid decarboxylation rate was dependent on potassium ion. Bocker (1964) tested demineralized wine. He also found that the four inorganic ions mentioned above plus calcium had to be added back to the medium

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for malo-lactic fermentation. The growth of the bacteria was influenced by magnesium. Radler (1966) found that 2 mM potassium ion was required for maximal growth in synthetic medium. Fornachon (1964) added a mixture of inorganic salts (Wright and Skeggs, 1944) to a complex medium used for growth of Leuconostoc. There probably is no universal medium for all malo-lactic bacteria. A major problem in development of a synthetic medium is the antagonism between certain growth factors. For example, small amounts of folic acid, copper, or purine deoxyribotides can inhibit growth of some lactobacilli (Rogosa and Sharpe, 1959; Morris and Williams, 1965). Even with nutritionally balanced media, the concentration of certain components can be surprisingly influential on the metabolism of the organisms. For example, Nossal (1952) found changes in pyruvate metabolism in the presence of glucose in a lactobacillus. To add to the difficulties, two or more strains growing together may show nutritional symbiosis by growth in medium in which neither could grow alone. Doctor and Couch (1954) showed that both Leuconostoc citrovorum, which required citrovorum factor and produced vitamin B12, and Lactobacillus leichmannii, which required vitamin B12 and produced citrovomm factor, could grow in medium lacking both of these biochemicals. We have been surprised to discover that some of the most fastidious organisms grow better in winery operations, or at least carry out malo-lactic fermentation faster, than do some of the least fastidious ones (Pilone et al., 1966). Perhaps the presence of certain natural components of the grape or wine overcome the nutritional handicap of the fastidious bacteria and then other factors become more influential. Red wines are more susceptible to malo-lactic fermentation than white wines. This has been attributed to the greater acidity and concentration of sulfur dioxide in white wines. Malo-lactic fermentation was found to be stimulated by the presence of skins during the alcoholic fermentation (Garino-Canina, 1943). This kind of result was also reported by Fornachon (1957) and by Kunkee (1966). Possibly some growth factors extracted from the grape skins are beneficial to malo-lactic bacteria and stimulate the fermentation in red wine. High glucose concentration had no effect on malo-lactic fermentation (Radler, 1958b).

2. p H Wine is highly acidic and bacteria isolated from it tolerate low pH. Malo-lactic fermentation readily occurs in wine at pH 3.4 and often

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at lower pH. It has been reported in wines with low alcohol at pH 3.0 (Rankine, 1963a). Optimal pH for the bacteria is much higher than the pH of wine, but it is generally lower than the optimal pH for other lactic acid bacteria (Ingraham et al., 1960).The pH range for growth varies with each organism, of course. We found it difficult to obtain growth of strains of P . cerevisiae in complex medium at pH below 4, even though the bacteria had been isolated from wine with pH lower than that (Pilone et al., 1966; Kunkee, unpublished results). The acidity of the medium influences malic acid fermentation as well as growth of the bacteria. Ribereau-Gayon and Peynaud (1961, pp. 478-479) found different pH thresholds for fermentation of malic acid than for the fermentation of sugars and the formation of volatile acidity. They suggested that the best organisms for malo-lactic fermentation in wine would be those which had the greatest differences between these pH thresholds, so that rnalo-lactic fermentation could be obtained with a minimum of other kinds of metabolism. The pH of the medium also greatly influences the activity of sulfur dioxide present (see below).

3. Alcohol The antiseptic quality of ethanol is well known, and its presence in wine, depending on the concentration, has an inhibitory effect on malo-lactic bacteria. The legal limit in California table wine is 10 to 14% alcohol, and malo-lactic fermentation is delayed, but not necessarily prevented, in medium with this much alcohol. At lower concentrations the inhibition is less but sometimes is still detectable to 6%. The tolerance to alcohol varies with bacterial strain and, again, generalizations are difficult to make. In California the lower alcohol limit in fortified wines is 19.5%, thus one is not concerned about malo-lactic fermentation in these wines. The only reports we have seen of malo-lactic bacteria which tolerate this amount of alcohol are a strain of Lactobacillus fermenti (Bidan, 1956) and another Lactobacillus sp. (Vaughn et al., 1949). Neither of these organisms was isolated from high-alcohol wine.

4 . Temperature Optimal growth temperatures for Leuconostoc spp. range from 20" to 25"C., for P . cerevisiae, 25" to 32"C., and for Lactobacillus spp., from 28" to above 37°C. (Breed et al., 1957). Malo-lactic fermentation is inhibited at 5" to 10°C. (Garino-Canina, 1943); but the fermentation

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25 1

occurs, more or less readily, at cellar temperatures of 10” to 15°C. We must be cautious in assuming that the lower temperature brings about merely a change in rates of growth and of malo-lactic fermentation. Temperature has been shown to have a profound effect on the regulation and activity of anabolic systems of prototrophic bacteria (Ng et al., 1962). Temperature could influence metabolic pathways of the fastidious lactic acid bacteria also. Lactic acid bacteria do not form spores and are relatively sensitive to heat. Two exceptions, with optimal temperatures about 45”C., are Lactobacillus bulgaricus and L. thermophilus, but these have not been reported as malo-lactic bacteria. Carr (1952) reported survival of cider Leuconostoc sp. at 60°C. for 10 minutes and Lactobacillus sp. at this temperature for 2.5 minutes. For bacteria in wine, less drastic heat treatment is required. Wine can be pasteurized by flash heat for 1 minute at 82°C. or by bottling hot at 54°C. (Ough and Amerine, 1966).

5. Oxygen and Redox Potential Malo-lactic bacteria are not obligate anaerobes, and they will develop in presence of dissolved oxygen (cf. Ribereau-Gayon and Peynaud, 1961, p. 490). Usually they will form colonies on surfaces of solid media. Oxygen seems to have a beneficial effect on their growth. Charpentie (1954) found malo-lactic fermentation started most rapidly when the redox potential was about t0.4 V; Bocker (1964) got malolactic fermentation in wine starting with redox potential between f0.1 and +0.2 V. (Obligate anaerobes usually require redox potentials less than -0.1 V.) However, Ribereau-Gayon and Peynaud (1961, p. 490) found saturation of new wine with air stimulated malolactic fermentation, but saturation with oxygen inhibited it. Thus microaerophilic (“love of small amounts of air”) is an apt description for malo-lactic bacteria.

6 . Sulfur Dioxide Sulfur dioxide is universally used for production of wine of all qualities. The antiseptic is strongly inhibitory to malo-lactic bacteria, if not lethal (Fell, 1961). Reports of malo-lactic bacteria as being sensitive to certain levels of sulfur dioxide are ambiguous because of the variety of forms of the compound in solution. A large fraction is “bound” (combined with aldehydes and pigments). The “free” form exists as sulfurous acid, bisulfite, or sulfite ion. Of the free form only sulfurous acid is directly active on metabolic systems of the bacteria-

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probably b y oxidation of disulfide group of proteins (Wyss, 1948). The bisulfite ion is indirectly inhibitory by its combination with acetaldehyde needed by the cell in alcoholic fermentation for reoxidation of reduced coenzymes. Thus the amount of active sulfur dioxide is dependent on the concentration of aldehydes and on the pH, both of which are variable. Instability of the bisulfite-acetaldehyde addition compound also adds to the difficulty. Fornachon (1963b)showed that lactic acid bacteria metabolized part of the addition compound and thus “freed” sulfur dioxide and increased the inhibitory effect. Sulfur dioxide is also relatively easily removed from solution by oxidation to sulfate or reduction to elemental sulfur. [Most lactic bacteria do not produce free acetaldehyde as do yeast (Wilkinson and Rose, 1963). Thus in laboratory cultures of bacteria, the inhibitory effect of sulfur dioxide ought to be greater than in mixed cultures with yeast.] Thus, the concentration of total, or even free, sulfur dioxide does not give a precise estimation of inhibitory activity. Nevertheless, practical concentrations for control can be arrived at empirically. Fornachon (1957) gave a table listing the proportion of wines which did not undergo malo-lactic fermentation at a variety of total sulfur dioxide concentrations and pH values.

7. Tannin We have already pointed out the inhibitory activity of wine against pathogenic organisms. Acid and alcohol are only partly responsible for the antiseptic qualities of wine. Masquelier (1959) showed that charcoal treatment of wine removed much of the antibiotic activity against salmonella, staphylococcus, and coliform bacteria. The tannins (phenolic compounds) were shown to be responsible. Lactic acid bacteria, however, are apparently resistant to grape tannins (Masquelier, 1959). Fornachon (1943) pointed out that if tannins were inhibitory to naturally occurring organisms of wine, bacterial activity should occur more readily in white wine than in red, but the opposite is true. Tannins from other plants are sometimes added to wine to facilitate fining and to improve flavor and protect color. Definite retardation of bacterial spoilage can b e found with tannin addition of 0.5 gm./liter, but the usual amount added is less than this. It would seem the effect of tannin is of minor influence on malo-lactic fermentation.

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E. METABOLISM

1. Sugar Fermentation Two distinct types of sugar fermentation are recognized in the lactic acid bacteria. One group produces mostly one product (lactic acid) from glucose and thus is called homofermentative. The other group, heterofermentative bacteria, convert about half of the sugar to lactic acid, one-third to ethanol, and one-sixth to carbon dioxide. (Heterofermentation is characterized by gas production from glucose.) A brief account of these two kinds of metabolic activity is necessary here. More thorough discussions can be found elsewhere (see especially Wood, 1961). The route of catabolism of glucose by the homofermentative bacteria is via the familiar glycolytic pathway (Embden-Meyerhof scheme). In glycolysis, hexose diphosphate is fragmented into two %carbon units which are oxidized to pyruvic acid and reduced to lactic acid. There is no net change in redox state of coenzymes, but there is a theoretical net formation of two high-energy phosphate bonds (in the form of adenosine triphosphate, ATP) per molecule of hexose. In contrast, the heterofermentative bacteria lack the enzyme aldolase which catalyzes the splitting of hexose diphosphate in two (Buyze et al., 1957). Thus complete glycolysis is blocked in heterofermentative bacteria, and glucose is fermented via the first part of the hexose monophosphate shunt (Warburg-Dickens-Horecker scheme). The phosphorylated glucose is oxidized and decarboxylated to give phosphorylated pentose. The latter is again phosphorylated and split into a %carbon and a 2-carbon compound (Heath et al., 1958). The 3-carbon fragment is converted to lactic acid in the way just described for the homofermentative bacteria. The 2-carbon unit, acetyl phosphate, is reduced to ethanol and inorganic phosphate. Again, there is no net change in the redox state of the coenzymes (two molecules of which are reduced in the formation of pentose and are reoxidized in the formation of ethanol). [Glucose oxidation not involving phosphorylation has been reported in Pediococcus spp. (Lee and Dobrogosz, 1965). There was no indication that this was a significant part of the energy metabolism of the organism.] The above pathways represent only the major routes of catabolism of hexoses. Other pathways are sometimes important. For example, in heterofermentative bacteria, the reduction of fructose to mannitol (Peterson and Fred, 1920) is important and is used as a diagnostic test for this kind of organism. Three molecules of fructose give

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approximately two molecules of mannitol and one of lactic acid, acetic acid (or ethanol), and carbon dioxide. This pathway apparently competes with the hexose monophosphate pathway, and various amounts of end products can result (cf. Wood, 1961). In the fermentation of pentoses, no reduction of coenzyme in the initial steps occurs; it is not necessary to use acetyl phosphate as a hydrogen acceptor. The high-energy phosphate of acetyl phosphate can be conserved by transfer to adenosine diphosphate (ADP) (Lipmann, 1944). This results in a greater yield of high-energy phosphate and in formation of acetic acid rather than ethanol as an end product. This fermentation of pentose occurs in both homo- and heterofermentative bacteria, It would seem that malo-lactic bacteria could use pentoses found in wine with greater efficiency per carbon atom than hexoses. Other sugars which should be considered here are those found in wine. Besides hexoses and pentoses (Melamed, 1962), other sugars have been detected (Esau and Amerine, 1964): altroheptulose, mannoheptulose, and glyceromannooctulose. Unfortunately, little is known about their metabolism by lactic acid bacteria. Sedoheptulose is involved in the pentose phosphate cycle of the hexose monophosphate shunt. However, studies with radioactive glucose show that the pentose phosphate cycle is not operative in lactic acid bacteria (Rappoport et al., 1951; Bernstein et al., 1955). It has been pointed out that malo-lactic bacteria are microaerophilic. If the reduced coenzyme formed during the oxidation of glucose in the heterofermentative bacteria could be reoxidized by oxygen as in aerobic organisms, then high-energy phosphate of acetyl phosphate produced could be conserved as in the fermentation of pentoses. There are hints of oxidative phosphorylation or other reactions with oxygen in lactic acid bacteria and other anaerobes (Davis, 1960; Wood, 1961; Gallin and VanDemark, 1964). Johnson and McCleskey (1957) tested a strain of Leuconostoc mesenteroides in which they obtained definite oxygen uptake- 1 mole oxygen per mole of glucose. This extra source of energy would be of great benefit to malo-lactic bacteria in wine which contains very low levels of energy sources. Many of the residual sugars, such as pentoses, which are found only in trace amounts in wine may not be available as energy sources for all malo-lactic bacteria. Often the machinery for metabolism of sugars (at least some hexoses) must be induced in the bacteria, and the induction may require concentrations as high as 2%, many times higher than that in wine (Rogosa and Sharpe, 1959).

