Advances in Applied Microbiology, Volume 45

ADVANCES IN

Applied Microbiology VOLUME 45

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ADVANCES IN

Applied Microbiology Edited by SAUL L. NEIDLEMAN Oakland, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 45

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CONTENTS

One Gene to Whole Pathway: The Role of Norsolorinic Acid in Aflatoxin Research J. W. BENNETT,P.-K. CHANG, AND D. BHATNAGAR I. 11. 111. IV. V. VI. VII.

....................... Introduction. . . . . . . . . . . . . . . . . . . . . . . . . ................... Discovery.. . . . . . . . . . . . . . . . . . . . , . . . . . Aflatoxin Biosynthetic Pathway. . . . . . . . . . . . * . . . . . .. . . . . . . . . Enzymolo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strain Degeneration. . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Genetics and Norsolorinic Acid. . , . . . . . . . . . .

.

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

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

...

1 2 3 6 7 10 11 12 13

Formation of Flavor Compounds in Cheese

P. F. Fox AND J. M. WALLACE 11. Glycolysis . . . . . . . . . . 111. Lipolysis . . . . . , . . V. Catabolism o

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

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

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

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

...

...

... ... ...

...

.. ...

17 20 27 31 45 70 75

The Role of Microorganisms in Soy Sauce Production

DESMONDK. O’TOOLE I. 11. 111. IV. V.

Introduction. . . , . . .............................. Types and Compos of Soy Sauce . . . . . . . . . . . . . . . . . . Aroma and Flavor of S h o p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Soy SauceProduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Water Activity on Microorganisms . . . . . . . . . . . . . . . . . . . . . .

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

VII. VIII. IX. X. XI.

...

...

...

... ... ...

Bacteria .......................................... ... Production and Metabolism of Amino and Organic Acids in Moromi . . . . Production and Fate of Other Substances. . . . . . . . . . . . . . . . . . . . . . . . . . . Possible Role of Metal Ions in Soy Sauce Production . . . . . . . . . . . . . . . . . Conclusion. . . . ... References . . . . . ...

87 89 100 101 119 122 125 126 130 133 140 143

vi

CONTENTS

Gene Transfer Among Bacteria in Natural Environments XIAOMING YIN AND

G. STOTZKY

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. 111. IV. V.

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

Transformation Conjugation. . . Transduction.. . Concluding Rem References , . . .

153 154 166 177 195 198

Breathing Manganese and Iron: Solid-state Respiration KENNETH H.

NEALSONAND BRENDALITTLE

Introduction. . . . .. ........................ Respiration: Organismal and Environmental. . . . . . . . . . . . . . . . . . . . . . . . . . Metal-Reducing Bacteria in Captivity . . . . . . . . ............ Reduction of Metals by Iron and Manganese Reducers Electron Transport In Metal Reducers . . . . . . . . . VI. Metal-Reducing Bacteria in Natural Environme VII. Summa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. 11. 111. IV. V.

..

.

213 214 219 221 220 231 235 237

Enzymatic Deinking

PRATIMA BAJPAI I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Enzymes Used in Deinking. . . . . . . . . . . . . . . . 111. Performance of Enzymes in Deinking . . . . . . .

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

IV. Effect of Enzyme on Pulp Yield and the Qualit V. Possible Mechanisms of Enzymatic Deinking . . . . . . . . . . . . . . . . . . . . . . . . VI. Factors Affecting Enzymatic Deinking. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . .

.

VIII. Current Research Needs IX. Conclusions. . .

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

241 242 245 256 258 261 263 263 265 266

CONTENTS

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Microbial Production of Docosahexaenoic Acid (DHA, C22:6) AJAYSINGH AND OWEN P. WARD I. 11. 111. IV. V. VI. VII.

Introduction ... ........ Biosynthesis ated ......................... Potential Microorganisms for DHA Production. . . . . . . . . . . . . . . . . . . . . . . . Kinetics of Microbial Growth and DHA Production. . . . . . . . . . . . . . . . . . . . Factors Affecting Microbial DHA Production . . . . . . . . . . . . . . . . . . . . . . . . . Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

271 2 78 283 289 2 94 304 305 307

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTENTS OF Pmvrous VOLUMES.. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . .

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One Gene to Whole Pathway: The Role of Norsolorinic Acid in Aflatoxin Research J. W. BENNETT Department of Cell and Molecular Biology Tulane University New Orleans, Louisiana 70118

P.-K. CHANG AND D.

BHATNAGAR

Southern Regional Research Center USDA Agricultural Research Service New Orleans, Louisiana 70179

I. 11. 111. IV. V. VI. VII. VIII.

Introduction Discovery Aflatoxin Biosynthetic Pathway Enzymology Molecular Genetics Strain Degeneration Reverse Genetics and Norsoloririic Acid Conclusions References

I. Introduction

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Although the mycotoxin problem is probably as old as agriculture (outbreaks of ergot alkaloid poisoning have been reported since medieval times (Bove, 1970)), contemporary interest in these toxic metabolites dates to the early 1960s and the discovery of aflatoxins from Aspergillus flavus and Aspergillus parasiticus (Goldblatt, 1969). The 15 years following the discovery of aflatoxins resulted in the identification of over 100 fungal toxins, an era described by Maggon et al. (1977) as a “mycotoxin gold rush.” Mycotoxins cause enormous economic losses in agriculture. One major study estimated that 25% of the annual food crop worldwide is mycotoxin-contaminated (Jelinek, 1987). Most mycotoxins are associated with veterinary diseases, which may be either acute or chronic (Richard and Thurston, 1986). For general reviews of 3 ADVANCES IN APPLIED MICROBIOLOGY,VOLUME 4 5 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. OO~~-Z~M $25 /W nn

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1. W.BENNETT, P.-K. CHANG, AND D. BHATNAGAR

mycotoxins, see Betina (1984), CAST (1989), and Bhatnagar et al, (1992);more recent aspects of the aflatoxin problem have been reviewed by Eaton and Groopman (1994). Aflatoxins are the most important and best-studied mycotoxins. They act as potent carcinogens in laboratory tests, and there is strong evidence that they can act as hepatocarcinogens in human populations (Eaton and Groopman, 1994). Because there are no good treatments against aflatoxin-induced diseases, the emphasis in aflatoxin research has been to find methods for prevention of aflatoxin contamination of food and feed (Lee et al., 1992).Good agronomic practices that reduce preharvest and postharvest mold growth, coupled with careful regulatory surveillance for aflatoxins in foods intended for human consumption, comprise the main methods in use. Since these methods are inherently limited, considerable basic research on aflatoxin production has been funded by various agricultural organizations. There is hope that an understanding of the fundamental mechanisms leading to aflatoxin biosynthesis may translate into practical methods for blocking the process (Keller et a]., 1992). The first known intermediate in the aflatoxin biosynthetic pathway is norsolorinic acid, a red-pigmented polyhydroxyanthraquinone. In this review, we discuss the important role this compound has played in our knowledge of aflatoxin biosynthesis, aflatoxin genetics, and aflatoxin molecular biology. II. Discovery

The decaketide 2-hexanoyl-1,3,6,8-tetrahydroxyanthraquinone was named norsolorinic acid after the lichen Solorina crocea, from which it was first characterized (Anderson et al., 1966).Soon thereafter, a norsolorinic acid-producing mutant fungus was isolated after ultraviolet light irradiation of an aflatoxigenic strain of A. parasiticus (Lee et al., 1971). This mutant, later designated nor-I, was easily identified in liquid and agar culture because of the orange-red color characteristic of norsolorinic acid accumulation. An 80% reduction in aflatoxin production, as well as a correlation between aflatoxin and norsolorinic acid production, was observed in this A. parasiticus mutant. In chemically defined media under continuous illumination, in zinc-deficient media, and in the presence of para-aminobenzoic acid, both norsolorinic acid and aflatoxin production were inhibited. Growth in enriched complex medium resulted in higher yields of both compounds (Bennett et al., 1971).This correlation was useful because the bright orange-red pig-

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

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ment of norsolorinic acid was an easy visual screen for the putative presence of aflatoxin. Other mutants of A . flavus and A . parasiticus that had lost the ability to produce aflatoxin were isolated on the basis of altered fluorescence under long-wave ultraviolet light. Mutants selected for altered fluorescence production also demonstrated abnormal pigment production. Likewise, mutants selected for lowered norsolorinic acid production had lowered aflatoxin production (Bennett and Goldblatt, 1973), suggesting that aflatoxin and mycelial pigment production might result from common precursors. Norsolorinic acid-accumulating mutants of aflatoxigenic species were also induced by nitrosoguanidine treatment in A . parasiticus (Detroy et al., 1973) and in A. flavus (Papa, 1982). Like nor-2, all of these norsolorinic acid-accumulating mutants were “leaky,” that is, they still produced aflatoxin, although in reduced quantities.

I l l . Aflatoxin Biosynthetic Pathway The polyketide origin of the carbon skeleton of aflatoxin B1 was established by 14C radioisotope labeling studies by Biollaz et al. (1968a,b). During the 1970s, several other anthraquinones, in addition to norsolorinic acid, were isolated from blocked mutants and shown to be aflatoxin precursors: averufin by Donkersloot et al. (1972) and versicolorin A by Lee et al. (1975). The role of sterigmatocystin as a late intermediate in aflatoxin biosynthesis was also established (Hsieh et al., 1973). When 14C labeling was used to follow incorporation of norsolorinic acid into aflatoxin, only 2.2% of label was recovered in aflatoxin B1, as opposed to 49.4% for averufin, 45.5% for versicolorin A, and 65.5% for sterigmatocystin (Hsieh et al., 1976). The availability of blocked mutants, coupled with the extensive use of both 14Cand 13C isotope studies, led to a generally accepted “canonical” scheme for aflatoxin biosynthesis in which the steps were usually summarized as acetate + malonate + anthraquinones 3 sterigmatocystins + aflatoxins B1. This pathway has been reviewed many times (see, e.g., Steyn et al., 1980; Bennett and Christensen, 1983; Turner and Aldridge, 1983; Dutton, 1988; Bhatnagar et al., 1992). In this scheme, norsolorinic acid is the decaketide product of a polyketide synthase and the first stable precursor in aflatoxin biosynthesis. Theoretically, the product of a polyketide synthase should be anthrone, but this compound has never been isolated, presumably due to its rapid oxidation to norsolorinic acid, a more stable conformation (Vederas and

4

J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR AFBl (312)

POLYKETIDE

I

1

I

&

NOR(370)

OMST (338)

AVN(372)

1

ST (324)

AVNN(370)

KQ?J

HO \

OHMe

1

0

0

AVF(368)

I

HO

HO

0

OH

VERA (338)

0

HO 0

0

VHA (400)

FIG.1. Proposed scheme for aflatoxin biosynthesis based on mutation analysis, precursor feeding studies, and isotopic labeling (mol. wt.]. NOR = norsolorinic acid; AVN = averantin; AVNN = averufanin; AVF = averufin; VHA = versiconol hemiacetal acetate; VERA = versicolorin A, ST = sterigmatocystin; OMST = o-methylstwigmatocystin; AFBl = aflatoxin Bl.

Nakashima, 1980). Recognizing that acetyl CoA is not always the starter unit for polyketide synthesis, Townsend et al. (1984, 1988) provided an alternative proposal in which the sidechain of norsolorinic acid arises intact from hexanoic acid. Brown and Salvo (1994) have shown that a n ascospore pigment of Aspergillus nidulans, ascoquinone A, has a strong resemblance to norsolorinic acid. After norsolorinic acid, the polyketide undergoes an estimated 1 2 to 1 7 enzymatic transformations, leading through a series of pathway intermediates, including averantin, averufanin, averufin, versiconol hemiacetal acetate, versiconol, versicolorins A and B, demethyl sterigmatocystin, and sterigmatocystin, to the final product, aflatoxin B1 (see Fig. 1). In this scheme, the intermediate following norsolorinic acid is usually given as averantin, another polyhydroxyanthraquinone with a

5

NORSOLOFUNIC ACID IN AFLATOXIN RESEARCH

0 orsoiorinic acid (NOR)

2

Reductase

\

NADPH

Dehydrogenase

\

\

Oxidation

Dehydrogenase (a)

0

0

Averufanin (AV")

Averantln (AVN)

0 Averufin (open form)

HO

0

Averufln (ketal form) WF)

FIG.2. Proposed pathways for conversion of norsolorinic acid to averufin. After Bhatnagar et al. (1992).

C-6 sidechain. The enzymatic step entails reduction of the carbonyl group in the sidechain of norsolorinic acid to a hydroxyl group. The reaction is variously labeled as a reductase or a dehydrogenase step. Averantin is then converted to averufin. Alternatively, norsolorinic acid is reduced to averufanin and then to averufin (McCormick et al., 1987). Another postulated scheme channels norsolorinic acid to averantin, averufanin, and then to averufin (Bhatnagar et a]., 1992). These alternative schemes are outlined in Fig. 2. Experimental evidence for a norsolorinic acid * averantin hydroxyaverantin 3 averufin conversion has been provided by Yabe et al. (1991). In order to better understand the mechanistic details of these proposed transformations, it is necessary to isolate the relevant enzymes.

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J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

IV. Enzymology

Compared to other cellular proteins, secondary metabolic enzymes are often present only in small amounts, rendering the purification of aflatoxin pathway enzymes technically difficult. Additionally, cell-free extracts containing these secondary metabolic enzymes seem especially sensitive to denaturing conditions. Consequently, most efforts to purify these enzymes result in crude protein homogenates instead of pure protein extracts (Dutton, 1988). The enzyme that catalyzes the conversion of norsolorinic acid to averantin is usually called norsolorinic acid dehydrogenase. Chuturgoon and Dutton (1991) developed an affinity chromatography method for the purification of this enzyme activity. Using reactive green 19-agarose and norsolorinic acid agarose affinity chromatography, the dehydrogenase was purified from the mycelia of 84-hour A . parasiticus. A molecular weight of 140 kDa was determined by gel filtration chromatography on a Sephacryl S-300 column. Additionally, the temperature and pH optima of the dehydrogenase were found to be 35°C and 8.5, respectively. Like other fungal dehydrogenases, a pH below 6.5 or above 9.0 resulted in a decline in enzymatic activity. Further studies of K, values for norsolorinic acid dehydrogenase, using norsolorinic acid and averantin as substrates with various nicotinamide cofactors, showed that norsolorinic acid dehydrogenase favored the reaction of norsolorinic acid to averantin somewhat more than the reverse reaction of averantin to norsolorinic acid. The K,,, for norsolorinic acid (3.45 mMj was lower than the K,,, for averantin (3.72 mM), indicating that norsolorinic acid dehydrogenase had a higher affinity for norsolorinic acid than averantin (Chuturgoon and Dutton, 1991). Yabe et al. (1991) studied the enzymatic conversion of norsolorinic acid to averufin in Emericella heterothallica, a non-aflatoxigenic species that produces the anthraquinones averantin, averufanin, and averufin. Cell-free experiments supported the reversibility of the norsolorinic acid dehydrogenase reaction. The presence of the reduced forms of the cofactors resulted in greater product formation in the forward conversion of norsolorinic acid to averantin, while the oxidized forms (NAD' or NADP+)were more efficient in the reverse reaction of averantin to norsolorinic acid. NADPH was preferred to NADH in the norsolorinic acid dehydrogenase reaction. Yabe et al. (1991) also reported that norsolorinic acid dehydrogenase activity was media-dependent. Activity was present when a complex culture medium containing 2% yeast extract and 20% sucrose was used, but when the sucrose was replaced with 20% peptone no norsolorinic acid dehydrogenase activity was detected. The presence of NAD under yeast extract-

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

7

peptone culture conditions restored low dehydrogenase activity (Yabe et al., 1991). Bhatnagar and Cleveland (1990) also worked on this enzyme activity, purifying a protein that catalyzed the transformation of norsolorinic acid to averantin from 3-day-old cultures of A. parasiticus by classical biochemical techniques. The multistep protocol involved ammonium sulfate precipitation and several chromatography steps, concluding with an HPLC BioScale anion-exchange column. The purified protein catalyzed the transformation of norsolorinic acid to averantin in the present of NADPH and had a pH optimum of 7.5, a temperature optimum of 3OoC, and an estimated molecular weight of 4 3 kDa. Using this purified enzyme, polyclonal antibodies were prepared; the prepared antiserum neutralized norsolorinic acid reductase enzyme activity significantly more than did control antisera (Lee et al., 1995). Because traditional biochemical approaches to enzyme purification have been so technically demanding, workers have turned to molecular genetics. Once relevant genes are isolated, it is possible to predict amino acid sequences from nucleotide sequences and engineer expression systems for producing purified proteins. In recent years, the tools of molecular genetics have been applied to the enzymes surrounding norsolorinic acid and other steps in the aflatoxin pathway. There have been some surprising discoveries. V. Molecular Genetics

Prior to 1989, a major obstacle to the study of these genes involved the need for efficient transformation systems that were met first for A. flavus (Woloshuk et al., 1989) and then for A. parasiticus (Skory et al., 1990). These transformation systems were developed for A. flavus by complementation of the pyr-4 gene of Neurospora crassa involved in pyrimidine biosynthesis, while the system developed for A. parasiticus used the homologous pyrG gene and involved the isolation and characterization of uridine auxotrophic mutants of A. parasiticus (Skory et al., 1990).

Almost simultaneously, another transformation method for A. parasiticus was developed based on complementation of nitrate reductase mutants with the homologous niaD gene (Chang et al., 1992). This complementation system was used to clone the first aflatoxin pathway gene. Specifically, a genomic library was constructed by inserting wildtype A. parasiticus DNA into a cosmid vector containing the nitrate reductase gene niaD. As a selectable marker, an aflatoxin-producing

8

J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

cosmid was isolated that complemented an aflatoxin-deficient (norsolorinic acid-positive), nitrate-nonutilizing mutant strain. The gene was called nar-1 for “norsolorinic acid related,” but later renamed nor-1. Further investigations showed that a 1.7-kb Bgl 11-Sph-I DNA fragment was responsible for the renewal of aflatoxin production (Chang et al., 1992). Shortly thereafter, a similar strategy was used to isolate a gene that encodes an activity associated with the conversion of versicolorin A to sterigmatocystin (Skory et al., 1992). This latter gene was named ver-1. The appearance of nor-1 and ver-1 transcripts was followed in batch cultures. The two genes were expressed at the end of trophophase. The timing of expression suggested that they were controlled by a common regulatory factor and that the accumulation of these transcripts partially regulated aflatoxin biosynthesis (Skory et al., 1993). Sequence analysis of nor-1 genomic DNA predicted a region containing three introns that encoded a putative protein of 294 amino acids with a molecular mass of 29 kDa. The deduced amino acid sequence of nor-1 had a high sequence similarity to other nucleotide-binding dehydrogenases-reductases (Trail et ul., 1994). A related nor-1 gene has been isolated from A. f l a w s (Brown-Jencoet al., 1994). Gene disruption was used to confirm the role of nor-1 in aflatoxin biosynthesis in A. parasiticus. Disrupted strains, like the original blocked norsolorinic acid mutations, accumulated norsolorinic acid, as well as retaining the ability to produce low levels of aflatoxin (Trail et al., 1994), supporting earlier biochemical hypotheses concerning alternative pathways from norsolorinic acid to averufin (Bhatnagar et al., 1992; Yabe et a!., 1991). There has been a burst of research activity concerning the molecular genetics of A . parasiticus and A. flavus. In addition to Complementation, genes have been isolated after purification of their relevant proteins (e.g., Yu et d., 1993) and by differential expression of transcripts during idiophase (e.g., Feng et al., 1992). See Bennett et al. (1994) and Trail et al. (1995a) for reviews of early aflatoxin molecular biology. Although classical parasexual studies had hinted that some of the aflatoxin pathway genes in A. flavus were linked (Bennett and Papa 1988), the high degree of linkage that has been revealed by molecular techniques was unexpected. Aflatoxin pathway genes in both A . flavus and A . parasiticus are clustered (Yu et a!., 1995; Silva et al., 1996). This fortuitous clustering has allowed “chromosome walking,” i.e., the identification of DNA sequences related to the pathway through the use of overlapping clones (Yu et a]., 1995). This technique has been exploited most fully in the study of the cognate polyketide pathway to sterigmatocystin in Aspergillus nidulans. This mold has been studied as a genetic model for over 50 years (Martinelli and Kinghorn, 1994),making

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

9

it more amenable to sophisticated genetic analysis than either A. flavus or A. parasiticus. Using standard molecular techniques, Brown et al. (1996a) have described a 25-gene cluster of approximately 60-kb DNA that contains most, if not all, of the enzyme activities required for sterigmatocystin biosynthesis. The A. nidulans homologue of nor-1 is called stcE. It encodes a putative reductase and shares 56% deduced amino acid identity with nor-1. Molecular studies have also clarified details concerning the starter chain. If, as proposed by Townsend et al. (1984, 1988), hexanoic acid initiates the polyketide, norsolorinic acid would be assembled from hexanoyl CoA (synthesized by a fatty acid synthase, FAS) and 7 malonate units, and not from 10 classical polyketide condensation reactions. The isolation and characterization of the A. parasiticus polyketide synthase gene p k s A on the gene cluster (Chang et al., 1995a; Feng and Leonard, 1995; Trail et al., 1995b) gives molecular support to Townsend’s postulate. Sequence analysis indicates that the p k s A gene encodes P-ketoreductase, acyltransferase, acyl carrier, and thioesterase domains, but no P-acyl ketoacyl reductase or enoyl reductase domains, suggesting that the pksA gene does not encode catalytic activities that are required for processing P-carbon (keto group). Moreover, a fatty acid synthase gene, fas-ZA, is necessary for norsolorinic acid production in A. parasiticus. The predicted amino acid sequence for fas-ZA shows a high degree of similarity with the P subunit of a fatty acid synthase from yeast (Mahanti e t al., 1996), suggesting that fas-ZA encodes a fatty acid starter unit. Subsequent characterization and disruption of the fas-ZA gene and another gene Ifas-2A) encoding a 6.5-kb transcript demonstrated that these genes were directly involved in early pathway functions (Chang et al., 1995b; Trail et al., 1994). Enoyl reductase domains were found in both genes. It appears that the two FAS genes are required for the production of the C6 sidechain of norsolorinic acid. Metabolite feeding studies with p k s A and fas disruptants provided evidence that the protein products of these genes may physically associate during normal pathway function (Watanabe et al., 1996; Linz et al., 1996). A unique fatty acid synthase dedicated to the synthesis of a hexanoate moiety also has been identified from A. nidulans (Brown et al., 1996b). A pair of fatty acid synthase genes, stcJ and stcK, are required for sterigmatocystin biosynthesis, but not for growth. Disrupted stcJ and stcKstrains are restored to sterigmatocystin biosynthesis by the addition of hexanoic acid (Brown et a]., 1996b).

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J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

In summary, these data indicate that the sterigmatocystin-aflatoxin pathway is indeed primed by specialized fatty acid synthases to generate a fatty acid (hexanoic acid), which in turn is converted by a polyketide synthase to norsolorinic acid, thus supporting Townsend’s early hypothesis. The ability of the polyketide synthase to use exogenous hexanoic acid also has important implications for engineering polyketide synthase diversity. VI. Strain Degeneration

Filamentous fungi are known for their morphological and metabolic variability. Traits such as virulence, sporulation, and secondary metabolite production may be unexpectedly lost, especially in cultures that have been maintained in the laboratory for long periods. Such strain degenerations are poorly understood and are particularly troublesome in industrial fermentations. Proposed theories for the cause of strain degeneration in filamentous fungi include chromosome instability, transposable elements, heterokaryosis, and cytoplasmic inheritance (Kale and Bennett, 1992; Li et a]., 1994). The norsolorinic acid mutant of A. parasiticus has been used to study experimentally induced strain degeneration. The loss of red pigment is easily identified in both liquid and solid culture. In an early study on the effects of zinc and barium on norsolorinic acid and aflatoxin production, unpigmented colonies were regularly detected. These unpigmented non-aflatoxigenic variants were associated with serial transfers of inocula that contained hyphae rather than conidia, but were not associated with either zinc deficiency or the presence of barium ions in the medium (Bennett, 1981). In addition to loss of norsolorinic acid and aflatoxin production, these strains sporulated poorly and gave an unusual “fan-like” growth on Petri plates. Similar degenerate forms were subsequently isolated from wild-type and genetically marked strains after 5-12 serial transfers of mycelial macerates. These strains were designated s e c for “secondary metabolite minus” (Kale eta]., 1994).The Kale et al. (1994) paper presented a color plate that showed the progression of variation from bright red “parental” colonies that accumulated large quantities of norsolorinic acid, to colonies observed after serial transfer in which there was no detectable production of this pigment. These variants were stable and did not revert back to norsolorinic acid or aflatoxin production even after several years of storage in the laboratory (Kale et al., 1994).

NORSOLORINIC ACID IN AFLATOXIN RESEARCH

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The reproducible isolation of sec- forms, and the pleiotropic nature of the changes observed, made them an attractive model system for studying strain degeneration. In another study, it was demonstrated that the s e c strains lacked the enzymatic ability to convert certain aflatoxin precursors to aflatoxin. Moreover, Northern blots probed with pathway genes demonstrated lack of message for both nor-1 and a regulatory gene associated with the aflatoxin pathway, while the polymerase chain reaction confirmed the presence of the structural gene for nor-2 (Kale et al., 1996). In other words, although these s e c forms retained genes involved in norsolorinic acid production, the genes were neither transcribed nor translated. In summary, the norsolorinic acid marker has been important in developing a model system for studying degeneration. These s e c strains display a pleiotropic phenotype that includes changes in conidiophore development and sporulation patterns, suggesting that the regulation of aflatoxin synthesis and condiogenesis may be interlinked. The norsolorinic acid marker may prove to be as useful for dissecting developmental pathway genes as it has been in studying polyketide pathway genes. VII. Reverse Genetics and Norsolorinic Acid

The most recent chapter in the history of norsolorinic acid concerns the “reverse genetics” approach to isolating genes. As described above, three different groups have studied the enzyme activity that transforms norsolorinic acid to averantin. Although Trail et a]. (1994) stated that “two different norsolorinic acid reductases associated with the conversion of norsolorinic acid have been purified,” it is not yet known whether the ability reported by Chuturgoon and Dutton is actually different from the one reported by Bhatnagar and Cleveland (1990). Nor is it clear how the Emericella enzyme studied by Yabe’s group is related to A. parasiticus enzyme(s). What is clear is that polyclonal antibodies prepared against the A. parasiticus enzyme exhibiting norsolorinic acid reductase activity purified by Bhatnagar’s group did not react with the 29-kDa nor-1 gene product isolated by complementation (Lee et al., 1995). A monoclonal antibody to norsolorinic reductase was therefore prepared and used to screen a cDNA library. A gene with two introns was isolated that exhibited 68% nucleotide identity to an aryl alcohol dehydrogenase gene known from Phanerochaete chrysosporium (Cary et a]., 1996). The gene was located in the aflatoxin cluster and was named norA, because monoclonal antibodies used to isolate the gene inhibited the conversion of norsolorinic acid to averantin and because

12

J. W. BENNETT, P.-K. CHANG, AND D. BHATNAGAR

the purified 43-kDa protein used to generate the monoclonal antibody demonstrated norsolorinic acid reductase activity. NorA shows high sequence similarity with an aryl alcohol dehydrogenase from A . nidulans designated stcV (Cary, personal communication). Gene disruption experiments have been difficult to perform with norA, presumably due to duplication of a 12-kb region of the gene cluster (Trail et al., 1995b), so it is not yet known whether disrupted strains will accumulate pathway intermediates. However, it is known that nor-2 and norA are under common regulation of the aflatoxin biosynthetic pathway regulatory gene aflB (Chang et al., 1995b; Cary et a]., 1996). These findings underline the importance of multiple approaches to molecular cloning. In this case, complementation and reverse genetics identified separate genes implicated in norsolorinic acid metabolism. The nomenclature of these genes is confusing, and we recommend that workers studying the aflatoxin pathway develop a standardized system in the near future. VII I. Conclusions

Norsolorinic acid is a C-20 anthraquinone that is the first stable intermediate in aflatoxin biosynthesis. Mutants of A . flavus and A. parasiticus that accumulate norsolorinic acid also produce low levels of aflatoxin, raising interesting questions about the early steps of the pathway. The development of transformation systems for the aflatoxigenic species A. flaws and A. parasiticus, and the rapid cloning, sequencing, and analysis of several genes involved in the pathway, have facilitated new insights into aflatoxin biosynthesis. A gene involved in norsolorinic acid (nor-1)was isolated by complementation. Disrupted strains accumulated norsolorinic acid, confirming the role of this compound as an intermediate. Additionally, disruption of a fatty acid synthase has confirmed the role of hexanoic acid as the starter unit initiating polyketide biosynthesis. Researchers initially had difficulty in purifying the enzymes of the aflatoxin biosynthetic pathway, a common problem for other secondary metabolites as well. Nevertheless, several of the enzymes have been successfully purified to homogeneity in the past few years. These include a norsolorinic acid reductase associated with the conversion of norsolorinic acid to averantin. When reverse genetics was used to isolate the gene corresponding to this enzyme activity, surprisingly, it was different from nor-1. The new gene is called norA. An understanding of the regulation and expression of these genes in aflatoxin-producing strains at the molecular level continues to expand

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our knowledge of fungal polyketide synthesis and may ultimately lead both to new ways of generating hybrid polyketides and to improved methods for controlling the production of aflatoxins. ACKNOWLEDGMENTS

We thank Nancy Keller and John Linz for sharing unpublished data, and Aaron Allen and Tarry1 Gallo for manuscript preparation. JWB and PKC acknowledge a cooperative agreement with the U.S. Department of Agriculture (532 243). REFERENCES Anderson, H. A., Thomson, R. H., and Wells, J. W. (1966). J. Chem. SOC.,pp. 1727-1729. Bennett, J. W. (1981).J. Gen. Microbiol. 124,429-432. Bennett, J. W., and Christensen, S. B. (1983).Adv. Appl. Microbiol. 29,53-92. Bennett, J. W., and Goldblatt, L. A. (1973). Sabouraudia 11,235-241. Bennett, J. W., and Papa, K. E. (1988). In “Advances in Plant Pathology” (D. S. Ingram and P. H. Williams, eds.), Vol. 6, pp. 263-280. Academic Press, London. Bennett, J. W., Lee, L. S., and Vinnett, C. (1971). J. Am. Oil Chem. SOC.48,368-370. Bennett, J. W., Chang, P.-K., and Bhatnagar, D. (1994). In “The Genus Aspergillus” (K. A. Powell, A. Renwick, and J. F. Peberdy, eds.), pp. 51-58. Plenum, New York. Betina, V., ed. (1984). “Mycotoxins: Production, Isolation, Separation, and Purification.” Elsevier, Amsterdam. Bhatnagar, D., and Cleveland, T.E. (1990). FASEB J. 4,A2164. Bhatnagar, D.,Ehrlich, K. C., and Cleveland, T. E. (1992). In “Handbook of Applied Mycology” (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, eds.), Vol. 5, pp. 255-286. Dekker, New York. Biollaz, M., Buchi, G., and Milne, G. (1968a). J. Am. Chem. SOC.90,5017-5020. Biollaz, M., Buchi, G., and Milne, G. (1968b). J. Am. Chem. SOC.90,5019-5020. Bove, F. J. (1970). “The Story of Ergot.” Barger, New York. Brown, D. W., and Salvo, J. J. (1994). Appl. Environ. Microbiol. 60,979-983. Brown, D.W., Yu, J.-H., Kelkar, H. S., Fernandes, M., Nesbitt, T. C., Keller, N. P., Adams, T. H., and Leonard, T. J. (1996a). Proc. Natl. Acad. Sci. U.S.A. 93,1418-1422. Brown, D. W., Adams, T. H., and Keller, N. P. (1996b). Proc. Nutl. Acad. Sci. U.S.A. 93, 14873-14877. Brown-Jenco, C. S.,Brewer, J. F., and Payne, G. A. (1994). Phytopathology 84,1098. Cary, J. W., Wright, M., Bhatnagar, D., Lee, R., and Chu, F. S. (1996). Appl. Environ. Microbiol. 62,360-366. CAST [Council for Agricultural Science and Technology] (1989). “Mycotoxins: Economic and Health Risks.” CAST, Ames, 10. Chang, P.-K., Skory, C. D., and Linz, J. E. (1992). Curr. Genet. 21,231-233. Chang, P.-K., Cary, J. W., Yu, J., Bhatnagar, D., and Cleveland, T. E. (1995a). Mol. Gen. Genet. 248,270-277. Chang, P.-K., Ehrlich, K. C., Yu, J., Bhatnagar, D., and Cleveland, T. E. (1995b). Appl. Environ. Microbiol. 61,2372-2377. Chuturgoon, A. A., and Dutton, M. E. (1991). Prep. Biochem. 21,125-140.

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Detroy, R. W., Freer, S., and Ciegler, A. (1973). Can. J. Microbiol. 19,1371-1378. Donkersloot, J. A., Matales, R. I., and Yang, S. S. (1972). Biochem. Biophys. Res. Comrnun. 47, 1051-1055. Dutton, M. F. (1988). Microbiol. Rev. 52,274-295. Eaton, D.L., and Groopman, J. D. (1994). In “Human Health, Veterinary, and Agricultural Significance.” Academic Press, San Diego. Feng, G. H., and Leonard, T. J. (1995). J. Bacteriol. 177, 6246-6254. Feng, G. H., Chu, F. C., and Leonard, T. J. (1992). Appl. Environ. Microbiol. 58,455-460. Goldblatt, L. A., ed. (1969). “Aflatoxin: Scientific Background, Control, and Implications.” Academic Press, New York. Hsieh, D. P. H., Lin, M. T., and Yao, R. C. (1973). Biochem. Biophys. Res. Commun. 52, 92 2-997. Hsieh, D. P. H., Lin, M. T., Yao, R. C., and Singh, R. (1976). J. Agric. Food Chern. 24, 1171-1174. Jelinek, C. F. (1987). Joint FAO/WHO/UNEP lnt. Conf. Mycotoxins, Znd, Bangkok, Thailand. Kale, S. P., and Bennett, J. W. (1992). In “Handbook of Applied Mycology” (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, eds.), Vol. 5, pp. 311-331. Dekker, New York. Kale, S. P., Bennett, J. W., and Bhatnagar, D. (1994). Mycol. Res. 98,645-652. Kale, S. P., Cary, J. W., Bhatnagar, D., and Bennett, J. W. (1996). Appl. Environ. Microbiol. 62, 3399-3409. Keller, N. P., Cleveland, T. E., and Bhatnagar, D. (1992). In “Handbook of Applied Mycology” (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, eds.), Vol. 5, pp. 287-310. Dekker, New York. Lee, L. S., Bennett, J. W., Goldblatt, L. A., and Lundin, R. E. (1971). J. Am. Oil Chem. Soc. 48,93-94. Lee, I,. S., Bennett, J. W., Cucullu, A. F., and Stanley, J. B. (1975). J. Agric. Food Chern. 23, 1132-1134. Lee, L.S., Bayman, P., and Bennett, J. W. (1992). In “Biotechnology of Filamentous Fungi” (D. R. Finkelstein and C. Ball, eds.). Butterworth-Heinemann, Boston. Lee, R. C., Cary, J. W., Bhatnagar, D., and Chu, F. S. (1995). Food Agric. Irnmunol. 7,Zl-32. Li, A., Begin, A., Kokurewicz, K., Bowen, C., and Horgen, P. A. (1994). Appl. Environ. Microbiol. 60,2384-2388. Linz, J. E., Townsend, C. A,, and Bhatnagar, D. (1996). In “Proc. Noble Foundation Symp.: The Biochemical and Metabolic Aspects of 3-Ketoacyl Synthases.” In press. Maggon, K. K., Gupta, S. K., and Venkitasubramanian, T. A. (1977). Bacteriol. Rev. 41, 822-855. Mahanti, N., Bhatnagar, D., Cary, J. W., Jourbran, J., and Linz, J. E. (1996). Appl. Environ. Microbiol. 62,191-195. Martinelli, S. D., and Kinghorn, J. R., eds. (1994). “Aspergillus: 50 Years On.” “Progress in Industrial Microbiology,” Vol. 29. Elsevier, Amsterdam. McCormick, S. P., Bhatnagar, D., and Lee, L. S. (1987). Appl. Environ. Microbiol. 53, 14-16. Papa, K. E. (1982). J. Gen. Microbiol.128,1345-1348. Richard, J. L., and Thurston, J. R., eds. (1986). “Diagnosis of Mycotoxicoses.” Martinus Nijhoff, Boston. Silva, J. C., Minto, R. E., Barry 111, C. E., Holland, K. A., and Townsend, C. A. (1996). J. Bid. Chem. 271, 13600-13608. Skory, C. D., Horng, J. S., Pestka, J. J., and Linz, J. E. (1990). Appl. Environ. Microbiol. 56,3315-3320.

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Skory, C. D., Chang, P.-K., Cary, J. W., and Linz, J. E. (1992). Appl. Environ. Microbiol. 58, 3 52 7-35 3 7.

Skory, C. D., Chang, P.-K., and Linz, J. E. (1993).Appl. Environ. Microbiol. 59, 1624-1646. Steyn, P. S., Vleggar, R., and Wessels, P. L. (1980). In “The Biosynthesis of Mycotoxins: A Study in Secondary Metabolism” (P. S. Steyn, ed.), pp. 105-155. Academic Press, New York. Townsend, C. A., Christensen, S. B., and Trautweink, K. (1984). J. Am. Chem. Soc. 106, 3868-3869.

Townsend, C. A., Brobst, S. W., Ramer, S. E., and Vederas, J. C. (1988).1. Am. Chem. Soc. 110, 318-319.

Trail, F., Chang, P.-K., Cary, J., and Linz, J. E. (1994). Appl. Environ. Microbiol. 60, 4078-4085.

Trail, F., Mahanti, N., and Linz, J. E. (1995a). Microbiology 141, 755-765. Trail, F.,Mahanti, N., Rarick, M., Mehugh, R., Liang, S.-H., Zhou, R., and Linz, J. E. (1995b).Appl. Environ. Microbiol. 61,2665-2673. Turner, W. B., and Aldridge, D. C. (1983). “Fungal Metabolites,” Vol. 2. Academic Press, London. Vederas, J. C., and Nakashima, T. T. (1980).J. Chem. Soc., Chem. Commun., pp. 183-185. Watanabe, C. M. H., Wilson, D., Linz, J. E., and Townsend, C. A. (1996). Chem. Biol. 3, 463-469.

Woloshuk, C. P., Seip, E. R., and Payne, G. A. (1989).Appl. Environ. Microbiol. 55, 86-90. Yabe, K., Nakamura, Y . ,Nakajima, H., Ando, Y.,and Hamasaki, T. (1991). Appl. Environ. Microbiol. 57, 1340-1 345. Yu, J., Cay, J. W., Bhatnagar, D., Cleveland, T. E., Keller, N. P., and Chu, F. S. (1993).Appl. En viron. Microbiol. 59, 35 64-3 5 71. Yu, J., Chang, P.-K., Cary, J. W., Wright, M., Bhatnagar, D., Cleveland, T. E., Payne, G. A., and Linz, J. E. (1995). Appl. Environ. Microbiol. 61, 2365-2371.

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Formation of Flavor Compounds in Cheese P. F. FOX AND J. M.

WALLACE

Department of Food Chemistry University College Cork, Ireland

I. Introduction A. Ripening Agents in Cheese 11. Glycolysis A. Metabolism of Lactose by Lactic Acid Bacteria B. Metabolism of Lactic Acid in Cheese C. Effect of Lactose Concentration on Cheese Quality D. Citrate Metabolism 111. Lipolysis A. Lipases in Cheese B. Catabolism of Free Fatty Acids IV. Proteolysis A. Contribution of Individual Proteolytic Agents B. Proteolysis in Cheese C. Free Amino Acids in Cheese V. Catabolism of Amino Acids A. Decarboxylation and Production of Amines B. Deamination/Formation of Ammonia and Neutral or Acidic Compounds C. Transamination, Strecker Degradation, and Production of Aldehydes D. Catabolism of Sulfur Amino Acids E. Catabolism of Phenylalanine, Tyrosine, and Tryptophan F. Other Important Flavor Compounds from Amino Acid Catabolism G. Reactions of Free Amino Acids with Other Compounds in Cheese H. Nonprotein Amino Acids VI. Chemistry of Cheese Off-Flavors References

I. Introduction

The quality of cheese is determined by its flavor (taste and aroma), rheological properties (commonly referred to as body and texture, including such parameters as hardness, cohesiveness, sliceability, meltability, and stretchability), and visual appearance (e.g., intensity and uniformness of color, presence or absence of eyes or slits, closeness, uniformity of mould growth, or smear, if relevant). The relative importance of these three quality attributes varies with the cheese variety, and they are interrelated, at least to some extent. The flavor impact of cheese 17 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 45 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. 0065-2164197 $25.00

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is very strongly influenced by its rheological properties, but, unfortunately, these attributes are rarely studied in a single investigation. The flavor of cheese has been the subject of scientific investigation for nearly a century. Initially, it was believed that cheese flavor might be due to a single compound, but it soon became apparent that cheese flavor is due to the correct balance and concentration of a wide range of sapid and aromatic compounds that led to the “Component Balance Theory” (Mulder, 1952; Kosikowski and Mocquot, 1958). The development of gas chromatography (GC) in the 1950s and its interfacing with mass spectrometry (MS)permitted detailed and sensitive analysis of the volatile compounds in cheese believed to be responsible for its aroma and to be major contributors to its overall flavor. Nonvolatile compounds, for example, acids, amino acids, and small peptides, also make significant contributions to cheese flavor, especially its taste. Paper, thin-layer, and ion-exchange chromatography were initially used to study the nonvolatile flavor compounds in cheese, but have been superseded by the much more powerful technique of reverse-phase high-performance liquid chromatography (RP-HPLC).An extensive literature on various aspects of cheese flavor has accumulated and has been reviewed extensively: 57 reviews on various aspects of cheese flavor are cited by Imhof and Bosset (1994). Two general reviews have been compiled by Urbach (1993, 1995). Techniques for studying the volatile fraction of cheese have been reviewed by Imhof and Bosset (1994). More than 200 volatile compounds have been recovered from Cheddar cheese (Aishima and Nakai, 1987; Maarse and Visscher, 1989). The principal compounds are listed in Table I. Most of the water-soluble nonvolatile compounds are produced by glycolysis and especially proteolysis; methods for monitoring these have been reviewed by Fox et al. (1995b) and McSweeney and Fox (1997). About 200 peptides, some of which may have an impact on flavor, have been identified in Cheddar cheese (Singh et a]., 1995, 1997, unpublished data; Mooney and Fox, 1997). The water-soluble fraction of cheese contains considerable concentrations of free amino acids that probably contribute to the background flavor of cheese and serve as substrates for various flavor-generatingreactions. All cheese varieties that have been studied in sufficient detail contain essentially the same range of sapid compounds. Their flavors differ owing to the absolute and relative concentrations of these compounds. In spite of very considerable progress on identification and quantitation of volatile and nonvolatile flavor compounds in cheese, it is not yet possible to fully describe cheese flavor in chemical terms. It is much

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TABLE I VOLATILE FLAVOR COMPOUNDS THATHAVEBEENIDENTIFIEXI IN CHEDDAR CHEESE' Acetaldehyde Acetoin Acetone Acetophenone P-angelicalatone 1,2-Butanediol n-Butanol 2-Butanol Butanone n-Butyl acetate n-Butyl butyrate n-Butyric acid Carbon dioxide p-Cresol y-Decalactone &-Decalactone n-Decanoic acid Diacetyl Diethyl ether

Dimethyl sulfide Dimethyl disulfide Dimethyl trisulfide S-Dodecalatone Ethanol Ethyl acetate 2-Ethyl butanol Ethyl butyrate Ethyl hexanoate 2-Heptanone n-Hexanoic acid n-Hexanol 2-Hexanone Hexanethiol 2-Hexenal Isobutanol Isohexanal Methanethiol Methional

Methyl acetate 2-Methylbutanol 3-Methylbutanol 3-Methyl-2-butanone 3-Methylbutyric acid 2-Nonanone 6-Octalactone n-Octanoic acid 2-Octanol 2 &Pentanediol Z-Pentanol Pentan-2-one n-Propanol Propanal Propenal n-Propyl butyrate Tetrahydro furan Thiophene-2-aldehyde 2 -'hi decanone 2-Undecanone

'Adapted from Urbach (1993).

easier to chemically define flavor defects that are usually due to a single compound or a class of compounds. Cheese flavor is normally assessed by subjective sensory methods. A key task here is establishment or recognition of the flavor characteristic of a variety. No stable reference exists, and personal preferences can be very important. Another major task is the development of a vocabulary of terms with which to describe cheese flavor. Very interesting work on this subject has been published by McEwan et al. (1989) and Muir and Hunter (1992).For various reasons, there has been a longstanding desire to develop objective chemical and physical methods for describing and assessing cheese flavor. In this review we do not intend to review the literature on cheese flavor or the methodology used but to describe the biochemical pathways for the formation of known sapid and aromatic compounds in cheese, compounds believed to contribute to its flavor. Particular attention is focused on the catabolism of amino acids.