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The above pathways of sugar fermentation are the major pathways with the major end products of lactic acid, ethanol, and carbon dioxide. The extent of utilization of these pathways by the organisms depends not only on strain, but also on pH, substrate, redox potential, stage of growth of the organism, etc. (Gunsalus and Campbell, 1944; DeMoss et al., 1951). Even in homofermentative bacteria, as much as two-fifths of the glucose can go, under special conditions, to products other than lactic acid (cf. Wood, 1961). Davis (1963) pointed out that in practice, the distinction between homo- and heterofermentative bacteria often is indefinite. 2. Pyruvic Acid Pyruvic acid is present in wine in relatively small amounts, but it is implicated in malo-lactic fermentation; the concentration is lower in wine which has undergone the deacidification (Rankine, 1965). Pyruvic acid is of course extremely reactive biologically, and some of its reaction products are important flavor components. Already mentioned was the reduction of pyruvic acid to lactic acid. Here pyruvic acid acts as a hydrogen acceptor in the reoxidation of coenzymes. Pyruvic acid can a1so be oxidatively decarboxylated with an uptake of phosphate to give acetyl phosphate. In one case, another molecule of pyruvic acid acts as hydrogen acceptor, the hydrogen being transported via a flavoprotein carrier (Hager et al., 1954). In another case, oxygen is reduced and the resulting hydrogen peroxide oxydizes a second molecule of pyruvic acid to give acetic acid and carbon dioxide (Hager et al., 1954). In either case, high-energy phosphate, which can be transferred to ADP, is formed from two molecules of pyruvic acid. Acetyl phosphate itself can be a hydrogen acceptor in its reduction to ethanol and inorganic phosphate. Free acetaldehyde is not formed in this reaction as it is in yeast alcoholic fermentation (Wilkinson and Rose, 1963). Pyruvic acid is a precursor to three related 4-carbon compounds: diacetyl (2,Sbutandione), acetoin (3-hydroxy-2-butanone), and butylene glycol (2,3-butandiol). All three are found in wine. The pathway of their formation is not completely understood. For acetoin, two molecules of pyruvic acid react to give a-acetolactic acid and carbon dioxide (Juni, 1952). a-Acetolactic acid is then decarboxylated to acetoin. Rowatt (1951) and Moat and Lichstein (1953) showed quantitative conversion of pyruvic acid to acetoin and carbon dioxide at low pH, in presence of glucose. Using radioactive substrates, Shirakawa et aZ. (1964) explained the need for glucose by what they called

256

RALPH E. KUNKEE

“entangled” fermentation in homofermentative lactobacilli. In this scheme, acetoin is formed from a molecule of free pyruvic acid and a molecule of pyruvic acid from glucose fermentation. The other molecule of pyruvic acid from glucose goes to lactic acid. Another molecule of free pymvic acid is reduced to lactic acid to balance the coenzyme redox state. Acetoin can be reduced to butylene glycol (DeMoss et al., 1951; Charpentie et al., 1951). The relative amounts of acetoin and butylene glycol depend on the amount of available hydrogen. Diacetyl is an oxidation product of acetoin. However, research with cell-free extracts indicated that the pathway of formation of diacetyl in lactic acid bacteria is not by way of acetoin as was supposed. It apparently arises, as acetoin does, from pyruvic acid via acetaldehyde-thiamine pyrophosphate. The latter forms an addition product with acetyl coenzyme A which rearranges to give diacetyl and to regenerate thiaminepyrophosphate carbanion (Speckman and Collins, 1966). Another product of pyruvic acid metabolism, at least in some bacteria, is formic acid which comes from the splitting of pyruvic acid. High-energy phosphate is produced in this reaction in the form of the other product, acetyl phosphate (cf. Utter et al., 1944). Fornachon (cf. Rankine, 1965) obtained faster malo-lactic fermentation when pyruvic acid was added.

3. Citric Acid Citric acid is normally found in grapes at much lower levels than tartaric or malic acids, but it is sometimes added to acidify wines artificially. Not all lactic acid bacteria will attack citric acid. du Plessis (1964). found that malo-lactic strains of P. cerevisiae and Lactobacillus buchneri did not metabolize it. In addition to these two species, the same was found to be true with malo-lactic Lactobacillus delbrueckii (Pilone et al., 1966). Bacteria that can metabolize citric acid can use it as an energy source (Deffner, 1938; Campbell and Gunsalus, 1944; Charpentie et al., 1951). In the first step, citric acid is split to oxaloacetic acid and acetic acid. Decarboxylation of oxaloacetic acid gives pyruvic acid which is metabolized in the ways outlined above. Oxaloacetic acid formed from citric acid can also act as a hydrogen acceptor, if need be, and be reduced to succinic acid. Bacteria which do not metabolize citric acid probably lack citrate permease (Collins and Harvey. 1962). In Streptococcus diacetilactis, citric acid is metabolized but is not used as an energy source; apparently citric acid metabolism provides some sort of detoxification (Harvey and Collins, 1963).

MALO-LACTIC FERMENTATION

257

Resting cell fermentation of citric acid by several malo-lactic lactobacilli have been studied by du Plessis (1964). The major end products were lactic acid, acetic acid, and carbon dioxide. Also found were formic acid, ethanol, succinic acid, and acetoin. Charpentie et al. (1951)found butylene glycol also an end product of citric acid fermentation by malo-lactic bacteria.

4 . Malic Acid It is generally believed that malic acid is stoichiometrically converted to lactic acid and carbon dioxide by malo-lactic bacteria. du Plessis (1964) obtained 97% recovery of the carbon of malic acid as lactic acid and carbon dioxide in resting cultures of Lactobacillus buchneri. With other malo-lactic bacteria the recovery was less, as low as 85%. Ethanol was also formed. With the homofermentative bacteria some acetoin and diacetyl were found. The stoichiometry of the malo-lactic reaction is important in the consideration of the energetics of malo-lactic fermentation. This will be discussed further in Section V1,C. L-Malic acid is the acid found naturally in grapes. D - M a k acid is sometimes added to wine (in the form of the DL-malic acid mixture) to increase the acidity. Malo-lactic bacteria do not attack the D-isomer. Further, Radler (1966) reported the inhibition of the induction of “malic” enzyme by D-malic acid (see below, Section V1,B). However, malo-lactic fermentation was readily obtained in musts in which DLmalic acid had been added to supplement or replace the natural acid (Ough and Kunkee, 1967). D-Malic acid has little or no effect on the activity of malic dehydrogenase used in enzymic assay of L-malic acid (Kunkee and Combs, unpublished results).

F. END PRODUCTS During malo-lactic fermentation, lactic acid is the product formed most abundantly; it is formed from sugars, malic acid, and citric acid. Lactic acid exists in two optically active forms. The types formedfrom glucose - depend on the organism. Of characterized malo-lactic bacteria, the types producing D-lactic acid or a mixture of D- and L-isomers predominate: P . cerevisiae form DL-lactic acid; Leuconostoc spp. form D-; and of the malo-lactic lactobacilli only L. delbrueckii form just the L-isomer (Breed et al., 1957). There is some confusion about the isomers: L-lactic acid (“sarcolactic”) has the same configuration as L-glyceric acid. Solid L-lactic acid is levorotatory, but in solution the formation of a strongly dextrorotatory ethylene oxide bridge causes the acid to rotate polarized light to the right (Lockwood

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RALPH E. KUNKEE

et al., 1965). Thus the isomer is designated L(+)-lactic acid. The opposite is true with D(-)-lactic acid (“paralactic”). Some of the lactic acid in wine is produced by yeast during alcoholic fermentation and is of the D type. In wines that have undergone malo-lactic fermentation most of the lactic acid is produced by the bacteria from malic acid. It is surprising that examination of many malo-lactic wines revealed L(+)-lactic acid as the predominant form present (Peynaud et al., 1966a,b; Brechot et al., 1966). We have seen no information ab,out kinds of lactic acid produced by bacteria from malic acid as the substrate. Increase in volatile acidity (mainly acetic acid) was very early shown to accompany malo-lactic fermentation (Seifert, 1901). Ribereau-Gayon and Peynaud (1961, p. 437) suggested that most of the acetic acid comes from citric acid. Wines with no fermentation of citric acid had little increase in acetic acid. Addition of more malic acid after malo-lactic fermentation brought about a second malo-lactic fermentation with no additional formation of acetic acid. However, du Plessis (1964) found acetic acid in resting cell fermentations of malic acid. Lactic acid is also slightly volatile with steam distillation. Small amounts, depending on pH and type of equipment used, are distilled during the usual volatile acid determinations (Amerine, 1965; Pilone, 1965). Some of the increase in volatile acidity found after malo-lactic fermentation may be contributed by lactic acid, but there seems to be no doubt that an increase in acetic acid also occurs. There is one report of no increase in volatile acidity after malo-lactic fermentation (Bremond, 1937, p. 29). Acetoin and related compounds are usually produced in very small amounts, but their organoleptic effects may be large. Butylene glycol has a slightly sweet taste similar to glycerin (Amerine, 1965). Acetoin is rather odorless and tasteless, but its general sensory effect can be pleasant (Niven, 1952). Acetoin and butylene glycol have optical isomeric forms, but there is no information about organoleptic differences between them. Diacetyl has a potent odor often associated with dairy products. Vaughn and Tchelistcheff (1957) reported that addition of diacetyl and acetoin to wine gave a flavor and odor similar to sauerkraut. Diacetyl in very small amounts may improve wine (Fornachon, 1963a). Citric acid generally increases diacetyl production, especially under nonoptimal conditions (Davis, 1963). Homofermentative bacteria usually produce higher concentrations of diacetyl than heterofermentative types (Fornachon, 1963a; Davis, 1963; du Plessis, 1964). The concentrations of acetoin and diacetyl are usually determined as the total of both. A higher concentration

MALO-LACTIC FERMENTATION

259

of these products is found after malo-lactic fermentation (Radler, 1962b; Fornachon, 1963a; Dittrich and Kerner, 1964; Fornachon and Lloyd, 1965), but Kunkee et al. (1965)found no statistical significance between concentration and malo-lactic fermentation. Short-lived off-odors often accompany malo-lactic fermentation in wine. Undoubtedly part of this is hydrogen sulfide (Suverkrop and Tchelistcheff, 1949) produced from the sulfur dioxide added to wine but not to laboratory media. We should also mentioned the esterification of acids by ethanol and higher alcohols. Yeasts are probably the causative agents (Nordstrom, 1966), but acid products of malo-lactic bacteria are involved. The tastes of the various nonvolatile organic acids are nearly indistinguishable (Pangborn, 1963), but the esters are more volatile and may play an important role in flavor and odor of wine. We have noted that lactic acid exists in two forms which probably have very similar sensory properties but the various esters of each of the acids may have a distinctive sensory character. There may, of course, be other products arising from the presence of lactic acid. In the laboratory the substrates, organisms, and conditions can be controlled. In practical operations many of these factors are variable, and they can influence each other profoundly. du Plessis (1964) speculated that there was little difference between malo-lactic bacteria and spoilage bacteria; he believed the environmental conditions were of more importance than the bacterial strain. Luthi (1957) reported he found no case where an organism from wine with some special characteristic produced that same characteristic when used as inoculum in sterile wine. H e attributed this to symbiotic relationships existing between the several organisms in the natural, mixed culture in wine.

V. Detection of Malo-lactic Fermentation Changes in acidity and pH are not reliable indices of malo-lactic fermentation because they can be influenced by many other factors. The qualitative determination of malic acid by paper chromatography is simple and has proved to be invaluable for routine analysis. Several procedures using butanol-acid solvents have been described (Lugg and Overell, 1948; Dolle, 1958; Ribereau-Gayon and Peynaud, 1958). Acid spots can be detected by spraying the paper with acid-base indicator or by addition of the indicator directly to the solvent. Absence of the malic acid spot is satisfactory proof of malo-lactic fermentation (Ingraham and Cooke, 1960). The presence of a spot at the lactic posi-

260

RALPH E. KUNKEE

tion is not confirmatory proof, because in these systems succinic acid and lactic acid are not separated. Solvents for separation of succinic acid and lactic acid were reported by Hartley and Lawson (1962) and Blundstone (1963). These acids can also be separated by two-dimensional thin-layer chromatography (Higgins and von Brand, 1966). Quantitative measurement of malic acid can be made enzymically. In one method, suggested by Korkes and Ochoa (1948), manometric determination is made of carbon dioxide formed by the decarboxylating activity of “malic” enzyme on malic acid (Kolar, 1962). In the other, spectrophotometric measurement is made of reduced coenzyme brought about by the action of malic dehydrogenase on malic acid (Mayer and Busch, 1963). D-Malic acid is not active in these assays. We have already discussed (Section IV,B) the determination of total and L-lactic acid, and of D-lactic acid by difference. A simple and reliable method of quantitative determination of D-lactic acid would be very useful.

VI. Deacidification

A. MECHANISM Ochoa and co-workers obtained an enzyme preparation from malic acid-adapted Lactobacillus urubinosus (plantarum) that decarboxylated malic acid to lactic acid (Korkes and Ochoa, 1948).This enzyme was similar to the carbon dioxide-fixing enzyme, “malic” enzyme, which had been obtained from pigeon liver (Ochoa et al., 1948). (We will use the name “malic” enzyme for the bacterial enzyme, but it is now properly called L-malate: nicotinamide adenine dinucleotide oxidoreductase (decarboxylating) E. C. 1.1.1.38.) Nicotinamide adenine dinucleotide (NAD) was required, and the reaction was assumed to involve a redox change. In analogy to the pigeon liver enzyme, a two-step reaction involving pyruvic acid was suggested: malic acid

+ NAD+

-

pynivic acid

+ COn + N A D H + H +

(1)

Pyruvic acid as an intermediate is a convenient explanation for the coenzyme requirement. However, free pyruvic acid has not been found in the reaction. In exchange reactions with radioactive malic acid, no label was found in pyruvic acid (Kaufman et at., 1951).

MALO-LACTIC FERMENTATION

26 1

Apparently there is a very close association between “malic” enzyme and lactate dehydrogenase, the enzyme for reaction (2), and pyruvic acid. The latter is thus reduced to lactic acid before being released from the enzyme surfaces. Lactate dehydrogenase activity has not been separated from “malic” enzyme, although the former is a constitutive enzyme of the bacteria, and the latter is inducible. With highest purification of “malic” enzyme, L-lactate dehydrogenase activity is present, while in the more impure preparations both D- and L-lactate dehydrogenase activities were found (Kaufman et d.,1951). The reactions are undoubtedly reversible, as shown, but this is difficult to prove. The equilibrium of the second reaction is far to the right (Burton and Wilson, 1953), but its reversibility can be demonstrated (Hohorst, 1963). In the first reaction direct fixation cannot be shown with the bacterial enzyme because the substrates, pyruvic acid and NADH are rapidly removed from the solution by the lactate dehydrogenase activity. However, in exchange experiments, uptake of radioactive carbon dioxide was found in malic acid (Korkes et aZ., 1950). “Malic” enzyme decarboxylates oxaloacetic acid, but oxaloacetic acid is apparently not an intermediate in the decarboxylation of malic acid. If it were there would be no situation where malic acid would be decarboxylated faster than oxaloacetic acid, but this did occur at pH 6 (Korkes et al., 1950). Decarboxylation of oxaloacetic acid required only manganous ions as cofactor. Malic acid decarboxylation required NAD, specifically, as coenzyme, and for an unexplained reason phosphate was required for maximal activity (Ochoa, 1951). Recent studies with Bacterium “L” (a strain of LactobaciZZus plantarum) show different enzymes for decarboxylation of malic acid and oxaloacetic acid (Flesch and Holbach, 1965). “Malic” enzyme activity was inhibited by p-chloromercuribenzoate, but oxaloacetic decarboxylase was not. Other evidence was presented against the oxidation of malic acid to oxaloacetic acid before decarboxylation. This oxidation would be catalyzed by malic dehydrogenase which was shown to have optimal activity at pH 10. If the intracellular p H was anywhere near that of wine, it would seem malic dehydrogenase would be nonoperative under malo-lactic fermentation conditions. A malic-lactic transhydrogenase in Micrococcus lactilyticus has been described (Allen and Galivan, 1965; Dolin et al., 1965). This enzyme reversibly converted malic and pyruvic acids to oxaloacetic and lactic acids. It had no dehydrogenase or decarboxylase activities and required no exogenous cofactors.