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A. RIPENINGAGENTS IN CHEESE Cheese curd is bland, and its characteristic taste and aroma, as well as texture, develop during ripening. The ripening of cheese involves a series of reactions catalyzed by: 1. Living microorganisms (primary and secondary starters, and nonstarter microorganisms). 2. Enzymes secreted by these microorganisms or released from their cells after death and lysis, and enzymes indigenous in milk or added to the milk or curd, especially the coagulant. 3. Chemical reactions, principally involving interactions between and modification of the products of biological and enzymatic reactions. The principal sapid compounds in cheese are produced through glycolysis, lipolysis, and proteolysis, which are now fairly well established. The secondary reactions involved in the evolution of cheese flavor are less well characterized, but several have been elucidated. II. Glycolysis

A. METABOLISM OF LACTOSEBY LACTICACIDBACTERIA Most (-98%) of the lactose in milk is removed in the whey as lactose or lactic acid, and that retained in the cheese curd is metabolized to lactate, partly during curd manufacture and partly during the early stages of ripening, normally by the starter. The pathway for lactose metabolism is characteristic of the type of starter used (see Cogan and Hill, 1993). Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris metabolize lactose to L(+) lactic acid. The glucose moiety is metabolized via the Embden-Meyerhof (EM) pathway, while galactose is metabolized via the tagatose pathway. In cheeses where a thermophilic starter is used, lactose is absorbed by S. thermophilus (which grows first as the curd cools) and hydrolyzed by P-galactosidase; the glucose moiety is metabolized via the EM pathway to L-lactate. S. thermophilus is unable to metabolize galactose, which it secretes. When the curd has cooled sufficiently, Lactobacillus spp. grow. The galactose-positive lactobacilli convert galactose via the Leloir pathway to Glu-6-P, which is then metabolized to DL-laCtate via the EM pathway. However, many strains of Lb. delbruekii ssp. lactis and Lb. delbruekii ssp. bulgaricus are galactose-negative. Lactic acid makes a major contribution to the flavor of acid-coagulated cheeses and contributes significantly to the flavor of young ren-

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net-coagulated cheeses. As the sapid compound present at the highest concentration, it probably makes some contribution to the flavor of all cheeses. However, the principal effect of the lactic acid fermentation on cheese quality is indirect: Cheddar cheese with a pH > 5.4 (which usually also has a high moisture content) has a high probability of developing off-flavors, while low pH cheese is crumbly (see Fox et al., 1990). A high pH also favors survival or growth of pathogenic and food-poisoning microorganisms. Starters for some cheeses include Leuconostoc spp., which isomerize the galactose moiety of lactose via the Leloir pathway to Glu-6-P and then metabolize it, and the glucose moiety of lactose via the phosphoketolase pathway to lactic acid, ethanol, and CO,. The lactose in brine- or dry-surface-salted cheeses is usually completely metabolized by the starter before salting, but in Cheddar-type cheeses, which are salted by mixing milled curd with dry salt, the salt concentration may rapidly reach a level that is inhibitory to the starter and residual lactose is then metabolized by salt-tolerant nonstarter lactic acid bacteria (usually mesophilic lactobacilli, but perhaps also micrococci and pediococci), some of which may be heterofermentative, producing lactic acid, ethanol, and CO,. Ethanol may contribute directly to cheese flavor or may be esterified with various fatty acids. The principal esters produced are ethyl hexanoate and ethyl octanoate, which are mainly responsible for the fruity off-flavors sometimes encountered in cheese. The CO, may be sufficient to cause pinholes and cracks in the cheese, generally considered undesirable in Cheddar-type cheese. B. METABOLISM OF LACTICACIDIN CHEESE

Young cheese contains 1.0-1.5% lactic acid, the fate of which depends on the variety (see Fox et al., 1990). In Cheddar, Dutch, and similar varieties, L-lactate is isomerized to DL-lactateby nonstarter lactic acid bacteria (NSLAB; Thomas and Crow, 1983). Racemization of L-lactate is not significant from the flavor viewpoint, but calcium D-lactate,which is less soluble than Ca-L-lactate, may crystallize in cheese, causing undesirable white specks, especially on cut surfaces (see Fox et al., 1990). Lactate can be oxidized by NSLAB in Cheddar, and probably in similar cheeses, to acetate and COz (see Fox et al., 1996). This reaction depends on the availability of 0,, which is determined by the size of the cheese and the oxygen permeability of the packaging material (Thomas, 1987). Acetate, which can also be produced by starter bacteria

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from lactose (Thomas et al., 1985), or citrate (see below), or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is usually present at fairly high concentrations in Cheddar and is considered to contribute to its flavor, although high concentrations may cause off-flavors (see Aston and Dulley, 1982). Thus, the oxidation of lactate to acetate may contribute to Cheddar flavor. The fermentation of lactose and lactate in Swiss-type cheeses has been described comprehensively by Turner et al. (1983). Typically, Emmental cheese contains about 0.4 and 1.2% D- and L-lactate, respectively, at 1 4 days, at which time the sugars have been metabolized completely. On transfer to a warm room, propionibacteria grow and preferentially metabolize L-lactate to propionate, acetate and CO,: SCH3CHOHCOOH + ZCH,CH,COOH Citric acid

Propionic acid

+ CHzCOOH + COZ + HZO Acetic acid

The COz generated is responsible for eye development, a characteristic feature of these varieties. Acetate, and especially propionate, contribute to the flavor of these cheeses. The metabolism of lactate is very extensive in surface mold-ripened varieties, for example, Camembert, Brie, and Carre de 1’Est (see Noomen, 1983; Lenoir, 1984; Karahadian and Lindsay, 1987). After manufacture, these cheeses contain 1.O% lactic acid, produced mainly or exclusively by the mesophilic starter. Secondary organisms quickly colonize and dominate the surface of these cheeses, principally Penicillium caseicolum, and to a lesser extent Geotrichum candidum and yeasts, and perhaps Brevibacterium linens. G. candidum and f? caseicolum rapidly metabolize lactate to C 0 2 and H,O, causing an increase in pH. Deacidification occurs initially at the surface, resulting in a pH gradient from the surface to the center and causing lactate to diffuse outward. When the lactate has been exhausted, E! caseicolum metabolizes proteins, producing NH3, which diffuses inward, further increasing the pH; the pH of mature Camembert may be 7.5 at the surface and 6.5 at the center. The concentration of calcium phosphate at the surface exceeds its solubility at the higher pH and precipitates as a layer of CaJPO,), on the surface, thereby causing a calcium phosphate gradient within the cheese. Reduction of the calcium phosphate concentration in the interior helps to soften the body of the cheese. The elevated pH favors the action of plasmin, which together with residual coagulant, is responsible for proteolysis in this cheese rather than proteinases secreted by the surface microorganisms, which diffuse into the cheese to only a very limited extent, although the products of their action on

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23

surface proteins may diffuse into the cheese. The combined action of increased pH, loss of calcium, and proteolysis are necessary for the characteristic softening of the body of Brie and Camembert. The metabolism of lactate and the concomitant increase in pH probably influence the flavor of these cheeses, which is rather mild and creamy, but their flavor is dominated by the products of other reactions. The ripening of surface smear-ripened cheeses, for example, Limburger, Munster, Tilsit, and Trappist, is characterized by a very complex surface microflora that includes Brevibacterium linens, Br. ammoniagenes, Arthrobacter spp., Coryniforms spp., G. candidum, and various yeasts. The surface of these cheeses is colonized first by yeasts, which catabolize lactic acid, causing an increase in pH. Deacidification is essential for the growth of Brevibacterium spp., which do not grow below pH 5.8, but probably has little direct effect on the flavor of smear-ripened cheeses, which is dominated by the catabolic activity of Brevibacterium spp. A common defect in many cheeses arises from the metabolism of lactate (or glucose) by Clostridium spp. to butyrate, H2, and CO, (see Fox et al., 1996). This reaction leads to late gas blowing and off-flavors in many cheese varieties unless precautions (e.g., good hygiene, addition of NaN03 or lysozyme, bactofugation, or microfiltration) are taken.

c. EFFECTOF LACTOSECONCENTRATION ON CHEESE QUALITY Although the concentration of lactose in milk decreases with advancing lactation, its concentration in bulked factory supplies from cows on a staggered calving pattern is essentially constant. However, when synchronized calving is practiced ( e g , New Zealand, Ireland, and Australia), substantial seasonal changes occur in the concentration of lactose in milk and, consequently, in fresh cheese curd. Variations in the concentration of lactose in cheese curd probably affect the final pH of the cheese, which affects cheese texture, enzyme activity, and perhaps the nonstarter microflora. Cheese flavor is likely to vary owing to variations in the concentration of lactic acid and catabolites therefrom, and due to variations in the metabolic activity of the cheese microflora. The concentration of lactose in cheese curd is affected by certain features of the manufacturing process. As far as we know, the concentration of lactose in cheese curd is not increased intentionally for any variety, but it is advertently increased in curd made from milk concentrated by ultrafiltration (UF),and the concentration of lactose may have to be reduced to an appropriate level by diafiltration. However, the concentration of lactose in the curd for several varieties, including

24

P. F. FOX AND J. M. WALLACE

1.5

1.o

0.5

0.0

0

4

8

12

16

20

24

28

32

36

Time, weeks FIG.1. Metabolism of lactose in Cheddar cheese curds during ripening: control (O), lactose-enriched cheeses: 1 ( A ) , 2 (A), whey-replaced cheese (01, and washed-curd cheese ( 0 ) .(Modified from Waldron, 1997.)

Gouda and Edam, is reduced by replacing part of the whey by warm water. This process, which was probably introduced as a simple method for cooking curds on farmsteads lacking jacketed cheese vats, effectively controls the pH of the cheese. Curds are washed in the washed-curd variant of Cheddar cheese and perhaps in the production of low-fat cheese (to increase moisture content). The effect of variations in the concentration of lactose in cheese curd on the quality of mature cheese has received very little attention. In an attempt to vary the concentration of lactose in Cheddar cheese curd, Huffman and Kristoffersen (1984) added lactose to the curd-whey after cutting the coagulum, but because there is a strong outflow of whey from the curd at that stage due to syneresis of the curd, the increase in lactose concentration within the curd was quite small. In a study by Waldron (1997), the lactose content of Cheddar cheese curd was reduced by removing 35-45% of the whey shortly after cutting the coagulum and replacing it with an equal volume of warm water

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

25

4.6 0

4

8

12

16

20

24

28

32

36

Time, weeks FIG. 2. Changes in the pH of lactose-modified Cheddar cheeses during ripening: control (0),lactose-enriched cheeses 1 ( A ) , 2 (A),whey-replaced cheese (01and , washedcurd cheese (). (Modified from Waldron, 1997.)

or by washing the curd after milling, or increased by adding lactose to the cheese milk up to a level of -8%. The concentration of lactose in the curd at milling ranged from 0.6 to 2.5%. Changes in the concentration of lactose in the cheese during ripening and the pH of the cheese are shown in Figs. 1 and 2. The lactose in the two types of washed-curd cheese was completely metabolized within -2 weeks, but it persisted in the high-lactose cheeses throughout ripening. Not surprisingly, the pH of the cheeses was inversely proportional to the concentration of lactose in the curd. The pH of high-lactose cheeses continued to decrease (to -4.8) throughout ripening, whereas in the washed-curd cheeses the pH increased once the lactose had been exhausted, as is common for many varieties of cheese, probably due to proteolysis and production of NH,. Flavor development was substantially faster in the high-lactose cheeses than in the washed-curd cheeses, although it was considered to be rather harsh, perhaps due to the low pH. The flavor of the low-lactose cheeses was clean and mild. The rate of growth and final number of NSLAB

26

P. F. FOX AND J. M. WALLACE

were not affected by the concentration of lactose, suggesting that NSLAB do not depend on lactose as a growth substrate. They probably utilize amino acids or are physically constrained within the curd, or colony size is restricted by local production of toxic substances. The types of NSLAB in the various cheeses may have varied, but this was not studied. The results of this study suggest that the concentration of lactose in cheese curd has a substantial effect on the quality of Cheddar and probably other cheeses. Replacing some of the whey with water or washing the cheese curd might be considered when a mild, clean flavor is desired. Normal variations in the lactose content of milk from mixedcalving herds are probably not significant but may have a substantial effect when synchronized calving is practiced. The effect may be very substantial when UF-concentrated milk is used due to low levels of curd syneresis. Diafiltration of the cheese milk or washing of the curd would appear to be desirable. Cheese made from milk with a high content of fat and casein may have a reduced lactose content, as may cheese produced from milk, the protein content of which is increased by adding UF retentate. The foregoing discussion indicates that the metabolism of lactose and lactate in cheese is well understood. In quantitative terms, these changes are among the principal metabolic events in most cheese varieties. However, compared to other biochemical changes during cheese ripening, conversion of lactose to lactate may have relatively little direct effect on the flavor of mature cheese, but since it determines its pH, it is of major significance in regulating the various biochemical reactions that occur during ripening. The isomerization of lactate probably has little impact on cheese flavor, but its conversion to propionate and/or acetate is probably significant, and, when it occurs, the metabolism of lactate to butyrate has a major negative effect on cheese quality.

D . CITRATE METABOLISM Bovine milk contains -1.8 g liter-' citrate (-8 mM), about 95% of which is soluble and lost in the whey. Citrate is not metabolized by Lc. lactis or Lc. cremoris, but it is metabolized by Lc. lactis ssp. lactis biovar diacetylactis and Leuconostoc spp. with the production of diacetyl and GO2 (for reviews, see Cogan, 1985; Cogan and Daly, 1987; Cogan and Hill, 1993). It is not metabolized by S . thermophilus or by thermophilic lactobacilli (Hickey et al., 1983), but several species of mesophilic lactobacilli metabolize citrate with the production of diacetyl and formate (Fryer, 1970).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

27

Production of COz from citrate is responsible for the characteristic eyes in Dutch-type cheese, and for undesirable openness and floating curd in Cheddar and Cottage cheese, respectively. Diacetyl is very significant for the aroma-flavor of cottage cheese, Quarg, and many fermented milks, and also contributes to the flavor of Dutch-type cheeses. Cheddar contains 0.2 to 0.5% (wt/wt) citrate, which is metabolized at a rate dependent on the NSLAB microflora. Diacetyl occurs at a considerable concentration in Cheddar and is generally regarded as a significant contributor to its flavor. Acetate from citrate may also contribute to cheese flavor. Diacetyl can be converted to acetoin, 2,s-butylene glycol, and 2-butanone. The latter is considered to be a major component of Cheddar cheese flavor (see, e.g., Dimos et al., 1996).

I I I . Lipolysis The fat fraction of cheese has a major effect on cheese texture and is important for the perception and development of cheese flavor. Poor development of flavor and texture in low-fat cheeses is a considerable technological problem that limits the market for such products. In addition to serving as a direct (fatty acids) or indirect (methyl ketones, lactones, esters) source of flavor compounds in cheese, fat serves as a solvent for sapid compounds produced from other constituents. It has been reported that a natural milk-fat emulsion yields better cheese than milk fat emulsified in skim milk, suggesting that the fat-water interface plays an important role in the development of cheese flavor; perhaps enzymes in the natural fat globule membrane (see Andrews et al., 19921 are important in flavor development. The fat in cheese can undergo degradation via lipolysis (enzymatic) or oxidation (chemical]. The degree of lipid oxidation in cheese is limited, probably due to the low redox potential of cheese and the presence of natural antioxidants. Many highly flavored compounds are produced via lipid oxidation, but the possible contribution of lipid oxidation to cheese flavor/off-flavor has been largely ignored. Fatty acids released upon lipolysis contribute directly to cheese flavor, especially in hard Italian and mold-ripened varieties and, probably, to a lesser extent in other varieties, especially when extra-mature, provided they are properly balanced by products of proteolysis and other reactions. However, extensive lipolysis is considered undesirable in most cheese varieties: Cheddar, Gouda and Swiss-type cheeses containing even a moderate level of free fatty acids (FFAs) would be

28

P. F. FOX AND J. M. WALLACE TABLE I1 TYPICAL. CUNCENTRATIONS OF FREE FATTY Acros (FFAs) IN SOME CHEESE VARIETFES

Variety

Sapsago Edam Mozzarella Colby Camembert Port Salut Monterey Jack Cheddar

FFA (mg1kg-I)

FFA (mg/kg-’)

Variety

Gjetost Provolone Brick Limburger Goat’s milk Parmesan Romano Blue-mould (US) Roquefort