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RALPH E. KUNKEE

B. ENZYMEINDUCTION The original studies on bacterial “malic” enzyme showed that malic acid decarboxylating ability of the bacteria was inducible. The enzyme developed in bacteria grown on malic acid and even in noninultiplying cells incubated in presence of malic acid (Blanchard et al., 1950). Biotin and an energy source were required for “malic” enzyme induction. Flesch and Holbach (1965) showed “malic” enzyme synthesis was inhibited by avidin, an antibiotin factor. Nathan (1961) used enzvme induction to separate malic acid and oxaloacetic acid decarboxylating activities in three malo-lactic bacteria. She found L-malic acid induced both these activities, but oxaloacetic acid induced only oxaloacetic acid decarboxylase activity. Oxaloacetic acid permease was induced only by oxaloacetic acid. Isolation of the enzymes is required before one can say whether malic acid was an inducer for one enzyme with two activities or for two separate enzymes, one of which can also be induced by oxaloacetic acid. Whiting and Coggins (1963) studied “malic” enzyme induction in several malo-lactic bacteria isolated from cider. Some of the bacteria were found to have inducible “ma1ic”- enzyme, but in others it was constitutive. In the bacteria studied, citric acid fermentation occurred only in those strains in which “malic” enzyme was inducible. I n the first studies on induction of “malic” enzyme, relatively high concentrations of inducer were used [150 mM DL-mahc acid (Blanchard et d.,1950)l. Other workers have used concentrations of inducer approximating that found in grape juice. Nathan (1961) and Flesch and Holbach (1965) used 30 mM L-malic acid. Results showing the dependency of “malic” enzyme induction on concentration of malic acid would be of special interest to enologists. Strains of malolactic bacteria that required high concentrations of malic acid for induction of “malic” enzyme would not carry out malo-lactic fermentation in wines with low malic acid. These strains should not be used for inoculation of wine for malo-lactic fermentation. The presence of these bacteria would explain situations where wines become biologically stable after incomplete malo-lactic fermentation. If the bacteria began to multiply again during the secondary fermentation, say because the wine was racked, then the newly formed bacteria might not be able to make the induced enzyme because of the lower level of inducer (malic acid) present.

263

MALO-LACTIC FERMENTATION

C. ENERGETICS In his excellent monograph Schanderl (1959, p. 164) described the malo-lactic reaction as one which yields no energy. This statement, which has been widely quoted, was based on a consideration of the difference between the molar enthalpies (AH) of the substrate, malic acid, and the products, lactic acid and carbon dioxide. [AH, determined as the heat of combustion, for malic acid is usually given as -320.1 kcal./mole and for lactic acid as -326 (Kharasch, 1929). (The heat of combustion of carbon dioxide is, of course, 0.) More recent evaluation gives the heat of combustion for L-malic acid as -318.0 kcal./mole (Wilhoit and Shiao, 1964) and for L-lactic acid as -321.2 (Saville and Gundry, 1959). Calorimetry experiments with malic acid and the enzymes and cofactors required for conversion to lactic acid resulted in a slight rise in temperature (Schmidt, 1959).] Thus the conversion, in the solid state, of malic acid to lactic acid and carbon dioxide is endothermic and requires an enthalpy input of 3.2 kca1.l mole. However, whether energy is required for the reaction depends not only on the enthalpy difference but also on the change in entropy at the temperature of the reaction; a large increase in entropy would overcome the increase in enthalpy to make the reaction actually exergonic. The change in free energy (AG) (Lewis and Randall, 1961; Lehninger, 1965) tells us whether the reaction will proceed and if it is a potential source of energy for the b a ~ t e r i a . ~ “Standard”8 free energy changes (AG’) for these reactions have been calculated by Burton and Krebs (1953): malate-2

+ NADP+ = pyruvate-* + NADPH + C O P-2.0 pyruvate-’

+ NADH + H+ = lactate-’ + NAD-

AG‘ kcal.,mo,e

(4)

6.0

(5)

Under “standard” conditions -at pH 7 -the acids are completely ionized, as written. Note that at this pH, hydrogen ion is not involved in the decarboxylation equation (4). The free energy changes for oxidation and reduction of NAD and ‘Under conditions of constant temperature heat cannot be converted to work, and any change in enthalpy is not available as energy anyway. ‘“Standard” conditions, as used by Burton and Krebs (1953), are 25”C., pH 7,0.05 atm. carbon dioxide and 0.2 atm. oxygen, and 0.01 M aqueous solutions of other reactants and products.

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RALPH E. KUNKEE

NADP (nicotinamide adenine dinucleotide phosphate) are not exactly equivalent (Burton and Wilson, 1953): NADPH NAD+

+ Hr = NADP+ + 2[H]

4.1

+ 2[H] = NADH + H+

-4.3

(6)

(7)

Summation of equations (4) through (7) gives: malate-2

+ H+ = lactate-’ + GO2

AG’ = -8.2 kcal./mole

(8)

Equation (8) shows that the reaction actually is exergonic and is therefore a potential source of energy for the bacteria. However, the above calculations must be modified to consider the reaction in wine rather than at “standard” conditions. Let us examine the reaction at (a) low pH in alcohol, (b) in presence of carbon dioxide, and (c) at cellar temperature. Because of the uptake of a proton by the substrate [cf. equation (S)], it would seem the high concentration of hydrogen ion in wine would tend to drive the reaction toward the formation of products. But at low pH one must also consider the change in ionic form of the acids.Y The p&’s for L-malic acid are 3.40 and 5.13 (Jones, 1962). The p& for lactic acid has recently been redetermined and found to be lower than previously given (Lockwood et al., 1965). For either isomer of lactic acid, the plc, is 3.73. The thermochemical equilibrium constants for these acids in alcoholic solution were not found in the literature; but for tartaric acid, in 10% alcohol at 20°C., there is an increase of p&, of 0.05 and of p&, of 0.20 (Berg and Keefer, 1958). With the use of these values as estimates of the alcohol effect on the dissociation of L-malic and DL-lactic acids, the approximate thermodynamic p&’s are: for L-malic, p&, = 3.45 and p&%= 5.33; for lactic acid, p& = 3.78. Then at the pH of wine, say pH 3.4, 52.5% of the malic acid is not ionized, 46.9% is malate-’, and 0.55% is malate-‘; and 70.6% of the lactic acid is not ionized and 29.4% is lactate-’. At pH 3.4, equation (8) would be represented by the following four reactions: (0.55%) malate-2

+ H+ (pH 3.4) = lactate-’ + COz

b)

3Thermodynamically it is probably unimportant which ionic forms of the acids are actually involved in the enzymic reactions as long as the change in protonation is consistent, but the change in free energy in equation (8)was obtained from reactions with acids in completely ionized form (pH 7 ) .

MALO-LACTIC FERMENTATION

(18.1%) malate-' (28.8%)

+ H+ (pH 3.4) = lactic acid + COZ malate-'

= lactate-'

+ COP

malic acid = lactic acid

(52.5%)

265 (b)

(4

+ COz

(d)

The free energy of equation (a) can be obtained from equation (8) by considering the change in pH at 25°C. ( R is the gas constant, T is the absolute temperature): AG,

= AG'

10-7 M H+ + RT In 10-3,4 K+ = -8.2

- 4.9 = -13.1 kcal./mole

For the other equations the free energy of ionization of the organic acids is required. For 1 molal solutions, AG = 2.3 RT PI(, (see Fruton and Simmonds, 1961). Substitution with the above thermodynamic equilibrium constants gives:I0 malic acid = malate-'

+ Hf AGM,= 2.3 RT (3.45) = 4.7 kcal./mole

+ H+ AG,wn,= 2.3 RT (5.33)= 7.3 lactic acid = lactate-' + H+ AGl, = 2.3 RT (3.78) = 5.2 malate-' = malate-2

(9) (10) (11)

Thermodynamic equilibrium constants were used, thus the free energies of ionization in kcal./mole are the same at 0.01 M concentration. Addition of equations (a) and (10) and subtraction of (11)gives equation (b): AGb = AG, AGW, - AG,, = -13.1 7.3 - 5.2 = -11.0 kcal./mole. For equation (c), the pH of equation (10) is changed from pH 2 (0.01M ) to pH 3.4 (on right side of equation): malate-l= malateP2 H+ (pH 3.4). The change in free energy

+

+

+

RT In

M H+ H+ = 7.3 - 1.9 = 5.4 kcaI./mole

Addition of this last equation to (a) gives equation (c): AGc = AGM~ kcal./mole. Equation (d) is calculated from equations (c), (11)and (9): AGd = AG, - AGL AGM,=-7.7 - 5.2 + 4.7 = -8.2 kcal./mole. By combination of the proper proportions of the changes in free energies as represented by equations (a), (b), ( c )and (d),the overall change in free energy at pH 3.4 is:

+ AGa = 5.4 - 13.1= -7.7

AG,

+

-13.1 x 0.55% = -0.07 kcal./mole

'OThe activity coefficient for hydrogen ion in water is nearly 1 at these concentrations (Kielland, 1937).

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RALPH E. KUNKEE

AC& AGc AGd

-11.0 X 18.1%= - I . % -7.7 x 28.8%= -2.22 -8.2 X 52.5%= -4.30 AG” = -8.6 kcal./mole

The pH effect is not great. Even though free energy changes for reactions involving hydrogen ion, (a) and (b), are larger than (c) and (d), the relative amount of the former is smaller than the latter. If carbon dioxide were released in the form of bicarbonate ion this would not be the case. Hydrogen ion would appear on the right side of equations (c) and (d) and the equilibria would be shifted toward the left. This is a very important consideration. Schmidt (1959) calculated the change in free energy, at pH 0, for the following reaction: COz

+ H2O = HCO3- + H+

11.2 kcal./mole

(12)

At pH 3.4, the change in free energy would be 11.2 - 4.6 = 6.6 kca1.l mole. Addition of the latter to equation (8)would give a reaction much less exergonic, -2.0 kcal./mole. Carbonic anhydrase studies on decarboxylation of pyruvic acid by yeast pyruvic carboxylase indicated carbon dioxide, not bicarbonate ion, as the product (Krebs and Roughton, 1948). This has also been assumed to be true for malic acid (Harary et al., 1953). The carbonic anhydrase studies ought to be repeated with “malic” enzyme. Carbon dioxide concentration in wine is higher than at “standard” conditions. Newly fermented wine has about 1 gm./liter carbon dioxide from alcoholic fermentation (see Amerine, 1958). The head space above the wine in storage tanks is often blanketed with carbon dioxide gas at about atmospheric pressure. The concentration of carbon dioxide in new wine could and probably does reach saturation. The maximum concentration of carbon dioxide in wine in equilibrium with 1 atm. of the gas is about 2 gm./liter (see Amerine, 1958)or about 26.8 times, say 30 times, the concentration under “standard” conditions” used in equation (8). The adjusted free energy change is at least: AG“‘ = AG“ RT In 30 =-8.6+2.0=-6.6 kcal./mole. Peynaud (1955) observed that at low temperature, where solubility of carbon dioxide is higher, malo-lactic fermentation can occur with no detectable gas production. More often effervescence is a noticeable aspect of malo-lactic fermentation in new wine. Carbon dioxide loss would

+

“Henry’s Law constant for solubility of carbon dioxide in water at 25°C. is 1.25 X

lo6mm Hg per molar fraction (Loomis, 1928). At 0.05 atm. carbon dioxide, the concentration of carbon dioxide in water would be 0.0745 gm./liter.

MALO-LACTIC FERMENTATION

267

tend to make the reaction more exergonic. The high acidity of wine also decreases the solubility of carbon dioxide; carbonic acid pK1 is 6.4. The decreased solubility because of acidity may be overcome by the increased positive charge on the proteins and pigments of wine at low pH which tend to cause electrostatic attraction of bicarbonate ion (Amerine et al., 1967, p. 214). At any rate, the actual concentration of carbon dioxide at the pH of wine was considered in calculation of AG"'. l 2 Finally, cellar temperature is different from "standard" temperature and must be considered. The change in free energy with temperature is related to temperature by the entropy change (AS) of the reaction (Lewis and Randall, 1961):

(g) =-AS=-aAT

AG - AH--6.6 - 3.2- -0.011 kca1.imole-" T 298

Thus the correction for change in free energy at cellar temperature of 18°C. is 0.08 kcal./mole, and the adjusted free energy change for the reaction at this temperature is -6.5 kcal./mole. (At 18"C., the concentration of carbon dioxide would be higher than at 25"C., but the actual concentration found under cellar conditions was used for calculation of AG"'). Several uncertainties in the calculations were noted above, but an estimate of change of free energy in the malo-lactic reaction in wine under cellar conditions (pH 3.4, 18"C., 1 atm. carbon dioxide) is -6 kcal./mole. From this estimate, the reaction is favorable and the equilibrium is on the side of the products. Whether this potential energy is actually available to the bacteria is another question. The potential energy is not available to the bacteria whenever malic acid is completely converted to lactic acid. Useful energy for the cell requires the formation of high-energy phosphate. We see there is no direct formation of ATP in the malo-lactic reaction. Reoxidation of reduced coenzymes can lead to formation of high-energy phosphate, but in malo-lactic fermentation there is no net change in redox state of l2 We might consider malo-lactic fermentation which occurs after bottling. Bottled wine has about 0.5 gm./liter carbon dioxide (see Amerine, 1958), and the concentration is less than in new wine blanketed with carbon dioxide. Even after malo-lactic fennentation, the additional carbon dioxide produced from, say 0.4% malic acid, would make the final concentration of entrapped carbon dioxide only about 2 gm./liter, Iess than the maximum concentration in wine in equilibrium with carbon dioxide at atmospheric pressure. Higher concentrations might be obtained when alcoholic or other fermentations occurred after bottling.

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coenzymes. The conversion of malic acid to lactic acid, however, is not necessarily complete. Early work (Ribereau-Gayon and Peynaud, 1938) has been cited to show the reaction to be complete, but the lactic acid found was only 75% of that required for complete conversion of malic acid. Furthermore, substantial amounts of lactic acid found were undoubtedly from sources other than malic acid. du Plessis (1964) reported 10 to 15%of malic acid was recovered in products other than lactic acid and carbon dioxide in resting cell siisnensions of malo-lactic bacteria. One might speculate on the conversion of the intermediate, pyruvic acid, to acetyl phosphate. The hydrogen produced in the formation of pyruvic acid and acetyl phosphate could be used for the reduction of acetyl phosphate to ethanol (Section IV,E). The reduced coenzyme might also be reoxidized b y reduction of another molecule of malic acid to succinic acid. In spite of these possibilities, it has not been shown that malic acid can be used as an energy source for malo-lactic bacteria. Two exceptions have been reported. Krasil’nikova (1965) found growth of Lactobacillus delbrueckii on wort was stimulated somewhat by malic acid; Bidan (1966) reported some growth of malo-lactic bacteria on malic acid. Radler ( 1 9 5 8 ~ found ) no loss of malic acid unless carbohydrate was fermented simultaneously. Melamed (1962) reported a decrease in residual sugars of wine after malo-lactic fermentation. Peynaud (1955)found addition of glwose stimulated malo-lactic fermentation in wine. The best proof of complete malo-lactic conversion was with tracer experiments; Schmidt et al. (1962) got nearly complete recovery of lactic acid and carbon dioxide from 1,4-malic acid-W. Unfortunately it was necessary to use DL-malic acid, and the disappearance of the substrate was greater than could be accounted for by fermentation of L-malic acid only. The average recovery of lactic acid was 97% with a range from 90% to 106%. It is strange that malic acid is not used for energy in the schemes outlined above. Perhaps pyruvic acid is available only for reduction to lactic acid. We have seen that free pyruvic acid is not formed as an intermediate. It is apparently bound tightly to the enzymes and reduced to lactic acid before being released. For citric acid to be used as an energy source, the oxaloacetic acid formed from citric acid may be decarboxylated to give unbound pyruvic acid. Kaufman et al. (1951) did not test oxaloacetic acid in their exchange experiments with pyruvic acid. Mutants of malo-lactic bacteria might be obtained in which enzymes were altered such that there was less affinity for pyruvic acid. In these mutants, pymvic acid from malic acid would not be bound as tightly

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as in the wild type and would be available for exergonic reactions. This hypothesis could be tested by searching for mutants of malolactic bacteria which could grow on malic acid as an energy source. The ruison d’etre of malo-lactic fermentation is puzzling if the reaction does not provide energy for the organism. From an evolutionary point of view, the increase in p H of weakly buffered solutions provided by malo-lactic fermentation would be advantageous for bacterial growth, but growth of all organisms present in the primitive medium would be favored. Another explanation might be that enzymes involved are used for other reactions and malo-lactic fermentation is an accidental expression of their activity. Additional studies on inducer and substrate specificities of “malic” enzyme are necessary. Metabolism of malic acid may provide some sort of detoxification as has been shown with citric acid (Harvey and Collins, 1963).