21 1 356 363 550 681 700 736 1028 ~~

~~~

1658 2118 2150 4187 4558 4993 6754 32230 32453

~~

Date from Woo et a1 (1984)and Woo and Lindsay (1984).

considered rancid. The total concentrations of FFAs in some major cheese varieties are summarized in Table 11. A. LIPASESIN CHEESE Lipases in cheese originate from milk, rennet paste (some varieties), starter, adjunct starter or nonstarter bacteria, or exogenous sources. Milk contains a well-characterized lipoprotein lipase (LPL) that liberates fatty acids from the sn-1 and 3 positions of mono-, di-, and triglycerides and the 1 position of glycerophospholipids (Olivecrona and Bengtsson-Olivecrona, 1991; Olivecrona et al., 1992). In milk fat, highly flavored short-chain acids that are esterified predominantly at the sn-3 position and hence even low LPL activity can have a significant effect on flavor. LPL is associated mainly with the casein micelles and is incorporated into cheese. LPL probably causes significant lipolysis in raw milk cheese but probably contributes little in pasteurized milk cheese. Good-quality rennet extracts are free of lipolytic activity, but the rennet paste used in the manufacture of some Italian varieties (e.g., esterase Romano, Provolone) contains a potent lipase-pregastric (PGE)-that catalyses the extensive lipolysis responsible for the “piccante” flavor characteristic of such varieties. The literature on PGE, also called lingual or oral lipase, was comprehensively reviewed by Nelson et al. (1977) and updated by Fox and Stepaniak (1993).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

29

PGE is highly specific for short-chain acids esterified at the sn-3 position. Slight differences in specificity between calf, lamb, and kid PGEs permit the manufacture of Italian cheeses with slightly different flavor characteristics. Most other lipases are unsuitable for the manufacture of Italian cheeses because of incorrect specificity, but it has been claimed that certain fungal lipases may be acceptable alternatives (see Fox and Stepaniak, 1993). The use of PGE to accelerate the ripening of other cheese varieties was discussed by Fox (1988-89). L Q C ~ O C O Cspp. C ~ Sare weakly lipolytic, but in the absence of strongly lipolytic microorganisms they probably make a significant contribution to the low level of lipolysis in Cheddar and Dutch varieties. The intracellular esterase-lipase of two Lactococcus strains has been isolated and characterized (Holland and Coolbear, 1996; Chick et al., 1997). Little is known about the genetics of these enzymes. Isolation of lipase/esterasenegative variants of Lactococcus would permit the significance of these enzymes in cheese ripening to be assessed. Both mesophilic and thermophilic lactobacilli possess (mainly) intracellular esterolytic-lipolytic activity (Khalid et al., 1990; Gobbetti et al., 1996). The esterolytic-lipolytic activity in cell homogenates of a number of lactobacilli was characterized by El-Soda et al. (1986), and an intracellular lipase and an intracellular esterase from Lb. plantarum were purified and characterized by Gobbetti et al. (1997a,b). Micrococcus spp., which constitute part of the nonstarter microflora of cheese, especially the surface microflora, produce lipases that may contribute to lipolysis during ripening (Bhowmik and Marth, 1990a,b). The nonstarter microflora of cheese may also include Pediococcus spp. that are weakly esterolytic and lipolytic (Tzanetakis and LitopoulouTzanetaki, 1989; Bhowmik and Marth, 1989). Propionibacterium shermanii possesses a lipase(s) (Paulsen et al., 1980) that contributes to lipolysis in Swiss varieties. An intracellular lipase of I! shermanii was partially characterized by Oterholm et al. (1970). Brevibacterium linens, a major component of the surface of smear-ripened cheeses, possesses intracellular lipases and esterases (Smhaug and Ordal, 1974; Foissy, 1974). The intracellular esterase of Br. linens ATCC 91 74 has also been purified and characterized (Rattray and Fox, 1997a). Very extensive lipolysis occurs in mold-ripened cheese, particularly blue mold varieties in which up to 25% of the total FFAs may be liberated (see Gripon, 1987, 1993). However, the impact of FFAs on the flavor of blue mold-ripened cheeses is less than for hard Italian varieties, possibly due to neutralization as the pH increases during ripening and the dominant influence of methyl ketones on the flavor of blue

30

I? F. FOX AND 1. M. WALLACE

mold cheeses. Lipolysis in mold-ripened varieties is due primarily to the lipases of Penicillium roqueforti or I! camemberti, which secrete potent extracellular lipases that are well characterized (see Kinsella and Hwang, 1976; Gripon, 1987, 1993). Psychrotrophs, which dominate the microflora of refrigerated milk, are potentially an important source of potent lipases in cheese if their numbers exceed lo7 ml-I (Cousins et al., 1977). Many psychrotrophic lipases are heat stable and may cause rancidity in cheese over a long ripening time (Mottar, 1989; Smhaug and Stepaniak, 1997). Unlike psychrotrophic proteinases, which are largely lost in the whey, psychrotrophic lipases adsorb onto the fat globules and are concentrated in the cheese.

B. CATABOLISM OF FREEFATTY ACIDS The flavor and aroma of blue mold cheese are dominated by saturated alkan-2-ones (n-methyl ketones). A homologous series of alkan-Zones with odd-numbered carbon chains from C3 to CI7, and some with even-numbered carbon chains, occurs in these cheeses: heptan-2-one, nonan-&one, and undecan-2-one are dominant during most of the ripening period (Dartley and Kinsella, 1971). Some reduction of alkan-2ones to alkan-2-01s occurs during ripening, which has an undesirable effect on flavor. The metabolism of fatty acids in cheese by Penicillium spp. involves four main steps: (1)release of fatty acids by lipases, (2) oxidation to a-ketoacids, (3) decarboxylation to alkan-2-ones with one less carbon atom, and (4) reduction of alkan-Zones to the corresponding alkan-2-01; step four is reversible under aerobic conditions. Alkan-2-ones can also be formed by mold from the ketoacids naturally present at low concentrations in milk fat (-1%of total fatty acids) or by the oxidation of monounsaturated acids. The rate of alkan-Zone production in cheese is affected by temperature, pH, the physiological state of the mold, and the concentration of fatty acids (Adda et al., 1982). Both resting spores and mycelia are capable of producing alkan-Zones at a rate that does not depend directly on the concentrations of FFA precursors. Indeed, high concentrations of FFAs are toxic to I! roqueforti (Fan et al., 1976). Lactones are formed by the intramolecular esterification of hydroxyacids through the loss of water to form a ring structure. a- and 0-lactones are very reactive and unstable, but y- and S-lactones are stable and occur in cheese. Lactones possess a strong aroma, which, although not cheese-like, may contribute to overall cheese flavor. y- and S-lac-

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

31

tones may be formed spontaneously from the corresponding y- and 6-hydroxyacids following their release from triglycerides; their concentration in cheese should therefore correlate with the extent of lipolysis. Lactones may also be formed from ketoacids after reduction (Wong et al., 1975). The formation and significance of lactones in cheese have received relatively little attention.

IV. Proteolysis

Proteolysis is the most complex and perhaps the most important biochemical event during the maturation of most cheese varieties. The extent of proteolysis varies from very limited (e.g., Mozzarella] to very extensive (e.g., blue mold varieties), and the products range from large polypeptides, comparable in size to intact caseins, through a range of medium and small peptides, to free amino acids. Proteolysis is mainly responsible for softening of the texture of cheese during the early stages of ripening and influences the development of cheese flavor via the formation of amino acids and peptides that make a direct, although probably limited, contribution to flavor. Amino acids also serve as substrates for the formation of numerous flavor compounds. Proteolysis also affects the mouthfeel of cheese and the release of flavor compounds from cheese during mastication. Various methods have been developed for quantifying the extent and pattern of proteolysis in cheese. They are reviewed by Fox et al. (1995b) and McSweeney and Fox (1997). A. CONTRIBUTION OF INDIVIDUAL PROTEOLYTIC AGENTS Proteolytic enzymes in cheese originate from milk, coagulant, starter, secondary starter, and nonstarter microorganisms. Information on the relative importance of individual proteolytic agents has been obtained from studies on cheeses with controlled microflora, in which the coagulant, plasmin, starter, and nonstarter bacteria, in various combinations, have been excluded or inactivated. These studies, which have been summarized by Fox et (11.(19931, indicate that rennet and plasmin are mainly responsible for the initial hydrolysis of as1-and p-caseins, respectively, and for the production of most of the water- or pH 4.6-soluble N in cheese. Although the lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus) are weakly proteolytic, they possess a very comprehensive

32

P. F. FOX AND J. M. WALLACE

proteolytic system that has been studied extensively and reviewed (see Fox and McSweeney, 1996; Kunji et al., 1996). The proteolytic system of Lactococcus has been particularly well studied. They possess a cell wall-associated proteinase, several intracellular proteinases, at least two intracellular oligoendopeptidases (PepO, PepF), at least three aminopeptidases (PepN, PepA, PepC), a dipeptidylaminopeptidase (PepX), a tripeptidase, a general dipeptidase, and proline-specific dipeptidases. However, Lactococcus spp. and most Lactobacillus spp. lack a carboxypeptidase. They also possess a number of peptide and amino acid transport systems. This comprehensive proteolytic system is necessary to enable the lactic acid bacteria to grow to the requisite high numbers ( 1 0 ~ - 1 0cfu/ml) ~~ in milk, which contains only low levels of small peptides and free amino acids. The proteolytic system of Lactobacillus spp. has been studied less thoroughly than that of Lactococcus, but the two systems appear to be generally similar. The cell wall-associated proteinase contributes to the formation of small peptides in cheese, probably by hydrolyzing larger peptides produced from a,,-casein by chymosin or from p-casein by plasmin. The aminopeptidases, dipeptidases, and tripeptidases, which are intracellular, are responsible for the release of free amino acids after the starter cells have died and lysed. The proteolytic system of NSLAB in Cheddar, and probably in similar cheeses, appears to supplement the proteolytic action of the starter, producing generally similar peptides and amino acids (Lynch et al., 1997). The proteinases and peptidases of P roqueforti and l? comemberfi make a major contribution to proteolysis in mold-ripened cheeses (see Gripon, 1993). Br. linens secretes an extracellular proteinase and an aminopeptidase and possesses a number of intracellular peptidases, which may be released on cell lysis (see Rattray et al., 1995; Rattray and Fox, 1997b). These enzymes probably contribute to proteolysis, including the production of free amino acids, in the surface of smear-ripened cheeses. Propionibacterium shermanii, the characteristic secondary microorganism of Swiss-type cheeses, is weakly proteolytic but has high peptidase activity, especially proline-specific peptidases, and contributes significantly to proteolysis in Swiss cheese. The proteolytic specificity of chymosin, plasmin, and the cell wallassociated proteinase of several lactococcal strains on a,,-, as2-, p-, and K-caseins has been determined (see Fox et al., 1995a, 1996; Fox and McSweeney, 1996). The specificity of the extracellular proteinases of €? roqueforti, €? camemberti, and Br. linens on as1-and p-caseins has been determined (see Gripon, 1993; Rattray et al., 1996, 1997).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

33

B. PROTEOLYSIS IN CHEESE The extent and type of proteolysis in a number of the principal cheese varieties has been characterized (see Fox and McSweeney, 1996). Proteolysis in Cheddar is very well characterized and can be summarized as shown in Figs. 3, 4, and 5. a,,-casein is completely hydrolyzed by chymosin, typically within about 3 months, at the Phe2,-Phez4 bond. The larger peptide, a,,-CN f24-199, is further hydrolyzed extensively by chymosin at the Leulol-Lys,oz bond and to a lesser extent at Phe3,Gly33, Leug8-Leu,,, and Leulog-Glullo, probably not in sequence. Some of the Lyslo3-Tyrlo4 and Lyslo5-Vallos bonds are also hydrolyzed by plasmin. The large C-terminal peptides, a,,-CN f24-199,102-199,110199, 99-199, 33-199, 104-199, and 106-199, are detectable in the water-insoluble fraction (Fig. 6). The complementary N-terminal peptides (e.g., a,,-CN f24-101, 24-98, 24-109) have not yet been identified in the water-insoluble fraction, but since these are highly phosphorylated, they may be in the water-soluble extract. The large peptides, which have not yet been characterized, are also hydrolyzed by lactococcal CEP and possibly by lactococcal PepO and several small peptides produced therefrom and are present in the UF permeate or retentate of the water-soluble extract (WSE) (Fig. 3). The peptide a,,-CN fl-23 is hydrolyzed rapidly by the lactococcal CEP at bonds Glng-Glyl0, Gln13-Glu14, Glu14-Val15, and Leul6-Asnl7, and probably at other sites, depending on the specificity of the CEP. The peptides aSl-CNh-9,1-13, and 1-14 accumulate and dominate the UF permeate or 70% ethanol-soluble fraction of the water-soluble extract of Cheddar. According to Exterkate and Alting (1995), these peptides do not affect the flavor of cheese. The complementary C-terminal peptides (i.e., a,,-CN f10-23, f14-23, f15-23, and f17-23) have been identified in the UF permeate, and some of them have been partially hydrolyzed by an aminopeptidase, probably PepN, releasing amino acids (Fig. 3). Peptides from the N-terminal region of a,,-CN f24-199 are also hydrolyzed by lactococcal CEP, or possibly PepO or PepF. The N-terminals of some of these peptides do not correspond to known CEP cleavage sites, suggesting the action of aminopeptidase, PepO, PepF, or NSLAB enzymes. In Cheddar and most other cheeses, p-casein is much more resistant to hydrolysis than a,,-casein: only about 50% is hydrolyzed within 6 months. It is hydrolyzed mainly by plasmin, at Lys28-Lys,g, LyslOsGlnlos,and Lyslo7-Glulo8,yielding yl-,f-, and f-caseins (P-CN f29-209, 106-209, and 108-209, respectively), which are clearly evident in the

W ip

1

-19

93 -lY 14-

7

DF Rctcotatc I* -35 2s-u

IS-35

116-124

U n

IU

-am

115-121

15-Y 14

110-

3 4

!’

Cleavage sites uf crll-euvelupc pruteinas uf starter L.Clueoccur rpp.

14 1 ,

IW57

I

I

Y3

Zb-31

1-9

-

16-31

1) IJ

2J-m

-

?

IBS 1.1

*(-?

y ? 4l-?

17-7 18 - ?

!’

DF Pernienle

?

Jl-?

Water-hsoluble Fraction .................

2, .......... 21 .- ~

_ _ 33 .........

,

,

”......._..... ..

7y

,

-

199

.- . ~ . -. . ~ . . .................. 1...... 199 lu.-.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . lu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - IY, IIO.-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - I99 121 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 I*y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I99 .......... 1% ?

FIG.3. ~ - C a s e i n - d e r i v e dpeptides isolated from the water-insoluble (----I, the diafiltrate (DF), retentate (-1, and the DF permeate (-1 of the water-soluble fraction of a mature Cheddar cheese. (Modified from Singh et ol., 1997, and Mooney, 1997.)

I

DF Permeate

7

191175176-1

197

lU2 ZOC-207

Cleavage sites of Plasmin

61-71

61-70

2s

DF retentate

-=

2 7 -

41

FIG.4. aS2-Casein-derivedpeptides isolated from the DF retentate (-), of mature Cheddar cheese. (Data from Singh et a]., 1997.)

and the DF permeate (-)

of the water-soluble fraction

93

51 51 51

w

93

Q,

92

n-n

7 3 n

I

.

[-)

Zm

In .

109 W

FIG.5. p-Casein-derived peptides isolated from the water-insoluble fiaction (----I, the DF retentate (-1, and the D F permeate of the water-soluble fraction of a mature Cheddar cheese. (Modified from Singh et al., 1997, and Mooney, 1997.)

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

37

FIG.6. Urea polyacrylamide gel electrophoretogram of sodium caseinate (C) and the water-insoluble nitrogen fraction of Cheddar cheese after manufacture (slot 2) and ripening for 1, 2, 3, 4, 5, 6 , 8, 10, 1 2 , 14, 16, 18, and 20 weeks (slots 3-14, respectively). Peptides isolated from the 20-week sample were identified as follows: 1. P-CN f106-149, 2. P-CN flO6-128, 3. P-CN flO6-209 (y),P-CN f29-209 (r'),5. P-CN flO8-209 (y),6. 0-CN, 7. P-CN f1-187/192 (P-I-CN),8. P-CN fI-*, 9. asI-CN f99-199,lO. CX~I-CN f80-*, 11. asl-CN, 12. as1-CN f102-199, 13. asl-CN f24-199 (USi-I-CN), 14. as1-CN 33-*, 15. asl-CN f121199, 16. asl-CN fl29-199, 17. CXSI-CNf60-*, 18. asl-CN f110-199, 19. USI-CN f70-156. * = peptide not identified completely. (Modified from Mooney, 1997.)

water-insoluble fraction of Cheddar and most other varieties (Fig. 7), and proteose peptone 8 fast (P-CN fl-ZS), 8 slow (P-CN f29-105/107), and 5 (P-CN fl-105/107), which are present in the UF retentate or 70% ethanol-insoluble fraction of the WSE. Most of the peptides that have been identified in the UF retentate (or 70% ethanol-insoluble fraction) of the WSE are produced from p-casein by the action of lactococcal CEP, probably on proteose peptones rather than on intact p-casein, since none of the identified peptides contain a plasmin cleavage site (Fig. 5). The N-terminal of many of the identified peptides corresponds to the carboxyl residue of a plasmin or CEP cleavage site, but several appear to be 1 , 2 , or 3 residues short, indicating

38

P. F. FOX AND J. M. WALLACE

FIG.7 . Urea-PAGE of the water-insoluble fraction of a selection of cheese varieties. Lane 1 = Na-caseinate; lane 2 = Cheddar 1; lane 3 = Cheddar 2; lane 4 = Cheddar 3; lane 5 = Emmental 1; lane 6 = Emmental 2; lane 7 = Emmental 3; lane 7 = Maasdam; lane 9 = Jarlsberg; lane 10 = Edam 1; lane 11 = Edam 2; lane 1 2 = Edam 3; lane 13 = Gouda 1; lane 14 = Gouda 2; lane 15 = Gouda 3. (Modified from McGoldrick, 1996.)

aminopeptidase activity. The C-terminal of several peptides does not correspond to reported CEP cleavage sites, suggesting carboxypeptidase activity (which has not been reported in Lactococcus spp.) or unknown CEP cleavage sites, or the action of other proteinases, for example, from NSLAB or oligopeptidases (PepO, PepF). Thus, the large water-insoluble peptides in Cheddar are the C-terminal segments of asl-casein produced mainly by chymosin or of p-casein (y-CNs)produced by plasmin. Para-K-casein (K-CN fl-105) is very resistant to proteolysis and is not hydrolyzed during ripening. The concentration of a,,-casein appears to decrease during ripening, but its fate is

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

39

unclear. No large peptide derived from a,,-casein has yet been reported, and only eight small as2-derived peptides have been identified in the WSE (Fig. 4). Perhaps a,,-CN, which represents only -10% of total casein, is hydrolyzed rather nonspecifically to several peptides, none of which are present at a high concentration, and hence have been overlooked. The small water-soluble peptides appear to be produced from chymosin- or plasmin-produced peptides by lactococcal CEP and perhaps endopeptidases. Some of the small peptides are hydrolyzed by aminopeptidases. Although NSLAB dominate the viable microflora in Cheddar for most of the ripening period, they appear principally to supplement the peptidolytic activity of the starter, especially in the production of amino acids (Lynch et al., 1997). The accelerated maturation of cheeses with added lactobacilli appears to be closely related to the concentration of , Ardo and free amino acids (Lloyd et al., 1980; Hickey et ~ l . 1983; Pettersson, 1988; Ardo et al., 1989; Puchades et al., 1989; Broome et al., 1990; Lee et al., 1990a,b;Lynch et al., 1997). Although most lactobacilli were found to increase the concentration of amino acids and the intensity of cheese flavor, the flavor was found to be atypical when cheese milk was inoculated with Lb. helveticus (Lloyd et al., 1980), Lb. brevis (Puchades et al., 1989),or Lb. casei MIL2A (Broome et al., 1990). Proteolysis in Cheddar- and Dutch-type cheeses, and in many other varieties, is generally similar (Fig. 7). The high-cooked varieties, for example, Emmental and Parmesan, differ, partly because chymosin is extensively or totally inactivated during cooking while plasmin activity is increased, and partly because the thermophilic starter lactobacilli are more peptidolytic than Loctococcus spp. In addition, Swiss-type cheeses contain Propionibacterium, which has very high proline-specific peptidase activity. Commercially mature Emmental undergoes relatively little primary proteolysis: only -50% of the aSl-casein is hydrolyzed at Phe,,-Phe,, (by chymosin) and/or Lys103-Tyr104 and Lyslo5-Vallo, (by plasmin); the Leulol-Lysloz bond is not hydrolyzed. Although primary proteolysis is relatively limited in Parmesan, very extensive peptidolysis occurs; most of the WSE occurs as very small peptides or free amino acids.

c. FREEAMINOACIDSIN CHEESE The final products of proteolysis are free amino acids, the concentrations of which have been used as indices of ripening for many years

40

P. F. FOX AND J. M. WALLACE

(Harper and Swanson, 1949; Kosikowski, 1951; Law, 1981; Hickey et al., 1983; Wood et al., 1985; Amantea et al., 1986; Puchades et al., 1989; Wilkinson, 1993; Resmini et al., 1993). Resmini et a]. (1993) reported that the concentration of free amino acids in Grana Padano cheese correlated with flavor intensity but not with ripening time. However, others reported that amino acids appeared to have no effect on cheese flavor (Dacre, 1953; Mabbit et al., 1955; Law, 1966; Law and Sharpe, 1977). As discussed in Section IV.B, ripening and flavor development appear to be accelerated when high levels of free amino acids are produced early in the ripening process. Although catabolic products of amino acid degradation are thought to be mainly responsible for Cheddar flavor, small peptides and free amino acids contribute to the flavor of most cheese varieties (Urbach, 1995), for example, “brothy,” “nutty,” and “sweet” tastes (Biede and Hammond, 1979). The flavor characteristics and flavor threshold of free amino acids are summarized in Table 111. High levels of proline contribute to sweetness in Swiss cheese (Reinbold, 1973; Langsrud and Reinbold, 1973; Lloyd et al., 1978, 1980) and to a sweet Swiss cheeselike flavor in experimental Cheddar (Lloyd et al., 1980). Arginine has been associated with an unpleasant bittersweet taste (Puchades et al., 1989).Free amino acids can also contribute to bitterness in cheese (Ney, 1971). About 20 to 30% of the total N in a mature Cheddar cheese is soluble in water, while about 2 to 5 % is soluble in 5% phosphotungstic acid (PTA). The levels of peptides and free amino acids soluble in 5% PTA in cheese are considered to be reliable indicators of the rate of flavor development (Aston et al., 1983; Pham and Nakai, 1984; Amantea et al., 1986; Ardo and Pettersson, 1988); however, the composition of the amino acid fraction and the relative proportions of individual amino acids are thought to be most important for the development of a typical characteristic flavor (Broome et al., 1990). The release of specific amino acids, particularly Glu, Met, and Leu, coincides with the development of Cheddar cheese flavor (Broome et a]., 1990; Marsili, 1985). Leu and Met are considered to be major contributors to cheesy flavor in the water-soluble extract of Cheddar (Kowalewska el al., 1985; Marsili, 1985; Aston and Creamer, 1986). The concentration of free amino acids in cheese at any stage of ripening is the net result of the liberation of amino acids from casein and their transformation to catabolic products. The concentration of individual amino acids in a number of cheese varieties are compiled in Table IV. Comparison of free amino acid profiles in different varieties is limited for a number of reasons. Cheeses are ripened at different rates,

41

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE I11

TASTEDESCFXPTORAND THRESHOLD VALUESOF AMINOACIDS'

Q" Amino acid

Gly Ser Thr His Asp Glu

(cal mol-'I

0

-300

Taste threshold (mg 100 ml-')

Concentration'in Cheddar (wg g-'1

Perceptiand

Taste Sweet

130

371

***

150

1210

**I

***

400

260

649

500

20

436

0

3

606

0

5

5075

750

50

1737

A% Ala Met

500

60

337

1300

30

869

LYs

1500

50

2330

Val Leu Pro Phe

1500

40

2022

Salt

Sour

Bitter

* ***

** ***

**

*** ***

***

**

** ***

1800

190

4610

2600

300

389

2500

90

2400

*** *** ***

607

***

Tyr

2300

Ile

2950

90

466

3li.p

3400

90

-

Umami

***

*** ***

"Adapted from O'Callaghan (1994). bHydrophobicity values of Bigelow and Channon (1976). Wilkinson (1992); 6-month-old Cheddar; total concentration = 24.1 mg g-' cheese. dAmino acids in Cheddar are deemed to be perceived if their concentration in a water extract (50 g cheese containing 37% moisture to 100 ml water) is greater than their threshold concentration.

even within a single variety. The age of the cheeses in Table IV vary, and in some cases are unknown. Different analytical techniques lead to variations in results. The rate of amino acid degradation differs between and, to a lesser extent, within varieties due to different manufacturing and ripening conditions and variations in cheese microflora. Without monitoring protein-bound as well as free amino acids, no conclusions as to the exact concentrations of amino acids released may be drawn since some of the free amino acids may have been degraded prior to analysis. The relative proportions of individual amino acids appear to be similar in many varieties. Leu, Glu, and Lys are the principal amino acids in Cheddar (Wood et al., 1985). Glu has been described as an important contributor to Cheddar flavor by many authors (Harper and Wang, 1981; Marsili, 1985; Nsar and Younis, 1986; Broome et a]., 1990).

42

P. F. FOX AND J. M. WALLACE TABLE IV

CONCENTRATION (mg kg-' CHEESE) OF FREEAMINOACIDS AND THEIR RELATIVI?.PROPORTIONS (% TOTAL FREE AMINO ACIDS) I N DWI%RENT CHEESE VARIETIES Chnese [Ripening time]

(:hedrlm (1)

Cheddar (2)

I8 niol

19 mol

mg/ kg

'&of FAA

CYS

44.7

029

Asp

1532 8

9.94

Amino acid

1.99

-

Thr

-

SW

41fi 3

2.7

GlU

:i144 1

20.39

Pro

336.2

2.18

GlY

30fi.n~

mg/ kg

Edam (3)

% of FAA

mg/

Gouda (4)

HnrrgArd (5) [.I4wkl

Emmental (6) [fi mol

mg/

mg/ kg

% of FAA

% of FAA

mg/

%of

kg

kg

FAA

-

-

0.65

20.5

0.9

91.7

1.4

1.4

1fifi.S

0.9

1.11

144.5

6.0

272.9

4.2

2.3

688.5

3.7

1.85

71.1

3.0

167 3

2.fi

2.7

548.9

2.9

6.21

351.7

1 2 3!

992.1

15.1

14.3

26no.s

14.3

i5y.n

54

345.6

5.3

4.8

2535.2

13.5

2.32

34.ti

1 2

108.G

1.6

2.7

430.8

23

-

~

k

nf FAA

Oh

-

Ala

3Sfi.2

2.31

-

3.67

68.6

24

iun.9

2.8

2.7

568.2

3.0

Val

1096.4

7.11

-

1 5 26

167.4

6.8

47fi.s

7.3

7.0

1561.5

8.4

Met

434.8

2.82

-

8.14

60.3

2.3

176.1

2.7

1.n

502.7

27

1Ie

-

8 46

48 1

2.4

200.5

3.1

2.3

1051 1

5.G

26.90

428.0

17.5

1056.9

16.2

14 3

17949

9.6

89.2

3.4

165.3

2.5

2.3

2859

1.5

7.56

291.9

11.6

645.9

9.9

56

1279.1

fi.8

49.7

1.7

143.6

2.2

3.8

RGR.2

4.6

11 00

245.5

9.1

fi71.4

10.3

12 LJ

22198

11.9

0 00

130.5

4.2

167.3

2.6

11 7

192

0.1

~

i.PI1

2774.1

'Tyr I'hn

464.1

3.01

-

1472.6

9.55

-

17.99

IIis

-

-

LYS

1127.2

7.31

Arg

1096.4

7.11

Total

~

~

~

~

258'~

05211

Asrr Gln

7.7 18616

'The concentrations of amino acids were either given by, or calculated from tho results of 1: Wood et a]. (19851; 2: Puchades ot a]. (1989); 3,4,6: Antila and Antila (1968);5: Ardo and Gripon (1995).

High levels of Asp and Met were also observed by these authors. Comparatively low levels of Glu (as a percentage of total FAAs) were found in Cheddar by Puchades et al. (1989), who found that Met represented >8% of total FAAs; in contrast, Wood et ~ l(1985) . found that Met was only 2.8% of FAAs. According to Wood et al. (19851, -10% of total FAAs in Cheddar is Asp, while Puchades et al. (1989) reported a value of only 0.65%.Large variations within single varieties have been reported for Emmental, Gruyere, Parmagiano Reggiano, Camembert, Stilton, Danablue, Cabrales, and Mahon (Table IV). There are, however, clear similarities within, and differences between, different cheese varieties. High concentrations of free amino acids in Parmesan are thought to be due to high numbers of lactobacilli and a long ripening time. High

43

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE IV continued Cheese (Ripening time1 Gruyere (7)

Amino acid

mgi kg

70of FAA

Gruyhrr (HI

Appenznller (9)

mgl kg

B of FAA

mgi kg

%of FAA

23

Grana (101 Padano [60samples 10-20 wkl

mgl kg

*A of FAA

Parmigino (111 Parinigino (121 Reggiano Reggiano [mean of 60 cheeses1 mgl

kg

-

%of FAA

mgl kg

“A of FAA

-

115.0

0.3

3.1

3241.9

4.3

2231.9

fi.3

2.4

3.2

4033.3

5.3

1031.3

3.2

0.76

4.4

4459.5

5.9

1580.5

5.0

20.42

18.0

14489.0

19.0

7580.2

21.4

3299.3

9.87

9.5

-

-

3649.6

10.3

74Y.O

2.2

2.4

2115.6

2.8

667.3

1.9

2.65

925.65

2.8

2.7

2260.2

3.0

1028.2

2.9

2381.5

7.3

3594.4

10.8

7.2

6011.9

7.9

2330.9

6.6

868.3

2.7

922.0

2.8

2.5

2351.5

3.1

970.7

2.7

6.1

1901.8

5.97

1YY6.3

6.0

6.1

5205.2

6.H

1762.5

5.0

1778.7

9.1

3765.6

12.5

4176.1

12.5

9.1

7290.4

9.6

2905.6

9.1

321.3

1.6

992.9

2.7

884.2

2.7

2.7

2054.7

2.7

1963.7

5.5

1366 7

7.0

2839.4

7.6

2548.7

7.6

5.1

4314.9

5.7

1753.0

4.9

CYS

-

-

ASP

328.9

1.7

834.8

2.6

774 6

Thr

7R1.2

4.0

1017.2

3.1

1317.1

Snr

446.1

2.2

979.4

3.0

253.2

GI”

2963.5

15.2

6415.9

19.7

6823.4

Pro

2817.3

14.4

3546.9

10.9

Gly

493.0

2.5

716.0

2.2

Ala

597.6

3.1

865.1

Val

1817.9

8.3

Met Ile

532.1

2.7

1189.2

Leu TYr Phe His

680.6

3.4

1276.8

3.2

1081.3

3.2

3.3

-

-

1034.5

2.9

LYS

2227.8

11.3

4038.7

13.7

4573.7

13.7

12.2

10091

13.3

2876.9

8.1

‘4%

21.3

0.1

158.5

0

0

0

-

791.4

1.0

977.1

2.8

Tulal

19342

32598

33419

76100

34459

The concentrations of amino acids were either given by, or calculated from the results of 7: Antila and Antila (1968); 8: Lavanchy and Buhlmann (1983);9: Lavanchy et 01. (1979); 10: Resmini et al. (1993); 11: Resmini et al. (1988); 12:Mariani et a1. (1993).

concentrations of free amino acids are also present in mold-ripened and Swiss cheeses. Of the cheeses tabulated, Edam and Gouda contain the lowest levels of free amino acids. However, an Edam-type cheese made from goat’s milk contained high levels of FAAs, as did another goat’s milk cheese, Rumelia (Baltajieva et al., 1985). Resmini et al. (1993) attempted to “fingerprint” cheeses using chemometric models based on free amino acid profiles. These models allowed Parmigiano Reggiano, Grana Padano, and Fontina to be distinguished from each other and from similar cheeses. In another comparative study, Engels and Visser (1994) found that the amino acid profiles for Cheddar, Edam, Gouda, Gruyere, Maasdam, Parmesan, and Proosdij cheeses were

44

€? F. FOX AND J. M. WALLACE

TABLE IV

continued Cheese [Kipening time] Danish 1131 Camembert

Camnm- 114) hrt

Stil- (15) ton

Stik- (16) ton IMarketl

170 days]

Amino acid

mgl kg

YO of FAA

rngl kg

% uf FAA

mgl

%of

mgf

kg

FAA

kg

Stil- (17) tnn

of FAA

mgl kg

%of FAA

Oh

Dana- (18) blue [Market] rngl

kg

"h of FAA

-

fi.4

0.1

-

-

1020

10.4

-

5.9

99.7

4.0

5390

28

626.77

2.2

340

3.5

1098.1

Thr

-

2.5

134.8

6.9

751.6

4.0

837 5

3.5

1811

1.8

1526.6

5.4

Ser

-

3.2

56.2

27

761.0

4.0

621.4

2.6

57u

58

896.6

3.2

Glu Pro

-

22.5

566.6

20.5

1750.0

8,2

4238.4

175

1090

11 1

in302

6.5

3.1

192.3

6.6

1446.3

7.6

1267.2

5.2

510

52

332.0

12

G~Y

-

1.2

36.9

1.3

216.4

1.1

650.6

2.7

140

1.4

485.2

1.7

Ale

-

1,8

93.8

3.3

876.9

4G

793.8

3.3

350

357

13194

47

VAI

-

5.8

169.1

5.7

1664.6

8.8

2687.4

in 7

fiiu

6.22

3353.~~ 11s

cys Asp

~

~

3.9

Met

-

3.6

65.6

3.1

603.6

3.2

929.7

3.8

410

4.2

1679.R

Ile

-

5.8

134.2

4.2

848.4

4.5

1716.3

7.1

310

3.2

2434.G

8.6

LCU

-

11.8

317.6

11.5

3015.9

15.9

2920.3

12.0

1220

12.5

3708.6

13.1

TLr

-

5.3

160.1

5.9

1495.6

7.9

1733.2

7.1

260

2.7

2340.9

17.3

PllB

-

6.4

241.8

8.7

1776.5

9.4

2386.2

9.8

610

6.2

2fi89.9

9.5

2.6

175.1

6.6

6337.7

3.4

463.7

1.9

490

5

996.0

3.5

93

209.1

6.5

1975.8

10.4

2456.7

10.1

1560

15.9

2760.9

9.7

36.2

1.5

616.9

3.3

279.2

1.2

130

1.3

919.3

3.2

His

4 s

*u Tulal

~

~

~

1.4

2790

18980

24275

YB00

5.9

28375

The concentrations of amino acids were either given by, or calculated from the results of 14: Antila and Antila (1968); 13,17:Ismail and Hansen (1972); 14: Antila and Antila (1968); 15,16,18:Madkor et a]. (1987); 17:Ismail and Hansen (1072); 17: Zarmpoutis (1995).

similar, with Glu, Leu, and Phe being the principal amino acids; Val, Pro and Lys were also quite abundant in these varieties. Maasdam, Cheddar, Gouda, and Edam contained similar concentrations of free amino acids in the UF permeate (MW < 500 Da), but higher concentrations were found in Gruyere, Proosdij, and Parmesan cheeses. The authors concluded that flavor and the concentration of free amino acids could not be correlated since Cheddar, Maasdam, Gouda, and Edam have very different flavors, although the concentration and relative proportions of free amino acids were generally similar. Although the concentration of free amino acids depends on the cheese variety and the dairy plant where the cheese is manufactured, they generally increase during ripening, with the exception of Arg, the

45

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE IV continued Cheese [Ripening time] Dana- (20) blue 18.3 mol

Dana- 1191 blue

-____ Amino acid

mg/ kg

%of FAA

mgl kg

Gorgon- (21) zola

Cashel (22) (Blue1

~

%of FAA

mg/

kg

~ % of FAA

Chetwynd (23) Gamonedo (24) (Blue) (Blue) 190 days1 _ _

-

kg

%of FAA

mg/ kg

%of FAA

mg/

mg/ kg

% of FAA

CYS

1160

9.48

-

-

1380

5.3

740

13.7

590

10.2

-

ASP

300

2.5

-

2.9

1020

3.9

140

2.6

190

3.3

3.7 6.4+gly

Thr

190

1.6

-

3.8

530

2.0

80

1.5

100

17

Ser

1020

8.3

-

3.5

1570

6.0

330

6. 1

390

68

4.2

Glu Pro

1730

14.2

-

16.1

3940

15.1

620

11.5

850

14.7

18.5

2.77

530

4.3

2320

1.2

160

3.0

160

160

1.3

-

10.3

G~Y

1.6

390

1.5

70

1.3

100

1.7

+Thr

Ala

340

2.R

-

5.8

1140

4.4

120

2.2

150

2.6

6.2

Val

610

5.0

-

6.1

2220

8.5

350

6.5

360

6.2

7.8

Met

500

4.1

-

37

760

3.0

180

33

200

3.5

2.5

Ile Leu

300

2.5

-

6.5

1300

5.0

2 70

3.1

220

3.8

6.1

1530

12.5

-

10.1

2910

11.2

690

12.8

650

11.3

11.5

5 r

520

4.3

3.9

650

3.3

290

5.4

270

4.7

7.4

Phe

680

5.6

6.0

1590

6.1

320

5.9

310

5.4

5.9

His

filO

5.0

1.5

800

3.1

240

4.4

270

4.7

3.5

LYS

1540

12.6

-

7.5

3050

11.7

650

12.0

960

16.6

4.5

510

4.2

-

1.5

280

1.1

260

4.8

-

Told

12.230

~

~

26070

5410

10

5770

The concentrations of amino acids were either given by, or calculated from the results of 19,21, 22, 23: Zarmpoutis (1995): 20: Isrnail and Hansen (1972); 24: Gonzalez de Llano et 01. (1991).

levels of which are reported to decrease during the later stages of ripening (Puchades et a]., 1989; Broome et a]., 1991; Wilkinson, 1993). V. Catabolism of Amino Acids

The general catabolism of free amino acids is summarized in Fig. 8 (Hemme et al., 1982). A. DECARBOXYLATION AND PRODUCTION OF AMINES

Amines, including biogenic mines, are produced in cheese by enzymatic decarboxylation of free amino acids (Joosten and Stadhouders,

P. F. FOX AND J. M.WALLACE

46

TABLE IV continued Cheese [Ripening time]

Cabrales (251 (BhlC)

[4 rnol

Amino

mgl

acid

kg

%of FAA

Cabrales @ti) (Blue) 14 mol

rngl kg

'% of FAA

Danbu (27) 18.3 mu] mgl kg

of FAA

'Yo

Maribo (281 (rindless) [8.4 mo] ing/ kg

of FAA

Oh

Maribo (291 (rind) In.4 mol

mgl kg

% of FAA

Havarti (30) [Smear]

mg/

kg

Oh of FAA

-

-

-

-

ASP

2800 0

ti.7

2880.2

5.0

1.9

1.1

1.7

1.7

Thr

4190.0

13.6

3039 fi

52

27

1.5

3.0

2.0

Ser

1230 0

4.0

790.5

1.4

22

1.0

2.6

2.3

Glu

4140.0

13.4

7604.9

13.1

21.0

11.3

19.2

21.2

4.7

3.1

4.1

7.ti

Gly

590.0

1.9

1041.n

1.8

1.R

1.7

1.7

1.8

Aln

1970 0

6.4

1893.6

3.3

2.8

5.6

2.8

2.n

Val

2040.0

6.8

4504.1

78

90

10.0

8.7

78

Mct

11R0.0

38

2696.3

9G

3.4

4.3

3.4

33

110

1310.0

4.2

4124.1

71

6.2

5.9

5.8

5.0

LOU

2640.0

8.6

7525.2

13.0

14.0

17.8

1ti.0

13 2

Cyr

PR,

TY~

540.0

18

333Y.8

5.8

1.7

1.7

1 6

33

I'hn

1730.0

5G

4406.0

7.6

7.9

8.3

R.0

7.6

llis

1140.0

3.7

1403.3

2.4

1.Y

2.0

13

1.5

1.ys

3270.0

10.6

71335.5

13.2

8.8

6.8

7.4

R.8

AX Total

870.0

2.R

281.9

0.5

0.1

0.4

0.2

0.1

30890

57897

The concentrations of amino acids were either given by, or calculated from the results of 25: Gonzalez de Llano et al. (1988);26: Tuckey and Sahasrabudhe (1958);27,28,29,30: Ismail and Hansen (1'372).

1987). Although amines are thought to be important contributors to cheese flavor, some may cause bitterness (Ney, 1971). Amines are potentially toxic to humans due to their ability to react with nitrogen oxides, forming carcinogenic nitrosamines, and their ability to induce symptoms of hypo- or hypertension in certain individuals (Rice et al., 1978). Concentrations of amines in Cheddar are generally too low to cause adverse effects (Joosten and van Boekel, 1988; Flynn, 1992). The principal amines in most cheeses are tyramine and histamine, produced by decarboxylation of Tyr and His (Fig. 9) (Fox et al., 1995a). The concentrations of tyramine and histamine in cheeses inoculated with lactobacilli were twice as high as in control cheeses, indicating that decarboxylases of lactobacilli play a major role in their production

47

FORMATION OF FLAVOR COMPOUNDS IN CHEESE TABLE IV continued Cheese [Ripening time] Lim- (31) burger 110 wkl

Brick (32) I10 wkl

Roma- (33) dour

Hovi (341 (Gervais)

Kreivi (35) (Tilsit)

Kesti (36) Wlsit mil Kummeln~satc)

~

Amino acid

mg/ kg

% of FAA

mg/ kg

% of FAA

mg/ kg

% of FAA

mg/ kg

Yn of FAA

mg/ kg

% of FAA

mg/ kg

% of FAA

CYS

450

3.5

-

-

-

-

-

ASP Thr

450

3.5

430

7.2

1ofi.n

2.0

7.5

3.4

96.5

1.2

114.6

600

4.7

in0

3.0

75.1

1.4

6.3

2.6

457.4

5.9

375.6

5.1

Ser

290

2.3

210

3.5

61.6

1.2

6.7

3.0

276.0

3.5

242.2

3.5

Glu

2580

20.1

600

10.1

519.0

9.7

53.2

23.8

117R.7

15.2

782.9

13.1

Pro

320

2.5

244.6

4.6

14.0

6.4

534.3

6.4

368.5

5.6

Gly

380

3.0

500

8.4

111.2

2.1

3.0

1.3

131.1

1.6

113.2

1.7

Ala

890

6.9

460

7.7

171.7

3.2

6.2

2.8

213.0

2.5

180.2

2.7

Val

lain

14.1

190

3.2

610.6

11.4

6.1

2.7

608.9

7.4

491.0

7.5

Met

1100

8.6

480

8.0

230.0

4.3

3.1

1.3

229.4

2.8

192.5

2.9

333.9

6.2

5.6

2.2

288.0

3.2

215.9

3.3

13.8

-

Ile

1.8

Leu

1900

14.8

lion

in4

1039.1

19.4

16.6

7.3

1161.7

15.6

919.8

TYr

R90

6.9

420

7.0

a3 o

1.6

7.1

3.2

327.2

4.0

257.8

3.9

Phe

100

0.8

520

87

468.1

8.8

13.0

5.7

796.1

10.0

658.2

10.0

His

260

2.0

223.6

4.2

5.9

2.6

575.1

7.2

148.9

2.3

LYs

370

29

726 8

13.6

24.0

10.7

974.8

11.8

748.0

11.5

34 n

0.6

11.9

5.3

161.8

2.1

235.2

3.7

-

Arg

Total

12818

4624

224.2

799n

6461

The concentrations of amino acids were either given by, or calculated from the results of 31, 32: Tuckey and Sahasrabudhe (1958); 33,34, 35, 36: Antila and Antila (1968).

(Broome et al., 1990). The concentration of tyramine in cheese often exceeds that of histamine, with lesser amounts of phenylethylamine (from Phe), tryptamine (from Trp), cadaverine (from Lys), and putricine (from ornithine) (Tokita and Hosono, 1968); the relative proportion of amines depends on the cheese variety and the nonstarter microflora. Lactobacilli appear to be responsible for the production of tyramine, histamine, and putricine in Gouda cheeses. In normal Gouda and Maasdam produced under hygienic conditions from pasteurized milk, concentrations of biogenic amines were low, indicating the inability of starter cells to produce them (Joosten and Stadhouders, 1987). A similar conclusion applies to Edam and Emmental (Antila ef al., 1984). Enterococci and coliforms in Gouda are capable of producing biogenic amines (Gripon et al., 1991);Br. linens is capable of producing tyramine, hista-

P. F. FOX AND 1. M. WALLACE

48

TABLE IV continued Cheese [Ripening time) Trap- (37) pisttype 135 days]

Ras (38) (Egyptian) [4 mol

Kashkaval (39) Kopanisti (40) (Egyptian) (Greek) [4 mol 146 days]

Yo of

1.6

1217.3

4.2

1642.4

4.9

1403.8

4.0

2.0

2146.3

64

10615

64

211.0

2.3

71322

21.2

59159

212

7.3

815.3

8.8

18978

57

1719.0

5.7

2.8

286.6

3.1

12778

3.8

1244.7

3.8

8.1

1022.8

11.1

1221.0

3.6

970 1

36

44

1280.4

14.0

30742

92

26543

92

54

11670

35

936 8

3.5

11 1

2077.0

6.2

1524.2

G.2 12.0

mgi kg

% of

rngl

I'AA

kg

18.1

0.5

182.5

7.7

56.3

1.3

103.0

2.9

109.4

4.6

143.1

4.2

99.9

28

41.7

Gl"

784.2

22.8

700.0

I'J 8

401.0

Pro

m8.n

5.5

167.0

4.7

Gly

59.2

1.7

83.3

2.4

Ald

204.0

5.1)

279.0

7.g

Val

290.6

8.5

307.0

8.7

Me1

32.3

09

2530

7.2

66.5

2.8

500.4

Ile

12'J 7

38

261.0

7.4

81.5

34

1026.6

LCU

400.8

% uf FAA

Cye

12.4

0.4

-

Asp

130.0

3.8

Tlir

44.4

Ser

mgl

Edam- (42) type (goat's)

rngi kg

% uf I'AA

mgi kg

kg

Amino acid

Rumelia ( 4 1 ) (goat's)

rngi kg

FAA

0.6

530.8

462.1

5.0

1.8

181.1

16.9

173.7 67.7 191.1 1043

-

~

% uf FAA

~

% vf

FAA

~

14.3

345.0

9.8

2600

11.0

2033.1

22.1

4014.7

12.0

3281.2

Tyr

57

n

17

75.3

2 1

130.9

55

25.1

0.3

521.0

1.n

38S.5

1.6

Phe

270 3

7 9

315.0

8'1

295 0

12.4

11as.9

12 0

1850.6

5.5

1475.7

5.5

His

74 fi

22

87.1

2.5

62.2

2.6

98.4

1.1

863.5

2.6

1202.5

2.6

LYS

480.6

140

1590

4.5

126.1

5.3

106.3

1.2

3921.0

11.6

4103.4

11.6

Arg

46 4

14

279.0

7.9

80.3

3.4

-

-

254.4

0.8

216.5

0.8

Total

3439

3530

2380

m n

33594

29450

l'hc concentrations of amino acids were either given by, or calculated from the results of 37:Ades and Cone (1969); 38:Omar (1984); 39: Omar and El-Zayat (1986); 40: Kaminarides et aJ. (1990); 41: Baltajieva et al. (1985);42:Ramos et aJ. (1987).

mine, monomethylamine (Fig. 10a), monoethylamine (Fig. lob), trimethylamine (Fig. lOc), dimethylamine (Fig. lod), cadaverine (Fig. loe), monopropylamine (Fig. 100, dipropylamine (Fig. log), tripropylamine (Fig. loh), triethylamine (Fig. l O i ) , and piperidine (Fig, loj) in model systems (Tokita and Hosono, 1968; Hosono and Tokita, 1969). Simple decarboxylation can explain the formation of most amines found in cheese, but there is no readily available explanation for the formation of secondary and tertiary amines and n-butylamine in cheese (Adda et a]., 1982). No relationship has been found between the concentration of free amino acids and the production of amines in cheese (Smith, 1981), probably due to differences in the rate of decarboxylation of individual

49

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

TABLE IV continued Cheese [Ripening time1 Turunmss (43) (Finnish) Amino acid

rngi kg

% of I'AA

Sir0 (44)

(Finnish) rngi kg

Loustari (45) (Port Salut)

Mahon (46) (traditional)

Mahon (47) (Industrial)

%of

rngi

FAA

kg

% of FAA

1.9

60

1.3 5,5+gly

rngi kg

% of FAA

% of FAA

-

-

-

ASP

53.3

0.9

70.3

1.3

113.2

Thr

349.2

7.3

354.6

6.5

185.6

8.7

5.7+gly

Ser

139.2

3.0

186.8

2.9

+Ser

tSer

5.5

3.8

Clu

740.5

159

51G.G

95

942.0

17.2

17.2

15.3

Pro

253.3

6.1

237.9

4.4

176.1

4.4

9.7

9.0

tily

63.4

14

110.7

1.U

73.1

1.4

+Thr

+Thr

CYS

-

rngi kg

-

Ala

110.8

2.0

181.1

3.3

176.2

2.8

3.4

3.2

Val

372.1

8.4

420.4

7.7

420.2

7.6

13.8

12.7

Met

125.6

2.8

197.9

3.6

157.7

3.1

0

2.9

Ile Leu 5 r Phe

123.8

2.4

lfi4 9

3.0

218.7

3.6

9.1

18.8

-

707.2

16.6

704 4

12.9

800.1

15.5

242.0

1.9

164.7

3.0

161.1

3.1

-

-

500.1

11.7

600.9

11.0

567.5

11.3

22.2

20.3

His

63.3

0.8

114.1

2.1

100.4

1.8

-

-

Lys

434.8

7.8

542.1

9.9

527.8

96

A%

95.5

2.4

352.6

6.5

51.4

1 5

0 74

1.0

Total

4733

5453

-

5076

The concentrations of amino acids were either given by, or calculated from the results of 43,44,45: Antila and Antila (1968);46,47:Polo et al. (1985).

amino acids or in the rate of deamination of resulting amines (Polo et a]., 1985). B. DEAMINATION-FORMATION OF AMMONIA AND NEUTRAL OR ACIDICCOMPOUNDS

Deamination of free amino acids leads to the production of ammonia and a-keto acids (Hemme et al., 1982). Bassett and Harper (1956) identified a number of a-keto acids, including pyruvic and p-hydroxyphenyl pyruvic acids, in cheese. However, more recent studies have not identified a-keto acids, perhaps due to their instability when measured

50

P. E FOX AND J. M. WALLACE Amino acids

Transamination

I

Oxidative deamination

I

\ Amines

Amino acids

I

a-keto acids

_i,,

Aldehydes

v

Phenols

Indole

I

Other sulphur compounds

Alcohols

Acids

FIG.8. Catabolism of free amino acids in cheese. (Modified from Hemme et al., 1982.)

H~N-CH-COOH Tryptophan

H,N- JH, T ptamine

FIG.9. Production of biogenic amines from tyyosine and tryptophan.

FORMATION OF FLAVOR COMPOUNDS IN CHEESE (a)

51

(C)

NH,

CH,

CH,

N

I

I

C'H,

C$

Monomethyl amine

Trimethylamine

(d)

(0 H,N

CH,

I

N -CH,

CH,

I

CH,

H

Dimethylamine

Monopropylamine

(g)

(9

cY 7' CH

I I

I

CH,

I I

N-

NH

/: ' CH,

CH,

CHI-CH,

CH,

I

CH, CH,

Dipropylamine

Triethylamine

ti)

0 HI

Pipiradine

FIG.10. Amines that have been identified in cheese (Tokita and Hosono, 1968; Hosono and Tokita, 1969).

by GLC (Urbach, personal communication). Ammonia is an important constituent in many cheeses, such as Camembert, Gruyere, and Comte (Fox et a]., 1995a). G. candidurn is capable of deamination, and Br. linens strains possess very active deaminases and produce large quantities of ammonia via the catabolism of serine, and to a lesser extent of glutamine, asparagine, and

52

P. F. FOX AND J. M. WALLACE

COOH

OH

I

I

Isovaleric acid FIG.

Isobutyricacid

Acetic acid

COOH

I

Propionic acid

11. Volatile fatty acids produced by amino acid catabolism.

RCOOOH II 0

RCH + C Q II 0

0

Strecker aldehyde

FIG.12. Strecker degradation pathway. (Modified from Urbach, 1995.)

threonine (Hemme et al., 1982). Ammonia can also be formed by oxidative deamination of amines, forming aldehydes (Hemme et al., 1982) or volatile fatty acids. Isovalerate (Fig. l l a ) , isobutyrate (Fig. I l b ) , acetate (Fig. l l c ) , and propionate (Fig. I l d ) are produced by oxidative deamination of isoleucine, valine, glycine-alanine-serine, and threonine, respectively.

53

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

0

0 II CH

x I

0 II CH I

CH,- CH

I

CH, CH3

Phenylacetaldehyde

Isobutanal

3-methylbutanal

0

II

CH

I

3-methylthiopropmal

Methional

FIG.13. Aldehydes produced by Strecker degradation of amino acids.

c.

TRANSAMINATION, STRECKER DEGRADATION, AND PRODUCTION OF ALDEHYDES

Production of aldehydes from free amino acids can result from decarboxylation, deamination, transamination (Fox et al., 1995a), or via Strecker degradation (Keeney and Day, 1957; Dunn and Lindsay, 1985; Urbach, 1995) (Fig. 12). Enzymatically catalyzed transamination of a free amino acid results in the formation of an intermediary imide that is subsequently degraded by decarboxylation or the Strecker reaction, resulting in the formation of an aldehyde (Keeney and Day, 1957; Polo et al., 1985). Phenylacetaldehyde (Fig. 13a), isobutanal (Fig. 13b), 3methylbutanal (Fig. 13c), and 3-methylthiopropanal (Fig. 13d) and methional (Fig. 13e) can be formed by this mechanism from Phe, Leu-Ile, Val, and Met, respectively (Adda et al., 1982). Transamination also leads to the production of other amino acids (Fox et ai.,1995a), such as Glu from Phe (as in Section V.E.1) (Lee and Desmazeaud, 1985) Interconversion of amino acids has been demonstrated in Tallegio and other Italian cheeses by using radiotracers (Cicchi et al., 1979), giving

54

P. F. FOX AND J. M. WALLACE

rise to various metabolites such as a-ketoglutaric and pyruvic acids that could participate in further interconversion reactions (Polo et al., 1985). Aldehydes are thought to contribute to the flavor of many cheese varieties. In Parmesan, where they accounted for 2% of the total volatiles, their production is assumed to be via Strecker degradation (Barbeiri ef al., 1994). The low pH of Parmesan during ripening is thought to be responsible for the decarboxylation of amino acids to amines that are subsequently oxidized to aldehydes when there is an increase in pH during the later stages of ripening (Belitz and Grosch, 1987). When the concentration of Strecker-derived compounds exceeds a certain threshold in Cheddar, unclean flavors develop (Dunn and Lindsay, 1985).Poor flavors were noted when the concentrations of two Strecker aldehydes (3-methylbutanal/2-methylbutanal or 2-methylpropanal) exceeded 200 pg/kg in Cheddar (Dunn and Lindsay, 1985). Isovaleraldehyde, 2-methylbutanal, isobutanal, and methional were formed by Strecker degradation of Leu-Ile, Val, Phe, and Met, respectively, in aqueous extracts of cheese and cultures of cheese microorganisms (Griffith and Hammond, 1989). Dunn and Lindsay (1985) found no correlation between the concentrations of Strecker-derived compounds and concentrations of individual free amino acids in Cheddar cheese. D. CATABOLISM OF SULFUR AMINOACIDS

The catabolic products of sulfur amino acids have been implicated as major contributors to Cheddar cheese flavor (Manning, 1974; Manning et al., 1976; Adda et al., 1982; Dunn and Lindsay, 1985; Kristoffersen, 1985; Barlow et al., 1989; Gripon et al., 1991; Alting et al., 1995) (Fig. 14). Certain smear surface-ripened cheeses exhibit flavors described as “cabbagey,” “garlic,” or “putrid” (Adda eta]., 1978; Hemme et al., 1982; Ferchichi et al., 1985; Manning and Nursten, 1985). These flavors are attributable to sulfur compounds derived from methionine and perhaps to a lesser extent from cysteine. Sulfur compounds in cheese (e.g., methanethiol (CH,SH), hydrogen sulfide (H2S), dimethylsulfide (CH3SCH3), dimethyldisulfide (CH3SSCH3), dimethyltrisulfide (CH3SSSCH3),and carbonyl sulfide (O=C=S)) are thought to interact with each other and with other compounds in cheese, generating typical cheese flavors (Kim and Olson, 1989). Sulfur compounds have been identified in many cheese varieties, but their importance in smear surface-ripened cheeses appears to be accentuated by their high concentrations at the surface (Ferchichi et al., 1985; Gripon et al., 1991).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

a

Homoalanine

CH,SH Methanethiol

3

Homocy steine

a-keto-thiornethy1 butyrate dernethiolase

Sulphur derivatives FIG.14. Catabolism of methionine. (Modified from Hemme et al., 1982.)

55

P. F. FOX AND J. M. WALLACE

56

(b)

I

L-Methionine

a-Ketoglutarate

L-Cy steine

FIG.15. Chemical structures of methionine, a-ketoglutaric acid, and cysteine.

The principal microorganisms in the smear of smear-ripened cheeses are coryneform bacteria, particularly strains of Brevibacterium linens. Sharpe et al. (1976) were the first to report the ability of Br. linens to produce methanethiol enzymatically. Br. linens is also found on the surface of many other cheeses, such as Camembert (Ferchichi et a]., 1985; Hemme and Richard, 1986) or Gruyere (Law, 1981), the distinct flavors of which have been attributed to the formation of sulfur compounds, particularly methanethiol, at the surface (Manning and Price, 1982).

The enzyme demethiolase, involved in methanethiol production, is highly dependent on the availability of Met (Fig. 15a) (Ferchichi et al., 1987) and the ability of cells to transport it into the cytoplasm (Forss, 1979; Ferchichi et af., 1985). The production of methanethiol involves a-ketoglutarate (Fig. 15b), which is used by the bacteria as a carbon source, as an intermediate (Hemme and Richard, 1986). Uptake of Met into the cells is strongly inhibited by high concentrations of Cys (Fig. 15c) (Ferchichi et al., 1987), as is its conversion to methanethiol (Urbach, 1995). Some authors (Adda et a]., 1982) claim that the enzyme has a preference for free methionine, but others (Ferchichi et a!., 1986b) have demonstrated higher activity on Met in the dipeptides L-Ala-L-Met and L-Met-L-Ala than on free Met. Methionine and methanethiol may affect characteristics of the cheese other than flavor. The production of a red-orange pigment by Br. linens appears to depend on the concentrations of methanethiol and dissolved O2 (Ferchichi et a]., 1986a). Methionine inhibits the germination of

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

57

certain mold spores; the active compound is thought to be methanethiol (Beattie and Torney, 1986). Manning et al. (1976) correlated the concentration of methanethiol with the flavor of Cheddar cheese but pointed out that this finding may only apply to experimental cheeses manufactured in their laboratory. Other authors (see Aston and Dulley, 1982) have since found that the concentration of methanethiol is a poor indicator of flavor, particularly in Cheddar cheeses subjected to accelerated ripening (Aston et a]., 1983). In cheeses such as Cheddar, which lack a surface microflora, flavor is produced by starter and nonstarter bacteria and their enzymes (Urbach, 1995). Since coryneforms do not occur in Cheddar, the production of methanethiol was thought to be a chemical process (Urbach, 1995). Certain NSLAB can produce methanethiol enzymatically from methionine, but the enzymes involved are thought to be very unstable in ripening cheese and are therefore unlikely to influence the concentration of methanethiol during ripening (Law and Sharpe, 1978). In cheese varieties such as Cheddar, the role of microorganisms in amino acid catabolism may be indirect. Key chemicals, such as methanethiol, may be produced chemically in the cheese as a result of starter-induced conditions (low pH and Eh) rather than enzymatically (Law, 1981; Kim and Olson, 1989). However, Urbach (1995) proposed that the secondary flora, particularly in Cheddar and Emmental, is likely to be more important than chemical reactions for the formation of sulfur compounds. This hypothesis was supported by Alting et al. (1995), who isolated an enzyme (similar to cystathionine P-lyase) from L. lactis ssp. cremoris B78 that was capable of producing methanethiol from sulfur amino acids under cheese ripening conditions (pH 5.2-5.4 and 4% NaCl). Engels and Visser (1996) produced typical Gouda flavor by adding methionine to cell-free extracts of this strain. Fermentation of lactose during manufacture reduces the pH to -5.1 and the redox potential from about +300 mV to -130 mV or lower (Law and Sharpe, 1977; Adda et al., 1982). In Swiss cheese, in which the highly reductive propionic acid fermentation results in a further decrease in Eh, the concentration of active sulfhydryl groups is twice that in Cheddar (Kristoffersen, 1973). Law and Sharpe (1977) found low levels of sulfur compounds in starter-free Cheddar cheeses (chemically acidified with GDL) and attributed this to the high redox potential of the GDL cheeses. Manning (1979a), who artificially reduced the Eh of GDL-acidified cheese using dithiotreitol and glutathione, demonstrated the nonenzymatic formation of methanethiol. Although the reducing conditions were created artificially, Manning concluded that the reduc-

58

P. F. FOX AND J. M. WALLACE

ing conditions produced by starter bacteria would be adequate to cause the same effect. Ponce-Trevino et al. (1987, 1988), who manufactured chemically and microbially acidified cheese slurries, found that the production of H2S,carbonyl sulfide, methanethiol, and dimethyl sulfide was highest in cheese slurries with added starters. When starter cells were inactivated by antibiotics, no H2S or (CH3)2Swas produced. When the Eh of chemically acidified slurries was reduced by using NADH, O=C=S and CH3SH were produced but H2S and (CH3)2Swere not. Samples (1985) commented that, while a negative redox potential may be necessary for the production of sulfur compounds in cheese, it is not sufficient. He felt that glutathione, which is stored in the cells of certain lactococcal strains (Fernandes and Steele, 1993), plays an important role. A low Eh does, however, have a stabilizing effect on sulfur compounds (Law et al., 1976a,b). Although the exact pathway for the nonenzymatic formation of CH,SH has not been established, a mechanism proposed by Manning (1979a,b) is generally accepted by most investigators (Law, 1981; Adda et al., 1982; Hemme et al., 1982; Barlow et a]., 1989; Fox et al., 1995a). The proposed pathway involves the release of H2S from cysteine in the presence of a reducing agent, followed by the reaction of H2S with methionine, in which the C-S bond is cleaved, with the release of CH3SH. This proposed pathway is supported by the fact that H2S is released initially in the curd prior to the formation of CHoSH(Manning, 1979a,b; Adda et a]., 1982). Green and Manning (1982) showed that low concentrations of H2S in cheese during the early stages of ripening coincided with the production of low levels of CH3SH. This appears to indicate that H2S is essential for the formation of CH3SH. However, Cys is an inhibitor of CH3SH production and the release of H2S from Cys may simply remove the inhibitor for the enzymatic production of CH3SH (Urbach, 1995). The thiol-producing enzyme system is thought to be inactivated by high heat treatment of milk (Samples, 1985), and indigenous milk enzymes may be responsible for the production of CH3SH in cheese (Urbach, 1995). The production of both CH3SH and H,S was increased by the addition of reduced glutathione to cheese (Samples, 1985), and glutamyltranspeptidase, which acts by desulfurating glutathione, is thought to be partially responsible for the formation of H2S, and possibly of CH3SH. Other enzyme(s) are also involved (Samples, 1985; Urbach, 1995). SH groups are a limiting factor in the formation of H,S (Samples 1985). Since there are few cysteine-cystine residues in casein, it seems likely that at least some of the sulfur groups arise from the

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

59

action of y-glutamyltranspeptidase on glutathione, which is introduced into the cheese in starter cells (Fernandes and Steele, 1993; Wiederholt and Steele, 1994; Folkertsma and Fox, 1996). The formation of H2S is thought to be influenced by Eh and the concentration of glutathione. The presence of H2S in fresh curd indicates that some may also be formed during pasteurization of cheese milk (Law et al., 1976a). Concentrations of H2S in cheeses with added NSLAB showed that some lactobacilli are capable of desulfurating Cys and producing H2S, while Group D streptococci, staphylococci, micrococci, and pediococci are not (Sharpe and Franklin, 1962; Law, 1981). While the concentration of H2S in cheese increased during ripening, Aston et al. (1983) found no correlation between its concentration (or that of other sulfur compounds) and flavor development. Similar observations were reported by Lawrence (1963) and Manning (1978). On the other hand, Kristoffersen and Nelson (1955)found that cheeses with the highest concentration of H2Sreceived the highest flavor scores. In later studies (Kristoffersen and Gould, 1960; Kristoffersen, 1967), fluctuations in H2S were observed throughout ripening, and the authors suggested that the ratio of H2S to short-chain fatty acids was important for flavor development. Barlow et al. (1989) found a high correlation between flavor and the concentration of H2S, particularly when the value for H2S was combined with either the concentration of water-soluble nitrogen or lactic acid. The authors concluded that these parameters (H2S + WSN/lactate) were better predictors of the way in which young cheeses would mature than their flavor or composition at an early age. A notable feature of Parmesan cheese is that, although a number of sulfur compounds have been identified, this cheese is characterized by low concentrations of H2S and CH3SH (Barbeiri et al., 1994). This may be due to inactivation of indigenous milk enzymes by the relatively high cooking temperature during manufacture (Barbeiri et al., 1994). Dimethylsulfide (DMS), dimethyldisulfide (DMDS), and dimethyltrisulfide (DMTS) are thought to be important contributors to cheese flavor (Manning et al., 1976; Barbeiri et al., 1994). DMS is a component of Swiss cheese flavor and is a metabolite of propionibacteria (Langler et al., 1966; Adda e f al., 1982). DMS levels remained constant in Cheddar cheese for up to 6 months of ripening and decreased thereafter (Aston et al., 1983).Manning et al. (1976) proposed that the concentration of DMS is seasonal and originates in the cheese milk rather than from the catabolism of sulfur amino acids. Variability of the concentrations of DMS and H2S throughout ripening rendered these two compounds of little value as indices of ripening (Aston and Douglas, 1983). DMDS is formed as an end-product of Strecker degradation (Belitz and Grosch, 1987). Strecker degradation involves a reaction between an

60

P. F. FOX AND J. M. WALLACE

---

CH,

I

Streckex Degradation

S

I

CH,

I

CH,

S

I

FH2 CH,

I

I

S N - CH - O H

o= CH

Methionine

Methional FIG.16. Structure of methional.

amino acid and a diketone, resulting in the formation of an aldehyde with one carbon atom less than the original amino acid (Aston and Dulley, 1982; Keeney and Day, 1957; Urbach, 1995) (Fig. 12). 3-Methylpropanal is formed in the first step of this reaction. DMDS has been identified in Parmesan (Barbeiri et al., 1994), Cheddar (Aston et al., 1983; Barlow et al., 1989), and surface-ripened cheeses (Jollivet et al., 1992). In Cheddar it was found to correlate fairly well with flavor scores (Barlow et al., 1989). DMTS is another potent aroma compound and has been associated with the aroma of cooked cabbage, broccoli, or cauliflower. It has been identified in Parmesan (Barbeiri et a]., 1994) and Cheddar (McGugan, 1975; Wood, 1989). Although DMDS is formed by Strecker degradation of methionine, the principal product of this reaction is methional (P-methylmercaptopropionaldehyde) (Keeney and Day, 1957; Aston and Dulley, 1982) (Fig. 16). Methional contributes positively to cheese only when present at low concentrations; a sweet corn-like aroma is detectable at high levels (Barbeiri et al., 1994). Methional is present in the volatile fraction of many cheeses, including Cheddar (Wijensundera and Urbach, 1993). Dunn and Lindsay (1985) noted that methional plays a role in flavor suppression in bland curds and proposed that this compound may play a critical role in masking off-flavors in cheese. Methional can also be produced enzymatically by many strains of lactic acid bacteria in enriched media (Tracey and Britz, 1989). Several other sulfur compounds have been identified in cheese volatiles. Thioesters, produced by esterification of methanethiol and acetic or propionic acid, have a cheesy aroma (Dumont and Adda, 1978). S-methylthioacetate (Fig. 17a) can be produced under certain conditions by selected strains of Br. linens (Ferchichi et al., 1986a,b) and has

61

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

-N

II

S-Methylthioacdate

Benzothiazole

0

II

FH

2-methyl propanal

FIG.17. Chemical structures of some volatile compounds derived from methionine.

CH,SH

Methanethiol

+

+ Formaldehyde->

Bis(methy1thio)methane

FIG.18. Production of bis(methylthio)methae by condensation of methanethiol with formaldehyde.

been found in Limburger cheese (Parliment et al., 1982). The mechanism of thioester formation is unknown, but it is thought to be enzymatically controlled as the presence of micrococci was required for their formation in model systems (Adda et al., 1982). A reported component of Parmesan aroma is the methionine-related benzothiazole (Meinhart and Scheier, 1986; Barbeiri et a]., 1994) (Fig. 17b). No mechanism for its formation has been reported. A sulfur-containing aldehyde, 3-methylpropanal (Fig. 17c), presumably formed by transamination of methionine, followed by decarboxylation of an intermediate imide, has been identified in Cheddar (Adda et al., 1982) and Parmesan (Barbeiri et al., 1994) and may coiitribute to the aroma of these varieties. Methylthiopropionate, which has a cheesy aroma, has been found in Cheddar (Cuer et al., 1979). Bis(methy1thio)methane (Fig. 18),an important component in Camembert flavor, can be produced by the condensation of methanethiol with formaldehyde (Dumont et al., 1976) (Fig. 18). The concentration of carbonyl sulfide increases throughout ripening and is less variable than other sulfur compounds (Aston and Douglas,

P. F. FOX AND J. M. WALLACE

62

CH, I

Phenylmethanol

c=o

OH

I

Phenylethanol

I

I

CHZ

I

0

CH,

CHZ

I

COOH

CH,

Phenylpropanone

Methylphenyl hydroxyacetic acid

CH, I

OH-CH-CH2 -COOH

O=CH

Phenylacetaldehyde

Pheny lpyruvic acid

Phenylethanol acetic acid

FIG.19. Some phenylalanine-derived catabolites that have been identified in cheese.

1983; Aston et al., 1983). It has not been established whether the concentration of carbonyl sulfide correlates with cheese flavor.

E. CATABOLISM OF PHENYLALANINE, TYROSINE, AND TRYPTOPHAN 1, Phenylalanine

Phe is released at high concentrations, particularly during the early stages of cheese ripening. Initial cleavage of the Phe,,,-Metlo6 bond of K-casein by rennet and of Phe,,-Phe,, of aSl-casein during early ripening provides terminal Phe, which could be easily released by bacterial exopeptidases (Dunn and Lindsay, 1985). A number of flavor compounds arising from Phe have been identified in cheese (Adda et al., 1982; Dunn and Lindsay, 1985; Lee and Desmazeaud, 1985) and in model systems (Jollivet et al., 19921, that is, phenylmethanol (Fig. 19a), phenylethanol (Fig. 19b), phenylpropanone (Fig. I ~ c ) methylphenyl , hydroxyacetate (Fig. 19d), phenylacetaldehyde (Fig. 19e), phenylpyruvate (Fig. 19f), and phenylethanol acetate (Fig. 19g). L-G~u(0.93 moll and phenylpyruvate (1.0 mol) were pro-

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

Phenylalanine

+

2-0xyglutaric acid

+

63

Phenylpyruvic acid + Glutamic acid

FIG.20. Transamination reaction with the production of glutamic acid from phenylalanine.

duced when Phe acted as amino group donor and 2-oxyglutarate as acceptor in a transamination reaction (Lee and Desmazeaud, 1985) (Fig. 20). Br. linens rapidly catabolyzes Phe and other aromatic amino acids and can utilize them as nitrogen sources. The enzymes involved in the initial breakdown of Phe are aromatic amino acid amino transferase, L-amino acid oxidase, L-phenylalanine ammonia lyase, L-aromatic amino acid decarboxylase, and phenylalanine dehydrogenase. Many of these enzymes have been identified in Br. linens (Jollivet et al., 1992, 1994), but their activity appears to be strain-dependent (Lee and Richard, 1984). Removal of the amino group from Phe by Br. linens is thought to be by oxidative deamination. A general L-amino acid oxidase is considered to be responsible for the ability of coryneform bacteria to utilize amino acids for nitrogen (Coudert and Vandcastelle, 1975). Surface yeasts in Camembert are thought to possess at least a transaminase and a decarboxylase active on Phe (Roger et al., 1988). In Cheddar (and other varieties with only primary starters),Phe is thought to be degraded by Strecker degradation (Dunn and Lindsay, 1985). The typical aroma of Camembert has been attributed to phenylethanol (Fig. 19b) produced by surface yeasts (Adda et al., 1982) involving transamination, decarboxylation, and reduction reactions (Dunn and Lindsay, 1985). Yeasts have been implicated as the main producers of phenylethanol in mold/smear-ripened cheeses (Roger et al., 1988; Jollivet et al., 1992); however, Br. linens degrades phenylethanol further (Jollivet et al., 1992). Phenylacetaldehyde (Fig. 19e) is considered to be an important flavor compound in many cheeses (Dunn and Lindsay, 1985; Jollivet et d., 1992). In Cheddar, phenylethanol and phenylacetaldehyde can be pro-

64

P. F. FOX AND J. M. WALLACE

duced by Strecker degradation of Phe (Dunn and Lindsay, 1985). Concentrations of phenylethanol in good- and poor-quality Cheddar cheeses (-100 kg/kg) were comparable, but the concentration of phenylacetaldehyde ranged horn 4 0 to 400 pg/kg, the highest levels being present in poor-quality cheese (Dunn and Lindsay, 1985). Both phenylethanol and phenylacetaldehyde have rosy-floral aromas and have been associated with off-flavors produced by Brewer’s yeast (Saccharomyces cervisiae) in beer (Sentheshanmuganathan, 1960). Unclean rose-like flavors observed in some poor-quality Cheddar have therefore been attributed to the presence of these compounds (Dunn and Lindsay, 1985). High concentrations of phenylacetaldehyde also contribute astringent, bitter, and stinging sensations to Cheddar flavor. The concentrations of these compounds were highest and their effects greatest in young and mid-ripened cheeses. In mature Cheddar cheese with more intense flavors, the effects of phenylethanol and phenylacetaldehyde are diluted, and off-flavors are either combined with, or masked by, other aroma compounds (Dunn and Lindsay, 1985). An important component of Camembert flavor is hydroxyphenylacetic acid, thought to be formed via initial deamination of phenylalanine followed by hydroxylation (Simonart and Mayaudon, 1956). Phenylethanolacetate (Fig. 19g)from Phe catabolism has been identified in many cheeses (Jollivet et al., 1992).Although phenylpropanone (Fig. 19c) is present in cheese (Jollivet et al., 1992), the mechanism for its formation has not been established. 2. Tyrosine and Tryptophan

Tyrosine serves as a precursor for three compounds in cheese: tyramine, formed by decarboxylation, and p-cresol (Fig. 21a) and phenol (Fig. 21b) by atypical Strecker degradation (Elsden et a]., 1976). Phenol and indole, pyruvate, and ammonia can be produced by the action of C-C lyases on Tyr and Trp (Hemme et al., 1982). Tyrosol (Fig. 21c) and tryptophol (Fig. 21d), two classical Strecker-type alcohols, have been reported to impart slightly bitter flavors to beer (Dunn and Lindsay, 1985);however, they have not been identified in cheese. p-Cresol and phenol have strong flavor potencies. While phenol (100225 mg k g * ) appears to have no detrimental effect on Cheddar flavor, p-cresol has been implicated in putrid “utensil” aromas (Dunn and Lindsay, 1985) when concentrations exceed 100 mg kg’ in Cheddar. Lactobacilli in Gouda and Vacherin cheeses are thought to be responsible for producing p-cresol (Badings et a]., 1968; Dumont et al., 1974). The concentration of phenol is unusually high in Limburger (Parliment eta]., 1982).

65

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

YCOH

p-Cresol

Phenol

Tyrosol

H&H,

Tryptophol

FIG.21. Aroma compounds produced by the degradation of tyrosine and tryptophan.

F. OTHER IMPORTANT FLAVOR COMPOUNDS FROM AMINO ACIDCATABOLISM Other compounds, thought to be important in cheese flavor, produced by Strecker degradation include benzaldehyde (Fig. 22a) and acetophenone (Fig. 22b) from Phe, 2-acetylthiazole (Fig. 22c) from Cys, and methylglyoxal alkypyrollizines from Lys. Amines can be acetylated, yielding to N-isobutyl acetamides, which have been identified regularly in Camembert (Dumont and Adda, 1978). Aldehydes are usually reduced as soon as they are formed (Polo et al., 1985) with the concomitant production of alcohols: isobutanal (Leu-Ile) is reduced to isobutanol (Fig. 22d), 3-methylbutanal (Val) to 3-methylbutanol (Fig. 22e), phenylacetaldehyde (Phe) to phenylethanol (Fig. 22f), and 3-methylthiopropanal (Met) to 3-methylthiopropanol (Fig. Zag) (Adda et al., 1982). 2,5-Dimethylpyrazine is produced in significant quantities for flavor perception in model systems containing Br. linens. This compound is present in many cheeses and can give a nutty, toasted note to the flavor. It could be produced by degradation of Thr to amino-acetone, the condensation of two amino acetone molecules forming one molecule of 2,5-dimethylpyrazine (Jollivet et al., 1992). Isobutanal (Leu-Ile) is reduced to isobutanol (Fig. 22d), 3-methylbutanal (Val) to 3-methylbutanol (Fig. 22e), phenylacetaldehyde (Phe) to phenylethanol (Fig. 22f), and 3-methylthiopropanal (Met) to 3-methylthiopropanol (Fig. 22g) (Adda et al., 1982). G. REACTIONSOF FREEAMINOACIDSWITH OTHER COMPOUNDS IN CHEESE

Cheesy flavors have been noted when free amino acids react with other molecules in the cheese. Addition of calcium or magnesium to

66

P. F. FOX AND J. M. WALLACE

0

0

II

II

L-’c-cH3 sYc-cH3 Benzaldehyde

Acetylthiazole

Acetophenone

(0 OH

OH

I

I

CK

x

CH3

CH,

Isobutanol

3-methylbutanol

Phenylethanol

(g) OH 1

CH-CH,SH I

YK CH3

Frc. 22. Some minor amino acid catabolic products found in cheese.

cas-amino acids enhanced their sweetness (Biede and Hammond, 1979), suggesting that Ca (or Mg) amino acid complexes may contribute to the sweet flavor of Swiss cheeses. A number of mono- and dicarbonyls are produced from free fatty acids in cheese (Reps et a]., 1987), particularly by lactobacilli. Kowalewska et al. (1985) showed that much of the flavor in the nonvolatile water-soluble fraction was generated from amino acid-carbonyl reactions. Glyoxal, methylglyoxal, dihydroxyacetone, and ethanal were prominent among the carbonyls, and Val, Leu-Ile, Met, Cys, Phe, Pro, and Lys were the principal amino acids that react chemically with them (Griffith and Hammond, 1989). Although the exact pathways for carbonyl-amino acid reactions are not known, Griffith and Hammond (1989) proposed a pathway for the

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

+

67

CH3

I

c= 0 I

H

Cysteine

Methylglyoxal

T

COOH I II KC- CH-N =CH- C-CH3 + CO,

I

SH

I

KC- C K - W C H - C-CH, + CO,

c=0

I

SH

2- Acdylthiazole FIG.23. Production of 2-acetylthiazole by reaction of cysteine with methylglyoxal; chemical pathway proposed by Griffith and Hammond (1989).

reaction of Cys with methylglyoxal (Fig. 23). The reaction of Lys with glyoxal or dihydroxyacetone can give rise to 6-valerolactam (Fig. 24a) or 2-acetyl pyrroline (Fig. 24b). Proline can react with ethanal, methylglyoxal, or dihydroxyacetone to produce 2-acetyl-1-pyrroline (Fig. 24c), 2-methyl benzaldehyde (Fig. 24d), and isomers of 2,3-dihydropyrolizine, respectively (Griffith and Hammond, 1989). These compounds

68

H I

+

c= 0 I c=o I

H

Ly sine

0)

-(2)-

P. F. FOX AND J. M. WALLACE

+

‘i“. CHZ +

CHZ

I

H2N- CH

6 -valerolactam (2-peperodine)

I

CHZ

‘iHz

-

H

I

I

Glyoxal

- COOH

I

c=o

CCH,

I H

COOH

Proline

+

Ethanal-

2- acdyl-1-pyrrolhe

FIG.24. Compounds formed by the reaction of amino acids with carbonyls. (Modified from Griffith and Hammond, 1989.)

were identified in aqueous extracts of cheese (Kowaleska et al., 1985) and Lb. bulgaricus cultures (Kowalewska et al., 1985; Reps et al., 1987).

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

69

COOH ;,H

""\/ \/" + YC

R;3

CH,-C-C,

H

- CH,

+ Methylglyoxal

Proline

I I

-

2 Methyl b d d e h y d e

(f 1

I

I I

I

yH3

7H3 S

I

I I

S

I

I

I

I I I I I

CH, I 0 II yH2 CH,C&C-NH-CH COOH

-

CH,

I

li' y".

I

CH,- C- NH-CH-COOH

I

I I I I

N-Propionyl methionine

I

N-acetyl methionine

I I

I

N-Ropionyl leucine

I I

I

N-Propionylphenylalanine FIG24d-h.

70

P. F. FOX AND J. M. WALLACE

Roudot-Algaron et al. (1993) isolated N-propionylmethionine (Fig. 24e), N-acetylmethionine (Fig. 24f), N-propionylleucine (Fig. 24g), and Npropionylphenylalanine (Fig. 24h) from the water-soluble fraction of Gruyere de Comte cheese: the first compound was cheesy, while the others had bitter flavors. AMINOACIDS H. NONPROTEIN

A number of nonprotein amino acids have been found in most cheese varieties, the principal being y-aminobutyric acid, formed by decarboxylation of Glu (Kaminarides et al., 1990) (Fig. 25a) and ornithine by arginase activity on Arg (Fig. 25b) (Broome et al., 1991). y-Aminobutyric is present at high concentrations in mold-ripened cheeses (Ismail and Hansen, 1972; Gripon et al., 1991). In Kopanisti cheese, y-aminobutyric acid levels increased 120-fold during ripening with a concomitant decrease in the concentration of Glu (Kaminarides et al., 1990). a-Aminobutyric acid has also been identified in many cheese varieties: Cabrales (Gonzalo de Llano et al., 1987; Ramos et al., 1987), Stilton (Madkor et al., 1987), Kopanisti (Kaminarides et al., 1990), and Gamonedo (Gonzalez de Llano et a]., 1991). However, its concentration was generally lower than that of y-aminobutyric acid. Enzymatic hydrolysis of the guanidino group of Arg leads to the formation of ornithine or citrulline (Hemme et al., 1982). Decreases in the concentration of Arg, particularly in the later stages of ripening, have been reported by a number of workers (Puchades et a]., 1989; Broome et a]., 1991). Certain strains of L. cremoris are capable of degrading arginine to a limited extent (Broome et a]., 1990). Low correlations between nonprotein amino acids and ripening time have been reported (Resmini et a]., 1969; Gonzalez de Llano et a]., 1991). VI. Chemistry of Cheese Off-Flavors

In addition to the characteristic desirable flavor of cheese, cheese frequently suffers from specific flavor defects. While normal desirable flavor has been difficult to define in chemical terms, the specific causes of many of the principal defects have been established more or less definitively and are described below. Bitterness in cheese results from the accumulation of hydrophobic short peptides that can originate from both asl-and p-caseins. Certain sequences in the caseins are particularly hydrophobic and when excised by proteinases can lead to bitterness. The action of chymosin has

71

FORMATION OF FLAVOR COMPOUNDS IN CHEESE

Glutamkacid

decarboxylation

*

y -amino butyric acid

(Kaminarides et al., 1990)

(b)

W-CH

Arginine

Arginase

- COOH

> Ornithine

+

Urea

(Broome ad.,1991)

FIG.25. Formation of nonprotein amino acids.

been implicated in the formation of bitter peptides in cheese (see Lemieux and Simard, 1991, 1992), and thus factors that affect the retention and activity of rennet in the curd may influence the development of bitterness. Lawrence et al. (1972) pointed out the importance of both starter strain and rennet type and suggested that the major role of rennet in the development of bitterness may be the production of long peptides that are subsequently degraded to small bitter peptides by starter proteinases. These authors found a higher concentration of free amino acids in cheeses made with nonbitter starters, suggesting greater

72

P. F. FOX AND J. M. WALLACE

peptidase activity in these strains. Certain strains of starter and Penicillium spp. are associated with the development of bitterness (see Adda et nl., 1982), and it would appear that bitterness in cheese results from the action of chymosin on casein with the release of bitter peptides. These peptides accumulate in bitter cheese due to the inability of “bitter” starters to hydrolyze them to nonbitter peptides due to a deficiency in peptidase activity (Stadhouders and Hup, 1975), or perhaps by degradation by bacterial enzymes of peptides that otherwise would be too large to be perceived as bitter (Lemieux and Simard, 1991). The development of bitter taste is common in low-fat cheeses (Banks et a)., 1992). In full-fat cheese, a certain proportion of bitter peptides, being hydrophobic, probably partition into the fat phase, where they are less likely to be perceived as being bitter. The literature concerning bitterness in dairy products has been reviewed by Lemieux and Simard (1991, 1992). The majority of studies in which bitter peptides were identified were conducted in model systems consisting of isolated casein incubated with various enzymes. Bitter peptides have been identified in hydrolyzates of a,,-, as2-, and P-caseins. Para-K-casein is a potential source of bitter peptides (Visser, 1981), although in the few studies in which the hydrolysis of para-lc-casein was investigated it was found to be resistant to proteolysis. Bitter a,,-casein-derived peptides identified in hydrolyzates include f22-43 and f45-50 (see Sullivan and Jago, 1972), f23-34, f9l-100, and f145-151 (Hill and van Leeuwen, 1974). LeBars and Gripon (1989) considered that three short peptides produced from the C-terminal region of a,,-casein by plasmin (f198-207, f182-207, and f189-207) may be bitter. Bitter peptides found in hydrolyzates of p-casein include f203-208, fl95-209, f201-209 (see Sullivan and Jago, 1972), and f53-79 (Clegg et al., 1974). A number of bitter peptides isolated hom cheese or casein hydrolyzates are summarized in Table V. A number of authors have described the isolation of bitter peptides from cheese (e.g., Harwalkar and Elliott, 1971; Visser, 1977), but there is some disagreement as to the source of these peptides (Visser, 1981). However, unlike peptides produced from a,,-casein, the initial peptides produced from p-casein by chymosin are probably bitter, and thus the limited hydrolysis of this protein may contribute to bitterness. The concentration of NaCl has a major effect on the hydrolysis of p-casein by chymosin in solution (Fox and Walley, 1971) and in cheese (Kelly, 1993), and thus may be a factor in the development of bitterness. The formation of p-casein f193-209 (the primary product of chymosin action on p-casein and potentially bitter) was inhibited by increasing NaCl

73

FORMATION O F FLAVOR COMPOUNDS IN CHEESE TABLE V BITTERPEPTIDES ISOLATED FROM CHEESE‘ Cheese

Origin

Cheddar

aS,-CN fl4-17 a,l-CN fl7-21 a,,-CN f26-32 CX,~-CN26-33 8-CN f46-67 P-CN f46-84

P-CN f193-209 Gouda ondh

P-CN f84-89 P-CN f193-207 P-CN fl93-208 P-CN fl93-209

Sequence

Hydrophobicity Q, cal residue-’

E.V.L.N N.E.N.L.L A.P.F.P.E.V.F A.P.F.P.E.V.F.G Q.D.K.I.H.P.F.A.Q.T.Q.S.L. V.Y.P.F.P.G.P.1.P Q.D.K.I.H.P.F.A.Q.T.Q.S.L. V.Y.P.F.P.G.P.I.P.N.S.L.P.Q.N. 1.P.P.L.T.Q.T.P.V.V.V Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P. 1.I.V

1162.5 1074.0 1930.0 1688.8

V.P.P.F.L.Q Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P.1 Y.Q.Q.P.V.L.G.P.V.R.GP.F.P.I.1 Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P. 1.I.V

1983.3 1686.7 1766.9

1580.5

1508.5 1762.4

1762.4

Alpkase

aSl-CN fl98-199

L.W

2710.0

Butterkase

P-CN f61-69

P.F.P.G.P.1.P.N.S

1792.2

“Adapted from Lemieux and Simard (1992).

concentrations (Kelly, 1993). The primary action of plasmin on p-casein probably does not produce bitter peptides. The fate of as2-casein in cheese is unclear, but plasmin can release potentially bitter peptides from this protein in solution (LeBars and Gripon, 1989). Guigoz and Solms (1974) found the bitter dipeptide, asl-CN fl98-199 (Leu-Trp), in Alpkese cheese. Stepaniak and Fox (1995) demonstrated the production of this peptide from asl-CN fl65-199 by a lactococcal endopeptidase (PepO). In addition to peptides, a number of other compounds can contribute to bitterness in cheese, including amino acids, amines, amides, substituted amides, long-chain ketones, and some monoglycerides (Adda et

d., 1982).

Rancidity, which is occasionally encountered in cheese, is due to excessive or unbalanced lipolysis caused by lipases-esterases from starter or NSLAB, psychrotrophs in the cheese milk or indigenous milk

74

P. F. FOX AND J. M. WALLACE

lipoprotein lipase. As discussed in Section II.C, a high concentration of lactic acid, perhaps arising from the retention of an excessive level of lactose in the cheese curd, imparts an acidic, harsh taste to cheese. Graders frequently use the term “over-acid” to describe a flavor defect in overripe cheese. The precise cause of this defect is not known, but it is probably due to excessive or unbalanced proteolysis rather than to a high concentration of lactic acid, The principal compounds responsible for fruitiness in Cheddar are ethyl butyrate and ethyl hexanoate, formed by esterification of FFAs with ethanol. Production of ethanol appears to be the limiting factor, as FFAs are present in cheese at relatively high concentrations (Bills et al., 1965). Ethyl esters are present at low concentrations in nonfruity cheeses and thus the fruity defect occurs as a result of increased production of ethanol or its precursors. The origin of “unclean” and related flavors in Cheddar was investigated by Dunn and Lindsay (1985), who quantified a number of Strecker-type compounds, including phenylacetaldehyde, phenethanol, 3-methylbutanol, 2-methylpropanol, phenol, and p-cresol. Phenylacetaldehyde concentrations were elevated in cheeses with an “unclean rosy” off-flavor, and the addition of this compound to clean-flavored mild Cheddar reproduced this defect. At higher concentrations (>400 pg/kg), phenylacetaldehyde imparted astringent, bitter, and stinging flavors to cheese. Concentrations of phenethanol were similar in most of the cheeses studied (-100 pg/kg). p-Cresol was found to impart a putrid “utensil”-type flavor when present in high concentrations (>lo0 pg/kg-l). Dunn and Lindsay (1985) also discussed the potential of short-chain fatty acids to potentiate the flavor impact of p-cresol. Branched-chain Strecker-type aldehydes (3-methylbutanal, 2methylbutanal, and 2-methylpropanal) did not cause flavor defects when added at concentrations below 200 pg/kg to clean-flavored cheese. Phenol (100-225 pg/kg-l) appeared to have no detrimental effect on cheese flavor and indeed enhanced the sharpness of Cheddar flavor (Dunn and Lindsay, 1985). Dunn and Lindsay (1985) added quinine to cheese to simulate the bitterness often associated with the development of Strecker-type compounds. It enhanced the flavor impact of these compounds. ACKNOWLEDGMENTS

The authors would like to express their thanks to Ms. Anne Cahalane for her assistance in preparing the manuscript.

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Manning, D. J., Chapman, H. R., and Hosking, Z. D. (1976). The production of sulphur compounds in Cheddar cheese and their significance in flavour development. J. Dairy Res. 43,313-320. Mariani, P., Bonomi, A., Sabbioni, A,, Lucchelli, L., Blanco, P., Zanzucchi, G., and Florentini, L. (1993). Caratteristiche organolittiche e chimico-bromatologiche des formaggio Parmigiano-Reggiano prodottd con il latte delle razze bruna e frisono Italiana. Rev. Sci. Alinientazione 22, 91-102. Marsili, R. (1985). Monitoring chemical changes in Cheddar cheese during aging by high performance liquid chromatography and gas chromatography techniques. J. Dairy Sci. 68, 3155-3161.

McEwan, J. A. Moore, J. D., and Colwill, J. S. (1989). The sensory characteristics of Cheddar cheese and their relationship with acceptability. J. Soc. Dairy Technol. 4, 112-1 17.

McGoldrick, M. A. (1996). “Intervarietal Comparison of Proteolysis in Cheese.” MSc. Thesis, National University of Ireland, Cork. McGugan, W. A. (1975). Cheddar cheese flavour. A review of current progress. J. Agric. Food Chem. 23, 1047-1050. McSweeney, P. L. H., and Fox, P. F. (1997). Chemical methods for characterization of proteolysis in cheese during ripening. Le Lait 77,41-76. Meinhart, E., and Scheier, P. (1986). Study of the flavour compounds from Parmigiano Reggiano cheese Milchwissenschaft 41,689-691. Mooney, J. S. (1997). “Isolation and Identification of the Water-Insoluble Peptides in Cheddar Cheese.” M.Sc. Thesis, National University of Ireland, Cork. Mottar, J. F. (1989). Effect on the quality of dairy products. In “Enzymes of Psychrotrophs of Raw Foods” (R. C. McKellar, ed.), pp. 227-243. CRC, Boca Raton, FL. Muir, D. D., and Hunter, E. A. (1992). Sensory evaluation of Cheddar cheese: The relation of sensory properties to perception of maturity. J. SOC.Dairy Technol. 45,23-30. Mulder, H.(1952). Taste and flavour forming substances in cheese. Neth. MilkDairy J. 6 , 15 7-1 67.

Nakae, T., and Elliott, J. A. (1965). Volatile fatty acids produced by some lactic acid bacteria, I: Factors influencing production of volatile fatty acids from casein hydrolyzate. J. Dairy Sci. 48, 287-292. Nelson, J. H., Jensen, R. G., and Pitas, R. E. (1977). Pegastric esterase and other oral lipases-a review. J. Dairy Sci. 60, 327-362. Ney, K. H. (1971). Prediction of bitterness of peptides from their amino acid composition. Z. Lebensm. Forsch. 147,64-68. Noomen, A. (1983). The role of the surface flora in the softening of cheeses with a low initial pH. Neth. Milk Dairy]. 37,229-232. Nsar, M. M., and Younis, N. A. (1986). Effect of adding alanine, phenylalanine and proline on the properties of Romi cheese. Egypf I. Food Sci. 14,385-388. O’Callaghan, D. M. (1994). “Physicochemical, Functional and Sensory Properties of Milk Protein Hydrolysates.” Ph.D. Thesis, National University of Ireland, Cork. Olivecrona, T., and Bengtsson-Olivecrona, G. (1991). Indigenous enzymes in milk: Lipase. In “Food Enzymology” (P, F. Fox, ed.), Vol. 1, pp. 62-78. Elsevier, London

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Olivecrona, T.. Vilaro, S., and Bengtsson-Olivecrona, G. (1992). Indigenous enzymes in milk, 11: Lipases in milk. In “Advanced Dairy Chemistry” (P. F. Fox, ed.), Vol. 1, pp. 292-310. Elsevier, London. Omar, M. M. (1984). Microstructure, free amino acids and free fatty acids in Ras cheese. Food Chem. 15, 19-29. Omar, M. M., and El-Zayat, A. I. (1986). Ripening changes of Kashkaval cheese made from cows milk. Food Chem. 22, 83-94. Oterholm, A., Ordal, Z. J., and Witter, L. D. (1970). Purification and properties of glycerol ester hydrolase (lipase) from Propionibacterium shermanii. Appl. Microbiol. 20, 16-22.

Parliament, T. H., Kolor, M. G., and Rizzo, D. J. (1982). Volatile components of Limburger cheese. J. Agric. Food Chem. 30, 1006-1008. Paulsen, P. V., Kowalewska, J., Hammond, E. G., and Glatz, B. A. (1980). Role ofmicroflora in production of free fatty acids and flavor in Swiss cheese. J. Dairy Sci. 63,912-918. Pham, A.-M., and Nakai, S. (1984). Application of stepwise discriminant analysis to high performance liquid chromatography profiles of water extract for judging ripening of Cheddar cheese. 1. Dairy Sci. 67, 1390-1396. Polo, C., Ramos, M., and Sanchez, R. (1985). Free amino acids by high performance liquid chromatography and peptides by gel electrophoresis in Mahon cheese during ripening. Food Chem. 16, 85-96. Ponce-Trevino, R., Richter, R. L., and Dill, C. W. (1987). Influence of lactic acid bacteria on the production of volatile sulphur containing compounds in Cheddar cheese slurries. J. Dairy Sci. 70, 59 (Abstr.). Ponce-Trevino, R., Richter, R. L., and Dill, C. W. (1988). Observations on effects of oxidation-reduction potential on volatile sulphydryl production in Cheddar cheese slurries. J. Dairy Sci. 71, 278 (Abstr.). Puchades, R., Lemieux, L., and Simard, R. E. (1989). Evolution of free amino acids during ripening of Cheddar cheese containing added lactobacilli strains. I. Food Sci. 54, 885-888, 946.

Ramos, M., Caceres, I., Polo, C., Alonso, L., and Jaurez, M. (1987). Effect of freezing on soluble nitrogen fraction of Cabrales cheese. Food Chem. 24, 271-278. Rattray, F. P., and Fox, P. F. (1997a). Purification and characterization of an intracellular esterase from Brevibacterium h e n s ATCC 9174. Int. Dairy J. In press. Rattray, F. P., and Fox, P. F. (1997b). Purification and characterization of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174. Le Lait 77, 169-180. Rattray, F. P., Bockelmann, W., and Fox, P. F. (1995). Purification and characterization of an extracellular proteinase from Brevibacterium linens ATCC 9174. Appl. Environ. Microbiol. 61, 3454-3456. Rattray, F. P., Fox, P. F., and Healy, A. (1996). Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine a,,-casein. Appl. Environ. Microbial. 62, 501-506. Rattray, F. P., Fox, P. F., and Healy, A. (1997). Specificity of an extracellular proteinase from Brevibacterium linens ATCC 91 74 on bovine p-casein. Appl. Environ. Microbiol. 63, 2468-2471.

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Reps, A., Hammond, E. G., and Glatz, B. A. (1987). Carbonyl compounds produced by growth of Lactobacillus bulgaricus. J. Dairy Sci. 70, 559-562. Rice, G. H., Stewart, F. H. C., Hillier, A. J., and Jago, J. R. (1978). Uptake of amino acids and peptides by Streptococcus lacfis.J. Dairy Res. 45,93-107. Roger, S.,Degas, C., and Gripon, J.-C. (1988). Production of phenylethyl alcohol and its esters during ripening of traditional Camembert. Food Chern. 28, 129-140. Roudot-Algaron, F., LeBars, D., Einhorn, J., Adda, J., and Gripon, J.4. (1993). Flavour constituents of aqueous fraction extracted from Comte cheese by liquid carbon dioxide. J. Food Sci. 58,1005-1009. Samples, D. R. (1985). “Some Factors Affecting the Production of Volatile Sulphydryl Compounds in Cheddar Cheese Slurries.” Ph.D. Thesis, Texas A&M University. Cited in Urbach, 1995. Sentheshanmuganathan, S . (1960). The mechanism of the formation of higher alcohols from amino acids by Saccharomyces cervisiae. Biochern. J. 74,568-576. Sharpe, M. E., and Franklin, J. G. (1962). Production of hydrogen sulphide by lactobacilli with special reference to strains isolated from Cheddar cheese. In “Proc. 8th Int. Congr. Microbiol. BJI,” Vol. 3, p. 46. Sharpe, M. E., Law, B. A., and Phillips, B. A. (1976). Coryneform bacteria producing methanethiol. J. Gen. Microbiol. 94,430-435. Simonart, P. and Mayaudon, J. (1956). Chromatographic analysis of cheese, 3. Aromatic Acids. Ned. Melk Z. 10, 261-267. Singh, T. K., Fox, P. E , and Healy, A. (1995). Water soluble peptides i n Cheddar cheese: Isolation and identification of peptides in the UF retentate of water-soluble fractions. J. Dairy Res. 62,629-640. Singh, ’T. K., Fox, P. F.,and Healy, A. (1997). Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese. J. Dairy Res. 64.In press. Smith, T.A. (1981). Amines in food. Food Chern. 6, 169-200. Ssrhaug, T., and Ordal, Z. J. (1974). Cell-bound lipase and esterase of Brevibacterium linens. Appl. Microbiol. 27,607-608. Ssrhaug, T., and Stepaniak, L. (1997). Psychrotrophs and their enzymes in milk and dairy products: Quality aspects. Trends Food Sci. Technol. 8,35-41. Stadhouders, J., and Hup, G. (1975). Factors affecting bitter flavour in Gouda cheese. Neth. Milk Dairy J. 29, 335-353. Stepaniak, L., and Fox, P. F. (1995). Characterization of the principal intracellular endopeptidase from Lactococcus lactis ssp. lactis MG 1363. Int. Dairy]. 5,699-713. Sullivan, J, J., and Jago, G. R. (1972). The structure of bitter peptides and their formation from caseins. Aust. J. Dairy Technol. 27,98-104. ’Thomas, T. D. (1987). Acetate production from lactate and citrate by non-starter bacteria in Cheddar cheese. N. Z. J. Dairy Sci. Technol. 22,25-38. Thomas, T. D., and Crow, V. L. (1983). Mechanism of D(-)-lactic acid formation in Cheddar cheese. N . Z. J. Dairy Sci. Technol. 18, 131-141. Thomas, T.D., McKay, L. L., and Morris, H. A. (1985).Lactate metabolism by pediococci isolated from cheese. Appl. Environ. Microbial. 49,908-91 3.

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The Role of Microorganisms in

Soy Sauce Production DESMONDK. O’TOOLE Department of Biology and Chemistry City University of Hong Kong Kowloon, Hong Kong

I. Introduction 11. Types and Composition of Soy Sauce A. Types of Shoyu B. Amino Acid Composition of Soy Sauce C. Organic Acids in Soy Sauce D. Sugars in Soy Sauce 111. Aroma and Flavor of Shoyu IV. Soy Sauce Production A. Composition of Raw Materials B. The Manufacture of Soy Sauce C. Koji D. Moromi E. Use of Continuous Fermentation Processes to Produce Soy Sauce V. Effect of Water Activity on Microorganisms VI. Yeasts A. Effect of Oxygen on Yeasts B. Alcohol Production by Zygosucchuromyces rouxii VII. Bacteria VIII. Production and Metabolism of Amino and Organic Acids in Moromi A. Role of Tetrugenococcus halophilus B. Interaction Between Organic Acids and Soy Sauce Organisms IX. Production and Fate of Other Substances X. Possible Role of Metal Ions in Soy Sauce Production A . Metal Ions i n Metabolism B. Investigation of Metal Ions i n Soy Sauce and Its Manufacture C. Adequacy of Minerals in Raw Materials XI. Conclusion References

I. Introduction

Soy sauce is a salty condiment commonly used in Eastern Asia that is made from soy beans with varying amounts of wheat or no wheat at all. It is known as shoyu in Japan, chiang-yu (or -yi) in China, kecup in Indonesia, kunjang in Korea, toyo in The Philippines, and see-ieu in Thailand (Djien, 1982; Beuchat, 1985; Fukushima, 1989). It provides 67 ADVANCES IN APPLIED MICROBTOLOGY, VOLUME 45 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. 0065-2164/97$25.00

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flavor in an otherwise bland diet, and nutritionally it provides salt (NaCI) and predigested proteins in a diet that is traditionally protein poor. It has been made for centuries on a small scale in many towns and villages in Asia, but since 1950, particularly in Japan, the manufacturing process has been studied and modernized so that its manufacture is now concentrated in large factories using modern, controlled methods of production (Sasaki and Nunomura, 1993). In Japan, soy sauce fermentation is a major food manufacturing activity. More than 1.1 million kiloliters of soy sauce was produced in 1986 by 3000 producers, and the Kikkoman Company supplied 30% of the market (Fukushima, 1989).By 1990 there were 2871 manufacturers, five of which produced about 50% of the total production (Sasaki and Nunomura, 1993). While modern methods are used for most of the soy sauce produced in Japan, and factory production in other Asian countries is growing, soy sauce is still produced by methods involving no modern technological inputs (Roling et a!., 1994a).