D. SECONDARY EFFECTS OF DEACIDIFICATION We have seen that malo-lactic fermentation causes loss of acidity and increase in pH in wine. These changes in turn can bring about other changes. The color of anthocyanin pigments of red wine is dependent on pH and oxidation state. Malo-lactic fermentation can cause up to one-third loss in red color. Part of the color loss is from the change in pH. However, Vetsch and Liithi (1964) found that fermentation of citric acid, rather than malic acid, by malo-lactic bacteria caused a color change by providing hydrogen as reductant. They also claimed loss of color of anthocyanins by NADH2 in absence of bacteria. If the acidity loss is SO large that an unusually high pH is obtained, not only is there loss in color, but the quality of the color changes from a natural full red to a bluish hue. With decreased acidity, wine is a more favorable medium for microbial growth and thus more susceptible to spoilage. This results directly from the change in pH and also from the indirect effect on sulfur dioxide (Section IV,D). The midpoint between p&’s for tartaric acid (Berg and Keefer, 1958) is about 3.6. Thus if wine is saturated with potassium bitartrate and malo-lactic fermentation brings about an increase in pH which approaches p H 3.6, the potassium bitartrate will precipitate. The precipitation causes turbidity and further loss of acidity. Fornachon (1963a) pointed out that wine fined with an excess of gelatin may become hazy after malo-lactic fermentation because of the increase in pH.

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VII. Control of Malo-lactic Fermentation

A. DESIRABILITYOF CONTROL Winemakers from regions where wines of high acidity are produced have praised malo-lactic fermentation and declared it an essential ingredient for premium quality wine (e.g., Ferre, 1928; RibereauGayon, 1946; Peynaud and Domercq, 1959; Marques Gomes et aZ., 1956). Much of the utility of the fermentation in these wines is, of course, the deacidification, but other benefits may result. In warmer regions, as in parts of California, the deacification effect is usually not desirable. Nevertheless, malo-lactic fermentation was found to occur most frequently in California wines of highest quality, vintage and varietal wines (Ingraham and Cooke, 1960). It has been suggested that by-products of malo-lactic fermentation make subtle flavor changes to give a distinctiveness and complexity the wine would otherwise not have (e.g., Suverkrop and Tchelistcheff, 1949; Vaughn and Tchelistcheff, 1957; Ingraham and Cooke, 1960). Bacteria associated with certain wineries (see Section IV,C) may add distinctive characteristics to their wines (cf. Marques Gomes et al., 1956; Webb, 1962). Pilone and Kunkee (1965) made sensory tests of wines especially fermented with different malo-lactic bacteria. Differences in the organoleptic qualities of the wines were found, but the differences were not as striking as might have been expected. In the chemical analyses of the volatile components of these wines, small differences were found in acetoin (plus diacetyl), volatile acidity, and diethyl succinate (Pilone et al., 1966). The authors suggested that use of grapes with higher varietal character than used by them might provide substrates for the bacteria for greater differences in metabolic end products. Biological stability is another beneficial aspect of malo-lactic fermentation. As long as malic acid is present, nonsterile wine should be considered unstable, although wines over 2 years old rarely undergo malo-lactic fermentation. However, gassy wines of Burgundy and Italy are found on the American market. Malo-lactic fermentation may not be of benefit to less-than-premium wines (Ough and Amerine, 1963). Some of these wines are ready for consumption about the time of the secondary fermentation. Malolactic fermentation would be considered spoilage in these wines. Furthermore, the changes in quality provided by the fermentation would probably be unappreciated in these wines. Undesirable aspects relating to deacidification caused by malo-

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lactic fermentation were discussed in Section VI,D. Other disadvantages are gassiness, the temporary formation of off-odors, and the increase in some divalent ions. Malic acid, the most potent chelating agent of the organic acids found in wine, complexes copper and iron ions (Rankine, 1960). Loss of malic acid brings about release of the ions which often cause haziness in wine and are oxidation catalysts. This effect is partly compensated by the increase in chelating activity of the other acids because of the increase in pH. Off-odors such as diacetyl are sometimes removed by refermentation by yeast (see Dittrich and Kerner, 1964). The decision to encourage or discourage malo-lactic fermentation can be difficult to put into practice. The fermentation has been described as “capricious.” Moreover, wines which need the fermentation most - the high acid wines -are the very ones in which it is most difficult to induce because of the low pH, and vice versa. T h e usual suggested procedures are helpful, but the best control requires a better knowledge of the strains of bacteria most likely to be involved and a thorough knowledge of the wine components.

B. STIMULATION Laboratory descriptions of induction of malo-lactic fermentation in wine by bacterial inoculation began with the first studies on malolactic fermentation (Koch, 1900) and now are prevalent (see Peynaud and Domercq, 1959; Sudraud and Cassignard, 1959; Domercq et al., 1960; Webb and Ingraham, 1960; Fell, 1961; Fornachon, 1963a; Kunkee et al., 1964). It is difficult to make an estimate of the degree of commercial use of bacterial inoculation. It is apparently used in France, Germany, Switzerland, and Portugal. We know of at least one California winery which has used the procedure consistently and successfully and of others which encourage natural malo-lactic fermentation for production of premium quality wine (Tchelistcheff, 1966). For stimulation of fermentation by inoculation, careful selection of bacteria is important at this state of the art. We recommend Leuconostoc citrouorum ML 34 because of its pH and temperature tolerances and its negligible formation of off-flavors. (The volatile acidity of the wine is increased about 200 mg./liter.) From the references listed, and the author’s observations, the following procedure can be suggested: The selected strain can be propagated in sterile grape juice containing 0.05% yeast extract and titrated to pH 4.5. The presence of malic acid in the propagating medium will assure

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adaptation of enzymes for malic acid fermentation, but adaptation probably also will occur during growth of the bacteria in the wine. Grape juice at pH 3.5 with low levels of sulfur dioxide has been used in an attempt to adapt the bacteria to some of the conditions of wine. Inoculations of 0.01% to 1%have been used. Wine which has recently undergone malo-lactic fermentation can also be blended with other wine to induce the fermentation. Here large amounts of “inoculum” are used: 15% to 50%. Some workers suggest the inoculation be made at the beginning of alcoholic fermentation, before the uptake of micronutrients by the yeast; others suggest the middle of alcoholic fermentation when free sulfur dioxide is lowest (because of the high concentration of acetaldehyde formed b y the yeast at this time); and still others at the end of alcoholic fermentation, when yeast autolysis begins and when sugars are at low concentrations to minimize formation of metabolic end products. With relatively large inocula, the speed of malo-lactic fermentation is independent of time of inoculation (Kunkee et al., 1964). Good data on the effect of size of inoculum and time of inoculation on the quality of the wine are not available. Natural malo-lactic fermentation can be encouraged by use of low levels of sulfur dioxide (less than 50 mg./liter), delay in addition of acidifying or fining agents, and storage at warmer temperatures. Aeration can stimulate malo-lactic fermentation (see Section IV,D), but Domercq et al. (1960) found it impaired fermentation. These conditions are also those which encourage bacterial spoilage, and the wine should be frequently examined. It is nearly impossible to obtain malo-lactic fermentation in wines with very high acidity -approaching pH 3.0. In regions producing these wines, it is often legal to increase the pH by addition of chemicals, by amelioration with water, or b y ion exchange. Further deacidification can then be made by malo-lactic fermentation. Increase in pH can also be obtained by blending the wine with another wine which has undergone malo-lactic fermentation and has less acidity. The pH of the blended wine may then allow further malo-lactic fermentation. After malo-lactic fermentation, wines have less acidity, are turbid, and often have temporary off-odors. Good winery practice suggests addition of extra sulfur dioxide; settling or fining, aeration if hydrogen sulfide is high (Rankine, 1963b), and storage before bottling.

C. INHIBITION If absolute prevention of malo-lactic fermentation is desired,

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sterilization of the wine is required. Pasteurization (Ough and Amerine, 1966) has been used, but undesirable changes in flavor from heat do occur. Sterile filtration is gaining acceptance. Removal of lactic acid bacteria by commercially available membrane filters with 0.65 p porosity has been reported (Tchelistcheff et al., 1964). Chemosterilants for wine such as sorbic acid and diethyl pyrocarbonate are relatively inactive against lactic acid bacteria (Peynaud, 1963; Mayer and Luthi, 1960) and are not used for control of malo-lactic fermentation. Secondary fermentation can b e discouraged, if not always prevented, by attention to good winery practices. Scrupulous care should be made to maintain clean filling and pumping Iines and storage containers, especially wooden cooperage. Sulfur dioxide is an effective inhibitor if cleanliness and low pH are maintained. Low acid wines should be acidified by chemical addition or by ion exchange -as much as legally permitted and economically feasible. Because of the variability in activity of sulfur dioxide, it is unrealistic to make specific recommendations as to the amount of sulfur dioxide to use. Addition of 100 to 200 mg./liter, depending on the condition of the grapes, has been suggested for red wine (Amerine et al., 1967, p. 363). Free sulfur dioxide ought to be determined regularly and maintained at some empirically selected concentration. Removal of gross lees soon after alcoholic fermentation and early fining with high concentrations of fining material also have been suggested, but these procedures may delay malo-lactic fermentation rather than prevent it (Fornachon, 1957; Kunkee, 1966). Ribereau-Gayon and Peynaud (1961, p. 496) discussed antagonisms between malo-lactic fermentation and certain yeasts used in alcoholic fermentation. Apparently some yeasts can inhibit the secondary fermentation by exhaustion of micronutrients of the medium or by excretion of some unknown inhibitory materials. Practical use could be made of these observations. Radler (1958~) found no symbiotic relationship between the yeast and bacteria as long as adequate amounts of amino acids were present. VIII. Conclusions

True today as it was 60 years ago is the statement of Alfred Koch (19OO), “. . . acid reduction in wine is a completely normal process [and] therefore the bacteria play a very important role in the normal development of wine.” Malo-lactic fermentation is necessary not only to deacidify some wines and to stabilize others, but also to improve some wines apparently by addition of certain products of metabolism

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which make the flavor more complex. Information on the latter is still scant. Metabolism of malo-lactic bacteria has been well studied, but more information about the organisms under conditions of malo-lactic fermentation in wine is needed. Malic acid is not used as an energy source, but the reason for this seems to be biochemical rather than thermodynamic. Malo-lactic fermentation is most likely to occur under conditions which also favor wine spoilage. However, with knowledge of the wine and the bacteria, the fermentation can be controlled - stimulated or inhibited -as desired. ACKNOWLEDGMENT The author thanks G. J. Pilone and Mrs. Leslie Westergaard for their help with some of the literature. REFERENCES Adams, A. M. (1964). Ontario Dept. Agr., Rept. Hort. Expt. Sta. Prod. Lab., Vineland Sta. p p . 108-111. Allen, S. H. G., and Galivan, J. H., Jr. (1965). Federation Proc. 24,531. Amerine, M. A. (1958).Adoatt. Food Res. 8, 133-224. Amerine, M. A. (1965). “Laboratory Procedures for Enologists,” p. 21. Associated Students Store, Univ. of Calif., Davis, California. Amerine, M. A., and Singleton, V. L. (1965). “Wine: An Introduction for Americans,” pp. 61-62. Univ. of Calif. Press, Berkeley, California. Amerine, M. A., Berg, H. W., and Cruess, W. V. (1967). “The Technology of Wine Making,” 2nd Ed., pp. 285-288,584-587. Avi Publ., Westport, Connecticut. Amthor, C. (1889).2. Angew. Chem. 2,5-6. Arena, A. (1936).Rev. Fac. Agron. Vet. Univ. Buenos Aires 8,155-325. Astruc, H. (1925).Ann. Fals. Fraudes 18,198-204. Balard, A. J. (1861).Compt. Rend. 53,1226-1230. Beech, F. W., and Carr, J. G. ( 1 9 5 5 ) Cen. ~ Microbiol. 12,85-94. Berg, H. W., and Keefer, R. M. (1958).Am.J.Enol. 9,180-193. Bernstein, I. A., Lentz, K., Malm, M., Schambye, P., and Wood, H. G. (1955).J.Biol. Chem. 215,137-152. Berry, J. M., andvaughn, R. H. (1952).Proc.Am. SOC. Enologistspp. 135-138. Berthelot, M., and d e Fleurieu, A. (1864).Compt. Rend. 58,720-723. Bidan, P. (1956).Ann. Technol. Agr. 5,597-617. Bidan, P. (1966).Bull. Ofic. Intern. Vin 39,1081. Blanchard, M. L., Korkes, S., del Campillo, A., and Ochoa, S. (1950). J . Biol. Chem. 187,875-890. Blundstone, H. A. W. (1963).Nature 197,377. Bocker, H. (1964).Zentr. Bakteriol. Parasitenk. Abt. ZZ 118,249-264. Boussingault, J. (1868). In “Agronomie, Chimie Agricole et Physiologie” (M. Boussinaault, ed.), 2nd Ed., Vol. 4, p p . 228-230. Gauthier-Villars, Paris. Brechot, P., Chauvet, J., Croson, M., and Irmann, R. (1966). Compt. Rend. 262, 16051607. Breed, R. S., Murray, E. G. D., and Smith, N. R. (1957). “Bergey’s Manual df Determiniative Bacteriology,” 7th Ed. Williams & Wilkins, Baltimore, Maryland.