BKIEFHISTORY Soy sauce “was derived from a Chinese food called chiang,” which was basically a mash (Fukushima, 1989). First recorded in a book published around 1200 B.c.,chiang was made by growing yellow aspergilli on millet (to produce the equivalent of modern-day koji),then the molded grain, animal, poultry, or fish flesh and salt were mixed with a “good liquor” in a bottle for 100 days. The use of a liquid similar to soy sauce from this type of product was first mentioned during the Han dynasty (25-220 A.D.). The first mention of the use of soy beans in this product was in 535 A.D., and the first record of the use of all the materials used in modern soy sauce production was published between 1271 and 1368 A.D. The method for making chiang-yuwas not described in detail until the sixteenth century (Wang and Fang, 1986). From China, the fundamental principles of soy sauce production spread to other Southeast Asian countries and to Japan about the seventh century (Fukushima, 1989). During the seventeenth and eighteenth centuries, large quantities of soy sauce were exported from Japan by the Dutch to other parts of Asia and to Europe. During the twentieth century, the manufacture of soy sauce has spread from Japan to the United States, where a plant was first opened in 1909, which was not successful. However, production continued, and in 1972 a more modern plant was opened by the Kikkoman Company. Soy sauce production, as well as the production of a number of other traditional Asian foods (e.g., miso and rice wine) is based on a two-step

MICROORGANISMS IN SOY SAUCE PRODUCTION

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microbial process that has some similarities to beer production (Fukushima, 1989; Yong and Wood, 1974). Fungi are allowed to grow on the pretreated raw material, soy bean and wheat, and the molded grain is then steeped in brine. The enzymes from the mold break down the constituents (proteins, starch, and cellulose) in the legume and grain, and subsequent bacterial and yeast growth produce the characteristic flavor of the soy sauce. Beer production depends on enzymes (amylases) produced by the sprouting of the barley grain to break down the cooked starch in the endosperm to mono- and disaccharides that are subsequently fermented by yeasts. Our scientific knowledge of soy sauce production and manufacture has increased rapidly over the last 45 years, and there have been several published reviews about soy sauce. Most of the important work has been done and published in Japan. Some idea of the quantity of Japanese work can be gleaned from two Japanese reviews entitled “Review of Research Reports on Soy Sauce and Miso in 1986” (Mori, 1987), and “Reports on Soy Sauce and Miso, Published in Japan in 1981” (Mori, 1982), in which 534 and 743 references, respectively, to soy sauce and miso are listed. Also, 539 of the 1092 papers in which soy sauce occurs in Food Science and Technology Absfracfsfrom 1969 to December 1996 were published in Japanese. An early English-language review of soy sauce is that of Yokotsuka (1960), while non-Japanese perspectives on the topic were presented by Yong and Wood (1974) and Wood (1982). Recent English reviews from Japan include those of Yokotsuka (1985, 1986) and a really comprehensive review from Fukushima (1989). However, no critical reviews in English seem to have approached soy sauce from a microbiological perspective, as other aspects of soy sauce such as the biochemical, chemical, and technological aspects have attracted more attention. The purpose of this review is to examine soy sauce in relation to the role of microorganisms in its production with a view to how they produce the soluble components and flavors in the sauce, and what factors can and do influence the quality of the final product.

II. Types and Composition of Soy Sauce

The spread of the manufacture of soy sauce to many parts of Southeast Asia and the subsequent growth of cottage-style production led to differences in production techniques from place to place that ultimately impinged on the composition and types of soy sauce. In some countries

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DESMOND K. O'TOOLE

TABLE I THEGENEKAL COMPOSITION" Product Koikuchi shoyu, Japan lisukuchi shoyu, Japan Soy sauce, Taiwan Soy sauce, Korea Soy sauce, Hong Kong Soy sauce, Philippines Soy sauce, Singapore Soy sauce, Malaysia Soy sauce, USA

OF SOME SOY SAUCES FROM VARIOUS PARTS OF THE WORLD

BB

NaCl

TN

23.6 22.2 25.6 21.9 28.5 23.3 30.1 23.9 22.8

17.0 18.0 15.6 17.3 26.2 24.7 24.1 18.3 16.5

1.70 1.18 2.05 1.50 1.54 0.76 1.97 1.17 1.65

RS(1S) Alcohol 5.07 4.00 5.95 2.10 4.22 1.06 4.81 8.50 3.70

2.50 2.00 0.86 0.39 0.00 0.01 0.00 0.03 2.07

Color

++

+ ++ ++ if+

++ +++ +++ ++

aBB = spccific gravity, dcgrees Baum6; NaCl = sodium chloride (g 100 ml-'I: TN = total nitrogen (g 100ml-I); KS(1S) = reducing sugar (invert sugar) (g 100 rn1-l): alcohol = ethanol (ml 100 nil-'). bAdaptcd from Table 2 of Yokotsuka (1985).

these interregional differences have been formalized into systems that define the various types of sauce. Although there are similarities in the composition of soy sauce, as, for example, there are similarities between cheeses, there are significant variations in the composition of the soy sauces produced. The Indonesians add sugar to the soy sauce, so that they have two basic kinds of soy sauce, kecap asin and kecap rnanis (the sugared sauce) (Roling et al., 1994a). The general composition of some soy sauces from different parts of the world is shown in Table I. A. TYPESOF SHOYU Japan has an organized system of classification to define types of soy sauce. Such systems probably exist outside Japan, and other countries do set standards for soy sauce (see, e.g., Chang et a]., 1987). In Chinese societies, dark and light soy sauces are marketed (Anon., 1984). The types of soy sauce recognized by the Japanese Government and their typical composition are listed in Table 11. However, the typical ethanol levels in Table I1 for tamari, saishikomi, and shiro shoyu differ significantly from values published by Sasaki and Nunomura (1993), who reported typical values for ethanol in tamari, saishikomi, and shim shoyu of 3.41, 2.59, and 3.85% (v/v), respectively. These differences may be due to better control of the process used in the production of

91

MICROORGANISMS IN SOY SAUCE PRODUCTION

TABLE I1 TYPICAL PERCENTAGE

COMPOSITIONS OF

FIVEVARIETIES OF SOY

RECOGNIZED BY THE JAPANESE

Sauce

(shoyul

Koikuchi Usukuchi Tamari Saishikomi Shiro

NaCl w/v

Total nitrogen w/v

16.9 18.9

1.57 1.19

19.0 18.6

2.55 2.39

19.0

0.5

Formol nitrogen w/v 0.94

0.80 1.05 1.11 0.24

SAUCE

GOVEFNMENT~ Reducing Alcohol sugar wlv vlv

Color

3.0 4.2

2.3

Deep brown

2.1

5.3 7.5

0.1 trace trace

Light brown Dark brown Dark brown Yellow-tan

20.2

‘Specific gravity is from 22.0 to 29.9°Be; pH is from 4.6 to 4.8. Adapted from Table 2 of Fukushima (1989).

the minor types of shoyu through the application of results obtained from research on koikuchi shoyu, or the direct addition of ethanol. The differences between these types of soy sauce are quite significant, and they come about primarily from the proportions of raw materials, soy beans, and wheat used to make the sauce. Koikuchi is made with about equal quantities of wheat and soy protein, whereas usukuchi is made with more wheat (Yokotsuka, 1985), but Fukushima (1989) says that the difference between these two sauces is due only to the manner in which the fermentation is conducted to reduce the development of color. Tamari is made using a soy:wheat ratio of about lO:l, while saishikomi is made using equal quantities of soy and wheat, with the variation that raw soy sauce is used and not freshly made brine. Shiro is made using a very high level of wheat in the soy-wheat mixture (Fukushima, 1989);hence the high level of reducing sugar in this shoyu. The most popular Japanese soy sauce is koikuchi shoyu, which made up 82.9% of the shoyu produced and checked by the Japan Agricultural Standard in 1990; tamari shoyu comprised only about 1.9% of production (Sasaki and Nunomura, 1993). Koikuchi shoyu has quite a high alcohol content (=3%), which has long been considered important for stabilizing the quality of and adding flavor to Japanese shoyu (Iwasaki ef a]., 1991a). As Table I shows, ethanol is not a significant constituent in soy sauces from other parts of Asia. Tamari-type soy sauce is the most widely produced soy sauce type in Southeast Asia (Fukushima, 19891, and it is also the main Chinese type of soy sauce (Sasaki and Nunomura, 1993), because the cereal-legume mixture for

92

DESMOND K. O'TOOLE

tamari production contains about 10% wheat, which is similar to the proportion used in Chinese soy sauce (Fukushima, 1989). However, high-wheat sauces (e.g., sauces made with wheat:soy bean ratios of 1:2 and 1:l) are also produced in China (Butler, personal communication, 1997). In addition, Fukushima (1989) says that the process now used in China involves a defatted soy bean meal-wheat bran mixture at a 6:4 ratio that is incubated for 24 h for mold growth and is then held for 3 weeks at a temperature in the moromi of 40-45OC, which is for a very much shorter period and at a much higher temperature than that used traditionally by the Japanese. On the other hand, Chiao (1986) does not mention such a shortened method of production in China. B. AMINOACIDCOMPOSITION OF SOY SALJCE The amino acid composition of the five types of Japanese shoyu and some Chinese and Korean soy sauces are shown in Table I11 (Ren et al., 1993a). The data for koikuchi shoyu are from ten samples, and the data for the Chinese and Korean soy sauces are from eight samples (data from one of the published Chinese samples have been dropped from the data herein because the raw materials for the sauce included monosodium glutamate). These data show that there is great variation in the amino acid content between the various Japanese types of shoyu, but the variation between various samples of koikuchi shoyu was not very great. The variation in amino acid contents between samples of Chinese sauce and between samples of Korean sauce was almost as great as the variation between the various types of Japanese shoyu, which may reflect variation in the raw materials used and in the processes used in Chinese and Korean sauces similar to those from Japan but which are not formally recognized in those countries as they are in Japan. The total nitrogen data for these samples reflect this, as the values for koikuchi, usukuchi, tamari,saishikomi, and shiro are 1.49, 1.02, 1.49, 1.45, and 0.51 mg . g-l, respectively, while the figures for Chinese and Korean sauces fall in the ranges 0.82-2.03 and 0.86-1.21 mg . g-l, respectively (Ren et al., 1993a). However, the above data from Ren et al. (1993a) differ significantly from those published by Sasaki and Nunomura (1993) (Table IV). Even after taking into account the different methods for expressing the concentrations, mg . g-' versus mg . ml-l, there is significant variation, particularly with the data from tamari shoyu. Fukushima (1989) says that the amino acid composition of the final product is almost the same as the original mixture of soy beans and wheat, with some exceptions. To assess whether soy sauce contains

MICROORGANISMS IN SOY SAUCE PRODUCTION

93

TABLE 111 THEAMINO AclD COMPOSITION (mg . g-') Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Wine Methionine Isoleucine Leucine Tyrosine Phenylalanine

OF SHOYu AND CHINESE AND

Usukuchi

Tamari

Saishikomi

3.71-4.31

2.76

0.31

2.13

1.00-1.32

1.29

0.95

1.04

1.93-2.39

2.60

5.41

2.53

3.35-5.42

2.15

1.87

2.77-2.93

1.60

3.61-4.16 12.38-1 7.33

Koikuchi

Shim

KOREANSOY

SAUCES

Chinese

Korean

1.15

1.15-4.44

0.97-4.53

0.25

0.42-1.21

0.51-1.59

0.72

0.61-3.41

0.34-4.53

4.95

0.50

1.85-4.85

1.88-7.45

1.63

2.35

0.53

0.95-2.37

1.33-2.81

1.92

2.07

3.44

0.79

1.31-3.58

1.87-3.76

4.20

3.90

7.82

5.15

2.28-9.26

5.85-15.36

3.464.15

2.01

0.28

2.91

1.37

1.20-3.91

2.00-4.07

2.03-2.58

0.98

1.34

1.84

0.36

0.55-3.45

0.92-3.50

4.15-6.49

1.69

1.86

3.48

0.80

1.59-5.65

2.08-5.06

0-0.05

0.06

0.95

0.12

0.09

0-0.20

0-0.09

3.73-4.27

2.05

1.98

3.36

0.70

1.49-4.00

1.59-3.48

0.94-1.14

0.82

0

0.92

0.24

0.42-0.91

0.43-0.85

3.05-3.68

1.91

2.14

3.00

0.58

1.29-2.61

1.46-2.59

4.61-5.65

2.48

3.46

4.50

1.00

2.23-3.96

2.37-4.53

0.66-0.81

0.76

0.35

0.91

0.64

0.56-1.83

0.35-1.35

1.22-2.14

1.48

1.95

2.34

0.83

0.59-1.50

0.28-1.56

'Adapted from Table 2 of Ren et of. (1993a).

amino acids in the proportions found in the raw materials, the data of Sasaki and Nunomura (1993) and Ren et al. (1993a) for the amino acid compositions of koikuchi and tarnari shoyu and the calculated values for amino acids from published values of amino acids for soy bean and wheat (Table IV) were used to determine the ratios of each amino acid in soy sauce and in the raw materials used. When the values are compared (Tables V and VI), we can see that the equivalence of the amino acid content with the constituents in the raw materials might not be the case and the values obtained really depend on the analytical results for the amino acids one chooses to use. In koikuchi shoyu, the ratio of amino acid concentrations in the shoyu to the theoretical amino acid levels in the raw materials is significantly higher for nine of the amino acids based on the results of Sasaki and Nunomura (1993), but only six are higher based on the results of Ren et al. (1993a). For tarnari shoyu, the discrepancy between the raw materials and the shoyu seems not so great by the results of Sasaki and Nunomura (1993), but by the results of Ren et al. (1993a) there are significant discrepancies between the s h o p values and the theoretical values for the raw materials.

94

DESMOND K. O'TOOLE

TABLE IV THEAMINO ACIDCOMPOSI'I'ION 01' SHoW (mg. rn-'), SOYBEANS (g . kg-l SED), DEFATTED SOY BEAN(g kg-'1, OF HARD R E D WINTER COMMERCIAL MILL WHEATMix (g . kg-' DRYWEIGHT) Soy beans

Shoyu"

Wheatb

Dc-

KOComponent Lysine Histidine Arginine Aspartic acid 'l'hreonine Serine Glutamic acid Proline Glycine Almine Cystine Wine Methionine Isoleucine Leucine Tyrosine Phenylalanine Protein Milling yield, 70

Tamari

Whole

fatted

kuchi

beansC

flourd

4.09

6.44

26.5

1.67

1.80

10.5

4.34

5.41

2.58

9.15

3.12

~

Whole grain

Flour

28.8-29.3

4.0

2.68

7.97

11.2-11.5

3.06

3.17

4.96

30.1

32.4

5.59

4.27

11.33

48.6

-

7.32

5.12

12.92

4.91

16

18.1-1 8.9

4.52

6.20

4.40

6.71

21.3

24.5-25.2

6.78

3.66 5.86

11.88

18.18

77.7

43.62

45.02

36.82

4.07

6.08

22.8

-

13.43

14.03

12.21

2.50

3.76

17.4

18.9-22.9

5.72

4.27

9.74

7.05

7.71

17.7

-

4.92

3.54

8.67

ND

0.28

5.5

6.8-6.9

2.0

1.95

-

4.82

6.73

20

22.5-23.4

6.12

5.12

9.03

Bran

8.14

1.17

1.17

5.3

6.3-6.9

1.R6

1.83

2.48

4.24

4.80

18.9

22.0-23.9

5.19

4.76

6.73

6.57

6.08

32.3

34.2-35.2

9.58

8.78

11.86

1.23

2.28

13

12.8-1 5.8

2.66

2.81

3.72

4.43

5.92

20.6

22.0-22.7

5.99

5.73

7.08

-

-

I22

177

-

-

133

-

-

100

72.8

21

ONunomura and Sasdki (1993). 'Mattern (1991).Table 40, calculated to g kg-' dry weight from g kg protein dry wcight using protein values in tahle above. 'Considine and Considine (1982). ' k d d r o u p and Smith (1989).

Three conclusions can be drawn from these data. The first is that amino acid values published for the various soy sauces may be very unreliable. This could reflect the difficulty of determining the amino acids in a soy sauce matrix, or the inherent difficulty of determining certain amino acids, The second is that a detailed study of the raw ingredients of batches and of the resulting soy sauce would be necessary to determine any equivalence between the amino acids in the raw

95

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE V ACIDSIN KO~KUCHI SHOYU AND IN THE RAW MATERIALS A COMPARISON OF AMINO OF SOUBEANAND WHEAT

Concentration of amino acids

Ref. 2

%, raw materials

Ref. 1

Ref. 2

6.0

6.7

4.79

125

140

2.5

2.2

2.45

100

90

6.4

3.6

5.84

109

62

Yo I

Ref. l n mg . ml-'

Ref. 2 m g ' g-'

Ref. 1

Lysine

4.09

3.97

Histidine

1.67

1.32

Amino acid

Amino acid ratios, sh oyu :raw materials, YO

shop

Arginine

4.34

2.16

Aspartic acid

2.58

5.42

3.8

901

8.78

43

104

Threonine

3.12

2.77

4.6

4.7

3.69

124

127

Serine Glutamic acid Proline

4.4

4.17

6.5

7.0

5.20

124

135

11.88

13.0

17.4

21.9

26.09

67

84

4.07

4.3

6.0

7.2

7.89

76

91

Glycine

2.5

2.85

3.7

4.3

4.31

85

100

Alanine

7.05

4.77

10.3

8.0

4.05

256

198

Cystine

0

0.05

0

0.1

1.44

0

6

Valine

4.82

4.23

7.1

7.1

4.79

148

148 132

Methionine

1.17

1.08

1.7

1.8

1.36

126

Isoleucine

4.24

3.05

6.2

5.1

4.30

145

119

Leucine

6.57

4.54

9.6

7.6

7.61

127

100

Tyrosine

1.23

0.81

1.8

1.4

2.61

69

54

Phenylalanine

4.43

1.22

6.5

2.1

4.81

135

44

68.16

59.44

100

100

100

100

100

Total

'Ref. 1 is Sasaki and Nunomura (1993):Ref. 2 is Ren et al. (1993a).The data from the shoyu samples with around 6 g . 100 ml-' of amino acids were used for this calculation.

materials and the resulting soy sauce. Finally, it is almost certain that amino acids are metabolized by microorganisms (see below), and, if so, the levels of amino acids are probably in part the consequence of microbial activity and not a straight conversion of protein in the raw materials to amino acids. If so, the levels of amino acids in soy sauce will never mirror the levels found in the raw materials and may vary depending on particular strains of microorganisms present in the brine stage.

96

DESMOND K. O'TOOLE

TABLE V1 A COMPARISON OF AMINOAcrns IN TAMAItl SllOYU AND IN T I E RAW MATERIAI.S 01'SOYBEANAND WHEAT

Concentration of amino acids Ref. I

Ref. 2

%, raw materials

Yo,

~

Amino acid

Ref. '1 mg. nil-'

Ref. 2 rng

. g'

shoy

~

Amino acid ratios, shoyuxaw materials, YO Ref. 1

Ref. 2

Lysine

6.44

0.31

6.6

1.02

6.20

107

17

Hi sti dine

1.80

0.95

1.9

3.12

2.57

72

121

Arginine

5.41

5.41

5.6

17.77

7.12

78

250

Aspartic acid

9.15

1.87

9.4

5.98

11.37

83

53

Threonine

4.91

1.63

5 .O

5.35

3.90

129

137

Serine

6.71

2.07

6.9

6.80

5.25

131

130

Glutamic acid

18.18

3.90

18.7

12.81

20.60

91

62

Proline

6.08

0.28

6.2

9.20

6.09

102

151

G1y cin e

3.76

1.34

3.9

4.4

4.31

90

102

Alanine

7.71

1.86

7.9

6.11

4.31

184

142

Cystine

0.28

0.95

0.3

3.12

1.38

21

226

Valinti

6.73

1.98

6.9

6.5

4.92

141

132

Methionine

1.17

0

1.2

0

1.32

91

0

Isoleucine

4.80

2.14

4.9

7.03

4.60

107

155

Leucine

6.08

3.46

6.2

11.34

7.92

79

143

Tyrosine

2.28

0.35

2.3

1.15

3.10

76

37

Phenylalanine

5.92

1.95

6.1

6.4

5.04

121

127

Total

97.41

30.45

100

100

100

100

100

'Rel'. 1 is Sasaki and Nunomura (1993);Ref. 2 is Ren ef ul. (1993a)

c.

ORGANIC

ACIDSIN SOY SAUCE

A variety of organic acids is found in soy sauce. Ushijima et al. (1982) reported levels of pyroglutamic, lactic, acetic, formic, citric, succinic, pyruvic, and malic acids found in individual batches of koikuchi and usukuchi shoyu, as shown in Table VII. More recent average figures (Sasaki and Nunomura, 1993) for some of these acids found in the five basic types of Japanese s h o p are shown in Table VIII. Lactic acid in koikuchi shoyu varied from about 3 to 1 2 mg . ml-l, with a median

97

MICROORGANISMS IN SOY SAUCE PRODUCTION

TABLE VII CONCENTRATIONS OF ORGANIC ACIDSFOUND IN T W O TYPES OF JAPANESE SHOYU (mg . m1-I)' PyrogluSample

Koikuchi s h o p A B C

D E F G H

tamic

Lactic

Acetic

Formic

Citric

Succinic

Total

3.30 3.65 3.78 2.78 7.89 5.17 3.59 5.11

10.22 8.81 9.18 6.39 2.30 7.15 12.64 12.49

1.92 1.93 1.70 1.43 1.09 1.74 1.97 2.14

0.16 0.18 0.15 0.19 0.22 0.22 0.20 0.25

0.18 0.40 0.18 0.81 2.15 0.84 0.12

0.34 0.37 0.37 0.50 0.59 0.43 0.31 0.34

16.20 15.40 15.40 12.30 14.70 15.60 18.80 20.40

1.63 2.67 1.62 5.64 2.87 4.90

6.74 6.74 7.94 2.29 4.70 3.92

1.25 1.22 1.60 0.70 1.73 0.67

0.08 0.09 0.17 0.08 0.14 0.12

0.39 0.35 0.33 0.42 0.29 0.43

10.10 11.70 12.10 10.70 10.60 11.90

trace

Usukuchi shoyu I

7 K L M N

trace 0.39 0.45 1.52 0.87 1.86

aAdapted from Table 5 of IJshijima et al. (1982). Malic acid concentration was either 0 or trace, except for two samples of koikuchi shoyu in which it was 0.12 and 0.42 mg . mlP. Pyruvic acid concentration ranged from 0 to 0.04, but one sample of usukuchi shoyu contained 0.27 rng . ml-'.

concentration of about 7 mg . rnl-l, and acetic acid was about 1.5 to 2.0 mg . ml-l. When the data in Tables VII and VIII are compared, we can see that the more recent levels of pyroglutamic acid are lower for koikuchi shoyu and for usukuchi shoyu, which may reflect improvements in the control of microbial and enzymic processes. Acetic and lactic acid concentrations are comparable, but formic and citric acids have virtually disappeared from the modern shoyu. Succinic acid has also been reduced when the 1982 data are compared with the recent data. In contrast to the above data, Ren et al. (1993b) reported significantly different levels of some of these acids in Japanese shoyu even after taking into account the different bases on which the levels have been calculated (mg . 8-l versus mg . ml-l). Table IX shows that the levels of citric and succinic acids were much greater and levels of acetic much lower than those reported by Sasaki and Nunomura (1993).

98

DESMOND K. O'TOOLE

TABLE VIII ORGANIC ACIDS IN THE

FIVE'rYI'ES

OF JAPANESE SHOYU

(mg .

PyroShop

glutamic

Lactic

Acetic

Formic

Koikuchi Usukuchi Tamari Shiro Saishikomi

3.67

7.59 7.61 9.01

1.81 1.19

0.09 ND

2.47

0.14

1.31 1.55

ND

N D ~ ND ND ND

0.10

ND

1.79 4.79 0.82

ND 4.12

8.18

Citric

succinic __ 0.26 0.25 0.30 ND 0.28

"Adapted from Table 5 of Sasaki and Nunomura (19931. hNon~ detected.

TABLE IX ORGANIC ACIDS IN THE FIVE TYPES (IF JAPANESE SHOYri AND RANGES OF THE IN

ACIDSFOUND

CHINESE AND KOREAN SOYSAUCE (mg . g-')' ~

SOY SdUCC

Koikirchi shoyu lisukuchi s h o p Tumari shoyu Shiro shoyir Saishikoirii s h o p

Chinese Korean

Pyroglutarnic 2.75-5.24 2.97 5.71 2.18 9.19 i.o3-~.2ti 1.48-5.24

Lactic

Acetic

Formic.

Citric

succinic

10.78-10.49

0-0.69 0

1.21-1.59 0.84

0

4.81

0 0 0-1.23 0-13.20

0 1.43

3.08-7.61 1.14 20.34 0 33.6 1.19-5.nn 0.94-7.61

9.10-11.15 18.4 18.49 4.21 24.27 1.99-18.33 1.45-5.26

8.08 19.39

4.66 13.48 3.72-19.67 3.19-11.5n

04.09

04.m

UAdaptedfrom Table 2 of Ken et nl. (1983b).

Chinese and Korean soy sauce samples (Ren et al., 1993b) showed great variation in the levels of organic acids, in line with the figures reported for the Japanese shoyu samples (Table IX). The level of lactic acid in old baceman (Table X) is comparable to the shoyu levels, but the level of acetic acid is lower.

D.

SUGARS IN SOY SAUCE

There are two sources of the sugars found in soy sauce. The first is from the sugars that arise from the breakdown of carbohydrates in the

99

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE X SIMPLE ORGANIC COMPOUNDS FOUND I N DIFFERENT BRANDS OF INDONESIAN BACEMAN(mg . ml-l)a Manufacturer and baceman age Ikan Lele at 28 d a y s Manggis a t 5 d a y s Purwokerto Libra a t 1 7 days

Lactic

Acetic

Ethanol

Glycerol

1.98 0.04 7.66 4.88

0.35 0.04 0.86 1.22

0.95 0.37 0.001 0.03

1.4 0.64 0.84 0.84

“Adapted from Table 1 of Roling et al. (1994a)

TABLE XI SUGARS IN THE FIVETYPESOF JAPANESE SHOYU(mg

Shop

Koikuchi Usukuchi Tamari Shiro Saishikomi

Mannose

Fructose

Arabinose

0.23 0.08 1.02 0.13 0.65

m 16.62 trace trace trace

0.83 1.72 0.39 1.30 0.25

rn1-’la

Galactose

Xylose

Glucose

2.26 2.66 3.80 trace 2.61

0.51 1.46 0.23 11.45 0.34

9.42 19.97 12.55 105.58 13.19

“Adapted from Table 5 of Sasaki and Nunomura (1993). ND = None detected. Sucrose at 1.88 mg . ml-’ was measured in shiro shoyu, and ribose was negligible in all shoyus.

mororni stage; the second is from the direct addition of sucrose, as occurs in Indonesian kecap manis. As seen in Table I, reducing sugars in soy sauces from around the world vary from about 1% to as much as 8.5%. Sasaki and Nunomura (1993) reported the levels of eight sugars in shoyu: namely, the pentoses ribose, arabinose, and xylose; the hexoses mannose, fructose, galactose, and glucose; and the disaccharide sucrose. They found that sugar levels in shoyu varied with the type of shoyu and that the levels of some sugars were relatively significant. Glucose varied from 9.42 mg . ml-1 for koikuchi shoyu to 105.6 mg . ml-1 for shim shoyu. Galactose was also present in significant quantities in most shoyu at a level of about 3 mg . ml-I (Table XI). In one baceman sample, about 4.4 mg . ml-l of glucose, fructose, and galactose combined was measured, but generally very little of any sugars is found in kecap asin (Roling et a]., 1994a).

100

DESMOND K. O’TOOLE

Ill. Aroma and Flavor of Shoyu

The aroma and flavor of shoyu are important aspects. Much of the Japanese work has been directed to improving and maintaining good flavor and aroma on a consistent basis while improving efficiency and yield. About 300 compounds have been identified as contributing to the final flavor and aroma of shoyu (Yokotsuka, 1985). Yokotsuka (1985) identifies some of them as follows: 1. methionon (3-methylthio-1-propanol) 2. 3.

4. 5.

6.

7.

4-ethyl guaiacol and 4-ethyl phenol phenol esters between phenolic compounds, such as $-ethyl guaiacol, vanillic acid, and vanillin, and organic acids such as benzoic acid and acetic acid maltol (3-hydroxy-2-methyl-4-pyrone; Zapsalis and Beck, 19861, and 5-hydroxy-malt01 hydroxy-furanones: 4-hydraxy-2(or5)-ethyl-5-(or 2)-methyl-3(2H)-furanone(HEMF) 4-hydroxy-5 -methyl4 (2H)-furanone (HMMF) 4-hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) cyclothen: 2-hydroxy-3-methyl-2-cyclopentene-l-one lactones: 4-butanolide 4-pentanolide 2-methyl-4-butanolide 4-hexanolide

This list, however, omits ethanol and glutamic acid, which are also considered significant for the organoleptic qualities of shoyu (Fukushima, 1989). Typical levels of significant compounds that contribute to flavor and aroma that are found in Japanese s h o p are shown in Table XII. The compound considered the character impact substance contributing to the flavor and aroma of shoyu is HEMF because it has an intense sweet aroma similar to shoyu, it occurs in high concentrations in shoyu, and it has a threshold detection limit in water of <4 x mg . liter1 (Sasaki and Nunomura, 1993). However, it does not occur in tamori shoyu, which is made with very little wheat (Fukushima, 1989). The optimum level in shoyu of another volatile aromatic compound, 4-ethyl guaiacol, is 1-3 mg . liter1 (Hamada et al., 1990), but Ishihara et al.

MICROORGANISMS IN SOY SAUCE PRODUCTION

101

TABLE XI1

THEMAJORFLAVOR COMPONENTS AND THEIR CONCENTRATION (mg . kg-’) FOLJNDIN S H O Y ~ P Concentration

Flavor component Ethanol Lactic acid Glycerol Acetic acid 4-Hydroxy-5-methyl-3( 2H)furanone (HMMF) 2,3-Butanediol Isovaleraldehyde 4-Hydroxy-2(or 5)-ethyl-5(or 2)methyl-3(2H)-furanone (HEMF) Methanol Acetolb Ethyl lactate 2,6-Dimethoxyphenol Ethyl acetate Isobutyraldehyde Methyl acetate Isobutyl alcohol

31,501 14,347 10,209 2,108 256.4 239.6 233.1 232.0 62.4 24.6 24.3 16.21 15.13 14.64 13.84 11.95

Flavor component Furfuryl alcohol Isoamyl alcohol Acetoin n-Butyl alcohol 4-Hydroxy-2 ,s-dimethyl3(2H)-furanone (HDMF) Acetaldehyde 2-Phenylethanol n-Propanol Acetone Methionol 2-Acetylpyrrole 4-Ethylguaiacol Ethyl formate 4-Butanolide Methional 4-Ethylphenol Dimethyl sulfide

Concentration 11.93 10.01 9.78 8.69 4.83 4.63 4.28 3.96 3.88 3.65 2.86 2.77 2.63 2.02 1.42 0.34 0.04

OSasaki and Nunomura (1993j.Reprinted with permission from Academic Press. bThisis a registered trade name, the substance may be 1-hydroxy-2-propanone(Budavari,1989).

(1996) report the preferred concentration of 4-ethyl guaiacol to be “0.8 ppm” and of 3-methylthio-1-propanol “3.9 ppm.”

IV. Soy Sauce Production

The traditional production methods for soy sauce rely heavily upon natural microflora that respond to the environmental changes imposed on the raw materials. Soy beans are boiled, mixed with cracked wheat or flour, left covered for a few days while natural mycoflora grow, and then the molded beans are steeped in brine for many days or months (Wang and Fang, 1986). This process ultimately produces soy sauce. Scientific studies have explained the major components in the process, so that the process is better understood and can be controlled to give a more consistent product.

102

DESMOND K. O'TOOLE TABLE XI11 CCIMPOSPI'ION or SOYBEANS,SOME SOY BEANPRODUCTS, WHEATGRAIN, WHEAT FLOUR, ANU WHEAT BIUN (g . kg-l) Soy beans

Component

Whole bean"

Defatted flourh

Flourb

Whole grain

52

115-140

-

37-177

153 19 18.5 717

140-180 9-24

119-22 9

-

30-68 38-96 619

703

650-740

141

21-26

12-21 3

352

24

431

86

72

343

4 70

Carbohydrate

187 51 316

62 384

Starch-amylose

4

Free sugar Cellulose Hemicellulose

=50 4.3

Water Protein Oil

Ash

Wheatr

12

345 206 45 352

Flour

4-14

10.8

Bran

76

OAugustin and Klein (19891. 'Haytowitz and Matthews (1989). CDecker and Hanners (1991). %onstituents of soy bmri carbohydrate from Snyder and Kwon (1987).

A. COMPOSITION OF RAWMATERIALS

The raw materials used are soy beans, wheat, salt, and water. Soy beans are the seeds of the plant Glycine max (L.) Merr., wheat is the common name of the seeds of Triticum aestivum L., T compacturn Host, and ?: durum Dest. (Mattern, 1991), and the salt is usually sea salt (Wood, 1982). The water must be of good quality, free of nitrates, which can lead to the production of N-nitrosamines (Sung et al., 1991), and low in Fe++and Mn++ (Noda, 1980). The proximate analysis of raw materials used in modern soy sauce production, namely, soy beans, defatted flour, and full-fat flour, and of wheat grains, flour, and bran, is shown in Table XIII. The moisture content of whole beans is between 10 and 13% depending on the length of time they have been stored (Weingartner, 1987), as is the water content of wheat. The proximate compositions of soy beans and wheat (Table XIII) are for indication only, so as to give some idea of the relative abundance of the different components, because these values vary depending on plant variety, analytical methods, and the growing conditions of the crop. Table XI11 shows that there is about 34% protein and 20% oil in the beans and, of

MICROORGANISMS IN SOY SAUCE PRODUCTION

103

course, that the protein level is higher after defatting. The oil component is not thought to contribute to the final composition or flavor of soy sauce, but, according to Yokotsuka, sauce made from whole beans is “more stable” and more resistant to “yeast invasion” (Yong and Wood, 1974). The carbohydrate content of soy bean is about 30% and comprises a soluble fraction and an insoluble fraction. The soluble fraction, about 10% of the bean, is a mixture of sucrose (5%),raffinose (l%),and stachyose (4%), and the insoluble fraction consists of structural carbohydrates such as pectin (30% of insoluble carbohydrate), hemicellulose (50%),and cellulose (20%), and the starch level is less than 1% (Snyder and Kwon, 1987). Soy hemicellulose consists mainly of arabinogalactan (Hisamatsu ef a]., 1995). The easily soluble fraction is a very small part of the whole bean and could easily be lost during rehydration of the beans before heating, and would almost certainly be lost during boiling and decantation of the water in the traditional process, so the reported levels in raw beans here may not be the same as in the cooked beans when inoculated to make koji. The insoluble carbohydrate is complex and is more resistant to degradation than starch, and requires a more complex enzyme mixture to degrade these structures to mono- and disaccharides (Coughlin and Ljungdahl, 1988; Singh and Hayashi, 1995). On the other hand, wheat contains at least twice the carbohydrate level of soy beans, but most of it is starch-amylose, an easily degradable carbohydrate. The protein content of wheat, at 153 g . k g l , is much less than the 343 g . kg-1 in soy beans. Of the amino acids in whole soy beans, wheat, and bran, about 19.2, 33, and 23%, respectively, consist of glutamic acid, but in absolute terms soy beans supply more glutamic acid than wheat (Table IV), which is not quite consistent with Yokotsuka’s reported claim that wheat is added “to serve as a rich source of glutamic acid” (Yong and Wood, 1974). B. THEMANUFACTURE OF SOY SAUCE

The soy sauce-making process can be stated simply as follows. Soy beans or defatted soy flakes are soaked and cooked, and wheat is either roasted and crushed or used as raw flour. The cooked beans and added wheat component are mixed in proportions that depend on the type of soy sauce being produced. Fungi are grown on the mixture of soy beans and crushed and roasted wheat for several days to produce what the Japanese call koji (Fukushima, 1989). The molded soy beans are then mixed with an 18-20% NaCl solution, producing a mixture called

104

DESMOND K. O’TOOLE

moromi in Japanese, and steeped for 6 to 9 months. Bacteria and yeasts grow in the moromi to develop the s h o p flavor, and then the raw shoyu (kiage in Japan) is separated from the residue, pasteurized, and bottled. In older systems of production the soy beans are boiled in water, but in modern systems the beans are cooked at a high temperature under steam pressure. A variation is the species of mold used to produce the koji, Aspergillus oryzae versus A. sojae. In other parts of Asia, different names are used and variations in the soy sauce-making process occur. In Indonesia the koji, called bungkil, is sun dried before steeping (to produce the baceman (moromi)stage) for 1 day or for as long as 5 months (Roling et al., 1994a). In Korea the equivalent of kojj is maeju, a cake of molded soy beans (Lee and Cho, 1971). The treatment of the molded beans in Korea is virtually the same as that published in China during the Yuan dynasty, 1271-1368 A.D. (Fukushima, 1989). The Indonesian soy is spiced and boiled before bottling, and Korean soy sauce is also boiled before bottling (Lee and Cho, 1971). In Hong Kong and China, before pasteurization and bottling, some manufacturers place the raw soy sauce in the sun for 1to 2 months to concentrate the sauce (Chiao, 1986). Each component and process contributes in various ways to the final product and its flavor. The severity and degree of heating of the soy beans affect the yield of nitrogenous compounds in the final soy sauce. The mold provides the enzymes that during the moromi stage break down the proteins and the wheat starch. In addition, the mold secretes such macerating enzymes as pectinase, cellulase, and hemicellulase that improve access of other enzymes to the protein and carbohydrate in the soy bean and wheat by breaking down cell walls (Sugiyama, 1984). The wheat supplies protein that can be degraded and carbohydrates that enhance the growth of yeasts in moromi (Roling et al., 1 9 9 4 ~ ) and that are converted into organic acids, such as lactic acid, and ethanol that are produced during fermentation. The soy beans supply the base protein that is hydrolyzed to peptides and amino acids, which give the nutritive value of the product. The bacteria produce lactic acid and lower the pH, which allows the growth of yeasts that produce the essential flavor constituents. The final pasteurization step stimulates the chemical development of essential flavoring agents. C. KO~I

The Japanese name koji is derived from the Chinese character for molded grain. In Chinese it is called shui, and in Korean it is ku (Lotong, 1985). Koji production in Japan is important for the production of soy

MICROORGANISMS IN SOY SAUCE PRODUCTION

105

sauce, rniso, and a range of alcoholic beverages. In soy sauce manufacture the grain is soy bean, and it usually includes wheat grain or flour, except for China, where wheat bran is now included (Fukushima, 1989). However, substitution of wheat bran for wheat flour results in an unsatisfactory soy sauce (Yong and Wood, 1974). Traditionally the koji has been produced by allowing the natural environmental inoculation of the prepared soy bean-wheat mixture, or prepared soy bean, and incubating aerobically in shallow trays (but with forced airflow in modern systems) for 2-3 days. Known strains of A . oryzae and A . sojae are now used, and Sugiyama (1984) stipulated that strains used for koji production must not produce mycotoxins, and should possess high levels of proteolytic, amylolytic, and macerating enzymes. 1. Seed Koji The use of pure fungal strains for koji production requires the production of seed koji (tune koji) to act as a starter culture for the process. Seed koji is a mass of spores, not mycelia, of the koji mold grown on a suitable carbohydrate base, usually rice or wheat bran. The carbohydrate base is sterilized, inoculated, and incubated and the mold grown until it has sporulated. The spore mass and carbohydrate base are inoculated at about 0.1 to 0.2% of the mass of raw material prepared for soy koji (Lotong, 1985). In traditional production, naturally high levels of fungal spores are relied on for the inoculation of the prepared materials for koji. 2. Materials and Conditions for Enzyme Production

In Japan, full-fat beans have been replaced with defatted beans because the nitrogen yield in the final soy sauce is greater and the soy oil harvested from the beans can be used for other purposes. Another significant effect on nitrogen yields in the sauce is the degree of heating used to cook the soy beans. The higher the temperature and the shorter the time of heating, the greater the yield of nitrogen from the original soy beans (Fukushima, 1989; Yokotsuka, 1985). Thus, the yield of nitrogen in the soy sauce obtained in the early part of the twentieth century by boiling the beans was about 60%, but today, when temperatures of 1 7 O O C for 15 sec are used to cook the beans (Fukushima, 1989), yields of 88% are obtained. When defatted soy flakes are used, there can be variation in the size of the flakes in batches produced by different techniques ( h i s h i , 1970).When batches contained 84% of the soy flake in the size range of >8 mesh (>2.5 mm) and 10% in the 8-20 mesh (2.5-0.9 mm) range, the moromi fermentation proceeded more rapidly than in a batch where 35% of the soy flakes were in the >8 mesh size

106

DESMOND K. O'TOOLE

First stirring Max. 38OC

40oC

30OC

$

Y

a L

pf. 20c

+-,

B

%"c:

Room humidity: saturated

b

lOOC

4-~

-Temp. of koji mixture --_-_ Temp. of koji room

0°C

0

9

54 Time course of incuhation (Hours) 18

27

36

45

63

72

FIG.1. Temperature changes in koji produced by a traditional small tray method. Adapted from Fukushinia (1989).

and 55% in the 8-20 mesh range (onishi, 1970), suggesting that the larger size promoted moromi fermentation. Yu et al. (1972) in Korea studied the substitution of corn gluten for the defatted soy bean and found that substitution of up to 15% of the total bean and wheat content with corn gluten produced good-quality soy sauce, as measured by the amylase and proteolytic activities in the koji. When more than 15% corn gluten was used, a soy sauce of poor taste and low nitrogen content resulted that was attributed to the low enzyme activity in high-corn-gluten koji. However, in a similar study, when corn substituted up to 60% of the wheat, or barley substituted u p to 70% of the wheat, good-quality soy sauce was produced from the koji (Lee et al., 1972). Substitution of the carbohydrate portion has a lesser effect than substitution of the soy protein. In the traditional small-tray method of making koji (Fukushima, 1989), the incubation temperature of the koji at the beginning is about 35OC. It quickly drops to about 25"C, after which the temperature rises, due to fungal growth, to about 38"C, at which point the mixture is stirred and the temperature drops to about 33OC (Fig. 1). During the first 24 hours, the humidity is controlled to about 1009'0, but during the rest of the time room humidity is lowered and the temperature drops below 29°C and is at 25°C after 72 h have elapsed. Solid matter is lost during the koji stage, as Takeuchi ef ~ l(1968), . in a study of koji production for

107

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE XIV THE PROTEIN DEGRADING ENZYMES IN KoJr AND THEIR ACTIVITY Enzyme Proteinases Alkaline Semialkaline Neutral I Neutral I1 Acid I Acid I1 Acid 111 Acid carboxypeptidases I I1 I11 IV

Optimum pH

Activity”

10.5 8.3 7

929 55

6

3

9 44 10 5

3 4 3-4 3 3 4

0.25 0.18 0.01-0.05 0.02-0.11

8.5

0.1 0.25 0.15 0.15 0.11 0.01 0.03

80

3.2 3

Leucine aminopeptidases

I I1 111

IV V v1 VII Ar ylaminadase

5-a 8

7 -

8.5

0

~

‘Unit against casein per g of koji. Adapted from Tables 1 0 , 1 1 , and 12 of Fukushima (1989)

tamari manufacture, reported a 9.6-21.4% loss of solid matter during 4 days of koji manufacture, a loss that increased with increased moisture content in the mixed koji raw materials. Yamamoto et al. [1972)reported that, when koji prepared for “white shoyu” was incubated at 27-29OC for up to 70 h, phosphatase activity reached a high point after about 40 h, after which there was very little increase, while acidic and alkaline proteases, and a- and P-amylases, reached maximum activities after 48 h, and neutral protease activity peaked after 60 h. They concluded that 48 h gave the most desirable result, because amylase activity was maximal at this point. Yong and Wood [1974) reported a similar pattern of enzyme production. The enzymes produced by fungi include pectinase, cellulase, and hemicellulase, and the starch-degrading enzyme amylase, as well as the protein-degrading enzymes such as proteinases and peptidases [Fukushima, 1989). The range of particular protein-degrading enzymes includes those shown in Table XIV.

108

DESMOND K. O'TOOLE

The proteinases, with peak activity in alkaline and also in neutral and acid conditions, are of the endo type, that is, they do not possess any amino- or carboxy-peptidase action and so hydrolyze protein to peptides only, not amino acids (Fukushima, 1989). The peptidases are of the exo type that liberate amino acids from the carboxy- or amino-terminals of the peptides and show maximum activity in slightly alkaline or acid conditions (Fukushima, 1989). The proteinases break down the protein, leading to an increase in total soluble nitrogen but with no concomitant increase in formol nitrogen, which is a measure of amino acid nitrogen. It is only with the inclusion of peptidases that formol nitrogen increases along with an increase in glutamic acid content. Proteinase production by A. oryzae NISL 1913 in chemostat cultures using a medium containing 2.6% soy flour as a nitrogen source was accelerated when soy bean oil was used as the carbon source. The specific protease production rate on a 1.8% oil mixture was 2.85 protease units . (mg dry weight of mycelium)-' . h-l, which was threefold greater than that with starch as the carbon source, but a-amylase production on the oil was only about a third of that when the organism was grown with starch as the carbon source. Soy oil could be replaced by C14 and Cl8 fatty acids (Fukushima et aJ., 1991). The starch from wheat is also important for the growth of the koji fungi. Roling et aJ. (1994~)found that the growth of fungi during the bungkiJ stage, which usually includes soy beans only, was faster and enhanced by the addition of 20% rice flour. The amount of fungal mass produced was not measured, but protease was increased about 30%, the leucine amino peptidase concentration doubled, and glucoamylase increased 136%. 3. Studies of Koji Fungi Studies of A. oryzae and A. sojae strains used for shoyu production have focused on comparing these two fungal species and improving their enzyme-producing abilities. A study of the enzymatic differences between 11 strains of A . oryzae and 20 strains of A . sojae showed that the activities of neutral, acid, and alkaline proteases, xylanase, pectin lyase, phosphatase, and aminopeptidase were not significantly different. However, acid carboxypeptidase activity and a-amylase activity were higher from A. oryzae when compared with A. sojae strains, while endopolygalacturonidase activity was much higher from A. sojae than from A. oryzae (Terada et QJ., 1980). The ratios between a-amylase activity and endopolygalacturonidase activity of 0.5-2 for A. sojae and 20-2000 for A. oryzae were suggested as a differentiation criterion for

MICROORGANISMS IN SOY SAUCE PRODUCTION

109

the species (Terada et al., 1980). Hayashi et a]. (1981) compared the performance of these two fungi in s h o p production. They found that the activities of protease, acetic carboxypeptidase, and a-amylase were lower and those of endopolyglucuronidase and glutaminase were higher in koji made with A. sojae. In the moromi stage, the proportions of NH3 nitrogen (N), glutamic acid N, and total N were higher, and viscosity and heat residue were lower with A. sojae. The resulting concentrations of citric and succinic acids in the shoyu were significantly higher ( p < 0.001) with A. sojae than with A. oryzae. Ishihara et al. (1996) compared the volatile components in commercial koikuchi s h o p s from different factories using either A. oryzae or A. sojae and found that the concentrations of 1- and 2-propanol, furfuryl and benzyl alcohols, ethyl-benzoate, and lactate, acetate, pyrazines, carbonyl compounds such as ethanal, maltol, and phenyl acetaldehyde, phenol, and others, were higher in the shoyu from factories using the latter fungus, but concentrations of &methyl- and 3-methyl-1-butanol, 2-phenyl ethanol, 2-methyl- and 3-methyl-butanoic acid, 3-methylthio-1-propanol, HEMF, 4-ethyl guaiacol, 4-ethyl phenol, and others were greater in shoyu from factories using the former fungus. These results have prompted factory managements to use A. sojae for koji production. Using an unusual system, Yasui et al. (1982) tested a range of koji fungal strains for glutaminase production and found that, when a strain showing 16% higher glutaminase activity than its parent strain was compared with its parent in the production of shoyu, the final glutamic acid concentration was 10% higher. In the early 1950s, A. sojae KS was irradiated with X-rays by Iguchi to produce strain X-816 of A. sojae (Sekine et al., 1970). Sekine et al. (1970) obtained seven strains with superior alkaline phosphatase activity (130-190%) and highly active protease, peptidase, cellulase, and amylase activities that were better at decomposing soybean protein. Yokoyama and Kadowaki (1983) UV-irradiated A. sojae strain H and obtained mutant strains with total protease activities 2.5 times that in wheat bran and soy sauce kojis. The mutant strains were diploidized and combined with natural mutants from M strains, and strains TH and D-15 were produced that possessed higher total protease activities than the M strains, and grew well. However, W irradiation may stimulate the production of toxic elements in otherwise safe fungi. Kalayanamitr et al. (1987)W-irradiated A. flavus var. colurnnaris Raper and Fennel (ATCC 44310) to obtain mutant strains with high protease and amylase activities, and light-colored conidia. Some selected mutant strains were found to be acutely toxic to weanling rats, even though they were negative for aflatoxin production. The investi-

110

DESMOND K. O’TOOLE

gators suggested that the toxic compound could be one of four substances: maltorhyzine, aspergillic acid, kojic acid, or cycoopiazonic acid. protoplasts Furuya et al. (19831 fused, with an efficiency of lo%, derived from two strains of A. oryzae, one with a high growth rate and the other producing high levels of protease. Ttyo strains derived from successful fusions showed high stability, fast growth, and abundant sporulation and produced 2. 3 times more protease than the parent fast-growth strain. The growth and development of microorganisms on defatted soybean and ground wheat koji prepared with A. sojae were studied by electron microscopy by Kitahara et al. (1980). Growth of the mold on the surface of the soybean was rapid up to 24 h, at which point formation of sporing bodies began, and spores were released within 40 h. However, very little fungal growth was seen on the wheat surface, but yeasts were seen growing on the wheat. Growth of Micrococcus species became noticeable after 16 h, as did multiplication of lactobacilli. These observations on the growth of the koji mold are at odds with the observation that 10-%0”/0of the dry matter in koji is lost in the koji stage (Takeuchi et al., 1968) and the observations below on the significant consumption of carbohydrate during the koji stage. I suggest that significant penetration of the wheat endosperm should have been seen. During koji production, carbohydrate is consumed by the fungus, thus leaving less carbohydrate available to provide flavor compounds for the final shoyu produced (Furuya et al., 1985). This carbohydrate consumption is positively correlated with a-amylase activity in koji culture. To overcome the depletion of carbohydrate before the moromi stage, Furuya et al. (1985) derived mutants that utilized 10-50% less carbohydrate during preparation of koji than the parent strain, with about 1/3, 1/20, and 1/150 of the a-amylase activity of the parent strain of A . oryzae. Significantly increased amounts of carbohydrate-derived compounds were found in the resulting shoyu made with these mutants. Enhanced glutaminase activity in koji is desirable to increase glutamic acid production in soy sauce, and reduced conidial production in the koji reduces contamination of the air with floating conidia (Ueki et al., 1994a). A mixed t a m koji of two koji fungi, A. oryzae strains K2 and HG, increased glutaminase activity of the mixture to 11.3 units g-l dry koji, which was higher than the 4.7 or 4.4 units. g-l dry koji produced by the K2 strain or HG strain, respectively, and conidia production was reduced tenfold (Ueki et a]., 1994a). The mixed tane koji was used in the manufacture of soy sauce, and the resulting mixed koji made with 3.6 tons of defatted soybean and of wheat grain showed 1

MICROORGANISMS IN SOY SAUCE PRODUCTION

111

high glutaminase activity (5.5 units . g1dry weight koji) when compared to strain K2 alone (1.8 units . g-l dry weight koji). In addition, the number of conidia in the mixed culture was 2.5 x lo7 g-l dry koji, which was lower than 1.3 x lo8 g-l dry koji produced by strain K2 alone. The glutamic acid content of the raw soy sauce was 1.25 times higher than the glutamic acid level found in normal soy sauce (Ueki et al., 1994b). Kim and Cho (1975) investigated soy sauce production in Korea using a soy-wheat koji prepared with A . sojae, using natto, a soy bean product prepared with Bacillus natto, and using a mixture of the two in varying proportions. The natto-brine mixture had protease activity twice as high as the koji alone, and this was reflected in the protease activities found in mixtures of the natto and koji. On comparing the organoleptic qualities of soy sauces fermented for 3 months, the kojinatto at a ratio of 6:4 had the best flavor, followed by koji alone.

D. MOROMI The name moromi is applied to the mixture obtained when koji is immersed in a liquid (Koseki et al., 1996), which could be water or a brine solution as used for shoyu production. For shoyu the koji is mixed with a brine solution to give a final salt concentration of about 3 MNaC1, which stops the fungal growth but does not stop the activity of the fungal enzymes that can continue the degradation processes. The degradation process for proteins is outlined in Fig. 2. Macerating enzymes, such as cellulase, hemicellulase, and pectinase, break down the cellwall components to make the proteins more susceptible to attack by the protein-degrading enzymes (Fukushima, 1989). These enzymes are essential to allow satisfactory filtration of the raw shoyu. At the same time, amylase and other starch-degrading enzymes attack the starch to break it down to glucose. Another important enzyme is glutaminase, which converts glutamine to glutamic acid. However, this enzyme is sensitive to an acidic pH and to salt, so that lower temperatures (15-20OC) are recommended for shoyu production during the first month (Fukushima, 1989) to slow acid production by the lactic acid bacteria to prevent loss of glutaminase. While the mold enzymes are doing their work, other microorganisms begin to grow. Traditionally, when added to the brine, the koji already contains a range of bacteria and yeasts, many of which are also killed by the high salt concentration, but in the traditional process in a wellestablished factory a wild microflora exists that adheres to the walls of tanks, often made of concrete in traditional plants. The walls of soy

112

DESMOND K. O’TOOLE Soy protein

Wheat protein

i I

Macerating enzymes: cellolase, hemicellulase, pectinase

Released protein Alkaline proteinases Neutral proteinases

Polypeptides

I

Acid carbox ypeptidases Leucine arninopeptidases

Amino acids

Glutamine

I

Glutaminase

Glutamic acid

FIG.2. The breakdown process of soy and wheat proteins in moromi by koji enzymes. Redrawn from Fukushima (1989).

sauce concrete brewing tanks in Japan had populations of 1.0 to 4 . 5 x lo5 m-2 Zygosacchuromyces species and 7.0 x lo4 to 5.6 x lo5 m-2 Cundida species adhering to them (Noda et al., 1 9 8 2 ~ )They . become incorporated in the brine mixture and begin to grow, and a complex fermentation takes place. This natural inoculum then follows growth cycles dictated by environmental factors, the presence of particular ingredients, and the byproducts of the growth of other microorganisms. In the first stage, a lactic fermentation takes place due to the growth of halotolerant lactic acid bacteria. These organisms produce lactic acid from the glucose released from starch and lower the pH of the mix from about 6.6-7.0 to <5.0. When the low pH has been reached, the natural yeasts can then begin to grow (see, e.g., Yong and Wood, 1976), and these organisms are responsible for the most important flavor components. The second yeast stage has two parts. In the first part, yeasts grow and produce alcohol; in the second part, other yeast species sometimes grow and produce essential flavor components. The time required for the moromi stage is about 6 to 9 months. A most significant advance in soy sauce manufacturing to control the flavor of the final product has been introduction of greater control of the moromi process. Parameters controlled include temperature, aeration, and inoculation of starter microorganisms dominating in the rnororni.

MICROORGANISMS IN SOY SAUCE PRODUCTION

113

The above applies to Japanese s h o p , but it does not apply completely to Indonesian kecap (Roling et al., 1996). Satisfactory kecap is produced after a short fermentation time of 1month, and yeast fermentation is not important for flavor development. Roling et al. (1996) concluded that Japanese process technology and techniques were not suitable for kecap production.

Temperature of the Brine Stage Temperature control in the early stage of s h o p production has involved two divergent approaches. The first approach is to raise the temperature at the beginning of the brine stage to about 50-55"C, so that degradation of the soy bean and wheat starch is accelerated, making them available for microbial growth more quickly. This is usually done in a stirred tank with a salt content of 10-12% (Osaki et al., 1985; Hamada et al., 1989). Pure microbial cultures must then be added for fermentation to proceed. The second approach is based on the old observation that moromi batches commenced in late winter, when temperatures in the moromi were low, produced the best-flavored shoyu (Sasaki and Nunomura, 1993). Consequently, in this approach the brine at 0°C is mixed with the koji to keep the temperature in the early stage of the moromi below 15°C for several days (Sasaki and Nunomura, 1993). Otsuki et al. (1981a) investigated the effect of temperature in the moromi stage. They set up seven treatments and used the temperature regime as shown in Table XV over a period of 5 months. Their results showed that, the total amount of soluble N released after 1 month was equal to 60% of the final total in treatment (i)but 92% of the final total for treatment (vii), that is, the higher temperature favored enzyme activity, which solubilized N. Ethanol was only produced at 30°C in any of the treatments, and the concentration was highest in treatments (i), (ii), and (v),Citrate levels were negligible in treatments (i). (ii), and (iv), and low in treatment [iii), but noticeable in treatments (v), (vi), and (vii); therefore, the prolonged and early high temperature of 30°C promoted the retention of citrate in the moromi, but the lower temperatures, especially in the early period, promoted the disappearance of citrate, presumably through its metabolism by the microorganisms. In a further report of the experiment, Otsuki et al. (1981b) showed that amylase activity decreased rapidly at 30°C, protease activity was stable at 20°C but decreased at 3OoC, and glutaminase activity increased at 20°C but decreased rapidly at 30°C but, irrespective of treatment temperature, glutamic acid concentration increased in months 1to 3 and then leveled off. Otsuki et al. (1982) showed later that glutaminase activity was lost 1.

114

DESMOND K. O'TOOLE TABLE XV

EXPERIMENTAL PLAN OF OTStJKI ETAL. (1981) FOR THE STUDY OF EFFECT OF TEMPERATURE ON MOROMI FERMENTATION Temperature, "C, in moromi at various times Treatment

Beginning

lil

5

1 month 10

Beginning

1 month

2 months

5 months

5 5 10 10

20 30 20 30 30 30

30 30 30 30 30 30

30 30 30 30 30 30

(ii) (iii) (iv) (v) (vi) (vii)

20

30

1.5 months 20

2.5 months 30

5 months 30

rapidly when the temperature was 30°C and that in the moromi glutamine conversion to glutamic acid ceased after about 1.5 months and excess glutamine was converted to pyroglutamic acid. Leucine-amino peptidase and acid carboxypeptidase activities increased at temperatures above 20aC but showed a steady decline at 30°C. From the same laboratory in further experiments (Hirakuri et al., 1982), conducted using a slightly different temperature regime (Table XVI), it was shown over a 6-month period that a lower temperature in the early part of the moromi stage favored the production of glutamic acid and reduced the production of pyroglutamic acid. Conversely, the higher temperature in the early part of the moromi stage favored the production of pyroglutamic acid over the production of glutamic acid. The temperature regime did not affect the concentration of acetic acid (130-150 mg . TN-') (TN = total nitrogen), whereas, contrarily, at the lower early temperature lactic acid production was favored. In treatment (i) the lactic acid content was 800 mg . TN-l after 5 months, but after the same time in treatment (iv) the content was 400 mg . TN-l. This shows that the lower temperature favored the growth of the added lactic acid culture (Hirakuri et al., 1982). The concentrations of 8-phenyl ethanol and +ethyl guaiacol were both higher at 20°C. On the other hand, temperature is not so important for kecap production. Roling et al. (1994b) investigated the effect of constant temperatures of 24, 30, and 37°C for 4 2 days on the composition of kecap and found little significant difference between the three temperatures. How-

115

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE XVI

EXPERIMENTAL PLAN FOR THE STUDY OF TEMPERATURE EFFECTS ON kfOROA41 FERMENTATION AND RESULTSFROM HlRAKURI ETAL. (1982)

Concentration at 5 months, mg . TN-'

Time, months 1

2

Treatment

(i) (ii] (iii] (ivl

3

4

5

6

Glutamate

Temperature, "C

Pyroglutamate Lactate

5

10

10

20

20 30

30 20

20 20

20 20

820 808

166 193

20 30

30 30

20 20

20 20

20 20

20 20

721 704

250 309

800

400

ever, the temperature did affect the rate of growth of bacteria. Halotolerant lactic acid bacteria grew much more quickly at 37OC, and numbers peaked within a week at about 3 x TO7 CFU . ml-1 and then declined within 35 days to lo3 CFU. m F , but at 24 and 3OoC the numbers reached about 5 x lo8 CFU . ml-1 within 2 weeks and declined very little over the rest of the 42 days. It seems that the effect of temperature is complicated by interactions with the carbohydrate added to the soy beans. 2. Aeration of the Brine Stage

Traditionally, moromi has been given brief bouts of occasional aeration to mix the contents and to promote microbial growth (Fukushima, 1989), but no element of time is mentioned in more recent reports (Sasaki and Nunomura, 1993). However, in Thailand, where Taiwanbased technology is being used, the moromi is aerated for several hours three times a week for the first month. Aeration is required by the yeasts, which are strict aerobes and need oxygen for growth. In addition, aeration in the early stage retards the growth of salt-tolerant lactobacilli and wild yeasts (Sasaki and Nunomura, 1993), and the stirring causes interaction between enzymes and substrates. A high NaCl concentration in water reduces the solubility of O2in the water and consequently also in the moromi. As the amount of NaCl in a water solution increased from 10 to 18%, the amount of dissolved O2 in the solution fell from 60 to 42% of the concentration in pure water, and the absorption rate coefficient of O2 also decreased with increasing NaCl concentration (Miyauchi et al., 1981). Similarly, Matsumoto et al. (1982) found the concentration of dissolved O2 in water was three times

116

DESMOND K. O’TOOLE

that in an 18% salt solution. In unaerated moromi, the amount of dissolved O2 was zero, but in aerated moromi the level of dissolved O2 after 1 month was 60% of that in 02-saturated pure water: the level dropped to zero 15 min after stopping the flow of O2 (Miyauchi et al., 1981), showing that the O z was being consumed either by microorganisms or in chemical reactions in the moromi. The latter possibility cannot be excluded from consideration as a contributor to flavor, good or bad, in the final product. Moromi contains a significant quantity of carbohydrate, but baceman contains no added carbohydrate apart from that present in the soy beans. During baceman production it is stirred daily for 3 weeks and thereafter about three times a week (Roling et a]., 1994a). Roling et al. (1994b) compared aeration or lack of aeration on baceman fermentation. They sparged air (30 liter . h-l) through the baceman or directed Nzover the baceman (anaerobic conditions) during fermentation. They found that under aerobic conditions staphylococcal and coryneform bacteria were the dominant bacteria up to 2 1 days. Yeasts reached only about lo3 ml-l, but under anaerobic conditions the staphylococcal and coryneform bacteria were present at the beginning, and by day 7 halotolerant lactic acid bacteria were the dominant and only bacteria present. In summary, control of temperature and aeration affects the manufacture of good-quality shoyu and influences the final content of some important components. 3.

Microorganisms in the Brine Stage

Apart from temperature, the presence or absence of 02,and the presence or absence of carbohydrate, a dominant factor influencing the growth of yeasts and bacteria in moromi, is the high level of NaCl that lowers the water activity of the mixture. The water activity is about 0.88 in a 3 M solution of NaCl (Yokotsuka, 1985), which restricts growth to those organisms able to grow at such a low water activity and prevents growth of many potential spoilage organisms. The growth of yeasts in the high concentration of NaCl is eventually reduced by byproducts of yeast growth such as guaiacol, vanillin, and ethanol (Yokotsuka, 1985). Another important factor is the presence of utilizable carbohydrate. The soy protein seems only important for provision of amino acids and peptides. In the natural situation, where a natural inoculum from the walls of the vessel starts the fermentation, the microbial growth pattern is as shown in Fig. 3. The organisms present come from the environment of the factory at which the soy sauce is produced. No doubt, unless the vessels are sterilized, the microorganisms adhere to the walls of the

MICROORGANISMS IN SOY SAUCE PRODUCTION

:b

.... i

i

n

1

2

3

4

:.

i

m

5

6

117

n

7

Months FIG.3. The changes in microflora in moromi during 7-month incubation: 1, wild yeasts; 2, Micrococcus species; 3 , Bacillus species; 4, Tetragenococcus species; 5, Z. rouxii; 6, Candida species. Adapted from Tamagawa et al., 1975 (Yokotsuka, 1985).

moromi vessel and are present when the freshly prepared koji and brine are added. However, bacteria and yeasts may also grow in the koji stage, and bacteria have been known to reach levels of 108to lo9 per gram. In modern Japanese koji production systems, the contaminating bacteria are mostly species of Micrococcus (Yokotsuka, 1985). These organisms are then present in the morami at the beginning hut generally do not grow. The microorganisms that grow in the moromi and produce the desirable flavors are lactic acid bacteria and yeasts. The predominant desirable lactic acid bacterium is the halotolerant Tetrugenococcus halophilus, formerly known as Pediococcus halophilus (Anon., 1994; Collins et al., 1990). The general properties of this organism that make it suitable for growth in the high salt concentration in moromi are shown in Table XVII. In the next stage, Zygosaccharomyces rouxii grows and produces alcohol followed by Candida versatilis and C. etchellsii, which grow and produce essential flavor components. The characteristics of these organisms that make them suitable for growth in moromi are shown in Table XVII. Realization of their importance has led to the use of cultures of these organisms as starter cultures in the production of shoyu (Noda

118

DESMOND K. O'TOOLE TABLE XVII

THECHARACTERISTICS OF YEASTSAND BACTERIA WHICHAREIJsm As SI'AKI'EKS IN hfOf1OMI FERMENTATION [SASAKI AND NUNOMURA, 1993) Growth in medium with

Z . rouxii Limits

C. versatilis

C. etchellsii

Tet. halophilus

Opt.

Limits

25 25

-

-

4 0

2042

25-30

4-5.5 4-5

3-7 3-7

5 4-5

-

-

5.5-9.0

7.0

5-24

5-10

Opt.

Limits

Opt.

Limits

25-30 25

<35 <35

25-30 25

4 0

3-7 3-7

Opt.

'18111 perat itre fur

growth. "C NaCi 0%

NaCl 18% PH

NdCl 0% NaCl 18%

<35 <42

3-7 4.5-6.0 3.5-5.5 4-5

NaC1, YO wlv

<24-26

-

<26

-

<26

-

Water activity

0.787-0.81

-

<0.787

-

0.787

-

0.9'+0.808 0 . 9 S 0 . 9 4

et al., 1982b). In Japan, when the moromi reaches pH 5.2-5.3, a culture of Z. rouxii is added to give a concentration of 5 x lo5 to loGcells per g of moromi (Ishihara et al., 1996). A different situation occurs during the production of baceman. The lack of excess carbohydrate does not stop bacterial growth but retards yeast growth, which eventually reaches levels comparable with those levels found in carbohydrate-augmented baceman (Roling et a]., 1994a,c). T halophilus also occurs in baceman, but equal numbers of staphylococcal and coryneform bacteria also grow in it (Roling et a]., 1994a). Yeasts in baceman form films on the top of some batches that can cause off-flavors in the kecap.

E. USE OF CONTINUOUS FERMENTATION PROCESSES TO PRODUCE SOYSAUCE Production of soy sauce on a large scale ties up large fermentation vessels for a long time. To speed up manufacturing, continuous processes and bioreactors have been studied. The first continuous method involved an enzymic process to hydrolyze the raw materials followed by lactic fermentation (Anon., 1982).The next step was the introduction

MICROORGANISMS IN SOY SAUCE PRODUCTION

119

of whole cells of T halophilus, Z. rouxii, and C. versatilis immobilized in calcium alginate gel to ferment the moromi (Osaki et al., 1985). Following enzymic hydrolysis and passage through a column bioreactor containing immobilized I: halophilus cells for lactic fermentation, the production stream was split in two and put through either a Z. rouxii or a C. versatilis reactor. Each stream was then heated, filtered, and combined. The production time was equivalent to 2 weeks. Sauce was produced continuously for 80 days through a 280-liter reactor, but it had an aroma pattern different from traditional sauce. However, the gel was unstable to heat and not resistant to a high-salt solution, so Horitsu et al. (1990) introduced a ceramic bead carrier for the whole cells and could produce satisfactory soy sauce within 8 days. Hamada et al. (1991) used a system based on “Chitopearl” to immobilize glutaminase and on alginate gel to immobilize the microorganisms. The koji mixture was hydrolyzed in 8.5% NaCl at 45OC for 3 days, filtered, and then put through the glutaminase reactor followed by the microbial reactors. Although production took about 2 weeks and the system ran continuously for 100 days, the product still did not completely match the traditional product (Hamada et al., 1991). Koseko et al. (1993), using a Microbe Immobilized Ceramic reactor, successfully fermented nonsterilized moromi mash with the microorganisms named above and were able to produce sauce with good taste and flavor, With a residence time of 30 h for Z. rouxii, 2% ethanol and 38 mg . liter-’ 2-phenyl ethanol was produced, and with a residence time of 48 h for C. versatilis the same level of ethanol and 3.7 mg . liter-l 4-ethyl guaiacol were produced. This is not an exhaustive look at the work done in this area. Other relevant work includes that of Iwasaki and Ueno (1990) and Iwasaki et al. (1991a,b, 1992). So far this apparently attractive approach to the problem of reducing the production time and thereby increasing productivity has not yet become a commercial reality. V. Effect of Water Activity on Microorganisms

Water activity (a,) is a physical chemistry term that describes the relationship between the vapor pressure of a solution over the vapor pressure of pure water. The aw of pure water is 1;on addition of NaC1, the water’s a, becomes a fraction of 1.Using the formula (Yoshii, 1979)

a,

=

-0.0093 (%NaCl) + 1.0214,

we find that a 10% NaCl solution has an a , of 0.92 and an 18% NaCl solution has an a , of 0.85 (Yoshii,1979). However, Tokuoka and Ishitani

DESMOND K. O’TOOLE

120

1.2

-

n 0

1.00

0.96

0.92 0.88 AW

0.84

0.80

FIG.4. (a) Effcct of water activity (aw)on the growth of bacteria found in rnororni as measured by optical density (OD) after a period of incubation. Names of bacteria and yeasts on the original figure have been changed to reflect current nomenclature. Adapted from Fig. 11 of Yoshii (1979). (b) Response of yeasts in moromi. Adapted from Fig. 1 2 of Yoshii (1979).

(1991) reported a graph showing that the a, against NaCl concentration plot was not linear, but that as concentration increased, a, decreased proportionately more. Tokuoka and Ishitani (1991) showed that the minimum water activity for growth of 2. rouxii varied according to the strain, and according to whether glucose, fructose, sucrose, or sodium chloride was used to lower the water activity; for example, in an NaCl medium the minimum a, for growth was 0.92, but in a fructose medium it was 0.67. When some ingredients in the NaCl medium, such as raw soy sauce or miso and koji extracts, were added or excluded, the minimum Q, for growth differed for Z. rouxii. In addition, the medium in which the organism was grown before the minimum a, test was done affected the minimum value obtained; for example, when Z . rouxii was pregrown on YM medium (g . kg-*:yeast extract 3, malt extract 3, peptone 5, glucose 10, agar 15 (Azuma, personal communication, 1996)), its minimum a, in a glucose-controlled a, medium was 0.85; however, when pregrown on a

MICROORGANISMS IN SOY SAUCE PRODUCTION

OS2

e I

121

0 C.etcheUsii I

I

I

I

l

I

I

I

'

I

1.00 0.96 0.92 0.88 0.84 0.80

Aw FIG4b.

50% (w/w) glucose medium, the minimum a , in a glucose-controlled a , medium was 0.80. When the a, of commercial shoyu is measured, the activity is about 0.80, which shows that a, is reduced by about 0.05 units during breakdown of the soy beans and wheat to smaller molecules. Nevertheless, Yoshii's (1979) figures indicate that the amount of growth of T. halophilus was consistent down to an a, of 0.90 (Fig. 4a), after which there was a rapid drop in total growth and then a decline to a minimum a, of about 0.81. For the yeasts tested by Yoshii (1979), C. versatilis and C. etchellsii were halophilic and good growth occurred down to an a, of about 0.85 (Fig. 4b), after which the growth dropped off to a minimum at an a, of <0.80 as the a, dropped. In effect, Yoshii's results seem to show that little growth of the organisms occurs at low a,, but they grow quite well at an a, of about 0.97-0.92. In his tables, he showed a minimum a, of 0.787 for Z. rouxii and C. etchellsii at 24-26% NaCl (w/v), of 0.81 for C. versatilis at 26% NaCl (w/v), and of 0.808 for T. halophilus at 24% (w/v) NaCl.

122

DESMOND K. O’TOOLE

VI. Yeasts

The names of yeasts important in shoyu production have been somewhat confused, and the nomenclature of yeasts in general is a significant problem (Barnett et al., 1990). The species of yeasts now considered the main source of the flavor components in shoyu are Z. rouxii and salttolerant Candida species such as C. versatilis and C. etchellsii (Hamada et a]., 1990). The early literature refers to Saccharomyces rouxii and Torulopsis species such as ?: famata (Debaryomyces hansenii). Where the original paper referred to Saccharomyces rouxii, I am using the more recent name of Z. rouxii. I am also using the Candida species names, but in other cases I am using the original names. Onishi (1960) found that in moromi containing 18% NaCl two yeast species dominated and that growth of the yeast was dependent on the pH of the moromi. The two main yeasts were I: famata and Z. rouxii, but neither grew until the pH of the moromi had fallen to pH 5.0. In the early stage of fermentation, the Z. rouxii dominated when the yeast cell count was about lo7 mlP, but after 1 year, when the count had fallen to lo4 ml-l, Torulopsis species dominated. onishi (1960) tested four strains, I:famata, C. polymorpha (anamorph of Pichia triangularis), and two strains of 2. rouxii, for growth in 18% NaCl over the pH range 3.0-7.0 and found that after 14 days T famata grew well but slowly and C. polymorpha grew not so well and slowly at pH 5-6, whereas Z. rouxii grew well and quickly in the pH range 3.8-5.0. The yeasts produce two important flavor compounds: 4-ethyl guaiacol and $-ethyl phenol. Asao et al. (1969) compared the flavor-producing abilities of Z. rouxii and Torulopsis species in moromi mash and found that 1 2 strains of Torulopsis species (C. halophilus, C. nodaensis, C. versatilis, C. etchellsii, and C. sake) could produce 4-ethyl guaiacol but that three strains of Z. ruuxii and I: fumuta could not. Sugiyama (1984) suggested that, when selecting strains of yeasts for shoyu production, those Z. rouxii strains that produce alcohol and excellent flavoring substances,and those C. versatilis and C. etchellsii strains that produce excellent flavoring substances in a high-salt-concentration brine should be used. In moromi made from koji containing Candida yeasts at a 4-ethyl guaiacol and 4-ethyl phenol at concentration of lo2 to lo3 gl, 5.2 and 0.9 mg . liter1, respectively, was found, but moromi made without Candida yeasts contained 2.1 and 0.1 mg . liter-l, respectively, of these substances (Noda et al., 1 9 8 2 ~ ) . In view of the importance of yeasts in shoyu production, it is remarkable that yeasts play no significant role in the production of typical p et a]., 1994a).In one batch of baceman (Roling Indonesian k e c ~ (Roling

MICROORGANISMS IN SOY SAUCE PRODUCTION

123

et al., 1994a),the dominant yeast was Debaromyces hansenii var. fabryi (D. hansenii is the sexual stage of C. famata (Barnett et a]., 1990)),which was replaced later in the fermentation by C. guilliermondii var. membranaefaciens (anamorph of Pichia ohmeri), a film-forming yeast. In a second batch, C. parapsilosis was the dominant yeast, but species of Debaromyces and Sterigmatomyces were also present. The addition of rice flour to the bungkil promoted yeast growth in baceman, so that levels of yeasts peaked 10 to 15 days sooner than in baceman made with soy bean only (Roling et al., 1994~). The importance of yeasts in the production of soy sauce depends on the type of sauce being produced. It is clear that yeasts are important for Japanese shoyu, but it seems they are not important for kecap production, and maybe that of other soy sauces as well. A. EFFECTOF OXYGEN ON YEASTS Nearly all yeasts are obligate aerobes and require O2 for growth. To test yeasts for their ability to grow in the presence or absence of 02, stringent conditions are needed (Visser et al., 1990). Visser et al. (1990) found that, although a fermentor was flushed with N2 and the tubes sealed, the dissolved O2 tension increased within 24 h, showing that, despite careful exclusion of 02,diffusion through the plastic tubes was occurring. Consequently, to maintain anaerobic conditions in the fermentor, continuous flushing with N2 was required to exclude O2 (Visser et al., 1990).In view of this, it is extremely unlikely that the experiments done in open tubes and paraffin oil topped tubes by Noda (1978a) to study the effect of O2 on the growth of yeasts in s h o p were a true comparison between oxygenated and anaerobic conditions. It is more likely, despite a contrary opinion that liquid paraffin completely eliminated O2 absorption (Matsumoto et al., 1982), that the paraffin-oiltopped tubes were just much less oxygenated than the open tubes, thus allowing some growth even though the level of O2 could not be measured. Matsumoto et al. (1982) found that some growth of Z. rouxii occurred in paraffin-topped polyethylene tubes immersed in moromi or water that was also paraffin topped, but not in tubes immersed in an Na2S0, solution, but Na2S03is an O2 absorbent. Under stringent conditions (Visser et al., 1990), 75 species of yeasts were examined. Of those, it was shown that C. tropicalis (found in miso) could grow anaerobically and produce ethanol under the conditions but that neither Z. rouxii nor Debaryomyces hansenii, also found in s h o p , could grow anaerobically (Visser et al., 1990). This indicates that O2 is essential for

124

DESMOND K. O'TOOLE TABLE XVIII

SOME EFFECTS OVER TIME OF ZYGOSACCHAROMYCES ROUxIr ADDITION ON pH, REDUCING SUGAR AND ETHANOL CONTENT OF MOROMI"

PH

Reducing sugar, g 100 ml-'

After 2 weeks

5.42

9.99

0.04

After 2 months, no yeast addition

5.01

7.03

0.67

After 2.5 months, with yeast cells added at 2 weeks Yeast cells not grown on o-vanillin Yeast cells grown on o-vanillin

4.86 4.90

2.06 3.18

2.56 3.28

Ethanol, g . inn mi-'

"Adapted from Noda (1978b)

the growth of some of the yeasts in moromi, so that aeration of the moromi is essential for successful growth.

B. ALCOHOL PRODUCTION BY ZYGOSACCHAROMYCES ROUXII Noda's (1978b) work demonstrates the effect on ethanol production in moromi of addition of Z. rouxii. In addition, he examined the effect of growing the organisms on an o-vanillin medium (10 ppm). To illustrate, some of his results are presented in Table XVIII. These results show that the addition of yeast boosted the metabolism of reducing sugars and production of ethanol, and when cells were grown on o-vanillin their ethanol production was boosted. Noda (1978a) also examined the effect of various chemical and physical factors on the growth of yeasts. He tested the effect of NaCl (15 and 19"/0),ethanol (1and 3%), an ether extract from soy sauce (1and 1.8%), and total nitrogen (1 and 1.8%). He found that Z. rouxii was almost equally inhibited by all factors except for total nitrogen, which had no effect, The physical factors included pH (5.5 and 6.5), temperature (20 and 3OoC),aeration (static or shaken flask), and NaCl (15 and 19%). For 2. rouxii and C. versatilis, aeration, or lack of it, and pH were the most important factors contributing to inhibition of growth. Koseko et al. (1993), using immobilized cells of C. versatilis and Z. rouxii in an airlift bioreactor, showed that, as the concentration of ethanol increased from about 1.7 to 2.2%, the productivity of ethanol in moromi decreased from about 0.8 to about 0.4 g . liter1 . h-l, and as residence time for C.

MICROORGANISMS IN SOY SAUCE PRODUCTION

125

versatilis in the bioreactor dropped from 48 to 40 h, and for Z. rouxii from 30 to 20 h, ethanol production by both organisms dropped from about 2% to about 1%,even though cell numbers of Z. rouxii in the effluent returned to their original level before the drop in residence time. For ethanol production to occur, a carbohydrate source must be added to the soy beans. Roling et al. (1994~) found that ethanol production by yeasts in baceman depended on the addition of rice flour to soy beans at the bungkil stage. VII. Bacteria

Yong and Wood (1974) give a brief history of bacteriological studies of soy sauce manufacture. They reported that Saito isolated a tetrad forming homofermentative lactic acid bacterium in 1907, and in 1930 Ishimaru isolated l? acidilactici var. soyae from shoyu and studied its effect on soy sauce brewing. Lockwood in 1947 isolated Lactobacillus delbrueckii from shoyu and in a series of experiments showed that good soy sauce could be produced using the organism. However, by about 1958 (Yokotsuka, 1985) the species l? soyae, which was subsequently reclassified as l? halophilus and has now been reclassified again as 7: halophilus, was recognized as the dominant organism in shoyu. However, the role in moromi of the tetrad-forming coccus continued to be studied until at least 1970 (Sugimori and Jose, 1970; Watanabe et al., 1970). Sugiyama (1984) said that T halophilus must produce lactic acid and other useful organic acids in high-concentration salt brine and must not possess tyrosine decarboxylase and histidine decarboxylase. If a dark-colored soy sauce is required, then the strain must not be capable of decolorizing the sauce as some strains are capable of decolorizing moromi (Kanbe and Uchida, 1984) and miso (Osawa and Yasuhira, 1988). EFFECTOF OXYGEN ON BACTERIA

Lactic acid bacteria are generally considered to be facultative anaerobes that do not consume oxygen: however, oxygen consumption by ?: halophilus growing in 10% NaCl media has been reported (Kanbe and Uchida, 1985). They found that resting cells of 7: halophilus could dissimilate glucose and fructose aerobically and anaerobically. Under anaerobic conditions, about 1.75 mol of lactate was produced per mol of hexose, while under aerobic conditions lactate and acetate were produced at a molar ratio of 1:1. However, the amount of O2 consumed

126

DESMOND K. O’TOOLE

by resting cells in a glucose medium depended on the medium in which the cells had been grown. Using cells grown in YPG medium (g . kg-*: yeast extract 3,polypeptone 10, glucose 10, K2HP0410, sodium glycolate l , NaCl 100, pH 7.2), the O2 consumption rate was much lower than in cells grown in MJ medium (filtered soy sauce mash containing 10% NaC1, pH 7.0). When resting cells were incubated with a range of sugars and acids, 0, consumption only occurred in the presence of glucose, L-lactate, and pyruvate, despite the ability of the cells to ferment other sugars that were tested. Even in the presence of NADH and NADPH, O2 was not consumed. VIII. Production and Metabolism of Amino and Organic Acids in Moromi

The soy bean and wheat proteins are broken down to amino acids by the koji enzymes, and the starch is converted to glucose, which is ultimately metabolized by yeasts and bacteria to organic acids, amongst other compounds. Organic acids are also present in the raw materials. The amino acids can be further metabolized, and the growth conditions can influence the organic acids. The sources of the acids vary, some arising from microbial activity (e.g., lactic and acetic acids), but others (e.g., citric acid) most likely come from the soy bean and wheat components, as defatted soybean 1985). Pyroglutamic meal contains about 1.4% citric acid (Osaki et d., acid is formed at low pH by conversion of glutamine to pyroglutamic acid instead of glutamic acid. A possible pathway for the breakdown is shown in Fig. 5. A. ROLEOF TETRAGENOCOCCUS HALOPHILUS

The fate and source of organic acids and amino acids in the final shoyu is somewhat complicated. The amino acid composition of the final product is almost the same as the original mixture of soy beans and wheat (Fukushima, 1989), with some exceptions, which are due primarily to the activity of some lactic acid bacteria found in moromi, as well as chemical and physical factors. If the lactic acid bacteria possess specific decarboxylases (Sugiyama, 1984), they can convert arginine to ornithine and tyrosine to tyramine, and they can degrade aspartic acid, histidine, phenylalanine, and threonine. Tryptophan and cystine are unstable in moromi, but some tyrosine can be precipitated because of its low solubility (Fukushima, 1989).

MICROORGANISMS IN SOY SAUCE PRODUCTION

-

Glutamic acid

Glutamine

-H,O

127

H /N\ O=f

-C-C H2

/cH-cooH H2

Pyroglutamic acid FIG.5. A possible pathway for the production of pyroglutamic acid from glutamine.

Citric acid is the main organic acid in the early moromi stage (Kanbe and Uchida, 1987b) along with malic acid, but they decrease substantially as lactic acid production increases (Kanbe and Uchida, 1982). A high positive correlation was found between lactate and acetate concentrations, but a negative correlation between lactate concentration and the combined concentrations of citrate and malate suggested a link between the two (Kanbe and Uchida, 1982). Most freshly isolated strains of T. halophilus could metabolize citrate and malate to lactic acid but required the presence of other organic energy sources such as glucose because the strains could not use citrate or malate as the sole carbon source (Kanbe and Uchida, 1982). Citrate metabolism, as shown for T halophilus strain 7117, arises from induction of citrate lyase, but, unlike the dairy streptococci that convert citrate to diacetyl or acetoin, these organisms convert citrate to pyruvate, with the release of COz, and subsequently to lactic acid, or to acetate, formate, and COz, depending on the absence or presence of glucose, respectively. In moromi the usual situation is for an excess of glucose, so that pyruvate is preferentially converted to acetic acid, a desirable product (Kanbe and Uchida, 1987b).The proposed pathway for the breakdown of citrate is shown in Fig. 6. However, acetic acid could arise from glucose fermented by Z . rouxii, as has been shown with strain NRRLY-1096, which also fixed COz to form formic acid at neutral pH (Yong et al., 1980).

128

DESMOND K. O'TOOLE COOH

I

ctha?ss

CH2 HO-C

I I

-

COOH

h1-

Acetic acid

1

NAD+

NADH

2xCoAsh

COOH

I

CH3

-

COSCoA

COOH Pyruvic acid

FIG.6. Pathway proposed for citrate metabolism by shoyu tetragenococci. Redrawn and rearranged from Kanhe and Uchida [1987b).

The data from 1982 and 1993 on citrate levels in the final commercial products as shown in Tables VII and VIII suggest that control of microbial activity can eliminate citric acid as a component of the organic acids in shoyu. In the moromi, L-malate can be converted to L-lactate with the release of carbon dioxide (Kanbe and Uchida, 1983). This has been demonstrated with ?: halophilus strain T187 (but not strain 34), isolated from moromi, from which Kanbe and Uchida (1983) isolated and partially purified the malolactic enzyme, which reduced L-malate but not pyruvate (in the presence of NADH2) to lactate, Their studies of the partially purified enzyme showed a requirement for NAD' M) and a metal

MICROORGANISMS IN SOY SAUCE PRODUCTION

129

ion. Amongst those tested, Co++and Mn++showed the greatest influence on activity. The partially purified enzyme showed maximum activity at pH 5.8, but activity was minimal at pH 7.0 and 4.0. However, when whole cells were grown at various levels of pH, the L-malate decreased quickest at pH 5.0-5.5, and at pH 6.0 the decrease in the L-malate concentration was slower. When the activity of partially purified enzyme was tested at NaCl levels from 0 to 20%, the activity decreased to 40% of the activity in a salt-free mixture. Partially purified malolactic enzyme from II: acidilactici, strain IF0 3076, showed no activity in a 20% NaCl concentration. Earlier it had been shown (Jose and Sugimori, 1971) that the activity of fructose-l,6-diphosphate-aldolasein the strain Tetracoccus 100 growing in a high NaCl concentration was reduced, and that catalase activity of the organism was reduced at an NaCl concentration greater than l o % , but the activities were restored by the addition of about 10 mM L-proline. Thus, L-proline enhanced the oxygen tolerance of the organism in a high-salt environment. B. INTERACTION BETWEENORGANIC ACIDSAND SOYSAUCE ORGANISMS

As early as 1969, it had been noted that a strong lactic fermentation depressed alcoholic yeast fermentation in moromi (Noda et al., 1980). Subsequent studies showed that, in a spent medium in which II: halophilus had grown and that contained 18% NaCl and an initial glucose concentration of lo%, the growth of Z. rouxii and C. versatilis was reduced to half that in an unspent medium. The cause of the partial inhibition was acetic acid produced by 'T: halophilus. The inhibition by acetic acid of a range of yeasts found in moromi paralleled the progression of those yeast populations in a normal fermentation. This suggested that the production of acetic acid helped to control the dominant yeasts in the moromi (Noda et al., 1980). Z. rouxii and C. versatilis, the dominant organisms, are relatively more resistant to acetic acid than others. Acetic acid at low pH was more inhibitory than at high pH, and in an 18% NaCl solution the acetic acid was even more inhibitory. In a medium containing 0.25% acetic acid, a level similar to that found in shoyu, and 18% NaC1, Z. rouxii grew at pH 4.5 but not at pH 4.0, while C. versatilis grew at pH 4.0 under the same conditions (Noda et al., 1982a). Yamamoto et al. (1972) showed that, as acetic acid concentration (range 10-500 mg . ml-I) increased in shiro shoyu, the medium was increasingly lethal for Z. rouxii, and as pH decreased (range pH 5.2-4.6) at constant acetate concentration, the medium be-

130

IIESMOND K. O'TOOLE

came even more lethal for the yeast. Using the medium shiro shoyu at pH 6 containing 15% NaCl and 300 mg . 100 ml-I citric acid, they showed that acetic and lactic acids were produced from citrate, and that the production of acetic and lactic acids was uncoupled from the growth of viable cells. When Z. rouxii and T halophilus were inoculated together into the medium, the complete breakdown of the citric acid depended on the relative inoculum sizes of the organisms. In a medium inoculated with T halophilus at los ml-I and Z. rouxii at lo4 ml-*, T halophilus grew very little, although Z. ruuxii grew well and very little citric acid was metabolized within 14 days. But when Z. rouxii was inoculated at about lo2 ml-l, both 'I: halophilus and Z. rouxii grew well and all of the citric acid was metabolized within 14 days (Yamamoto et al., 1972). These results show that T halophilus removes citric acid from the moromi and suggest that an inhibitor of T halophilus is produced by Z. rouxii, possibly Hz02. Inamori et al. (1984) examined the effect of 02,initial pH, and inoculum size on the growth and fermentative activity of T halophilus and Z. rouxii in shoyu koji extract mash in static cultures under aerobic and anaerobic conditions and found that, at pH 6.0 in mixed culture, growth and lactic acid production of T halophilus was inhibited by Z. rouxii under aerobic conditions and growth of Z . rouxii and alcoholic fermentation was inhibited by i? halophilus under anaerobic conditions. At an initial pH of 5.6, growth of T halophilus declined, irrespective of aeration conditions. Similar results were obtained using a shoyu moromi medium instead of a koji extract medium. Yong et al. (1978) examined the effect of various factors on the growth of Z. rouxii and found that, in an 18% NaCl growth medium acidified to pH 4.5 with HC1 or lactic acid, growth was better in the lactate-acidified medium. The organism was also shown to utilize lactic and citric acids at pH 4.5 in 10% NaC1. These results with lactic acid have not been confirmed in further studies by others. IX. Production and Fate of Other Substances

In this section we will deal with the origins of other substances found in soy sauce. Fungal enzymes produce sugars and amino acids, which, in addition to being metabolized by bacteria and yeasts, react in various ways to produce flavoring compounds. The color of shoyu, apparently due to a range of substances (Noda, 1980), including melanoidins and amino-carbonyl reaction-type pigments (Terasawa et al., 1996), is influenced by the presence of trace

MICROORGANISMS IN SOY SAUCE PRODUCTION

131

metals (see below). However, xylose and arabinose, produced from the hemicellulose fraction by xylanase and arabinofuranosidase, contribute to the browning of shoyu. The activities of the enzymes are significantly inhibited by Mn++and EDTA but were not stimulated by metal ions [Kimura et al., 1995). Significant levels of free radicals in soy sauce seem to arise from the carbohydrate or cellulose component and not from any phenol component that may be present (Gonis et d.,1995). Glycerol occurs in shoyu at a concentration of about 1%, and Onishi (1960) showed that it is produced by Z. rouxii while growing in a low-water-activity environment. Recent work has shown that concentrations of up to 3% of glycerol can be produced by Z. rouxii growing in a high-glucose medium [Groleau et ~ l .1995) , and that, when placed in a high-NaC1 environment, 2. rouxii responds with the accumulation of internal glycerol (and Na+,K+, and C1-) to maintain internal osmotic pressure equal to the external osmotic pressure (Yagi and Nishi, 1993). When Roling e f al. ( 1 9 9 4 ~ added ) rice flour to soy beans in the preparation of bungkil, they found that glycerol production depended on rice flour being present. The source of the glycerol produced by Z. rouxii and other halotolerant yeasts is the glucose produced from starch. 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), 4hydroxy-5-methyl-3[2H)-furanone [HMMF), and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) are furan-based substances. Furan is a cyclic compound containing four carbon atoms and one oxygen atom, with two double bonds. Recent work suggests that the precursor for HEMF is ~-xylulose-5-phosphate,a pentose that is produced not from the carbohydrate portion of the raw materials but from the protein fraction in soy bean or wheat, from which it is released by the protease and cellulase produced by the koji mold (Sasaki, 1996a). The yeasts then convert the D-xylulose-5-phosphate through a biosynthetic transformation to HEMF. (Curiously, HEMF production was greatest in NaCl concentrations of 17-18% (Sasaki, 1996b).) On the contrary, the homologues of HEMF, namely, HMMF and HDMF, arise by chemical reaction during heating of the raw soy sauce (Sasaki and Nunomura, 1993), and they are also produced when meat is cooked (Zapsalis and Beck, 1986). HMMF, also known for its ability to break DNA, arises from the heatpromoted Maillard reaction between pentoses and amino acid , (Hiramoto et ~ l .1996). Two other important phenolic character impact substances known to arise from yeast growth are 4-ethyl guaiacol and 4-ethyl phenol (Sasaki and Nunomura, 1993). More 4-ethyl guaiacol and 4-ethyl phenol were produced in summer than in spring or winter, apparently due to the better growth of Candida yeasts under warm conditions, and aeration

132

DESMOND K. O’TOOLE

reduced the production of these substances (Noda et al., 1 9 8 2 ~ )These . phenolic compounds are derived from ferulic acid and p-hydroxy-cinnamic acid in koji through the action of species of Candida yeasts (Sasaki and Nunomura, 1993). The 4-ethyl guaiacol arises when the carboxylic acid group is removed from ferulic acid (4-hydroxy-3methoxycinnamic acid), which is a component of the cell wall in wheat bound to the arabinoxylan in the hemicellulose (Budavari, 1989; Smith and Hartley, 1983) and which differs from p-hydroxy-cinnamic acid in having the 3-methoxy group. Apparently, 4-ethyl phenol arises via the same mechanism. Suezawa (1995) found that soy yeasts either broke down ferulic acid and p-hydroxy-cinnamic acid or they did not and those that did produced either 4-vinyl guaiacol and 4-vinyl phenol, respectively (D. hansenii, Pichia subpelliculosa, I? anomala, and C. famata), or +ethyl guaiacol and 4-ethyl phenol, respectively (C. versatilis, C. halophila, and C. mannitofaciens). C. etchellsii, C. nodaensis, and Z. rouxii, amongst other species, did not produce these volatile flavors. Hamada et al. (1990), in a continuous-culture study, showed that about 80% of added ferulic acid was converted to 4-ethyl guaiacol by C. versatilis. Some of the aldehydes that arise in the final soy sauce are derived from Strecker degradation during the pasteurization of the raw soy sauce (Sasaki and Nunomura, 1993).The Strecker degradation reactions involve combination and rearrangement upon heating of a dicarbonyl compound and an amino acid that is the direct source of the corresponding aldehyde (Zapsalis and Beck, 1986). Thus, for example, isovaleraldehyde, acetaldehyde, and methional can arise on bread crust from leucine, alanine, and methionine, respectively. Lactones, referred to by Yokutsuka (1985) as part of the family of compounds imparting the flavor to soy sauce, are cyclic esters that arise through the formation of rings by hydroxy-carboxylic acids either spontaneously or at elevated temperatures (Zapsalis and Beck, 1986). Ethyl acetate can be synthesized by 2. rouxii NRRL Y-1096 when grown under aerobic conditions on a medium containing ethanol alone but also on media containing glucose and ethanol, which were the main substrates for ethyl acetate production (Yong et al., 1981). Glucose or acetate alone was not suitable for synthesis. Furfuryl alcohol was produced by 22 strains of yeasts, including Z. sojae and I! farinosa, and molds (A. oryzae) during growth in koji extract media containing 0.1% furfural, a furan ring compound with an attached aldehyde group, which is produced when pentoses in oat hulls, and presumably wheat, are heated (Loudon, 1988). The yield following yeast growth of furfuryl alcohol from furfural was 29.42-

MICROORGANISMS IN SOY SAUCE PRODUCTION

133

103.5% (v/v) (Morimoto and Matutani, 1969). Z. rouxii also produces higher alcohols such as isoamyl alcohol, iso- and n-butanol, methionol, 3-methylthio-1-propanol, and 2-phenyl ethanol from amino acids (Aoki and Uchida, 1990; Sasaki, 1996b). Alcohols such as 2-phenyl ethanol arise from phenylalanine, 2- and 3-methyl-1-butanol arise from isoleucine and leucine, as do 2- and 3-methylbutanoic acids, and methionol and methional arise from methionine through yeast activity (Ishihara et a]., 1996). The sources and agents by which some of the substances in soy sauce are produced are summarized in Table XIX. X. Possible Role of Metal Ions in Soy Sauce Production

Metal ions are essential for the growth of bacteria, for example, a lack of metal ions in a growth medium can prevent growth of lactic acid bacteria (O’Toole, 1995), and Mn++stimulates acid production in lactic acid bacteria (Raccach, 1985). Some organisms secrete metal-binding proteins to survive in a low-metal environment, and the antibacterial properties of lactotransferrin are thought to be due to the iron-binding properties of the substance. Further, the high-NaC1 environment can influence the needs of the organisms for metals. The purpose of this section is to explore the possibility that certain metal ions may be limiting in koji and moromi. A. METALIONSIN METABOLISM