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Author Index Numbers in italics indicate the pages on which complete references are listed. A

Aalto, T. R., 31,35 Abelli, G., 61 Abrams, J. D., 25,26,35,36 Abuelo, J. G., 62 Ada, G. L., 48,51,65 Adams, A. M., 240,274 Adler, F. L., 57,63 Ahlgren, E., 117,125 Aiba, A., 166 Aha, K., 246,278 Albright, J. F., 51,57,65 Albury, M. N., 248,279 Aleksandrova, I. V., 172,177,183 Alin, K., 3,36 Allen, S. H. G., 261,274 Almin, K. E., 113,114,125 Altman, P. L., 40,63 Amemura, A., 116,125,159,168 Amerine, M. A,, 236, 240, 245, 247, 251, 254, 258, 266, 267, 270, 271, 272, 273, 2 74,2 75,276,2 78 Amies, C. R., 20,36,56,63 Ammer, U., 106,108,125,127 Amthor, C., 238,274 Andersen, C. G., 207,211,216,232 Anderson, H. D., 57,64 Anderson, K. F., 25,26,27,36 Anderson, T., 208,232 Angeletti, P. U., 61 Aoki, T., 55,63 Arena, A,, 240,241,274 Arnason, B. G., 39,63 Arnon, R., 61 Aronson, H., 273,279 Aronson, J. M., 138,141,166 Arquilla, E. R., 43,63 Arseculeratine, S. N., 63 Aschan, K., 115,124,125,128 Ashford, W. R., 22,38 Astruc, H., 239,274 Austen, K. F., 60 Austin, C. M., 48,51,65

281

Avers, C. J., 78,88 Avery, 0. T., 42,63 Avramov, J . A., 75,89 Axelos, M., 101,128 Axelrod, V., 56,67 B

Baba, T., 84,90 Bailey, I. W., 105,125 Balard, A. J., 237,274 Barber, C., 62 Barber, G. A., 99,100,125,137,167 Bard, R. C., 255,256,275 Barker, H. A., 254,278 Barr, N., 23,24,25,26,27,29,30,36,37 Barrett, J. T., 61 Barth, E. F., 187,213,214,232 Barton, J. C., 82,89 Basaca-Sevilla, V., 62 Basu, S. N., 109,125,155, 167 Baumann, J. B., 53,67 Bavendamm, W., 107,125 Bayley, S. T., 93,129,134,137,169 Beaman, A. J., 243,279 Beard, R. L., 62 Beck, A. J., 210,225,233 Beck, J. C., 61 Beech, F. W., 244,274 Beer, M., 132,135,136,167 Beernink, K. D., 60 Beiser, S. M., 61 Bell, M. K., 124,125 Beloian, A., 24,36 Benacerraf, B., 49,64,66 Ben-Hayyim, G., 100,125,139,167 Bennett, A. J., 42,65 Benziman, M., 133,134,167 Berenbaum, M. C., 57,63 Berg, H. W., 236, 240, 264, 267, 269,273, 2 74 Berglund, G., 59 Bergmann, F. H., 59

282

AUTHOR INDEX

Bergquist, L. M., 59 Berlin, B. S., 56,64 Bemheimer, A. W., 41,64 Bemstein, I. A., 254,274 Berry, J. M., 237,274 Berthelot, M., 237,274 Bibikova, M. V., 79,90 Bidan, P., 241,243,250,268,274 Birner, J., 16,36 Biro, C. E., 48,64 Bjorkman, E., 111,125 Blanchard, M. L., 262,274 Bloodgood, D. E., 206,232 Bloomhuff, R. N., 212,214,233 Blum, R., 112,125 Blundstone, H. A. W., 260,274 Blyth, W. A., 63 Bobak, M., 143,169 Bobrov, R. A., 228,232 Bock, R. M., 86,88 Bocker, H., 248,251,274 Bogoch, S., 60 Boiden, J., 181,183 Bourdel, G., 61 Boussingault, J., 237,274 Boyle, W., 62 Boyse, E. A., 62 Bradney, L., 198,234 Braun, W., 49,66 Brechot, P., 258,274 Breebaart, A. C., 45,64 Breed, R. S., 241, 242, 243, 245,250,257, 274 Bremond, E., 240,258,275 Brown, A. M., 135,136,167 Brown, G. C., 57,64 Brown, M. R. W., 24,25,26,28,36,38 Brown, W. R. L., 30,36 Brummond, D. O., 137,167 Bruno, C. F., 245,275 Budd, W. E., 208,216,233 Bui-Khac-Diep, 166,167 Burger-Rachamimov, H., 133,134,167 Burk, D., 72,88 Burman, N. P., 6,38 Burnet, M., 42,64 Burnett, J. H., 124,125 Burrell, H., 31,37 Burton, K., 261,263,264,275

Busch, I., 260,277 Buschke, W., 27,36 Buswell, A. M., 227,234 Butler, W. T., 58,64 Butschak, G., 77,89 Butterfield, C. T., 191,232 Buyze, G., 253,275 C

Calkins, E., 60 Cameron, M. P., 61 Campbell, D. H., 40,43,48,64,65 Campbell, J. J. R., 255,256,275,276 Canale-Parola, E., 134,167 Cannon, D. A., 18,37 Carey, R. J.. 226,234 Carlin, H., 7,37 Carmichael, J. W., 228,232 Carr, J. G.,241,242,244,251,274,275 Carra, J. H., 112,128 Carrasco, A., 240,278 Carroll, V. P., Jr., 59 Carson, J., 140,167 Cartwright, K. St. G., 108,125,147,167 Caso, L. V., 62 Caspari, E., 41,64 Cassell, E. A., 224,232 Cassignard, R., 271,272,275,279 Castor, J., 77,90 Cato, E. P., 245,275 Chaix, P., 77,89 Chance, B., 72,88 Chander, K., 5 , 3 7 Chang, P. W., 63 Charney, J., 51,64 Charpentie, Y., 251,256,257,275 Charpentier, M., 102,125,157,167 Chase, M. W., 39,47,64 Chauvet, J., 258,274 Chimenes, A. M., 79,88 Cho, Y. S., 70,72,85,89 Chopra, I. C., 5,37 Christie, A., 58,66 Christman, R. F., 173, 174, 175, 176, 177, 183 Christopher, J. A., 59 Cinader, B., 61 Clark, I. T., 145,169 Clarke, A. E., 108,125, 159,166,167

283

AUTHOR INDEX Clarke, W. M., 60 Clausen, L. W., 35,38 Cockburn, T., 208,232 Cocking, E. C., 124,125 Coggins, R. A., 244,262,279 Cohen, H. H., 17,38 Cohen, J. J., 60 Cohen, S. M., 17,36 Cole, F. E., 159,165,167 Collins, E. B., 256,269,275,276,279 Colvin, J. R., 100, 121,125,130, 132, 133, 134, 135, 136, 137, 139, 140, 167, 169, 170 Combs, G. F., 56,156 Combs, R. E.,240,246,259,276 Conchie, J., 123,125 Condie, R. M., 41,66 Constantin, T., 79,88 Cook, W. B., 171,181,183 Cook, W. H., 121,130 Cooke, G. M., 240, 242, 243, 244, 250, 259,270,276 Cooke, W. B., 201,232 Coons, A. H., 58,64 Cooper, J., 34,36 Cooper, J. W., 12,36 Copeman, S. M., 54,64 Corbett, N. H., 104,106,125 Cosgrove, F. P., 32,38 CBte, W. A., 141,167 Cotgrave, R., 275 Couch, J. R.,249,275 Courcon, J., 60 Courtois, H., 103,104,105,106,107,125 Courtois, J. E., 166,167 Cowling, E. B., 92,102,104,108,115,117, 121, 125, 127, 128, 147, 149, 161, 167, 169,178,179,181,183 Cox, C. B., 17,37 Craigie, J. S., 177,183 Cremer, N. E., 40,43,64 Criddle, R. S., 86,88 Crompton, D. O., 25,26,27,36 Croson, M., 258,274 Crowle, A. J., 51,64 Cruess, W. V., 236,240,267,273,274 Cryan, W. S., 52,64 Cuadrado, R. R., 63 Culbertson, C. G., 20,36

Curds, C. R., 191,232 Cushing, J. E., 41,66 Cutler, J. L., 56,64 D

Dacre, J. C., 242,275 Dancis, J., 53,65 Daniel, T. M., 63 Danon, D., 132,137,169 da Silva Babo, F., 240,270,277 Datta, P. K., 115, 121, 123,125,130, 157, 161,167,170 Davidovits, J., 96,125 Davidson, R. W., 108,129 Davies, D. A. L., 62 Davies, G. E., 11, 13,36 Davies, T. C., 26,36 Davis, H. S., 44,65 Davis, J. G., 244,245,254,255,258,275 Davis, N. C., 60 Davison, J. E., 10,13,36 Davisson, E. O., 20,36 Day, E. D., 62 Deardoff, D. L., 30,37 Deese, D. C., 103,125 D e f i e r , M., 256,275 de Fleurieu, A., 237,274 de Haan, P. G., 253,275 Deighton, L., 275 De Jager, E., 61 Del Campillo, A., 260,261,262,268,274, 276 Dementijeva, G., 226,234 De Moss, R. D., 255,256,275 Dennis, D. T., 100,125,133,134,135,167 Deters, W., 147,167 Diacumakos, E. G., 87,88 Dick, G. W. A., 18,36 Dickinson, D. B., 101,125 Diding, N., 3,36 Diena, B. B., 18,36 Dietrich, F. M., 59 Dimache, G., 62 Dimond, A. E., 103,111,126 Disraely, M. N., 243,279 Dittmer, D. S., 40,63 Dittrich, H. H., 259,271,275 Dixon, F. J., 56,64 Dixon, F. J., Jr., 51,56,64

284

AUTHOR INDEX

Dobrogosz, W. J., 253,277 Doctor, V. M., 249,275 D o h , M. I., 261,275 Dolle, H., 259,275 Domercq, S., 236, 243, 244, 246, 247, 270, 271,272,275,278 Donaldson, D. M., 59 Donaldson, W., 191,232 Dondero, N. C., 227,232 Dose, K., 245,278 Dougherty, M. H., 215,232 Douglas, H. C., 241,242,250,276,279 Downie, A. W., 42,52,64 Draper, L. R., 46,64 Driver, C . H., 179, 183 Droblyanets, E. E., 226,234 Dumonde, D. C., 60 Duncan, C. W., 112,126 du Plessis, L. de W., 240, 242, 247, 256, 257,258,259,268,275 Dupuy, P., 245,278 Durall, G. L., 41,66 Durdin, A. C., 211,233 Dyer, H. M., 71,89 E

Eagleton, A. J., 54,64 Eaton, M. D., 63 Eberhart, B., 157,169 Eckenfelder, W. W., 217,229,232 Edsall, G., 21,36,39,56,64 Edwards, G. P., 214,232 Ehrlich, R., 46,67 Eisen, H. N., 50,64 Eisentraut, A. M., 48,67 Eisman, P. C., 34,36 Elberg, S. S., 60 Elbien, A. D., 137,167 Eldering, G., 17,21,37 Elliker, P. R., 26,37 Ely, H. M., 206,224,232 Emanual, C. F., 177,183 Enebo, L., 102,125 Enevoldsen, B. S., 133,167 Engelbrecht, R. S., 213,232 Enger, M. D., 157,159,167 Ephrussi, B., 77,79,88,89 Eriksson, K.-E., 113, 114, 117, 123, 125 Erlanger, B. F., 61

Esau, P., 254,275 Eshelby, J . D., 136,167 Estrup, F., 63 Ettinger, M. B., 187, 213, 214, 216, 226, 232,233 Evan, J. B., 242,275 Evans, W. C., 182,183 Evers, M. A., 56,67 Everson, G., 134,167 Ezawa, K., 161,168 F

Faber, J. E., 60 Fadenko, P. S., 240,279 Fkhraeus, G.,92,102,110,121, 125 Falck, R., 106,125 Falk, G., 60 Farr, R. S., 51,56,64 Fasman, G., 61 Faust, L., 228,232 Fecsik, A. I., 58,64 Feduchy Marino, E., 240,245,275 Feingold, D. S., 133,168 Felix, A., 18,36 Felix, R. I., 10, 13,36 Fell, G., 236,244,245,251,271,275 Felton, E. A., 242,275 Felton, L. D., 42,64 Fernando, A. N., 45,64 Ferre, L., 239,270,275,279 Festenstein, G. N., 123,125 Fetner, R. H., 213,233 Fiala, A., 77,88 Fiala, S., 77,88 Fiedler, W., 35,36 Findlay, W. P. K., 108,125,147,167 Fink, M. A., 42,64 Finkelstein, R. A., 62 Finn, J., 43,63 Finstad, J., 41,66 Finstein, M. S., 219,232 Firman, M. C., 31,35 Fishburn, A. G., 11,36 Fitch, F. W., 54,66 Flaig, W., 108,126, 176,183 Flanigan, C. C., 70,71,89 Flanigan, J. F., 62 Flesch, P., 261,262,275 Flickinger, R. A., 60

AUTHOR INDEX

Flora, R. M., 112, 114, 116, 117, 127, 159, 166,168 Florenzano, G., 245,275 Ford, D. L., 217,229,232 Fornachon, J. C. M., 236, 240, 241, 242, 243, 244, 248, 249, 250, 252, 258, 259, 269,271,273,275,276,279 Forrester, P. C., 60 Forristal, J., 60 Fost, C. A., 62 Foster, J. H. S., 23,24,25,26,36 37 Fowler, I., 60 Frame, J. D., 187,212,233 Francis, T., Jr., 42,64 Fred, E. B., 253,278 Freeman, K., 77,89 Frei, J. V., 60 Frei, P. C., 49,64 French, D. W., 103,128 Freter, G. G., 53,66 Freudenberg, K., 95,126 Freund, J., 56,64 Frey-Wyssling, A., 96, 121, 126, 132, 142, 167 Fruton, J. S., 265,276 Fudenberg, H. H., 59 Fukuzumi, T., 173,175,181,183 Funatsu, M., 161,168 G

Gabel, L. F., 30,36 Gadd, O., 105,126 Gaisford, W., 53,66 Gaitskhoki, V. S., 75,76,89 Galivan, J. H., Jr., 261,274 Gall, D., 56,64 Gallin, J. I., 254,276 Gamble, C. N., 57,64 Garb, S., 42,52,62,64,65 Garcia, G., 48,64 Gardner, R. A,, 17,20,21,36 Garino-Canina, E., 240, 246, 249, 250, 2 76 Garnet, J. R., 16,36 Garnjobst, L., 87,88 Garrett, S. D., 103,126 Gamey, J. S., 40,43,64 Gascoigne, J. A., 92, 114, 126, 135, 155, 167

285

Gascoigne, M. M., 92, 114, 126, 155, 167 Gaudy, A. F., 213,222,232 Gaudy, E., 226,227,232 Cause, G. F., 69,78, 79,80,81, 82, 83,88, 89 Gawehn, K., 71,90 Geissler, A., 71,90 Geller, D. M., 255,276 Genetelli, E. J., 210,228,232 Gershenfeld, L., 23,24,25,26,27,37 Geschwind, I. I., 61 Chose, R., 109,125,155,167 Ghosh, B. L., 157,159,169 Gibbons, A. P., 137,167 Gibson, J., 218,232 Gilbert, R. J., 5,37 Gill, B. S., 56,64 Gilligan, W., 112, 113,126,128 Gini, B., 241,276 Ginns, J. H., 179, I83 Ginsberg, M., 27,37 Ginsburg, A., 253,276 Gioia, M. N., 240,278 Gitlin, D., 59 Glaser, L., 133,167 Glass, D. G., 19,37,38 Glenny. A. T., 54,64 Glimp, H. A., 61 Goebel, W. F., 42,63 Goi, H., 161,168 Goldman, L., 58,66 Gomer, R., 136,167 Good, R. A., 41,64,66 Goodfriend, T., 61 Goring, D. A. I., 95,126 Gottschall, R. Y., 57,64 Goudey, R. F., 224,232 Goullet, P., 55,64 Goverde, B. C., 61 Grabar, P., 60 Graffi, A., 77,89 Graham, B., 17,21,37 Graham, D. M., 63 Grandchamp, S., 87,89 Gray, J. G., 59 Greaves, H., 103,104,126 Greeley, S. A.,210,211,213,232 Green, D. E., 86,88 Greenberg, A. E., 187,212,232,233

AUTHOR INDEX

Greenberg, L., 18,36 Grgich, M., 273,279 Grimes, R. M., 112,126 Grohn, H., 147,167 Gromet, Z., 133,134,168 Grundy, W. E.,2,11,37,38 Guillot, M., 9,37 Guimarais, A. F., 240,270,277 Guimberteau, G., 258,278 Guld, J., 22,37 Gundelfinger, B. F., 50,66 Gundersen, K., 78,89,108,126 Gundry, H. A., 263,279 Gunn, C., 12,36 Gunsalus, I. C., 255,256,275,276 Gurvich, A. E., 56,65 H

Haber, E., 60 Hachiga, M., 124,126 Hadgraft, J. W., 14,37 Hager, L. P., 255,276 Haider, H., 108,126 Haldar, D., 77,89 Hale, R. D., 100,129 Hall, C. B., 101,126 Hall, D. A., 138,168 Halliwell, G., 112, 116, 120, 126, 162, 168 Halloran, M. J., 62 Halsall, K. G., 24,37 Hammarstrom, A., 103,128 Hana,I.,63 Handa, N., 124,128 Hanson, K. R., 115, 121, 123, 125, 130, 157,161,167,170 Hanstein, E., 113,126 Hanstein, E. G., 113,126,161,165,168 Happey, F., 138,168 Hara, T., 161,168 Harada, H., 142,170 Harary, I., 266,276 Hardy, 0.H., 63 Harmsen, L., 102,126,154,168 Harris, F. W., 208,232 Harris, H. H., 187,234 Harris, S., 59 Harris, T. N., 59 Harris, W., 34,37 Harrison, M. E., 228,232

Hartig, R., 104,126 Hartley, R. D., 260,276 Harvey, R. J., 256,269,275,276 Harvie, N. R., 60 Hasek, M., 51,64 Haseltine, T. R., 202, 206, 207, 209, 212, 214,218,232 Hashimoto, Y., 115,124,126,128 Hassid, W. Z., 99, 100, 125, 128,129, 133, 137,167,168,254,278 Hata, K., 108,126 Hawkes, H. A., 228,232 Hay, M., 59 Hayashi, K., 124,126,161,168 Hayward, A. C., 245,276 Haywood, R. W., 206,233 Heath, E. C., 253,276 Heidelberger, M., 52,54,64,66 Heidig, C. P., 37 Heller, P., 60 Helmers, E. N., 187,212,233 Henderson, C. N., 213,234 Henderson, M. E. K., 182,183 Herbst, J., 60 Hermann, E. R., 207,234 Hernandez, A., 240,276 Hess, B., 72,88 Hess, H., 10, 12, 13, 14,37 Hestrin, S., 132,133,134,137,168,169 Heukelekian, H., 195, 201,202, 208, 210, 211, 213, 216, 219, 221, 227, 228, 232, 233 Heyn,A. N. J., 97,126,132,168 Higgins, H., 260,276 Higuchi, T., 173,183 Hilleman, M. R., 39,44,51,64,65 Hingson, R. A., 44,65 Hirata, A. A., 48,65 Hirsch, H. M., 78,90 Hjorth, N., 33,37 Hochwald, G. M., 62 Hodgson, R., 20,36 Hohnl, G., 226,233 Hohorst, H.-J., 245,261,276 Holbach, B., 261,262,275 Holm, 181,183 Homan, J. D. H., 61 Hoobler, S. W., 60 Hook, W. A., 60

287

AUTHOR INDEX

Hopkins, S. J., 13, 14,23,37 Hoppert, C. A., 112,126 Horecker, B. L., 100,126,253,276 Horsfall, J. G., 111,126 Horton, J . C., 110, 111,126,157,168 Hosoi, M., 79,89 Hottinguer, H., 79,88 Houba, V., 63 Hsieh, B., 62 Hsiung, G. D., 63 Hugo, W. B., 24,37 Humphrey, A. E., 166 Hungate, R. E., 110,126 Hunter, J., 72,88 Hunvitz, J.. 253,276 Husa, W. J., 31,37,38 Husain, A., 102,103,126 Husfeld, B., 245,276 Hiiskens, C., 268,279 Hyde, R. M., 42,52,64,65 I

Ichihara, K., 171,183 Ichinose, K., 161,169 Ikeda, R., 161,168 Ikeda, S., 171,183 Imada, I., 157,168 Imai, S., 246,278 Ingols, R. S., 210, 211, 213, 216, 219, 233 Ingraham, J. L., 240, 242, 243, 244, 246, 250,251,259,270,271,276,277,279 Inove, K., 60 Ipsan, J., 21,36 Ipsen, J., Jr., 42,65 Irmann, R., 258,274 Irwin, M. R., 59 Isacson, P., 63 Isenberg, E., 208,216,233 Ishikawa, T., 160,169,173,175,183 Ito, T., 141,169 Ivanyi, J., 41,47,65 Iwazaki, T., 161,168 J

Jackson, A. L., 63 Jacob, F., 111,126 Jacobs, J., 48,65 Jaconia, D., 34,36

Jacot-Cuillarmod, H., 56,64 Jakob, H., 87,89 James-Witte, J., 45,64 Jansson, C., 113,114,125 Jarrett, W. F. H., 44,65 Jemski, J. V., 46,65 Jensen, R. S., 59 Jerchel, D., 268,279 Jermyn, M. A., 158,162,168 Johansson, M., 121,126 Johnson, A. C., 60 Johnson, M. K., 254,276 Jones, E. B. C., 106,126 Jones, K. M., 264,276 Jones, P. H., 228,233,234 Jonsson, J., 62 Julia, J. F., 53,65 Juni, E., 255,276 Juialek, L., 143, 146, 148, 149, 150, 152 164,165,168,169,170 K

Kiarik, A., 181,183 Kachmar, J. F., 215,224,226,234 Kadota, H., 102,126 Kaeberle, M. L., 53,66 Kagami, H., 240,277 Kaiser, A. D., 41,64 Kaiser, H. E., 82,89 Kaiser, S. J,, 52,64 Kaminski, M., 62 Kaplan, N. O., 124,126 Kasukawa, R., 60 Katsh, G. F., 62 Katsh, S., 62 Kaufman, S., 260,261,268,276 Kaufman, W. J., 212,232 Kaufmann, H., 55,64 Kawamura, I., 173,183 Kawamura, K., 160,169,173,183 Kayser, D., 71,W Kazakova, T. B., 75,76,89 Kazal, L. A., 40,66 Kazama, K., 240,277 Kedzia, W., 4,37 Keefer, C. E., 202,215,233 Keefer, R. M., 264,269,274 Keegan, H. L., 62 Keen,N.T., 110,111,126,157,168

288

AUTHOR INDEX

Keleman, M. V., 123,126 Keller, S., 48,67 Kelman,A., 102,126,127,181,183 Kemp, D., 61 Kendrick, P. L., 57,64 Kenney, D. S., 11,37 Kerner, E., 259,271,275 Ketchel, M. M., 46,155 Kharasch, M. S., 263,276 Kielland, J., 265,276 King, K. W., 92, 108, 110, 112, 114, 116, 117, 118, 127, 156, 159, 165, 166, 167, 168 Kirchmeyer, F. J., 2, 11,38 Kiselevskaya, R. M., 240,279 Kitahara, K., 255,279 Klarmann, E. G., 10,37 Klein, G., 62,212,232 Klein, M., 26,27,36,37 Kliewer, W. M., 248,277 Klima, J., 86,89 Klop, W., 165,168 Klungsoyr, S., 133,168 Knight, B. C. J. G., 227,233 Koaze, Y., 161,168 Koch, A., 238,239,243,247,248,271,273, 276 Kochan, I., 59 Kochetkova, G. V., 79,80,81,82,83,89 Kohan, S., 7,37 Kohli, J. D., 5,37 Kohn, S. R., 23,24,25,26,27,37 Kokumai, K., 159,168 Kolar, G. F., 260,276 Komberg, A., 260,277 Konno, N., 157,159,168 Kononova, M. M., 172,177,183 Kooiman, P., 165,168 Korey, S. R., 266,276 Korkes, S., 260,261,262,268,274,276 Koski, T., 24,36 Kostenbauder, H. B., 34,37 Kovachevich, R., 78,79,89 Krasil’nikova, E. N., 268,276 Kraus, L. S., 202,206,209,211,219,233 Krebs, H. A., 263,266,275,276 Krumperman, P. H., 237,276 Kulisch, P., 237,238,239,276 Kunkee. R. E.. 240. 243. 245. 246. 249.

250, 256, 257, 259, 270, 271, 272, 273, 276,278 Kupchyk, L., 51 Kuramoto, R., 30,34,38 Kurata, Y., 60 Kuryanova, G. V., 240,279 Kiirschner, K., 147,168 Kwapinski, J. H., 63 1

Lach, J. L., 13,37 Lackey, J. B., 191,201,225,226,227,229, 233,234 Lafon-Lafourcade, S., 243,247,258,278 Lagerberg, T., 104,127 Lamb, J. C., 224,232 Lambion, R., 236,243,277 Lamporte, D. T. A., 93,127 Landi, S., 21,22,37,38 Landsteiner, K., 48,65 Landy, M., 63 Lange, P. W., 144,168 Lanz, H. C., 48,67 Larsen, W. A., 223,233 Lascelles, J., 85,89 Law, K., 181,183 Lawrence, C. E., 23,24,25,26,27,37 Lawson, G. J.. 260,276 Lawson, L. R., 178,183 Leagus, M. B., 44,65 Leathenvood, J. M., 157, 168 Lebovitz, H., 56,65 Ledbetter, M. C., 99,127 Lee, C. K., 253,277 Lee, C. O., 35,36 Lehninger, A. L., 73, 74, 76, 84, 89, 263, 277 Lehrer, H. I., 61 Leibowitz, H. M. I., 45,66 Lengerova, A., 51,64 Lentz, K., 254,274 Lepper, M. H., 46,65 Leskowitz, S., 53,65 LeVeen, H. H., 60 Levi, M. P., 104,105,127,152,154,168 Levi-Montalcini, R., 61 Levine, L., 59,61,63 Levinson,H. S., 113,115,121,128,129 Levvy, G. A., 123,125

AUTHOR INDEX

Levy, J., 106,127 Levy, J. F., 103, 104,126,127 Levy, R. P., 61 Lewis, G. N., 263,267,277 Lewis, Y. $.,60 Lewon, J., 4,37 Li,L.-H., 112, 114, 116, 117,127,159, 166, 168 Lichstein, H. C., 255,277 Liese, W., 104, 106, 107, 127, 129, 143, 145, 146, 147, 148, 150, 151, 152, 154, 168 Lillie, S., 25,26,27,36 Lim, S., 108,126 Lin, F. H., 78,88 Lin, T.-Y., 99,129 Lindberg, B. P., 98,127 Lindeberg, G . , 108,121,125,127 Lindegren, C., 85,89 Lindegren, G., 85,89 Lindgren, L. B., 32,38 Lipmann, F., 254,255,256,276,277,279 Littlejohn, 0. M., 31,37 Littman, M. L., 220,233 Lloyd, B., 259,276 Lloyd, P. F., 138,168 Lockwood, L. B., 257,264,277 Loewi, G., 60 Logan, R. P., 208,216,233 Loginova, L. G., 157,168 Lomander, L., 78,89 Long, M. V., 261,275 Loomis, A. G . ,266,277 Loosemore, M., 10,37,38 Lopez-Saez, J. F., 142,167 Lorenz, S., 71,90 Luderitz, O., 19,37 Ludzack, F. J., 201, 212, 213, 214, 216, 226,232,233 Lugg, J. W. H., 259,277 Lundberg, G . , 104,127 Luthi, H., 247,248,259,269,273,277 279 Lyndeberg, 181,183 Lyr, H., 104, 107, 108, 109, 110, 113, 121, 127,157,158,168,173,183 M

McCall, M. S., 48,67 McCarty, M., 63

289

McCleskey, C. S., 254,276 McCollum, J. P., 101,125 McComb, J. A., 21,36 McCombs, C. L., 103,130 McConahey, P. J., 56,64 McDermott, G. M., 187,213,214,232 McDevitt, H. O., 43,65 McEwan, J. S., 13,14,37 MacFarlane, J. O., 20,36 McGarry, E. E., 61 MacGregor, D. R., 26,37 Machlowitz, R. A., 51,64 McIver, A. K., 24,37 McKinney, R. E., 187,191,233,234 Mackler, B., 87,89 Maclachlan, G. A., 101,127 McLachlan, J., 177,183 McLaren, A. D., 165,169 McLean, I. W., 19,21,37,56,67 MacLeod, C. M., 52,64 MacMorran, G. H., 13,14,37 McNary, R. R., 215,232 Macrae, R. M., 227,233 Madison, L. L., 48,67 Maejima, K., 159,169 Magis, J. M., 46,67 Magnus, K., 22,37 Magnusson, M., 22,37 Mahler, H. R., 87,89 Maitland, C. C., 119, 121, 122, 127, 129, 162,164,169 Makinodan, T., 51,55,57,65 Malm, M., 254,274 Malmgren, R. M., 70,71,89 Mandels, M., 101, 110, 115, 116, 123,127 128,129,156,159,169 Mange, A. P., 62 Manley, R. St. J., 96, 97, 122, 127, 132, 168 Mantyjarvi, R., 45,65 Marcovich, H., 79,89 Margerie, C., 101,127 Marques Gomez, J. V., 240,270,277 Marr, A. G., 251,277 Marsh, P. B., 112,114,127 Marx-Figini, M., 95, 96, 97, 100, 127, 138, 169 Mascoli, C. C., 4 4 6 5 Masquelier, J., 252,277

290

AUTHOR INDEX

Matchett, W. H., 101,127 Matheson, A,, 59 Matsurama, C., 159,169 Matthijsen, R., 61 Mathijsen, R., 61 Maumenee, A. E., 45,66 Maurer, P. H., 42, 43, 48, 49, 50, 52, 56, 65,66 Mayer, K., 260,273,277 Medawar, P. B., 49,65 Meer, W. A., 35,38 Mehler, A. H., 260,277 Meier, H., 93, 94, 95, 98, 106, 127, 144, 147,148,149,151,154,169 Meisel, J., 215,233 Melamed, N., 254,268,277 Melin, E., 104,111,127,128 Melnick, J. L., 20,37 Menzin, A. W., 39,65 Menzoian, J. O., 46,65 Mercer, N. H., 31,38 Mergenhagen, S. E., 56,65 Merola, G. V., 112,114,127 Merrill, W., 103, 108,128 Meskhi, A,, 236,243,277 Mestrezat, W., 238,239,277 Meynell, E., 244,277 Meynell, G. G., 244,277 Mickle, F. L., 248,277 Middlebrook, G., 45,65 Mifuchi, I., 79,81,89 Miles, A. A., 41,67 Milgrom, F., 60 Milisavljevic, D., 240,245,277 Miller, J. F. A. P., 55,65 Miller, T. A., 44,65 Millis, N. F., 166 Millman, B., 132,133,136,139,169 Millwood, E. G., 27,37 Mitchell, J. A., 243,279 Mitchell, R., 108,128 Miyawaki, G. M., 34,37 Moat, A. G., 255,277 Moeller, G., 53,65 Mohlman, F. W., 190,207,233 Monaco, A. P., 59 Monakhov, N. K., 75,89 Monier, R., 77,89 Monod, J., 111,126