Particular metal ions are of importance as ligands involved in the structural integrity and the activity of the enzymes required for metabolic processes. Antonov (1993) listed many proteolytic enzymes, including aminopeptidases, dipeptidases, carboxypeptidases, and metalloproteinases, from bacteria and molds that required either Zn++,Mn++,Ca++, Mg++,or Co++.Further, Mn++is found in enzymes involved in redox reactions that include manganese superoxide dismutase, which catalyzes dismutation of superoxide radicals, manganese ribonucleotide reductase, which is used in the production of amino acids and nucleotides, manganese thiosulfate oxidase, which is needed for the oxidation of thiosulfate to sulfate, manganese peroxidase, which is involved in the degradation of lignin, and manganese catalase, which is equivalent to the so-called “pseudocatalase” found in lactic acid bacteria in particular (Larson and Pecoraro, 1992). These metalloenzymes may be important in the two major stages of soy sauce production, the koji and the high-salt moromi stage. For example, Njoku (1989) found that, when compared with other metal ions, Co++ activated an aminopeptidase enzyme from A. oryzae the

134

DESMOND K. O'TOOLE TABLE XIX

FO~JNII IN SOY SAUCE AND THE SOURCES AND AGENTS BY WHICH THEY ARISE

SOME OF THE SUBSTANCES

Source

Agent

Glucose

Starch from wheat

Enzymes from A. oryzan or A. sojas

Galactose and pentoses

Non starch carbohydrate?

As above

Substance

Reference

Amino acids

Soy and wheat protein As above

%Hydroxy-Z(nr 5I-ethyl5(or 2)-methyl-3(2H)furanone (HEMF)

XyllllOSe-5-ptlosphdte, Muromi yeasts in protein fraction

Sasaki. 1993

Ethanol

Glucose

Z. rouxii

Noda, 1978b

Lactic acid

As above

Lactic acid bacteria

Sugiyama, 1984

Glycerol

As above

Ethyl acetate

Glucose and trthdn01

8r:id

z.rouxii z.rouxii

Onishi, 1960 Yong eta]., 1981

Glucose and citrate i n raw ninterinls

7: halupliilus

Kanbtt and Llchitla, 1982

4-Hydroxy-5-methyl3(PH)-furanonc (HMMF)

Pentoses and amino acids

Maillard reaction promoted by heating

Hiramoto st ol.,

Furfuryl alcohol

Furfural, from roasted wheat

Z. sujae, Pichiu

farinosa, A. oryzae

Morimoto and Matutani, 1969

4-Hydroxy-2,!i-diniethyl3(2H)-furanune(HDMF)

Pentoses and

Heating of soy sauce

Sasaki and Nunomura, 1993

2-Phenyl ethanol

p-hydroxy cinnamic acid; phenylalanine

Acetic

amino acids

z.rouxii, Candido spp.

1996

Horitsu e t a / . , 1990; Suezawa, 1995; Sasaki. 199Gb; Ishihara et al., 1996

+Ethyl guaiacol

Ferulic acid, in wheat cell wall

Cundida spp., C. versatilis

Hamada et al., 1990; Horitsu et a]., 1990

Isoamyl alcohol. isoand n-hiitannl, 3-inethylthio-1-propanol

Amino acids

2. couxii

Aoki arid Uchida, 1990;Sasaki, 1996h

Isovaloraldehyde, acctaldehyde and methional

Uicarbonyl compounds and amino acids

Streckcr degradation reactions promoted by heating

Zapsalis and Beck, 1986

Lactones

Hydroxy carboxylic acids

Spontaneous or by heating

Zapsalis and Beck, 1986

Shoyu yeasts

Ishihara at 01.. 1996

Shoyu yeasts

lshihara el al., 1996

2- and 3-Metliyl-1-butanol, Isoleucine and 2- and ~-1nethy1b11tanoic leucine ai:idh

Mcthionol and mcthional

Methionine

MICROORGANISMS IN SOY SAUCE PRODUCTION

135

most, and in a study (Scott et al., 1986a) of patulin production by Penicillium urticae it was found that, of eight metal ions tested, only Mn++was required for secondary metabolite production at concentrations of =1.5 to 20 pM (69 mg . kg-l to 1.1g . kg-l). Mn++has also been implicated in secondary metabolite activity in species of Aspergillus, Penicillium, and Bacillus (Scott et al., 1986b). Camarero et al. (1996) showed that, during solid-state fermentation of wheat straw by Pleurotus pulmonarius, the addition of Mn++to give a final concentration of 0.3 mM, equal to about 73 pg . g-l wheat straw, boosted mineralization of wheat straw by 125%, but with no increase in the amount of manganese peroxidase; the Mn++boosted the activity of the enzyme. Moreover, because of the extreme ionic environment for microorganisms in a solution containing 3 M NaCl, it can be postulated that the concentration in the microorganisms of some metabolically important ionic species may be altered. De MBdicis et al. (1986) examined the Mg++and Mn++concentrations in Vibrio costicola, a eubacterium, and found that, as the concentration of NaCl increased in the medium, the internal concentrations of Mn++and Mg++rose linearly. The bacteria accumulated about 0.4 mM Mn++per kg of cell water in an external solution containing 20 pM. kg-I water (1.1g . kg-l water), and when the external concentration had dropped to 1.25 pM. k g l water (=69 mg . kg-l water) the intracellular accumulation of Mn++dropped off to 1.7 mM . kg-l of cell water. This shows that an increasing NaCl concentration imposed a requirement on the organism for a greater amount of internal Mn++. A malolactic enzyme that requires metal ions for activity has been partially purified from a moromi strain of T halophilus (Kanbe and M showed Uchida, 1983). CO++and Mn++at a concentration of 5 x 100% and 97% activity, and Zn++at the same concentration showed 56% activity of that shown with CO++.Kumar and Raccach (1996) found that the wild population of lactic acid bacteria in a wheat flour fermentation (25 g in 50 ml water) showed no lag phase and at 30°C had a fermentation time (time for pH to drop to 5.0) of 1h, compared with 8.8 h, when Mn++was added at 3 pg . g-l (9 pg . g-I flour) to the brew. As the natural level of Mn++in wheat flour was likely to be much greater than the Mn++added (see Table XXII), Mn++availability may be the source of the stimulation by Mn++of lactic acid production.

B. INVESTIGATION OF METALIONSIN SOY SAUCE AND ITSMANUFACTURE Ren et al. (1993b) reported levels of major minerals in shoyu as well as in Chinese and Korean soy sauces. The levels of Na in Japanese shoyu

DESMOND K. O'TOOLE

136

TABLE XX

LEVELS OF MAJOR MINERALS FOUND IN JAPANESE Siiovri, AND CHINESE AND KOREANSOY SAUCES (mg 100g-')" ~~~

Soy sauce

Na+

~

Kf

Ca++

Mg++

c1-

PO;-

Koikuchi

7973-7724

401-1128

65-110

0-8

8302

llsukuchi

8793

283

47

45

9359

841

Tarnari Shim Saishikorni Chinese Korean

6997

1687

143

0

8440

1240 1263

824-1133

fi978

93

15

0

8586

10016

179

433

30

10165

758

5972-9473

157-2039

58440

0

7316-10915

263-1393

6018-9972

204412

48-131

0-31

6829-11721

533-1269

'Adapleil trriin TRhle 2 of Kcn et al. (1993b)

showed a similar range to that found in Chinese and Korean sauces, and the levels of K were higher in tamari and some Chinese sauces (Table XX). There was also great variation in the level of Ca in the sauces. There has been one study of the mineral content of the residue in rnororni from the production of koikuchi shoyu. Sonehara and Hayashi (1982) found that the dry matter in the residue formed in shoyu during dialysis contained protein (23.4%) and polysaccharides (73.2%) and significant amounts of Na+,Mg++,Ca++,and Fe++,as well as other metals. Fe++,Mn++,and Zn++were bound mainly to protein, and Mg++,Ca++, and phosphorus were bound mainly to polysaccharides. The metal content of sediment corresponded well with that of soy sauce. Noda (1980, 1981) examined the role of minerals in brewing water on soy sauce color and looked at the sources of minerals found in soy sauce. He concluded that the major source of the metals studied was the soy beans and wheat (Table XXI) but seems not to have considered the role of the salt. In addition, the Ca level reported is not consistent with the values for the other minerals, indicating that all of the Ca in the shoyu was not accounted for, possibly because it had precipitated as CaC03, or the reported values are wrong, or salt was a significant source of Ca and was not considered. Noda (1980) traced the fate of the added minerals in the colored material in soy sauce, which was separated into three fractions on the basis of molecular weight, and one was blackishred, another was brilliant red and contained the added minerals, and the last was yellowish red. Noda (1980) examined the metal content of brewing waters in Japan and found Fe++,Cu++,and Mn++at 10-700, 0-50, and 0-130 p g . kg-',

MICROORGANISMS IN SOY SAUCE PRODUCTION

137

TABLE XXI

THEPERCENTAGE OF MINERALS OBTAINED FROM THREE SOURCES OF MINERALS IN SOYSAUCE' Percentage of mineral in soy sauce from source Source of mineral

Fe

Water Soy bean Wheat

0.9

0.4

1.2

4.1

58.2 40.2

49.2 50.1

78.7 20.2

49.7 8.4

Mn

cu

Cab

"Compiled from Noda (1981). bThe Ca values do not add up to 100%.

and Cat+ at 5-150 mg . k g l . The use of either demineralized brewing water or water with addition of metal salts (FeCl,, FeC13, and MnC12 at 50 mg . kg-') in soy sauce production had no significant effect on the chemical composition or color of soy sauce made using the water, but the added minerals increased the oxidative color when the soy sauce was heated (Noda, 1981). Fe++and Mn++had previously been reported as darkening the color of unheated shoyu when added to shoyu from which these metals had been removed using Dowex-A-1 ion exchange resin (Hashiba et al., 1970). Hashiba et al. (1970) also found that Cut+, Zn++,Co++,and Ni++did not influence shoyu color.

c. ADEQUACY OF MINERALS IN RAW MATERIALS The question arises as to whether the level of minor minerals in the koji and the moromi are sufficient to meet the metabolic requirements of the fungi, bacteria, and yeast. When seed koji is produced using brown rice, the ash of twigs and leaves of certain plants is added at a rate of about 2% of the dry weight of the rice (Lotong, 1985). As the ash contains the mineral components in the plant, this is prima facie evidence that some metal elements are needed for adequate growth of the koji mold. The metal concentrations in soy bean, wheat, and sea salt are shown in Table XXII. Data on the metal concentrations in soy bean and sea salt were difficult to obtain. The soy bean data indicate that there are high levels of phosphorus in soy bean and wheat. Potassium levels are very high in soy, but wheat contains only about a third of the level found in soy bean. Cat+levels are low in wheat but high in soy beans, as are Mg++ levels. Levels of these minerals in wheat are variable depending on the

138

DESMOND K. O'TOOLE

TABLE XXII RANGE IN

MINERAL COMPOSITION FOUND IN SOY BEAN,WHEAT, AND IN SEASALT ON A DRYMATTLR BASIS Soy beans Wheat

~

DeWhiile fatted Wholc bean" flourb Flourb grain

Component Phosphorus, g

~

he'

Whole grain

Bran

Sea salt'

4.8

6.7

4.9

4.id

6.8"

1.5-5.4'

3.8-1.7~

0.00186

Pntnssium. g kg-'

IH.~

23.8

252

5.8

10.4

z.wi.2

3.5-4.11

11.98

11.65

Calrium, g kg-'

2.2

2.4

2.1

06

0.98

0.05-1.20.32-0.81

12.37

12.26

Magnesium. g kg-'

2.8

-

-

1.8

3.5

0.9-2.9

1.62.0

38.7

41 4

54.3

30

2.3

55

127

5-2tiu

50-70

0.006

0.06

Ciipper. rng kg-'

14.3

41

ZY

H

9.6

4-24

5.7-9.11

0.~075

Iron, mg . kg

am

92

ti4

fin

94

48-420

44-75

u.uti

0.31

69

200

130

46

9'

-

3.1-58

324,198

321,881

45

25

3.1)

44

68

19-100

36-55

0.15

0.31

Manganese. mg . kg-'

I

Sudiuin, mg kg

Zinc, mg kg?

'

0.0~21"

0.m

"Augustin and Klein (19R9). 'IIaytowitz and Mntthews (1989). "Calculated from published values for elements i n sea water. 'Bock (19911,in the dry matter. 'Mattern (1991),American wheat. Finnish wheat, in the dry mattcr. fBmk (1991), fiLide (1995). "Weast and Lide (1990). 'Poineranz (1987).

growing conditions or cultivar of wheat. Mn++and Zn++levels are about the same in soy bean and wheat. The levels of minerals in sea salt are calculated from published figures for the amount of minerals in sea water because published figures for sea salt were not found. When the two sources of information are compared, the figures show that concentrations are comparable for the major constituents, but there are significant variations in the levels of Mn++,CU++, Fe++,and Zn++reported for sea water. This suggests that the levels of these minor mineral constituents in sea salt could vary considerably from source to source, or it may indicate difficulties with analysis of sea water. Assuming that soy bean and wheat do not lose any minerals during the processing stage before the koji stage, assuming also a moisture content of 50% and 400 g of soy bean and 340 g of wheat i n the pre-koji mixture, and that all the minerals go into solution, then the calculated levels of the minerals in the water phase in koji at the beginning of the

139

MICROORGANISMS IN SOY SAUCE PRODUCTION TABLE XXIII

THECALCULATED LEVELSOF MINERALS IN KO]]MIXTURE BEFORE THE INCUBATION STAGE AND IN THE MOROMISTAGE’ Quantity i n koji i:omponents and mixture

Minerals, mg liter-’ H20, in: SOY

Wheat, mg

In wet koji Total, mix, (330 mg . liter-’ mlH,O) H,O

Soy bean

Sea salt

Total in moromi

Minera1

bean, mg

P K Ca

1,720

1,230

2,950

8,940

1,340

960

0.42

2,300

6,550

1,740

8,290

25,100

5,120

1,360

2,548

9,028

Wheat

800

180

980

2,970

630

140

2,656

3,426

Mg Mn

1,020

540

1,560

4,730

800

420

8,637

9,857

19.5

16.5

36

109

15

12.9

0.0072

28.2

CU

5.15

2.4

7.55

22.9

4

1.88

1.01

5.91

Fe Na Zn

31.2

18.0

49.2

149

24

14.1

0.04

38.5

24.8

13.8

38.6

117

19

10.8

69,656

69,686

16.2

13.2

29.4

89.1

13

10.3

0.05

23.1

“The calculations assume a mixture of 400 g soy beans and 340 g wheat containing 10% moisture, and 276 g sea salt and 1200 ml of deionized water. The mineral concentrations from Table XXII have been used, and the sea salt mineral concentration is the average of the tabulated figures.

incubation stage are as shown in Table XXIII. Calculated levels for minerals of possible metabolic importance for microorganisms in koji water and in moromi water for Mn++,Cu++,Fe++,and Zn++are 109 and 28.2, 22.9 and 5.91, 149 and 38.5, and 89.1 and 23.1 mg . ml-l, respectively. These calculations are only a guide and do not necessarily express the real concentrations of these minerals, as they could be bound to proteins from which they are not easily available for organisms, particularly as the minerals are more likely to be bound deep in pieces of solid wheat particles or soy bean. The calculated levels of Ca++and Mg++should be adequate, but, on the basis of reported levels in soy sauce (Table XX), not much goes into solution. It is probable that the COz formed by the bacteria and yeast combine with Ca++and Mg++to form carbonates that precipitate. Mg++ at 200 mg . liter-l was sufficient to grow halophiles and to maintain their viability (Gochnauer and Kushner, 1971), and this level is less than the levels found in soy sauces. Phosphorus and K+ are probably adequate. Kushner (1978) reported that K+ at 1000 mg . liter-’ was required by halobacteria to prevent K+ from limiting the growth of the organisms, and the levels calculated here are far in excess of that quantity, and the

140

DESMOND K. O’TOOLE

levels found in various soy sauces (Table XVII) reveal adequate levels are present in the brine stage. The adequacy of the levels of Mn++may be marginal based on two observations. First, based on the Mn++levels reported in halophilic organisms by de Medicis et al. (1986), the concentration of Mn++in the external saline solution when uptake levels were being affected by concentration of external Mn++was about 69 mg . liter-*. Second, the concentration of Mn++required for the activity of the malolactic enzyme in T halophilus was 275 mg . liter-l, although Zn++and Co++could substitute for Mn++.The calculated values to be found in moromi mash are much smaller than these figures, and in the case of the malolactic enzyme the calculated levels are only a tenth of that required for full enzyme activity. The evidence for a deficiency in trace elements in either the koji or the moromi stages is equivocal, and further work is needed to answer the question, especially given the traditional use of a mineral supplement in the form of ash to produce koji on rice. However, the inclusion of Mn++ (and Fe++)is not considered desirable because it causes a darkening in color of the final shoyu (Sasaki and Nunomura, 1993),but for tamari shoyu or Chinese style soy sauce a dark color is desirable and acceptable. So for soy sauce production in other countries the mineral requirements may differ from those for the production of Japanese shoyu. XI. Conclusion

The production of soy sauce depends on a combination of raw materials, physical factors, chemical environment, and microbial activity. The raw materials, soy bean and wheat, when reduced to their essential components for shoyu production, can be characterized as supplying soy protein and starch for the process. The processes that occur originated to break down these two fundamental constituents to extract the amino and organic acids, and other flavoring constituents found in the finished sauces, and to separate the extract from residual solid material for which the cellulases, hemicellulases, and pectinases are needed. If we place the Japanese work in a broader context, we can further reduce the raw materials down to soy protein only. Soy sauce produced in other countries is done with little or no carbohydrate source, although carbohydrate in the raw material promotes mold growth in the koji stage. The emphasis on Japanese shoyu production in the literature tends to overshadow soy sauce production as a whole, and, as Japanese s h o p is considered more desirable if ethanol is present, a characteristic that to

MICROORGANISMS IN SOY SAUCE PRODUCTION

141

other Asian groups appears not important, then the role of different constituents and microorganisms varies. Thus, soy bean and wheat are perceived as essential constituents for shoyu production but for production of other soy sauce wheat starch may not be important, at least not in large quantities. The production of soy sauce has five major steps. I. Selection of raw materials.

2. Cooking of raw materials. 3. Production of enzymes in the cooked material through the

growth of mold. 4. Steeping of the molded cooked material in a brine solution. 5. Treatment of the brine extract. These steps each influence the final product. Their effects can be summarized as follows. 1. Selection of Raw Materials

Whether or not soy flakes or whole beans are used is unimportant from the point of view of the quality of the final product. However, whole beans do result in a greater mass of waste product because the soy oil in the beans is not used and is ultimately discarded unless the spent beans are used in other products. Soy flakes do not contain oil. The use of wheat carbohydrate is variable, and soy sauce can be produced without carbohydrate being added to the mixture. So wheat is not essential to produce soy sauce. However, it can be argued that it is essential for the production of desirable flavors in soy sauce (Yong and Wood, 1974) because at least 4-ethyl guaiacol arises from a component in the cell walls of wheat. Further, the carbohydrate of wheat also promotes the growth of mold on the cooked raw materials, thus increasing enzyme production, which is a desirable outcome. The use of wheat bran instead of wheat grain or flour by Chinese manufacturers has the effect of reducing the amount of starch added but raises the amount of minerals, cellulose, and hemicellulose added (Table XIII).The mixing of wheat flour with cooked soy beans is also said by Yokotsuka to reduce the moisture content of the koji and so reduce the growth of undesirable bacteria (Yong and Wood, 1974), but the addition of dry wheat flour does not reduce the a , of the mixture, which remains high: for example, barley containing about 30% moisture has an a , of about 1 .O and in oily seeds the moisture level is even lower at the same a, (Flannigan, 1996).

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2. Cooking of R a w Materials

Cooking of the soy protein is essential, as raw soy protein is difficult to digest with enzymes. The discovery that a very high temperature for a very short heating time promoted a greater yield of soluble nitrogen in the final sauce boosted productivity and apparently had little effect on the organoleptic quality of the final product. The roasting of wheat cooks the starch, making it accessible to the amylase from the mold (Sasaki and Nunomura, 1993), but it also may have significant effects on the flavor of the final product, as heating of wheat promotes Strecker degradation reactions as well as the formation of furfural. 3. Production of Enzymes in the Cooked Material Through the

Growth of Mold

In traditional fermented soy sauce, mold growth is essential to provide the enzymes to break down the components in the raw materials. Simple growth of the molds is insufficient, as it is the enzymes produced and their activity that provide the means for breakdown. Factors influencing the production of enzymes include species and strain of mold, and the nutrient available for mold growth. Nutrients known to influence enzyme production include starch, oil, and fats. What has not been explored is the possibility that mineral elements may influence growth and enzyme production. 4.

Steeping of the Molded Cooked Material in a Brine Solution

The steeping stage is the final essential extraction stage. This stage can be simple or complex. In the simple process, steeping allows the mold enzymes to break down the protein and carbohydrate components and solubilize them. Then a microbial process may occur that will ultimately change the organoleptic qualities of the final sauce. The time of the simple process is usually short, as exemplified by Indonesian soy sauce. The complex process depends on the growth of bacteria and yeasts over a long period, and temperature and aeration of the brine stage should he controlled. The growth of lactic acid bacteria occurs irrespective of the presence of carbohydrate in the starting material, and these organisms lower the pH with the production of lactic and acetic acids. The pH limits their growth, but acid production can continue as acid production can be uncoupled from growth. The lactic acid bacteria reduce levels of citric acid, produce lactic and acetic acids from glucose and citric acid, and metabolize some of the amino acids.

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Whether or not yeasts grow is dependent on the presence of oxygen as well as the pH of the mixture. The high NaCl concentration depresses the pH range in which yeast growth occurs and shortens the pH range over which it occurs. During growth, yeasts produce ethanol, if the temperature reaches at least 30”C, and catalyze the production of flavor compounds from precursors. 5 . Treatment of the Brine Extract

Chinese soy sauce is usually exposed to the sun, supposedly to concentrate it (Chiao, 1986),but exposure to the sun subjects the extract to ultraviolet light, which will promote chemical reactions, and will allow the evaporation of volatile constituents that should alter the flavor of the sauce. The effect of this sunning step is unknown. All soy sauce is heated after extraction. This has the effect of improving the flavor of the final product and would destroy enzymes, thus stopping their activity, and kill bacteria and yeasts to stop their activity and prevent spoilage. The heating stage is essential to promote production of flavor compounds from precursor compounds in the final product by chemical reactions. ACKNOWLEDGEMENTS

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Sekine, H., Nasuno, S . , and Iguchi, N. (1970). Attempt to improve koji moulds for soy sauce manufacture by means of artificial mutation [in Japanese]. Seasoning Sci. [Chomi Kagaku] 17,41-46. Cited in Food Science and Technology Abstracts, 72-01"0065. Singh, A., and Hayashi, K. (1995). Microbial cellulases: Protein architecture, molecular properties, and biosynthesis. Adv. Appl. Microhiol. 40,1-44. Smith, M. M., and Hartley, R. D. (1983). Occurrence and nature of ferulic acid substitutions of cell wall polysaccharides in graminaceous plants. Carbohydrate Res. 118, 65-80. Snyder, H. E., and Kwon, T. W. (1987). Oriental soy food products. In "Soybean Utilization" (H. E. Snyder and T. W. Kwon, eds.), pp. 218-241. Van Nostrand Reinhold, New York. Sonehara, T., and Hayashi, K. (1982). Polysaccharides and metals as compounds of sediment produced from raw soy sauce during pasteurization [in Japanese]. J. Jpn. Soy Sauce Res. Inst. INihon Shoyu Kenkujo Zasshil8, 16-20. Cited in Food Science and Technology Abstracts, 85-02-TO072. Suezawa, Y. (1995). Bioconversions of ferulic acid and p-coumaric acid to volatile phenols by halotolerant yeasts. Studies of halotolerant yeasts in soy sauce making, Part I [in Japanese]. Nippon Nogeikagaku Kaishi 69, 1587-1596. Cited in Biological Abstracts 101,86495. Sugiyama, S. (1984). Selection of micro-organisms for use in the fermentation of soy sauce. Food Microbiol. 1, 339-347. Sugimori, T., and Jose, H. (1970). Studies on halotolerant lactic acid bacteria as related to brewing of soy sauce, I: Isolation and taxonomic studies of Tetracoccus spp. [in Japanese]. J. Fcrment. Technol. [Hakko Kogaku Zosshil 48, 218-27. Cited in Food Science and Yechnology Abstracts, 71-09-T0445. Sung, N. J., Klausner, K. A., and Hotchkiss, J. H. (19911. Influence of nitrate, ascorbic acid, and nitrate reductase microorganisms on N-nitrosamine formation during Korean-style soy sauce fermentation. Food Additives and Contaminants 8 , 291-298. Takeuchi, T., Joshida, M., Hosokawa, N., Nanha, T., Amano, T., Yoshii, H., Yoshida, T., and Murai, K. (1968). Studies on tamari brewing, I: On the factory coefficient lin Japanese]. Annual Reports of the Food Research Institute Aichi Prefecture [Aichi ken Shokuhin Kogyo Shikenjo Nenpol 9, 58-67. Cited in Food Science and Technology Abstracts, 73-07-T0330. Terada, M., Hayashi, K., and Mizununia, 1' : (1980). Distinguishing Aspergillus oryzoe and Aspergillus sojae by their production of hydrolytic enzymes lin Japanese]. , I Jpn. . Soy Sauce Res. Inst. [Nihon Shoyu Kenkujo Zasshil6, 75-81. Cited in Food Science and Technology Abstracts, 83-08-7'0488. Terasawa, N., Murata, M., and Homma, S. (1996). Comparison of brown pigments in foods by microbial decolorization. J. Food Sci. 61, 669-672. Tokuoka, K., and Ishitani, 'I: (1991). Minimum water activity for the growth of yeasts isolated from high-sugar foods. J. Gen. Appl. Microbiol. 37, 111-119. Ueki, T., Noda, Y., Teramoto, Y., Ohba, R., and Ueda, S. (1994a). Increasing glutaminase activity in shoyu koji using a mixed culture of two koji moulds. World J. Micrubiol. Biotechnol. 10,694-696. Ueki, T., Noda, Y., Teramoto, Y., Ohba, R., and Ueda, S. (1994h). Practical soy sauce production using a mixed koji-making system. J. Ferment. Bioeng. 78, 262-264. Ushijima, S . , Hamada, T.. and Kanbe, C. (1982). A new liquid chromatographic determination of organic acids in soy sauce [in Japanese]. J. Jpn. Soy Sauce Res. Inst. R , 58-63.

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Visser, W., Scheffers, W. A., Batenburg-van der Vegte, W. H., and van Dijken, J. P. (1990). Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56, 3785-3792. Waldroup, P. W., and Smith, K. J. (1989). Animal food uses of legumes. In “Legumes: Chemistry, Technology, and Human Nutrition” (R. H. Matthews, ed.), pp. 245-337. Dekker, New York. Wang, H. L., and Fang, S . F. (1986). History of Chinese fermented foods. In “Indigenous Fermented Food of Non-Western Origin” (C. W. Hesseltine and H. L. Wang, eds.), pp. 23-36. Mycological Memoir No. 11. J. Cramer, Berlin-Stuttgart. Watanabe, Y., Ishii, M., and Tazaki, R. (1970). Control of soy sauce fermentation, I: Addition of yeasts and lactic acid bacteria [in Japanese]. Seasoning Sci. [Chomi Kagaku] 17, 35-40. Cited in Food Science and Technology Abstracts, 72-01-T0050. Weast, R. C., and Lide, D. R., ed. (1990). “CRC Handbook of Chemistry and Physics,” 70th ed. CRC Press, Boca Raton. Weingartner, K. E. (1987). Processing, nutrition and utilization of soybeans. In “Soybeans for the Tropics” (S. R. Singh, K. 0. Rachie, and K. E. Dashiell, eds.), pp. 149-178. Wiley, Chichester. Wood, B. J. B. (1982). Soy sauce and miso. In “Fermented Foods, Economic Microbiology,” Vol. 7 (A. H. Rose, ed.), pp. 39-86. Academic Press, London. Yagi, T., and Nishi, T. (1993). Regulation of intracellular osmotic pressure and changes in intracellular proteins during the initial stages of salt stress in Zygosaccharomyces strains exhibiting differences in salt-tolerance. Microbios 74,155-166. Yamamoto, Y., Yanagida, F., and Suminoe, K. (1972). Changes of enzyme activities during preparation of the koji for white shoyu [in Japanese]. 1. Food Sci. Technol. [Nihon Shokuhin Kogyo Gakkai shi] 19, 29-30. Cited in Food Science and Technology Abstracts, 74-12-T0655. Yasui, T., Shikata, H., and Ishigami, Y. (1982). Glutaminase of soy sauce koji, 111: Screening koji moulds for high total glutaminase activity, and formation of glutamic acid in moromi [in Japanese]. J. Jpn. Soy Sauce Res. Inst. [Nihon Shoyu Kenkujo Zasshi] 8, 117-122. Cited in Food Science and Technology Abstracts, 83-11-T0714. Yokotsuka, T. (1960). Aroma and flavour of Japanese soy sauce. Adv. Food Res. 10, 75-134. Yokotsuka, T. (1985). Fermented protein foods in the Orient, with emphasis on shoyu and miso in Japan. In “Microbiology of Fermented Foods,” Vol. 1 (B. J. B. Wood, ed.), pp. 197-246. Elsevier Applied Science, London and New York. Yokotsuka, T. (1986). Soy sauce biochemistry. Adv. Food Res. 30, 195-329. Yokoyama, T., and Kadowaki, K. (1983). Breeding koji mould by a combination of mutation and diploidization [in Japanese]. 1.Jpn. Soy Sauce Res. Inst. [Nihon Shoyu Kenkujo Zasshi] 9, 137-142. Cited in Food Science and Technology Abstracts, 84-04T-223. Yong, F. M., and Wood, B. J. B. (1974). Microbiology and biochemistry of soy sauce fermentation. Adv. Appl. Microbiol. 17, 157-194. Yong, F. M., and Wood, B. J. B. (1976). Microbial succession in experimental soy sauce fermentations. J. Food Technol. 11,525-536. Yong, F. M., and Wood, B. J. B. (1977). Biochemical changes in experimental soy sauce moromi. J. Food Technol. 12, 263-273. Yong, F. M., Lee, K. H., and Wood, B. J. B. (1978). Study of some factors affecting the growth of soy yeast (Saccharomyces rouxii NRRLY-1096). J. Food Technol. 13, 385-396.

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Yong, F. M., Lee, K. H., and Wong, H. A. (1980). The production of volatile acids from glucose by soy yeast (Saccharomyces rouxii) NRRLY-1096. J. Food Technol. 15, 421-428. Yong, F. M., Lee, K. H., and Wong, H. A. (1981). The production of ethyl acetate by soy yeast Saccharomyces rouxii NRRL Y-1096. J. Food Technol. 16, 177-184. Yoshii, H. (1979). Fermented foods and water activity [in Japanese]. Nihon Jozo Kyokai Zasshi [I. Soc. Brewing, Japan] 74,213-218. Yu, J., Kim, Y.S., Lee, J. M., and Hong, Y.M. (1972). Studies on the substitution of raw materials for soy sauce, I: Use of corn-gluten [in Korean]. Komnn 1. Food Sci. Techno!. 4,106-111. Cited in Food Science and Technology Abstracts, 74-08-T0456. Zapsalis, C., and Beck, R. A. (1986). "Food Chemistry and Nutritional Biochemistry." Macmillen, New York.

Gene Transfer Among Bacteria in Natural Environments XIAOMINGYIN SRA Technologies,Inc. Rockville, Maryland 20850

G. STOTZKY Department of Biology Laboratory of Microbial Ecology New York University New York, New York 20003

I. Introduction 11. Transformation A. Mechanisms of Transformation B. Plasmid Transformation C. Transformation by Cell Contact D. Transfection E. Transformation in situ F. Conclusions from Transformation Studies 111. Conjugation A. Mechanisms of Conjugation B. Factors that Affect Conjugation in the Environment C. Conjugation in situ D. Conclusions from Conjugation Studies IV. Transduction A. Brief Introduction to Bacteriophages B. Types of Transduction C. Factors that Affect Transduction in the Environment D. Transduction in situ E. Conclusions from Transduction Studies V. Concluding Remarks References

I. Introduction

Griffith (1928) was the first to observe gene transfer among bacteria (Streptococcus pneumoniae), although he did not understand the mechanisms involved in this transfer (i.e., transformation). Alloway (1932) demonstrated transformation with cell-free extracts of pneumo153 ADVANCES IN APPLlED MICROBIOLOGY,VOLUME 4 5 Copyright 0 1997 by Academic Press Ail rights of reproductioii in any form roserved. 0065-2164/97$25.00

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cocci. Avery, MacLeod, and McCarty (1944) demonstrated that extracelMar bacterial DNA could change bacterial phenotypes. After this identification of the transforming factor (DNA), gene transfer in bacteria by conjugation (Lederberg and Tatum, 1946a,b) and by transduction by bacteriophages (Zinder and Lederberg, 1952) was successively discovered. Since then, these three major mechanisms for the transfer of genetic information among bacteria have been extensively studied and routinely used to transfer genes under laboratory conditions. Subsequently, other mechanisms of gene transfer have been identified, such as capsduction by a gene-transfer agent that is a small phage-like structure (Joset and Guespin-Michel, 1993; Scolnik and Marrs, 1987), protoplast fusion (Matsushima and Baltz, 1986), and transposition (Berg, 1989; Berg and Howe, 1989; Kingsman et al., 1988; Salyers et a]., 1995). However, gene transfer in the environment has been studied only relatively recently (see Levy and Miller, 1989). One major aspect that should be considered in attempting to stimulate, initiate, and promote research in this area is ecological safety. With the rapid growth of genetic engineering and biotechnology, more and more genetically engineered microorganisms (GEMs) are being used in industry, agriculture, medicine, and research. Genetic engineering and biotechnology have greatly promoted the development of many fields of basic science, and they have indicated the vast potential of their application for economic gain. However, their application may also result in deliberate or accidental release of GEMs into the environment that could cause harmful effects. Consequently, the potential ecological risks need to be evaluated, especially the possibility of GEMs transferring potentially dangerous novel genes to autochthonous microbes, which are probably more adapted to natural environments than the GEMs (Stotzky, 1989). Gene transfer among bacteria in nature can occur by three major mechanisms: (1)transformation, in which extracellular DNA is taken up by recipient bacteria; (2) conjugation, in which genetic material is transferred from one bacterium to another by cell-to-cell contact; and (3) transduction, in which the transfer of genetic information between bacteria is mediated by bacteriophages. Studies on the t h e e major mechanisms of gene transfer in natural environments are briefly discussed. II. Transformation Transformation (also called bacterial, genetic, or DNA transformation) was the first mechanism of gene transfer that was studied (Avery et al., 1944; Griffith, 1928; Katz and Marquis, 1991). Transformation has been

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categorized into natural transformation and artificial transformation. In natural transformation, recipient bacteria develop a physiological state of competence and actively take up and incorporate extracellular DNA into their genetic information. Natural transformation appears to occur only in bacteria. However, since the development of recombinant DNA technology, artificial transformation has been used with both prokaryotic and eukaryotic cells (see Lorenz and Wackernagel, 1994). Natural transformation (hereinafter referred to as “transformation”) can be distinguished experimentally from conjugation and transduction by the sensitivity of this process to nucleases of DNA (e.g., DNase). Inasmuch as gene transfer by transformation occurs via extracellular DNA, DNase can degrade the DNA and prevent transformation. In contrast, during transduction, transducing DNA is protected by the protein coat (capsid) of the phage and is never exposed extracellularly to DNase. In conjugation, DNA is transferred by cell-to-cell contact, and the transfer of DNA from donor to recipient cells also occurs without exposure of DNA to DNase. Therefore, the addition of DNase can be used to eliminate the possibility of transformation in experiments on transduction and conjugation. A. MECHANISMS OF TRANSFORMATION

The process of transformation in the environment consists of the following sequence of major steps (Lorenz and Wackernagel, 1994; Mazodier and Davies, 1991): 1. Release of DNA from cells. 2.

3. 4.

5. 6.

7.

Dispersal of the released DNA into the environment. Persistence of the released DNA in the environment. Development of competence in potential bacterial recipients. Interaction of recipient cells with the released DNA. Uptake of exogenous DNA and the processing of the entering DNA in cells. Integration of the exogenous DNA into the endogenote and the expression of its gene(s).

1. Release of DNA

Extracellular DNA can be derived from dead or viable cells. The death of bacteria can be caused by both abiotic and biotic factors in the environment. The abiotic factors include ionic strength, presence of chelators, salinity, pH, and temperature (Paul and David, 1989). The

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biotic factors comprise attack by bacteriophages (Bmsheim, 1993), protozoan (Heijnen and van Veen, 1991) and bacterial (Schlegel, 1986) predation, and bacterial autolysis (Leduc et a/., 1982). Many viable bacterial cells release DNA without cellular lysis (Lorenz and Wackernagel, 1994), suggesting that release may be a normal function of bacteria. However, the mechanisms of the release of DNA from living cells need to be clarified. 2. Dispersal of DNA In the environment, both bacteria and free or bound DNA may be moved by percolation of water (Madsen and Alexander, 1982; Trevors et a/., 1990), earthworm disturbance (Henschke et a]., 1989), transport by protozoa (Heynen etal., 1988) and fungi (Stotzky, 1989),and by flows of air, dust, wind, and water. Significant amounts of free DNA have been detected in both marine (DeFlaun et a/., 1987; Paul et a]., 1988) and freshwater environments (DeFlaun et al., 1986), soils, and sediments (Ogram etal., 1987; Steffen et al., 1988).For example, 0.2 to 44.0 kg/liter of extracellular DNA has been detected in different aquatic environments (Lorenz and Wackernagel, 1994). This DNA seems to originate mostly from microbes. Although it is assumed that free DNA is present in almost all environments, more research is needed to obtain a clearer picture of the distribution of DNA in the environment. 3. Persistence of DNA

Theoretically, transformation is probably the most general and nonspecific system for the transfer of DNA. However, it is subject to numerous constraints in the environment, and it was, therefore, thought to be unimportant in natural environments. Because nucleases are ubiquitous, free DNA will be rapidly digested in the environment. The turnover times (half-lives) of free DNA in the environment range from 0.017 to 235 hours in aquatic environments and from 9.1 to 28.2 hours in terrestrial environments (Lorenz and Wackernagel, 1994). However, DNA adsorbs rapidly and binds tightly on sand and clay minerals (Gallori et a/., 1994; Khanna and Stotzky, 1992; Lorenz et a/., 19881, other minerals (including quartz and feldspar), dead cellular material (detritus), soil and sediment (Lorenz and Wackernagel, 1994), and humic substances (Crecchio and Stotzky, 1997). The bound DNA is protected from degradation by nucleases, but it can transform recipient cells (Crecchio and Stotzky, 1997; Gallori et a/., 1994; Khanna and Stotzky, 1992; Lorenz et al., 1988). In addition, cell-contact transformation (Stewart et nl., 1983), discussed below, may also provide a means

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for the protection of transforming DNA by enzymatic degradation, even though this process is partially DNase-sensitive (Stewart and Sinigalliano, 1989).

of Competence Competence is defined as a physiological state in which a bacterial cell is capable of taking up molecules of extracellular DNA and transporting them into its cytoplasm (Birge, 1994). The development of competence is a complex process and is encoded by chromosomal genes (Stewart and Carlson, 1986).Not all bacteria are transformable in nature. Even among transformable bacteria, the development and duration of competence are variable. At least one organism, Neisseria gonorrhoeae, is constitutively competent (Sparling, 1966). In general, Gram-positive bacteria, such as Bacillus subtilis (Dooley et a]., 1971) and S. pneumoniae (Tomasz, 1966), induce competence by secreting a small polypeptide (competence factor) until it reaches a critical concentration. In contrast, Gram-negative bacteria, such as Haemophilus influenzae (Spencer and Herriott, 1965) and Azotobacter vinelandii (Page, 1982), regulate competence internally. Although the mechanisms involved in the development of competence are unclear, its appearance usually results from a nutritional shift-down (i.e., a decrease in nutrient concentration or available energy) and usually coincides with a decrease in or blockage of DNA synthesis (Joset and Guespin-Michel, 1993). The development of competence in the environment has yet to be clearly understood. It is assumed that the laboratory models for the induction of competence are valid in natural environments. It is also possible that some environmental conditions (e.g., limited nutrients) trigger competence in bacteria by shifts to unbalanced growth and starvation (Stewart, 1992). 4. Development

5. Binding of DNA

The process of the binding of DNA on cells varies among different transformable bacteria. Extracellular DNA can bind to proteins associated with the outer surface of cells. The specificity of these DNA-binding proteins differs among transformable bacteria. For Gram-positive bacteria, such as B. subtilis and S. pneumoniae, there is no specificity in the binding and uptake of double-stranded DNA (i.e., binding and entry do not require homologous DNA, and any double-stranded DNA can bind and enter these cells). However, double-stranded DNA is required for effective transformation (Morrison, 1977). Single-stranded DNA or RNA, RNA-DNA complexes, and glycosylated DNA are not

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bound or compete poorly for binding sites (Smith et al,, 1981). Gramnegative bacteria, such as H. injluenzae and N. gonorrhoeae, display a specificity in the DNA binding process. H. influenzae binds and takes up double-stranded DNA only from the same or closely related species, with an 11-base-pair (bp) DNA sequence in the double-stranded DNA being the specific recognition site (Danner et al., 1982). The cell surface bindinghptake system of N. gonorrhoeae recognizes a 10-bp DNA sequence (Goodman and Scocca, 1988). Both Gram-negative bacteria can bind and take up double-stranded and single-stranded DNA (Postel and Goodgal, 1966; Stein, 1991). However, only N , gonorrhoeae can be transformed with similar efficiency by single- and double-stranded DNA (Stein, 1991). DNA binding is usually subdivided into “loose” and “tight” binding stages. During the first stage (loose binding) DNA can be dissociated from the cell surface by salt washes (Ceglowski et a]., 1980). In the second stage (tight binding), DNA cannot be removed by washing, but it is still sensitive to digestion by nucleases (Seto and Tomasz, 1976). There appear to be no studies on the binding of DNA to cells in natural environments. Because binding can be affected by ionic strength, cations, nuclease concentration, and competition from other DNA under laboratory conditions (Ceglowski et al., 1980; Paul et al., 1987, 1989; Stewart and Carlson, 1986), these factors might also influence transformation in nature by affecting the DNA-binding step. 6 . Uptake of DNA

The uptake of DNA is defined as the transition of DNA bound on the cell surface into a DNase-resistant state (Lorenz and Wackernagel, 1994). In the well-studied transformation model of S. pneumoniae (Birge, 1994; Joset and Guespin-Michel, 1993), the double-stranded DNA, after binding on the cell wall, is cut by the endonuclease (located near the binding site) of the competence factor into 15-kilobase (kb) fragments that become bound to the cytoplasmic membrane. The fragmented double-stranded DNA molecules then undergo a second processing: either strand of the transforming DNA is hydrolyzed by an exonuclease located in the cytoplasmic membrane, and the other nonhydrolyzed strand is coated with proteins, also located in the cytoplasmic membrane, to form an eclipse complex that enters the cytoplasm. When the eclipse complex is formed (called the eclipse stage of transformation), fragments of exogenous DNA extracted from these cells have no transforming activity, because the DNA is single-stranded and lacks the requisite double-stranded form for binding (Smith et al., 1981).

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Some Gram-negative bacteria, such as H. influenzae and H. parainflnenzae, have a different mode of uptake. During the development of competence, H. influenzae forms numerous small membranous structures that protrude outside the cell (transformasomes) and increase contact between the external DNA and the internal chromosome (Kahn et al., 1983). The exogenous intact DNA first binds reversibly to specific receptors present at the surface of the transformasome and then is irreversibly internalized into the vesicle (Birge, 1994; Goodgal, 1982). This double-stranded DNA becomes protected very rapidly by binding with polypeptides in the transformasome, and it is then linearly translocated from the vesicle to the cytoplasm by the degradation of one strand (from the 5' end) (Joset and Guespin-Michel, 1993). After this singlestranded DNA penetrates into the cytoplasm, it is very quickly integrated into the chromosome of the recipient cell (Kahn and Smith, 1984).

The uptake of DNA requires not only energy, but also threshold concentrations of Mg2+,Na+,K', NH:, or Ca2+in Gram-positive bacteria and of Ca2+and Na+in Gram-negative bacteria (Romanowski et al., 1993; Set0 and Tomasz, 1976; Smith et al., 1981). Uptake can be prevented by the addition of ethylenediamine tetraacetic acid (EDTA),which chelates some of these cations (Noteborn et ~ l .1981). , Hence, the concentration of energy sources and cations and the presence of chelating agents in a natural environment may influence the uptake of transforming DNA and, thus, the efficiency of transformation. 7 . Hom ologo us Recombination

The integration of transforming DNA into the chromosomal DNA of the recipient seems to be similar in all transformable bacteria. The incoming single-stranded DNA pairs with the double-stranded DNA of the host chromosome and forms an unstable tri-stranded donor-recipient DNA complex. Homologous crossing-over between one strand of the chromosomal DNA and the incoming single-stranded DNA results in genetic recombination, which involves a series of Rec proteins (de Vos and Venema, 1982, 1983). After being covalently linked to the chromosome, the transformed DNA replaces the displaced strand of the chromosome of the endogenote and becomes inheritable, and the transformed gene(s) may be expressed (Smith et al., 1981). B. PLASMID TRANSFORMATION Transformation by a plasmid (an extrachromosomal, double-stranded, circular molecule of DNA capable of self-replication) is called plasmid

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transformation. In general, transformation by plasmids has a lower efficiency of transformation (number of transformants per pg of DNA) than by chromosomal DNA. This may be a result of the covalently closed circular structure of plasmids. For plasmid transformation of Gram-positive bacteria, double-stranded DNA must be converted to single-stranded DNA outside the cells. After the single-stranded DNA enters the recipient, it appears to be almost impossible for the linear single strand of a monomeric plasmid to recircularize and replicate. However, the separate entry of two complementary single strands of a monomeric plasmid that may reassemble into a complete plasmid molecule occurs occasionally (Behnke, 1981). Furthermore, when a single strand of oligomeric plasmid DNA and a shorter fragment of the opposite strand (which serves as a primer for DNA synthesis) simultaneously enter the cell, an intact duplex monomer of this plasmid may be reconstructed after the completion of an intramolecular recombination event (Saunders and Guild, 1981). Another route for the successful transformation of the plasmid requires a region of homology on the donor plasmid DNA molecule (which is being transformed) with the DNA molecule of the recipient (Canosi et d . ,1981).Transformation by monomers is very inefficient and appears to be unique to 3. subtilis (Canosi et a]., 1981; Gallori ef a]., 1994). Conversely, in Gram-negative bacteria, when the plasmid enters transformable cells, it may remain trapped in the transformasome and not penetrate into the cytoplasm, and its replication is prevented in the transformasome. Moreover, as a result of its circular structure, the plasmid DNA might not be released as easily as linear molecules from the transformasome vesicle, which may account for the lower efficiency of transformation of Gram-negative bacteria by plasmid DNA than by chromosomal DNA.