Montgomery, W. F., 24,37 Moore, A. T., 93,129 Moore, A. W., 176,183 Moore, W. E. C., 245,275 Moreau, L., 239,277 Moreland, A. F., 40,43,65 Morgan, E. H., 210,211,225,233 Morgan, H. R., 18,37 Morimoto, I., 124,128 Morita, T., 79,81,89 Morris, G. K., 249,277 Morris, H. P., 71,89 Morse, S. I., 63 Moslinger, 238,277 Mounolou, J. C., 87,89 Moustacchi, E., 79,87,89 Mueller, A., 55,67 Mueller, W. H., 30,37 Muhlethaler, K., 96,97,128,132,142,167, 169 Muir, H., 60 Mulder, E. G., 222,226,233 Miiller-Thurgau, H., 238,239,241,277 Munkres, K. D., 86,90 Munoz, J., 19,21,38,56,65 Murakami, W. T., 63 Murphy, D., 176,183 Murray, E. G. D., 241,242,243,245,250, 257,274 Murray, H. L., 31,38 Muschel, L. H., 60 Myers, F. L., 113,128 Myers, J., 60 Myers, M. G., 157,169

N Nair, A. D., 13,37 Nakamae, K., 141,169 Nakayama, S., 124,128 Nance, J. F., 101,127 Nannfeldt, J. A., 105,128 Nassar, A. R., 248,277 Nathan, H., 262,277 Navratilova, Z., 146,169 NeEesany, V., 143,169 Neeper, C. A., 51,65 Neess, J., 55,67 Neidig, C. P., 31,37 Neifakh, S. A., 75,76,89

291

AUTHOR INDEX

Neish, A. C., 95,128 Nell, E. E., 63 Nerd, F. F., 108,126 Nettesheim, P., 51,57,65 Neufeld, E. F., 133,168 Ng, H., 251,277 Nicholson, M. J., 19,38 Nilsson, H., 111,128 Nishida, M., 79,89 Nisizawa, K., 115, 124, 126,128, 160,169 Nissen, T. V., 102,126,154,168 Niven, C. F., Jr., 242,246,258,275,277 Niwa, T., 160,161,169 Nobles, M. K., 178,181,183 Noe, R. W., 96,128 Nonomura, H., 240,277 Nord, F. F., 173,183 Nordstrom, K., 259,277 Norkrans, B., 92, 101, 103, 110, 111, 112, 113, 114, 115, 116, 122, 123, 124, 125, 128,156,158,169 Norris, J. R., 63 Northcote, D. H., 93,94,98,99, 113,128 Northey, W. T., 57,65 Norton, D. A,, 24,25,26,28,36 Nossal, G. J. V., 48,49,51,65 Nossal, P. M., 249,277 Notkins, A. L., 56,65 Novak, E., 104,127 Nussberger, F. E., 214,232 0

O’Brien, R. A., 54,64 Ochoa, S., 260, 261, 262, 266, 274, 276, 277 Ogawa, K., 160,169 Oglesby, R. T., 173, 174, 175, 176, 177, 183 Ogur, M., 85,89 Ohad, I., 100, 125, 132, 137, 139, 167, 169 Ohara, Y., 240,277 Ohnishi, To., 75,89 Ohnishi, Ts., 75,89 Okabe, K., 84,90 Okada, G., 160,169 Okada, S., 60 Oki, T., 173,175,183 Okumoto, M., 26,38

Okun, D. A., 216,221,233 Old, L. J., 62 Olovnikov, A. M., 56,65 Olson, B. H., 17,2137 Olson, V. H., 45,66 Ohnishi, T., 75,89 Ono, T., 71,89 Ordonneau, C., 237,277 Orford, H. E., 208,216,233 Ortega Tello, H., 240,276 Osborn, J. J., 53,65 Ostenvalder, A., 239,241,277 Otsuka, K., 246,278 Otto, R. H., 11,37 Ouchterlony, O., 58,66 Ough, C. S., 251, 257, 270, 271, 272, 273, 2 76,2 78 Ovary, Z., 59,62 Overell, B. T., 259,277 P

Pace, M. G . ,54,66 Pal, P. N., 157,159,169 Palczuk, N. C., 49,66 Paleus, J., 62 Palmer, J. R., 206,233 Panda, B., 57,66 Panghorn, R. M., 259,278 Papermaster, B. W., 41,64,66 Pappenheimer, A. M., 54,66 Parish, H. J., 18,37 Parker, C. W., 62 Parks, J. J., 45,66 Paronetto, F., 56,613 Pasteur, L., 229,233,237,278 Patel, N. K., 34,37 Paul, W. E., 49,66 Pearce, J. H., 50,64 Pearse, L., 201,233 Peaud-Lenoel, C., 101,127,128 Pederson, C . S., 248,279 Perkins, E. H., 51,57,65 Perkins, F. T., 53,66 Perlin, A. S., 115,128 Pernis, B., 56,66 Perrault, J., 101,127 Pesigan, T. P., 62 Peterson, J. C., 58,66 Peterson, W. H., 253,278

292

AUTHOR INDEX

Peterson, W. J., 55,65 Petit,]. F., 77,89 Pettersson, G., 115, 117, 123, 125, 128, 159,161,169 Pew, J. C., 145,169 Peynaud, E., 236, 239,240,241,243,244, 245, 246, 247, 250, 251, 256, 257, 258, 259,266,268,270,271,273,275,278 Pfeffer, C. R., 78,88 Pfleiderer, G . ,245,278 Phaff, H., 77,90 Phares, E. F., 261,275 Phillips, G. B., 46,65 Phillips, J . H., 41,66 Phillips, R. A., 227,232 Pickett-Heaps, J. D., 94,98,99,128 Pilch, Y. H., 62 Pillai, S. C., 201,224,234 Pilone, G. J., 240,243,245,246,249, 250, 256,258,259,270,276,278 Pinchuck, P., 42,43,49,66 Pinkerton, W., 4 5 6 6 Pipes, W. O., 185, 201, 207, 218,228,234 Pitot, H. C., 70,71,72,85,89 Pittman, D., 85,89 Pittman, M., 17,20,21,36,37 Pivnick, H., 19,22,37,38 Plescia, 0.J., 49,66 Poittevin, M. E., 240,278 Pollock, M. R., 111,128 Pope, C. G., 61 Porath, J., 115,117,128, 161,169 Porter, B. R., 112,128 Porter, K. R., 99,127 Potter, V. R., 71,73,89 Powell, H. M., 20,36 Pressman, D., 60 Preston, R. D., 101, 104, 105, 127, 128, 152,154,168 Prickett, P. S., 31,38 Pridham, J. B., 99,128 Pringsheim, E. G . ,228,234 Proom, H., 227,233 Puls, D. D., 32,38 Purdy, W. C., 202,213,234 Q

Quinn, V. A , , 42,64 Qusten, K. F., 60

R

Racker, E., 133,169 Radler, F., 236, 242, 243, 246, 247, 248, 249,257,259,268,273,278 Rajam, P. C., 60 Ranby, B. G.,96,100,113,128,129 Rancourt, M. W., 78,88 Randall, M., 263,267,277 Rankine, B. C., 236, 250, 255, 256, 271, 272,278 Rappoport, D. A., 254,278 Ravnick, A., 7,38 Raymond, S., 56,67 Rdzok, E. J., 2, 11,38 Reese, E. T., 101, 108, 110, 112, 113, 115, 116, 121, 123, 126, 127, 128, 129, 155, 156,158,159,164,166,169 Reeve, P., 63 Reichlin, M., 59 Reilly, C., 85,89 Reinhart, H., 44,65 Repin, V. S., 75,89 Ribereau-Gayon, J., 236, 239, 240, 241, 245, 246, 250, 251, 256, 257, 258, 259, 268,270,273,275,278 Rice, A. C., 240,278 Richards, R. M. E., 24,26,28,36,38 Richter, M., 60 Ridenour, G . M., 213,234 Riegelman, S., 26,38 Rigler, N. E., 31,35 Riley, H. D., Jr., 39,66 Rishbeth, J., 179,183 Ritter, G., 111,129,157,169 Rizzo, A. A., 56,65 Robbins, K. C., 60,62 Robinson, B., 52,64 Robson, J. M., 27,37 Roelofsen, P. A., 93,98,106,129,132, 137, 138,139,140,141,169 Rogosa, M., 243,244,249,254,279 Rollins, M. L., 93,112,128,129 Rose, A. H., 252,255,279 Rose, B., 60 Rosen, M., 44,65 Rosenstiehl, A., 239,279 Rothbard, S., 60 Rouf, M. A., 226,234 Roughton, F. J. W., 266,276

AUTHOR INDEX

Rousseaux, E., 239,279 Rowatt, E., 255,279 Rowley, D. A., 54,66 Roy, C., 165,170 Royce, A., 8,9, 12, 13, 14,38 Ruchhoft, C. C., 190, 215, 224, 225, 226, 234 Rudman, P., 108,129 Rudney, H., 84,90 Ruesink, A. W., 124,129 Rumke, P., 62 Rushworth, M. A., 60 Russell, A. D., 5, 10,25,37,38 Russell, P. S., 59 RypaEek, V., 145,147,169

S Sabar, N., 108,128 Sachs, I. B., 145,169 Saenko, N. F., 240,279 Sakamoto, Y., 171,183 Sakurada, I., 141,169 Salotto, B. V., 187,213,214,232 Salvin, S. B., 63 Sampliner, J., 61 Sang, J. H., 57,66 Santer, M., 63 Sarkanen, K. V., 171,179,183 Sarukhanova, L. E., 79, 80, 81, 82, 83, 89, 90 Sassen, M. M. A., 101,129 Saunders, L., 7,38 Saville, G., 263,279 Savory, J. G., 104,129 Sawyer, C. N., 187,198,212,233,234 Saxl, H., 138,168 Schaffer, R. B., 212, 213, 214, 216, 226, 233 Schambye, P., 254,274 Schanderl, H., 236,263,279 Schani.l,L., 109,127,157,158,168 Schatman, B., 60 Schatz, G., 86 89 Schechter, B., 61 Scheele, C., 56,65 Schimmel, S.,31,38 Schmid, R., 104, 129, 146, 147, 148, 150, 151,152,154,168 Schmidt, H.-L.,263,266,268,279

293

Schneider, E. S., 77,89 Schramm, M., 132,133,134,168,169 Schubert,W. J., 108,126,173,183,184 Schuchardt, L. F., 19,21,38 Schukow, I., 238,279 Schulloff, H. B., 213,234 Schulz, G. V., 95, 96, 97, 100, 127, 138, 169 Schulze, K. L., 228,234 Schuurs, A. H. W. M., 61 Schwartz, R. S., 57,66 Scott, W., 210,234 Searcy, R. L., 59 Seastone, C. V., 51,65 Segre, D., 53,66 Seifert, K., 92,104,105,129 Seifert, W., 238,243,247,258,279 Sela, M., 43,50,65,66 Selby, K., 112, 119, 121, 129, 162, 164, 165,169 Sen Gupta, P. N., 5,38 Setterfield, G., 93,129,134,137,169 Sharp, N. C. C., 44,65 Sharpe, M. E., 242,244,249,254,275,279 Sherman, F., 78,85,86,89 Shiao, D., 263,279 Shibata, T., 113,129 Shirakawa, T., 255,279 Shive, R. A., 227,234 Short, P., 14,37 Shotton, E., 7,38 Shur, I. M., 240,279 Sibal, L. R., 45,66 Sidransky, H., 71,89 Siebenmann, C. D., 22,38 Sillen, L. G., 92,129 Simmonds, S., 265,276 Simpson, M. E., 112,114,127 Sims, G . ,62 Singer, P. C., 207,234 Singleton, V. L., 236,247,274 Siskind, G. W., 49,66 Siu, R. G. H., 92, 113, 115, 122,129, 155, 169 Skeggs, H. R., 249,279 Skerman, V. B. D., 226,234 Skinner, F. A., 102,129 Sleeper, B. P., 157,159,167 Slonimski, P. P., 77,78,86,87,89,90

294

AUTHOR INDEX

Sluyser, M., 62 Smart, C. L., 138,170 Smit, J., 210,225,228,234 Smith, D. E., 60 Smith, J. H., 50,66 Smith, M. W., 209,211,234 Smith, N. R., 241, 242, 243, 245, 250, 257, 274 Smith, R. S., 190,203,213,234 Smith, R. T., 51,66 Smolens, J., 40,66 Snell, E. E., 244,248,279 Sobey, W. R., 57,61,66 Sopko, R., 143,169 Sorensen, H., 182,184 Soulsby, E. J. L., 44,66 Sowden, L., 140,167 Speckman, R. A., 256,279 Spiegal, M., 211,233 Spiro, R. G., 59 Spitzer, R., 60 Spragg, J., 60 Stafford, E. S., 58,66 Stahl, W. H., 112,125 Stahmann, M. A,, 103,125 Stamer, J. R., 248,279 Stanier, R. Y., 102,129 Staub, A. M., 19,37 Steele, B. F., 42,64 Stein, A. A., 62 Stephens, J. M., 41,66 Stevens, K. M., 50,62,66 Stevens, M. G., 104,127 Stewart, C. M., 95,129 Stewart, M. J., 208,234 Stier, T., 77,90 Still, C. N., 178, 183 Stille, W. T., 50,66 Stjernsward, J., 55,67 St. John-Brooks,R., 50,66 Stokes, J., Jr., 40,44,65,66 Stokes, J. L., 226,234 Stone, B. A., 108, 119, 125, 129, 159, 166, 167 Stone, R. L., 20,36 Stone, W. H., 59,62 Stoner, R. D., 53,66 Strasdine, G. A., 157,169 Subrahmanyan, V., 201,224,234

Sudraud, P., 271,272,275,279 Sugimura, T., 84,90 Sulzberger, M. B., 47,66 Sulzer, F. T., 224,232 Sumi, Y., 100,129 Summaria, L., 60 Sussdorf, D. H., 40,43,46,48,64,65 Sutherland, I. W., 17,38 Sutliff, W. D., 42,64 Suverkrop, B., 236,240,259,270,279 Swafford,W. B., 35,38 Sykes, G . ,4,8,9, 10, 12, 13, 14,32,38 Sylvester, J. C., 2, 11,38 Symons, J. M., 187,234 T

Takebe, I., 255,279 Takeda, R., 124,128 Taliaferro, L. G . ,58,66 Taliaferro, W. H., 58,66 Talmage, D. W., 53,66 Tamanoi, I., 60 Tanaka, T., 161,169 Tao, T., 53,66 Tapleshay, J. A., 213,234 Tarantola, C., 240,279 Tashpulatov, Z., 157,168 Tatum, E. L., 87,88 Taub, A., 35,38 Taverne, J., 63 Taylor, E. W., 6,38 Tchelistcheff, A., 236, 240, 242, 258, 259, 270,271,273,279 Teller, M. N., 55,63 Tempelis, C., 55,67 Tempelis, C. H., 41,66 Terres, G., 53,66 Tervi,G., 116,125,157,159,168 Thimann, K. V., 124,129 Thomas, H. A., 207,233 Thomas, R., 110,130,157,170 Thompson, K. V. A., 119, 129, 162, 164, 169 Thompson, L. D., 61 Thompson, R., 45,66 Thomson, A., 53,66 Thorbecke, G. J., 49,62,64,66 Tice, L. F., 29,30,36 Tillett, W. S., 42,64

295

AUTHOR INDEX Tillman, A. D., 61 Tillman, W. J., 30,34,38 Timell, T. E., 93,95,126,129 Tisdale, H., 86,88 Tkachenko, N., 226,234 Tocantins, L. M., 40,66 Tomoda, K., 157,168 Tonduz, P., 240,279 Torpey, W. N., 208,234 Toyama, N.; 113, 115, 117, 124,128,129, 160,169 Tracy, J. M y ,19,37,38 Trader, F. W., 56 Trapani, I. L., 57,66 Treffers, H. P., 54,66 Treiber, E., 95,129 Trenina, G. A., 79,90 Triplett, E. L., 41,66 Tripp, V. W., 93,112,128,129 Trolle Larsen, C., 33,37 Troutman, H. D., 56,67 Tuppy, H., 86,87,89,90 Turner, B. G., 222,232 Turner, T. B., 58,66 Turovski, V. S., 75,89 Tytell, A. A., 51,64,67 U

Ueda, Y., 157,168 Uhr, J. W., 52,66,67 Ulbrich, M., 245,279 Ullrich, A. H., 209,211,234 Umbreit, W. W., 172, 184 Unestam, T., 103,129 Unger, R. H., 48,67 Utter, M. F., 256,279 V

Valentova, V., 41,47,65 Van Demark, P. J., 254,276 vanden Hamer, J. A., 253,275 van Hell, H., 61 van Hemert, P., 17,38 van Heyningen, W. E., 6 3 van Veen, W. L., 226,233 van Vunakis, H., 61 van Wezel, A. L., 17,38 Vassiletz, I. M., 75,89 Vaughan, D. G., 26,38

Vaughn, R. H., 236, 237, 241, 242, 243, 244, 245, 246, 250, 258, 270, 274, 276, 2 79 Verkhovtseva, V. I., 157,159,169 Verona, O., 245,275 Verwey, W. F., 19,21,38 Vestal, M. R., 105,125 Vesterberg, O., 117,125 Vetsch, U., 247,248,269,277,279 Villemez, C. L., 99,129 Vladimirova, G. B., 79,80,81,82,83,89 Vladoianu, I. R., 62 Vogt, A. B., 40,66 Vojtiskova, M., 51,64 Volk, V. K., 57,64 von Brand, T., 260,276 W

Waaler, H., 22,37 Wada, S., 157,168 Wagener, W. W., 108,129 Wagner, B. P., 71,89 Wahlstrom, L., 123,128 Waitz, S., 226,234 Wakazawa, T., 161,169 Waksman, B. H., 39,63 Waldmann, T. A., 56,65 Wallace, A. L. C., 61 Wallace, R., 18,36 Wallis, C., 20,37 Walseth, C. S., 113,116,129 Walther, W. W., 27,37 Wang, C. H., 134,170 Warburg, O., 70,71,90 Wardrop, A. B., 100,129,142,170 Warren, B., 60 Wasserman, E., 63 Watkins, J. H., 225,234 Watson, R. F., 60 Wattie, E., 201,225,226,227,229,233 Webb, A. D., 243,245,249,250,256,270, 278 Webb, R. B., 243,244,246,270,271,279 Webb, T. E., 135,170 Webber, M., 45,66 Webster, R. G., 46,67 Wedderburn, D., 34,38 Weibel, R. E., 44,65 Weichlein, R. G., 191,233

296

AUTHOR INDEX

Weidanz, W. P., 63 Weigle, W. O., 61 Weintraub, M., 56,67 Weisberg, E., 201,213,219,233 Werkman, C. H., 256,279 West, C. D., 60 Westphal, O., 19,37 Wetterlow, L. H., 21,36 Wheeler, M. W., 17,36 Whelan, W. J., 123,126 Whelton, R., 77,90 Whistler, R. L., 138, 170 Whitaker, D. R., 110, 113, 115, 118, 121, 123, 125, 126, 129, 130, 157, 158, 161, 165,167,168,169,170 White, G. A., 134,170 White, R. G., 39,56,67 Whitehead, R., 7,37 Whiting, G. C., 244,262,279 Whittemore, F. W., Jr., 62 Whittenbury, R., 242,279 Wigzell, H., 54,55,67 Wilcox, W. W., 104,130,179,181,184 Wildner, G., 87,90 Wilhoit, R. C., 263,279 Wilkinson, J. F., 227,233,252,255,279 Wilkinson, J. H., 124,130 Williams, R. C., Jr., 60 Williams, W. C., 58, 66 Williams, W. L., 249,277 Williamson, D. H., 87,89 Wilner, B. I., 56,67 Wilson, G . S., 41,67 Wilson, T. H., 261,264,275 Wing, W. T., 8,38 Winogradsky, S. N., 103,130 Winstead, N. N., 103,130 Wiseman, R. F.; 243,279 Wisniewski, T., 4,37 Wolfe, E. K., 46,65,67

Wolfe, H. R., 55 Wolfe, R. S., 134,167,226,227,228,232 Wolins, W., 53,66 Wolstenholme, G. E. W., 61 Wood, H. G., 254,274 Wood, M. L., 59 Wood, R. K. S., 92,130 Wood, W. A,, 253,254,255,279 Woods, M., 72,88 Woodward, D. O., 86,90 Woolridge, R. L., 50,66 Work, T. S., 77,89 Wortmann, J., 238,279 Wright, L. D., 249,279 Wu, H., 62 Wuhrmann, K., 221,222,234 Wyss, O., 252,279 Y

Yagi, Y., 60 Yakulis, V. J., 60 Yamada, Y., 161,168 Yamashiroya, H. M., 46,67 Yanagihara, Y., 79,89 Yardley, B. J., 41,66 Yatoko, J., 7,38 Ycas, M., 86,90 Yetts, R., 53,66 Yoder, D. E., 257,264,277 Yotsuyanagi, Y., 86,90 Youatt, G . , 124,130,166,170 Youngner, J. S., 56,67 2

Zaidela, F., 77,89 Zickler, F., 248,279 Ziegler, H., 107,127 Zienty, M., 257,264,277 Zimmering, P. E., 61 Zimmermann, M. H., 141,170

Subject Index A

Absorption of bactericides by rubber, 8 Acetoin, 256 Acriflavine 5-fluorouracil, 81 Activated sludge, 185 applied research on, 185 bulking of, 185,201,204 compaction problems, 201 competition for soluble substances, 220 deflocculation, 195 density problems, 196 dispersed growth, 193 dissolved oxygen, 216 floc formation problems, 191 inorganic content, 211 operating problems, 187 pH, 215 plant operation, 204 protozoan hypothesis, 223 settling problems, 189 temperature, 215 Active immunity, 15 Adjuvants in antibody formation, 56 Aeration tank environment, 214 Age of animal, antigen response, 54 Alcohol(s), 14 malo-lactic bacteria, 250 Alum-precipitated toxoid, 19 Amber glass, 8 Anaerobic sludge, 198 Antibacterial agents in immunological products, 16 Antibiotics, 1,39 Antibody production effects of route of immunization on, 46 factors affecting antibody production, 47 genetic, 42 Anticoagulants, 40 Antigen quality, 47 Antimicrobial agents, 1 Antisera, 16 Antiserum production, 39 animal species employed in, 41 routes of inoculation employed in, 43 Artificial acquired immunity, 15

Assay of cellulolytic enzymes, 112 Autoclave sterilization, 4 AIV, 219 B

Bacillus as bulking agents, 227 Bacterial equilibrium hypothesis, waste disposal, 224 Bacterial vaccines, 16 Bactericide, 6 , 9 Bacteriostats, 5 Basidiomycetes, 106 Beggiatoa, 227 Benzalkonium chloride, 26 Benzethonium chloride, 19 Benzoic acid, 14,31 Benzyl alcohol, 14 Billowing sludge, 196 Biophysical strain hypothesis, waste disposal, 224 Biosynthesis of cell wall polysaccharides, 98 Bleeding of animals, 40 Blue-staining fungi, 104 Bovine serum albumin, solubilization, 48 Brown rot, 106,147 C

C’-HydrocelIulase, 116 Cambial tissues, 93 Cancer, 69 Cell wall morphology, plants and chemistry, 92 Cellulases, 109,155 applications, 124, 166 comparison of, 163 inhibition of, 122 mode of action, 115 production of, 157 purification of, 112,158 sources, 157 Cellulolysis, 91 Cellulolytic organisms, 102

297

298

SUBJECT INDEX

Cellulose chemistry, 95 degradation of, 92,101,145 bacteria, 102,154 course of, 165 fungi, 103 microbial, 92 fibers, 131 elongation of, 136 inhibition, 122 microfibrils, 132 biosynthesis of, 132 resynthesis of, 101 synthesis, 99 animal, 138 bacterial, 132 by green plants, 137 fungal, 138 Cellulosic fibers, 141 degradation of, 131,141 formation of, 131 microstructure of, 141 Chemistry of antigens, 49 Chlorbutanol, 13,27 Chlorhexidine, 27 Chlorocresol, 13,24 Chloroform, 32 Cholera vaccine, 18 Citric acid, 256 Closures, 7 Collection of antiserum, 40 Competition for oxygen, wastes, 221 Containers, 7 Contamination, 11 Contractile protein, 75 Control of malo-lactic fermentation, 270 Control mechanisms in respiratorydeficient yeast, 77,83 in tumors, 70,72 Creams, 32 Cresols, 11 D

Diagnostic reagents, 21 Diphtheria vaccine, 19 Dry heat, 4 E

EDTA, 20

Elixir, 28 Emulsifying agents, 30 Emulsions, 34 Encrusting substances, 144 Energetics, malo-lactics, 263 Enzyme A, cellulose, 116 Esters of P-hydroxybenzoic acid, 31 Evaluation of bactericides, 10 Evolutionary perspective, tumors, 70 External use, 32 Extracellular production, cellulolytic enzymes, 109 Eye drops, 23 F

Fat sludge hypothesis, 222 Fiber decay, 109 Filamentous bulking, 202,217 organisms, 225 Filtration, 6 Floating sludge, 199 Foreignness of antigens, 47 Formaldehyde, 21 Formol toxoid, 19 Fungi as bulking agents, 229 0

Galenicals, 28 Geotrichium candidum, 228 Glass, 8 Glycerol, 20 Growth of microorganisms in fibrous materials, 145 H

Halogenated cresols, 12 Hemicellulases, 144 Hormonal balance and antigens, 57 Humification, 175 Humus, 171 8-Hydroxyquinoline, 22 Hypodermic injections, 3 I

Ideal preservative, 23 Immunization effects of specfic antibody on, 52 intravaginal route of, 46 ocular route of, 45

SUBJECT INDEX oral route of, 44 respiratory route of, 45 Immunological products, 15 Immunosuppressive drugs, 57 Incompatability, 1 Induced synthesis, cellulolytic enzymes, 110 Inducer-repressor mechanism, cellulolytic enzymes, 110 Infusion fluids, 3 Injections, 3 Intracisternal, 3 Intracutaneous, 3 Intradermal, 3 Intramuscular, 3 Intrathecal, 3 Intravenous, 3 J

Jellies, 34 K

299

inhibition of, 272 occurrence, 239 temperature, 250 Matrix substances, 144 Mechanism of formation of whole cellulase fibers, 140 Mechanisms of filamentous bulking, 218 of lignin biodegradation, 173 Mercurials, 13,20 Mercury compounds, 25 Merthiolate, 14 Metabolism of malo-Iactic organisms, 253 Microbial models of tumors, 69 Microfibrils, 136 Middle lamella, 92 Minimal deviation tumors, 71 Minimum amount antigen required, 51 Mitochondria, 73 in respiratory-deficient yeast, 85 Mitochondria1 DNA, 87 Multiple-dose vials, 7 Mutagens, 81

Keratoconjunctivitis, 45 N

1

Lactic acid, 235 Laxative hypothesis, waste disposal, 224 Leaky mitochondria, 76 Lignin, 95,144 decomposition of, 182 to humus, 171 Linctus, 28 Liniments, 32 Lotions, 32 M

Maintaining proper MLSS, 207 Malic acid, 257 “Malic” enzymes, 261 Malic-lactic transhydrogenase, 261 Malo-lactic bacteria, 241 growth conditions, 247 habitat, 246 induction, 262 enzyme, 262 isolation of, 243 Malo-lactic fermentation, 235 detection of, 259 end products of, 257

Native cellulose, 92 Natural immunity, 15 Nitrogen or phosphorus limitation, in wastes, 221 4-Nitroquinoline N-oxide, 81 Nonaqueous vehicles, 7 Normal sludge, 192 0

Ointments, 32 Operating with filamentous bulking, 205 Ophthalmic solutions, 23 Oral adminisbation, 28 Organic content, industrial wastes, 210 Organisms involved in humification, 182 Orientation of cellulose microfibrils, 139 Overaerated sludge, 198 Oxygen, malo-lactic, 251 P

Parebens, 19,24,31 Parental route, 2 Passive immunity, 15 Peridural, 3 Pertussis vaccine, 17

300

SUBJECT INDEX

pH in wine production, 249

Phagocytosis of antigens, 49 Pharmaceutical products, 1 Phenols, 11 Phenyl mercuric salts, 13 Physiologic balance and antigens, 57 Pin point floc, 196 Plant cell walls, 92 Poliomyelitis, 19 Polyuronides, 144 PPD, 21 Preservation of emulsions, 34 ofjellies, 34 of solutions, 33 of suspensions, 3 3 Preservatives, 1,23 efficacy of, 2 Prophylactics, 21 Purified toxoid, 19 Pyruvic acid, malo-lactics, 255 Q

Quantity of antigen, 50 Quarternary ammonium compounds, 14 R

Redox potential, malo-lactic, 251 Respiratory-deficient mutants, 69 Rising sludge, 197 Rubber closures, 9

Sterilization, 3 methods of, 4 tests for, 11 Storage tests, 2 Subcutaneous, 3 Substrates to assay cellulase, 112 Sulfites, 32 Sulfonamides, 1 Sulfur dioxide, 32 as wine antiseptic, 251 Supramolecular morphology, 95 Suspending agent, 30 T

Tannin, as wine antiseptic, 252 Tetanus, growth in tumor bearing animals, 71 Thiomerosal, 14,20 Time interval, antibody response, 57 Toxic compounds, influent waste, 212 Toxoid-antitoxin flocules, 19 Toxoids, 19 Tracheid, 92 Tuberculin, 22 Tumor mitochondria, 73 Typhoid-paratyphoid A and B vaccine, 18 V

Vaccines, 16 Vehicles, 6 Vials, 7 Viral vaccines, 19

S Sabin vaccine, 20 Salk vaccine, 19 Sealing, 8 Secondary antibody response, 58 Selective toxicity in wastes, 223 Shock loading, 213 Simultaneous rot, 149 Size of antigens, 48 Sludge return, 208 Smallpox vaccines, 19,20 Soft-rot, 105, 152 Sorbic acid, 32 Sphaerotilus, 201,225 State of aggregation of antigens, 48

W

Warburg’s theory of cancer, 70 Waste composition, 209 Waste for injection, 6 White-rot, 106, 151 fungi, 178 Wine, 235 deacidification in, 260,269 Wood decay, 104 X

Xylem fiber, 92

Z Zoogleal hulking, 201