c. TRANSFORMATION BY CELL CONTACT A special form of transformation is called cell-contact transformation. During contact between bacterial cells, genes may be transmitted in a manner that appears to have more features of transformation than of conjugation. This process needs cell-to-cell contact, but the transfer is partially sensitive to DNase I (i.e., not completely inhibited) and is bidirectional. The mechanisms involved in this process are not well known. Ephrati-Elizur (1968) first observed this phenomenon between intact cells of B. subtilis, and it has been observed subsequently in species of Haemophilus (Albritton et al., 1982), Pseudomonas (Stewart

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et a]., 1983), Streptococcus (Buu-Hoi et al., 1985), Acinetobacter (Rochelle et al., 1988), Vibrio, and Escherichia (Paul et al., 1992). Stewart et al. (1983) described this phenomenon in Pseudomonas stutzeri as a DNase I-sensitive transfer of chromosomal genes between intact donor and recipient cells that occurred in the absence of detectable phages. Cell-contact transformation can occur with either plasmid or chromosomal DNA and even with heat-killed donor cells (Buu-Hoi et al., 1985; Day and Fry, 1992b). Consequently, this form of transformation may be significant in gene transfer between intact cells in natural environments. D. TRANSFECTION An unusual variant form of transformation has been named transfection, in which the extracellular DNA is the naked DNA of bacteriophages, and the result is a virus-infected recipient cell (Birge, 1994). Transfection was first described by Foldes and Trautner (1964) in transformable bacteria. Although transfection has basic similarities with transformation, such as the requirement for competent cells, the mechanisms of transfection may be different from those of transformation, at least in the DNA-uptake process (Birge, 1994). The importance of transfection in the natural environment has apparently not been studied.

E. TRANSFORMATION IN SITU Even though transformation was the first described mechanism of gene transfer, transformation of only a limited number of bacteria has been studied in only a few environmental settings. 1. Animal Systems

Griffith (1928) first demonstrated transformation by conducting experiments in mice. He used two variants of S. pneumoniae (then called Pneumococcus or Diplococcus pneumoniae). One virulent variant has a smooth (S) cell surface (capsule formation) and can cause disease in mice, whereas the avirulent mutant has a rough (R) cell surface (no capsule) and does not induce disease. When the encapsulated S cells were heat-killed before injection into mice, their virulence was lost. However, when a mixture of living R cells and heat-killed S cells was injected into a mouse, the mouse soon died. The bacteria isolated from the dead mouse had capsules (S) and were virulent when injected into other mice. Griffith stated that the capsule-forming “transforming prin-

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ciple” was probably “that specific protein structure of the virulent pneumococcus which enables it to manufacture a specific soluble carbohydrate” (Griffith, 1928). This important experiment is a milestone in biology, because it led to the discovery that the “transforming principle” was DNA, the carrier of genetic information (Avery et al., 1944). 2. Soil

Graham and Mock (1978, 1979, 1981) first reported the exchange of chromosomal genes by transformation in B. subtilis in sterile soil. Spores of two strains, each carrying three different chromosomally located linked marker genes and one unlinked marker gene but no plasmids or transducing bacteriophages, were washed into sterile soil after they were heat shocked to induce germination and competence, as well as to release transforming DNA. After incubation for 8 days, about 79% of the bacteria exhibited a phenotype containing markers from both parents that were not detectable after the first day. Gene transfer in this system was not sensitive to DNase, and it was not reduced by the addition of DNA from calf thymus. These two tests are often used to ensure that transformation actually occurred (Stewart and Carlson, 1986), but they did not provide the expected results in this study. Graham and Istock suggested that the DNase had been adsorbed on soil particles and was no longer active or that the transforming DNA bound to proteins, thus preventing degradation by DNase, and that the DNA from calf thymus was bound on humus. These and other confusing results of these studies have been discussed (Stotzky, 1989). Transformation of chromosomal markers between B. subtilis and B. licheniformis also occurred in sterile soil (Duncan et al., 1989). Although the phenotypic stability of hybrids between these two bacterial species was variable, this study indicated that interspecific transformation may occur in natural environments. Transformation of chromosomal and plasmid genes coding for amino acid biosynthesis and antibiotic resistance among strains of B. subtilis was observed in sterile and noristerile soils (Lee and Stotzky, 1990). The transformation frequencies in sterile and nonsterile soils were significantly lower than in pure culture. The adsorption of nucleic acid materials (e.g., RNA, DNA, nucleotides, nucleoproteins) from various organisms on clays, sand, and soils has been studied by numerous investigators for many years (e.g., Bower, 1949; Conrad, 1939; Goring and Bartholomew, 1952; Greaves and Wilson, 1969, 1970, 1973; Iverson et al., 1982; Ogram et a]., 1988; Paget and Simonet, 1994; Pinck et al., 1946). Lorenz and coworkers (Aardema et al., 1983; Lorenz and Wackernagel, 1987, 1994; Lorenz et

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al., 1981,1988) showed that DNA from B. subtilis was adsorbed on sand and other components of sediments and acquired a higher resistance to degradation by DNase than free DNA. The sand-bound DNA was still capable of transforming competent cells. In addition, adsorbed DNA appeared to facilitate attachment of competent cells to sand. Their results supported the concept that transformation in habitats with a high so1id:water ratio occurs preferentially at the solid-liquid interface (Lorenz et al., 1988; Stotzky, 1986, 1989). However, even though these studies are interesting and informative, their relevance to transformation in soil is not clear, because these experiments were conducted with “clean” sand (Stotzky, 1989). Khanna and Stotzky (1992) observed that DNA binds on clay minerals, which protects the DNA from degradation by DNase. They studied the adsorption and binding of chromosomal DNA from B. subtilis on montmorillonite and kaolinite under different conditions and showed that montmorillonite had a higher affinity for DNA than kaolinite and that the bound clay-DNA complexes, as well as desorbed DNA, could transform genes coding for resistance to chloramphenicol and for amino acid biosynthesis into competent cells. The DNA bound on montmorillonite, but not on kaolinite, was capable of being amplified by the polymerase chain reaction (PCR) when a 14-mer random sequence was used as the primer (Alvarez et al., 1997).X-ray diffractometry indicated that DNA did not intercalate the clays. This was confirmed by transmission and scanning electron microscopy, which showed that DNA was bound primarily on the edges of the clays, although some DNA was also bound on the planar surfaces of the clays (Khanna et a]., 1997). This binding was probably primarily by H-bonding and electrostatic interactions between the positively charged edges of the clay and the negative charges on DNA (Stotzky, 1986), which changed the electron distribution and/or the conformation of the DNA and prevented nucleases from recognizing or interacting with appropriate binding or cleavage sites on the DNA. Moreover, the DNA extending from the edges of the clays could interact with receptor sites on competent cells and result in transformation (Khanna et d . , 1997). Gallori et ~ l(1994) . also reported the transformation of B. subtilis by DNA bound on clay in nonsterile soil. Chromosomal DNA and different forms of plasmid DNA (covalently closed circular, linear monomer, and linear multimer) were adsorbed and tightly bound on montmorillonite. The different forms of DNA, with the exception of linear monomeric plasmid DNA, retained the ability to transform competent cells. Their results also indicated that the tertiary structure and size of the DNA molecules influenced adsorption and binding of the DNA on clay.

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Moreover, the bound DNA was protected against the activity of restriction endonucleases, persisted in nonsterile soil, and transformed competent cells added to the soil. Methods for studying transformation in soil have been published (Stotzky et al., 1996). Vettori et al. (1996) reported that the chromosomal DNA bound on kaolinite and one type of montmorillonite could be amplified by the random amplified polymorphic DNA (RAPD) technique. However, amplification of DNA bound on another type of montmorillonite occurred only when the concentration of clay particles present in the clay-DNA complexes was decreased or when the amount of Tag DNA polymerase was increased. The inhibition of amplification by this clay appeared to have been caused by inactivation of the polymerase, possibly as a result of its binding on the clay particles or by interference of the particles with its catalytic site. DNA from B. subtilis also adsorbed rapidly and was bound tightly on humic acids extracted from soil (Crecchio and Stotzky, 1997). The bound DNA was protected against degradation by DNase and retained the ability to transform competent cells. In addition to Gram-positive soil-inhabiting strains of Bacillus, two Gram-negative soil-inhabiting bacteria, f! stutzeri and Acinetobacter calcoaceticus, have been shown to undergo transformation in sand and soil extracts (Carlson et al., 1983; Lorenz and Wackernagel, 1990; Lorenz et al., 1992). Transformation of F! stutzeri was more efficient when cells and DNA were supported on a solid surface than when in liquid (Carlson et al., 1983). Transformation of I? stutzeri by sand-adsorbed DNA (Lorenz and Wackernagel, 1990) indicates that uptake of particletiound DNA by I? stutzeri in soil could be possible. High efficiency of plasmid transformation of A. calcoaceticus was detected in extracts of fresh and air-dried nonsterile soil, which suggested that competent cells of A. calcoaceticus could take up extracellular DNA in natural soil and sediment environments (Lorenz et al., 1992). Although several studies have indicated that transformation can occur in soil, more studies with natural (i.e., nonsterile) soils and more bacterial species are necessary to verify these results, most of which were obtained under controlled laboratory conditions, and to determine the frequency and, hence, the relevance of this mode of transfer of genetic information in this habitat. 3. Aquatic Habitats

The existence of extracellular DNA with high molecular mass (M,) in marine environments suggests that transformation can occur in this

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habitat (DeFlaun et al., 1986; Paul et al., 1987). Jeffrey et al. (1990), using a transformable marine bacterium, Vibrio sp. DI-9, as a recipient and a broad-host-range (BHR),nonconjugative plasmid, pKT230, as the source of transforming DNA, showed that the plasmid was transformed into the recipient on both filters and in sterile sediment microcosms. The rate of transformation was increased when the transforming plasmid DNA was in multimeric forms. Paul et al. (1991) demonstrated that transformation occurred also in marine water columns by utilizing a high-frequency-of-transformation (HFT) variant of DI-9 (designated WJT-1C) and multimers of DNA from a BHR plasmid (pQSR50). The presence of the plasmid DNA in the presumptive transformants was confirmed by colony hybridization. Transformation was also observed in sterile sediment microcosms but not in nonsterile sediments. These results suggested that transformation is more likely to occur in water columns than in marine sediments in nature. Paul et al. (1992) also reported intrageneric and intergeneric transformation by plasmid pQSR50 among marine strains of Vibrio and between Escherichia coli and Vibrio species in sterile and nonsterile estuarine water. Transformation by the nonconjugative plasmid was via a DNasesensitive mechanism that required cell-to-cell contact. The authors suggested that this may be a mechanism for the spread of nonconjugative plasmids in the environment. Stewart and Sinigalliano (1990) showed the transformation of chromosomally encoded resistance to rifampin in strains of a marine isolate of l? stutzeri in sterile and nonsterile marine sediments. They also detected the transfer of chromosomal genes through cell contact among strains of l? stutzeri isolated from soil and marine environments (Stewart, 1992). Frischer et al. (1994) demonstrated the transfer of extracellular plasmid DNA to indigenous marine bacteria by transformation. This appears to be the first report of transformation of a natural mixed microbial community. The natural mixed microbial communities from water and extracted from various marine invertebrates (sponges and holothurians) were transformed. Transformants included members of the genera Vibrio and Pseudomonas, as well as members of unknown genera. The presumptive transformants were confirmed by colony hybridization and PCR. Transformation was not detected in the sediments, again indicating that the sediment environment is not conducive to transformation. The investigators suggested that transformation may be a mechanism responsible for the dissemination of small plasmids in aquatic environments and the alteration of the genetic structure of marine microbial communities.

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Baur et al. (1996) reported that strains of E. coli K-12 were transformed by plasmid pUC18 in filter-sterilized river water, spring water, and mineral water that contained between 0 to 11 mM Ca2+.Only 1 to 2 mM of Ca2+was sufficient to obtain transformants, suggesting the natural development of competence in wild-type E. coli. This is an important observation, as E. coli was thought to be unable to develop natural competence, that is, its ability to be transformed could only be obtained by artificial methods, such as the addition of high concentrations of Ca2+.This also suggests that some presumably untransformable bacteria can develop competence in natural environments, even though they do not show this ability under laboratory conditions. 4. Epilithon

An epilithon is the community of organisms within the slimy layer that develops on the surfaces of stones in aquatic habitats (Fry and Day, 1990).Rochelle et al. (1988)reported an identical 7.8-kb nonconjugative mercury-resistance plasmid in two strains of A. calcoaceticus from a riverine epilithic community. The plasmid isolated from intact cells of these two strains was successfully transferred in vitro via a DNase-sensitive process into a naturally competent strain of A. calcoaceficus isolated from soil, suggesting that it was a transformation-like process. Williams et al. (1992) further demonstrated the transformation of the nonconjugative plasmid to strains of A. calcoaceticus in the epilithon on river stones. Williams et al. (1996) also reported that strains of A. calcoaceticus incorporated into a river epilithon were transformed to prototrophy in situ by either free chromosomal DNA (lysates) or live donor cells. F.

CUNCLUSIONS FKOM TRANSFORMATION STUDIES

The results of these studies contradict the earlier assumption that transformation is a relatively unimportant method of gene transfer in natural habitats. They demonstrate that transformation could be important in the spread of genetic information through natural bacterial populations. 111. Conjugation

Conjugation (also called bacterial conjugation) was first discovered in E. coli by Lederherg and Tatum (1946a,b). Although half a century of intensive studies have been carried out in numerous laboratories around the world since this discovery, the mechanisms involved in

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conjugation, especially in the environment, are still not completely known. Conjugation is usually assumed to be primarily a plasmid-associated process in which conjugative plasmids carry transfer (tra)genes that not only mediate self-transfer but can also mobilize nonself-transmissible plasmids (tru-) and chromosomal genes to transfer unidirectionally from donor to recipient cell via cell contact. However, another type of nonplasmid conjugative element has been discovered: the conjugative transposon (Clewell and Gawron-Burke, 1986; Franke and Clewell, 1981; Salyers et al., 1995; Shoemaker et ul., 1980). In addition, another type of conjugation, retrotransfer, has been described (Mergeay et al., 1987; Powell et al., 1989) in which there is reciprocal genetic exchange, that is, donor bacteria can also obtain genes from recipient bacteria via the movement of nonconjugative plasmids and chromosomal genes into cells containing some types of conjugative plasmids. This direction of gene transfer is opposite to that in classical conjugation and mobilization. Conjugation has been demonstrated to be the primary method for the transfer of BHR DNA between different genera of bacteria. Moreover, conjugation is a very useful and powerful tool for the study of gene transfer and the construction of genetic maps. A. MECHANISMS OF CON1UGATION 1. Self-Transfer of Conjugative Plasmids

In Gram-negativebacteria, conjugation can be divided into two stages. The first stage involves the formation of a specific cytoplasmic bridge between the mating cells, whereby the plasmid-containing donor and the recipient make contact through extracellular conjugative pili, which have been broadly divided into two morphological groups: (1)long flexible pili and (2) short rigid pili (Bradley, 1980). After the tip of the F-pilus (encoded by an extrachromosomal fertility (F) factor) initiates contact with the recipient cell, a cellular mating aggregate is formed and stabilized between the two cells. A DNA-transport pore extends through the cell envelopes of the joined donor and recipient and connects the cytoplasm of the pairing cells. In the second stage, which involves the transfer and processing of DNA, one strand of the plasmid DNA in the donor is nicked at the specific site of the origin of transfer ( o r i n , and a mobilization protein, Mob, is covalently attached to one end (5’ terminus) of the nicked DNA strand and interacts with the proteins of the transport pore to anchor this end of the transferring DNA strand to the pore. At the oriT site, DNA replication (termed transfer replication) by

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the rolling-circle mode produces a single-stranded copy of the plasmid DNA that moves through the pore to the recipient cell. When the end (3’ terminus) of the transferring DNA strand reaches the pore, it joins covalently with the leading end (5’ terminus) of the same DNA strand, which is anchored there by the Mob protein-pore complex. As a result, a covalently closed single-stranded copy of the plasmid is introduced into the cytoplasm of the recipient, and its complementary strand is generated by DNA replication in the recipient. The double-stranded plasmid can then either replicate further or integrate into the recipient chromosome (Clewell, 1993; Day and Fry, 1992a; Ippen-Ihler, 1989; Salyers and Shoemaker, 1994). When a plasmid becomes integrated into the donor chromosome, it can also transfer chromosomal genes to a recipient cell. For example, the self-transmissible F-factor in E. coli is an episome, which can either exist autonomously in the cell or integrate into the bacterial chromosome at several different locations by recombination between homologous insertion sequences present on both the plasmid and the host chromosome. After integration, the tra operon of the F-factor is still functional, and the integrated F-factor can direct the synthesis of pili, rolling-circle replication, and transfer of part of the F-factor (leading or starting region of the transferred DNA) and part of the chromosome into an F- recipient cell. Such a donor is called a “high frequency of recombination” (Hfr) cell. In Gram-positive bacteria, the mechanism of conjugation is very different, as pili are not found, and no specific bridge between mating pairs of bacteria via a conjugative pilus is formed. At least two categories of conjugation that are mediated by self-transferable plasmids have been found. The third category involves conjugative transposons, which are discussed below. One of the plasmid-conjugation systems in Gram-positive bacteria is represented by the pheromone-responding plasmids (Clewell, 1981, 1990). These plasmids are confined to the enterococci and their close relatives. The size of the plasmids tends to be larger than 25 kb. During the transfer process, the recipient cells excrete sex pheromones composed of small peptides (also known as clumping inducing agents or CIAs), which trigger the plasmid-carrying donor cell to produce surface proteins that promote cell clumping. This increases cell-to-cell contact between donor and recipient cells. The necessary cellular and molecular events for the conjugal transfer of plasmids are then induced. These pheromone-responding plasmids often contain genes encoding hemolysins, bacteriocins, or resistance to antibiotics (Brantl et a]., 1990). The second system of plasmid transfer in Gram-positive bacteria has been observed in streptococci and staphylococci (Clewell, 1993). The

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transfer of these plasmids occurs at variable frequencies (generally in the range of to per recipient), depending on the plasmid type and the genotype of the mating pair, and requires that donor and recipient cells be co-cultivated on a solid surface (i.e., direct contact between donors and recipients). These plasmids do not show a response to pheromone signals, and the mechanisms of their transfer are unclear. The BHR plasmids of streptococci mostly encode genes for resistance to macrolides, lincosamides, and streptogramin B (MLS'), usually have similar restriction-enzyme digest patterns, and range from 20 to 35 kb. The host range of streptococcal plasmids include species of Streptococcus, Staphylococcus, Clostridium, Lactobacillus, Listeria, Pediococcus, and Bacillus. Conjugative plasmids of streptococci can mobilize nonconjugative plasmids. Staphylococcal conjugative plasmids encode resistance to gentamicin, appear to be stable, and are considerably more restricted in their host range. A model staphylococcal conjugative plasmid, pGO1, contains 52 kb, can transfer among staphylococcal species (possibly by single-stranded transfer), can mobilize small plasmids, and appears to contain an integrated copy of plasmid pUCll0 carrying several antibiotic-resistance genes (Clewell, 1993). 2 . Mobilization

Mobilization is a process by which smaller nonconjugative plasmids, which are unable to transfer themselves because they do not encode a self-conjugative system, may be transferred from donor to recipient with the help of self-transmissible plasmids. There are two ways by which this can be achieved. The nonconjugative plasmids can use the transfer system of a co-resident conjugal plasmid to move from the host cell into the recipient. In this case, nonconjugal plasmids first cause the production of proteins to initiate the transfer process by making a singlestranded nick at their own oriT site. The resulting DNA-protein complex then interacts with proteins of the mating pore encoded by the conjugative plasmid and continues the conjugal transfer process. The nonconjugative plasmids can also be mobilized by conduction (Clarke and Warren, 1979), in which the plasmid forms a co-integrate with a conjugal plasmid and enters the recipient cell as part of the conjugal plasmid. Subsequently, the co-integrate plasmid may separate into the original two plasmids. Mobilization has been observed in Gram-negative and Gram-positive bacteria. 3. Broad-Host-Range Plasmids

Although the host range of a conjugative plasmid may be limited to a relatively few closely related organisms, and some plasmids have a

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very narrow host range, many plasmids have a very broad host range. BHR plasmids of incompatibility (Inc) groups N, P, Q, and W can transfer to a wide variety of Gram-negative bacteria (see Clewell, 1993). Some plasmids originally found in Gram-positive bacteria can be transferred from Gram-positive to Gram-negative bacteria (del Solar et al., 1993; Trieu-Cuot et al., 19881, and vice versa (Trieu-Cuot et a]., 1987). Moreover, plasmids have been shown to be transferred from E. coli to the eukaryotic yeast Saccharomyces cerevisiae (Heinemann and Sprague, 1989).Even interkingdom transfer has been observed. For example, the Ti plasmid of the bacterium Agrobacterium tumefaciens can transfer a segment of its DNA (T-DNA)to plants (Winans, 1992). Goussard et al. (1996) demonstrated that the replicative plasmid pAT497 or the integrative plasmid pAT498 (prokaryote-eukaryote shuttle vector) was transferred from constructed E. coli BM2710 cells (containing the invasive plasmid pWRl10 of Shigella flexneri) to mammalian cells (HeLa, CHO, A549, or Cos cells) and stably inherited and expressed by cell progeny. This appears to be the first report of direct gene transfer from prokaryotes to mammalian cells. This type of DNA delivery by “suicidal” invasion of eukaryotic cells by bacteria has been designated “kamikazation. ”

4. Conjugative lhnsposons

A transposon (Tn) is a DNA segment that carries genes required for transposition and can move around on the chromosome and plasmid (Prescott et al., 1996). Conjugative transposons are discrete DNA elements that are normally integrated into a bacterial genome (Salyers et al., 1995). Since conjugative transposons were first discovered in Enterococcus faecalis (formerly called Streptococcus faecalis) strain DS16 (named Tn926 by Franke and Clewell (1981) and Gawron-Burke and Clewell (1982)) and Streptococcus pneumoniae (originally called the Qcathet elements and now Tn5253 by Shoemaker et al. (1980) and Ayoubi et al. (1991)), they have been found in numerous genera of Gram-positive and Gram-negative bacteria (Bertram et ul., 1991; Clewell, 1993; Scott, 1992, 1993; Salyers et al., 1995). Hence, they appear to be widespread in the bacterial world. Conjugative transposons are similar to (1)other transposons, as they excise from and integrate into chromosomal and plasmid DNA, but they appear to have a different method of excision and integration; (2) plasmids, as they have a covalently closed circular transfer intermediate and are transferred by conjugation, but the intermediate does not replicate; and (3) bacteriophages, as their excision and integration resemble those of temperate phages,

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but they do not form viral particles and are not transferred by phage transduction (Salyers et al., 1995). Based on current information, some properties of conjugative transposons can be summarized (Salyers et al., 1995; Salyers and Shoemaker, 1994): a. Size: range from 10 to >150 kb among different bacterial strains. b. DNA sequence: differs between bacterial strains. c. Mechanism of transposition: (1)the conjugative transposon is excised from the chromosome to form a covalently closed double-stranded circle of DNA, which is the transposition intermediate; and (2) the circular form can integrate either back into the chromosome or into a plasmid in the same cell (intracellular transposition), or it can transfer itself by conjugation and integrate into the genome of the recipient (intercellular transposition). However, the integration mechanisms are considerably different among the various DNA elements involved, although they share some common features: (1)their ends do not cross-hybridize with each other, which is different from standard transposons; (2) the excised circular form is the transfer intermediate, and it is transferred similarly to a plasmid from an internal oriT site, but the regenerated double-stranded form of the circle can integrate into the chromosome of both the recipient and the donor; and (3) they have the ability to mobilize plasmids by two means: the conjugative transposon can either integrate into the plasmid and produce a self-transmissible chimera, or it can mobilize plasmids by providing the mating pore, much as self-transmissible plasmids mobilize nonconjugative plasmids (Salyers and Shoemaker, 1994).

Because conjugative transposons have BHRs similar to the BHR plasmids, have relatively high frequencies of transfer to lo4 per recipient) under laboratory conditions, and are apparently involved in the spread of antibiotic-resistance genes among different bacterial pathogens and between pathogens and members of the resident microflora of the human body, they probably are important in gene transfer in the environment. A tetracycline-resistance gene (tetM), carried on Tn926, has been found in many natural isolates of Gram-positive and Gram-negative bacteria, suggesting that such gene transfer does occur (Salyers et a]., 1995) and that more studies on gene transfer mediated by conjugative transposons in the environment are needed. 5. Retrotransfer

This curious and unexplained phenomenon of reciprocal genetic exchange has been observed in numerous bacterial species (Mazodier and Davies, 1991; Perkins et al., 19941, and it has been mediated by

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IncP1, IncM, and some IncN plasmids, but not by plasmids of the IncW, IncFI, IncFII, or IncV incompatibility groups (Thiry et a]., 1984). This indicates that, although the process is not unusual, it is plasmid-specific and not a general property of conjugation systems. Although the mechanism of the retrotransfer process is not known, several models have been proposed. Mergeay et al. (1987) proposed a one-step model: retrotransfer occurred early in conjugation, and both partners simultaneously would gain a new plasmid from the other cell. Thus, bacterium A (the original donor) became a retrotransconjugant, and bacterium B (the original recipient) became a transconjugant. A two-step model for retrotransfer was suggested by Blanco et al. (1991): retrotransfer was not simultaneous but occurred after the conjugative plasmid from bacterium A had been transferred to bacterium B, after which the plasmid present in bacterium B before the transfer of the plasmid from bacterium A was retrotransferred to bacterium A. The final result is essentially the same as in the one-step model, in that each cell would now contain two plasmids. Perkins ef al. (1994) studied the kinetics of conjugation, retrotransfer, and mobilization between strains of Pseudomonas putida using plasmids pQKH6 and R300B. Retrotransfer occurred about 45 to 60 min after conjugation, but the process was neither delayed nor accelerated by breakage of the conjugation bridge (pilus) or by mixing transconjugants with the mating partners. Their results did not support either of the two models mentioned above and implied that another mechanism was involved. Hence, retrotransfer in natural environments may involve more than one mechanism to deal with different situations. Moreover, retrotransfer might enable bacteria that harbor large BHR plasmids in natural environments to capture foreign genes or small tra- plasmids from other bacteria, including GEMS (Perkins et a]., 1994).

B, FACTORS TIIAT AFFECT CONJUGATION IN THE ENVIRONMENT When studying conjugation in the environment, the biological and physicochemical factors of specific environments that affect this process must be considered (e.g., Day and Fry, 1992a; McIntire, 1992; Miller, 1992a; Stotzky and Babich, 1984, 1986; Stotzky et al., 1990, 1991; Wilkins, 1990). The biological factors include (1)surface (entry) exclusion: many conjugative plasmids encode products that interfere with the ability of a cell to pair and/or for DNA to move between cells carrying the same type of conjugative plasmid; (2) incompatibility: the inability of two closely related plasmids to coexist in the same cell; (3)

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restriction: a bacterial cell recognizes and degrades incoming foreign DNA by restriction endonucleases; (4) fertility inhibition: most conjugative plasmids inhibit their own transfer and that of other co-resident plasmids by repressing the expression of plasmid-encoded transfer functions; (5) expression: some foreign genes are not expressed or are expressed at different levels in new hosts; (6) mutation: mutation will affect the stability of a plasmid or the expression of its genes, as well as the structural integrity of plasmids; (7) lethal zygosis: killing of recipient cells caused by an excess of Hfr cells during conjugation, which is associated with metabolic changes in the recipient cell that could result from membrane alterations that accompany conjugation and that could result in a decrease in the number of viable recipient cells and an associated loss of recombinant transconjugants; (8) density factors: gene transfer is influenced by cell densities and the donorxecipient ratio; (9) physiological state: active bacteria may undergo conjugation with a higher frequency of recombinants than inactive cells; (10) survival: survival and growth of bacteria are usually reduced after inoculation into a nonsterile environment; and (11) microbial competition: the frequency of conjugation is usually decreased by an increase in competition from indigenous microbial populations. The physicochemical factors include temperature, nutrient status and energy sources, pH, water content, oxygen and Eh, electromagnetic radiation, ionic composition and concentration, surfaces, and time (Stotzky, 1989).

c.

CONJUGATION IN SITU

Because conjugation was thought to be the major mode of gene transfer in natural environments, most studies on gene transfer have concentrated on this mechanism. Conjugation has been demonstrated in many environmental settings. 1. Animal Systems

The conjugal transfer of different plasmids between bacteria of the intestinal tract and associated with the respiratory and/or urogenital tract was summarized by Roberts (1989). A conjugative transposon, TetM, was also found to be associated with the transfer and spread of antibiotic-resistance genes. These data indicated that both pathogens and the normal microflora could and did acquire antibiotic-resistance genes and plasmids in these habitats. Doucet-Populaire (1992)indicated that the streptococcal transposon Tn2545 could be conjugatively transferred from Enterococcus faecalis to Listeria monocytogenes and to E. coli in the gastrointestinal tract of gnotobiotic mice. Duval-Iflah et al.

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(1994) reported that recombinant plasmids that existed in donor strains of E. coli K12 could be transferred to wild-type E. coli strains from the human fecal flora (HFF) in the digestive tract of gnotobiotic mice. The HFF mice were initially axenic (germ-free) and received, intragastrically, 1 ml of a diluted fecal sample from a healthy volunteer and 1 ml of a culture of E. coli PG1 of human origin. As reviewed by Duval-Iflah (19921, most experimental studies of gene transfer in the guts of human beings and other animals have been mainly concerned with conjugation. Direct gene transfer from bacterial cells to mammalian cells by kamikazation was reported by Goussard et al. (1996). A variety of Gram-positive and Gram-negative bacteria are associated with insects. Jarrett and Stephenson (1990) detected conjugal plasmid transfer between strains of Bacillus tliuringiensis in the insects Galleria mellonella and Spodoptera littoralis. Armstrong (1992) reported that conjugation occurred among Enterobacfer cloacae, Erwinia herbicola, Klebsiella planticola, and Pseudomonas cepacia in the cutworm (Peridroma saucia). 2. Plants

Farrand (1989, 1992) demonstrated that conjugal transfer of bacterial genes can occur in plants. There is ample evidence that plant-associated bacteria harbor genetic elements that can be transferred by conjugation from one bacterium to another. The direct evidence for gene transfer by conjugation in planta has been documented in the bacterial genera Erwinia, Klebsiella, Pseudomonas, Rhizobium, and Xanthomonas (see Farrand, 1992; Stotzky and Babich, 1984). Members of the genus Agrobacterium can also transfer a portion of their genome into plant cells. This transferred DNA fragment can then be integrated into and be expressed in the plant chromosome. 3. Soil

In 1972, Weinberg and Stotzky demonstrated conjugal transfer of chromosomal genes from prototrophic to auxotrophic strains of E. coli in sterile soil (Weinberg and Stotzky, 1972). This was apparently the first report of conjugation in a natural habitat, and it not only showed the relation between bacterial genetics and microbial ecology but also led to the subsequent intensive studies of gene transfer in the environment. The transfer of chromosomal genes in E. coli by conjugation was further studied in nonsterile soil (Krasovsky and Stotzky, 1987; Stotzky and Krasovsky, 1981).The frequency of transfer was significantly lower in nonsterile than in sterile soils, indicating that the presence of the indigenous microbiota interferes with conjugal transfer. The results of

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these studies also indicated that the survival of donors and recipients and the frequency of chromosomal gene transfer increased as the soil pH was increased and that the frequency of conjugation was enhanced by the addition of the clay mineral montmorillonite. Conjugative or nonconjugative plasmid transfer in sterile and nonsterile soils has also been reported among and between introduced Gram-negative and Gram-positive bacteria, such as species of Alcaligenes (Mergeay et al., 1990), E. coli (Devanas and Stotzky, 1986, 1988; Devanas et al., 1986; Schilf and Klingmiiller, 1983; Trevors and Oddie, 1986), Enterobacter and Proteus (Stotzky, 1989; Stotzky et al., 1990), Klebsiella (Stotzky, 1989; Stotzky et al., 1990; Talbot et a]., 1980), Pseudomonas (Pertsova et al., 1984; Stotzky, 1989; Stotzky et al., 1990), Bacillus (van Elsas et al., 1987),and Streptomyces (Rafii and Crawford, 1988; Wellington et al., 1990). Furthermore, Henschke and Schmidt (1990) successfully transferred the plasmid of a laboratory strain of E. coli to indigenous bacteria in soil. Smit et al. (1991) showed, using a bacteriophage for counterselection of the donor, plasmid transfer from I? fluorescens to indigenous bacteria in soil, including species of Alcaligenes, Comamonas, Enterobacter, and Pseudomonas. Perhaps the best demonstration of gene transfer by conjugation in soil in situ was provided by the studies of Sullivan et al. (1995), which showed the apparent transfer of chromosomal genes for symbiosis from Rhizobium loti to nonsymbiotic rhizobia in a soil in New Zealand. R. loti had been used as an inoculant 7 years earlier on Lotus corniculatus, which is not native to New Zealand, and, therefore, the soil did not originally contain rhizobia capable of nodulating this legume. 4. Rhizosphere Lynch (1990) suggested that elevated microbial activity in the rhizosphere relative to the bulk soil could enhance the likelihood of gene exchange. He provided data for plasmid transfer by conjugation in the rhizosphere among the rhizosphere bacteria Agrobacterium, Pseudomonas, and Rhizobium. van Elsas and Trevors (1990)confirmed that the BHR self-transmissible plasmid RP4 could be transferred between introduced pseudomonads in the rhizosphere of wheat, and Smit and van Elsas (1992) showed that a plasmid from an introduced donor could be transferred into indigenous bacteria in the rhizosphere of wheat. This study also showed that donor counterselection methods are helpful when investigating gene transfer to indigenous bacteria by conjugation. 5. Fresh Water

Conjugal transfer of DNA has been studied in natural freshwater habitats, including lakes, ponds, and rivers (Saye and Miller, 1989).

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Gowland and Slater (1984) demonstrated conjugal gene transfer between E. coli strains incubated in environmental chambers in a pond; transconjugants were obtained after 192-360 h of incubation. O’Morchoe et al. (1988) studied the transfer of plasmids between strains of I! aeruginosa in lake water. The studies were performed in both the absence and the presence of the indigenous microbial population, and transfer frequencies were significantly reduced under the latter condition. 6. Marine Waters

Comparatively little is known about genetic exchange by conjugation in marine ecosystems. Stewart and Koditschek (1980) detected the transfer of tetracycline-resistance genes between E. coli donors and recipients in sterile seawater-sediment laboratory systems. Gauthier and Breittmayer (1990) also demonstrated the conjugal transfer of plasmids between E. coli strains in seawater and marine sediments. Goodman et al. (1993) showed conjugal transfer of a plasmid between marine bacteria in a model system using artificial seawater without added nutrients. In these experiments, the bacteria were starved for u p to 15 days before they were mixed on filters. Inasmuch as starvation is probably a normal condition in marine ecosystems, the results of these studies are ecologically significant. Sandaa and Enger (1994) reported gene transfer by conjugation in marine sediments under in situ conditions in microcosms containing a natural mixed bacterial population; a naturally occurring plasmid, pRAS1, from Aeromonas salmonicida that encoded multiple antibiotic resistance was transferred to bacteria in the sediments. 7 . Activated Sludge

Activated sludge is probably a good site for gene transfer by conjugation, as in this relatively nutrient-rich ecosystem the autochthonous microflora is likely to be well adapted to grow and survive and to act as recipients. Mobilization of catabolic plasmid pDl0 from I? putida and E. coli to indigenous bacteria in activated sludge has been reported, and the transconjugants were confirmed by comparing the restriction endonuclease digest patterns of the transferred plasmid with the original plasmid from donor cells (McClure et al., 1990). Doyle et al. (1995) have summarized other data that show that conjugation occurred among Pseudomonas strains in this habitat. 8 . Wastewater Treatment

Altherr and Kasweck (1982) reported the transfer of an antibiotic-resistance plasmid (R-plasmid) between E. coli strains in a sewage-treat-

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ment facility and observed that transfer was more efficient between 20 and 25°C than at 30°C. Mach and Grimes (1982) observed both intraand interspecies transfer of R-plasmids in a wastewater treatment facility. The transfer rates in situ were about 100-fold lower than under laboratory conditions. Gealt et al. (1985)used a transfer-proficient (Tra+) plasmid to mobilize the transfer of two transfer-deficient (Tra-) plasmids from one strain of E. coli to other strains of E. coli and to Enterobacter cloacae in artificial wastewater. Triparental mobilization of a Tra- plasmid was successfully done in a laboratory-scale waste treatment facility (Gealt, 1992; Gealt et al., 1988; Mancini et al., 1987). More transconjugants were found near the bottom of the primary clarifier of the facility, suggesting that the increase in surface area provided by concentrated particles in this quiescent setting enhanced conjugation (Gealt, 1992). 9. Epilith on Bale et al. (1987, 1988) demonstrated conjugal transfer of plasmid DNA between strains of I? aeruginosa on the surface of stones in rivers, as well as the transfer of mercury-resistance genes from indigenous epilithon organisms to a restriction-deficient strain of I? putida. The rate of transfer was enhanced linearly with an increase in temperature (Fry and Day, 1990). These studies were among the first to demonstrate both plasmid transfer in an unenclosed environmental system and interspecific plasmid transfer in controlled studies in situ.

D. CONCLUSIONS FROM CONJUGATION STUDIES There is little doubt that conjugation occurs in natural environments. It may be the major mode of exchange of genetic information among bacteria, at least under some environmental conditions. More diverse genetic elements, more complex mechanisms than those of other types of gene transfer, and a wider range of recipients are observed in conjugation. IV. Transduction

Transduction (also called genetic or phage transduction) was first observed in Salmonella typhimurium by Zinder and Lederberg in the , Zinder and Lederberg, 1952). early 1950s (Lederberg et ~ l . 1951; Transduction can be used to transfer genes and to construct genetic maps under laboratory conditions. Inasmuch as bacterial viruses or bacteriophages (phages) are the key elements in this process of gene

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transfer, it is necessary to have a clear understanding of these important elements and of the mechanisms involved in this process. A. BRIEFINTRODUCTION TO BACTERIOPHAGES 1. General Properties of Phages

Phages are predators or obligate intracellular parasites of host bacteria and have no independent metabolic ability. Phages were discovered independently by Twort and D’Herelle in 1919 (Duckworth, 1976).They have relatively simple structures in which their genetic material is packaged within an outer coat (capsid) that is composed mainly or entirely of protein subunits (capsomeres). The capsid protects the genetic material inside. Most phages have only DNA (usually doublestranded) as their genetic material, but some phages (which are nontransducing) may contain only RNA. Phages lyse bacterial lawns on plates to produce plaques, which are used to determine the concentration (titer) of the phages. Phage titer is expressed as the number of plaque-forming units (PFUs) in a given volume. 2. General Groups of Phages

Phages can generally be placed into the following morphological groups (Joset and Guespin-Michel, 1993: Prescott et al., 1996): (1) tailless icosahedral phages; (2) contractile tail-containing phages: (3) noncontractile tail-containing phages; (4) filamentous phages; and (5) phages with envelopes. Phages can also be categorized into three types according to their infection mechanism: (1)virulent phages always lyse the infected bacterial cell to release their progeny; (2) temperate phages can either enter the lytic cycle as virulent phages or enter the lysogenic cycle in which the phage genome is retained as a replicable entity (prophage) for a long time within the host; and (3) filamentous phages can attach to the sex pili of F’ Gram-negative bacteria and mediate production of progeny phage particles from the growing cell instead of lysing the cells, as the generated viruses are secreted through the cell wall (Duckworth, 1987). Both temperate and virulent phages can mediate transduction, although most successful transductions are mediated by temperate phages. The F+-specific filamentous phages can also mediate transduction of plasmid DNA (Ohsumi et al., 1978). 3. Types of Phage Life Cycles

There are two main types of phage “life” cycles.

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a. There are four steps in the life cycle of lytic phages. (1)Adsorption: phage particles (virions) adsorb on bacterial cells at specific receptor sites, which limits the host range of the phage to one or few species of bacteria. This step is affected by the ionic composition, pH, and temperature of the medium. It is possible for some cells to become resistant to specific phages during this step (Stent, 1963). (2)Injection: in this step, which is not well understood, the nucleic acid of the phage enters the host cell. In some phages, the tail may contract, acting as a microsyringe to inject phage nucleic acid into the cytoplasm of the host. The capsid remains outside the cell. Phage tails may also contain enzymes to weaken the cell envelope and, hence, facilitate the penetration process. (3) The latent period: after the viral nucleic acid is injected into the cells, no phages are released from the infected cells. During this period, phage proteins and nucleic acid are synthesized and assembled into nascent phage particles within the bacterial cell. Early in the latent period, which is called the eclipse period, no virions are present within cells. The rate of increase in the number of phages in the latent period is linear and not exponential. The length of the latent period is characteristic of individual phage species and is dependent on the metabolic state of the host and the ambient environmental conditions (Kokjohn and Miller, 1992). (4) The rise or burst period: during this period, infected cells begin to lyse and release mature virions. The burst of the cells is a very rapid process, and a single infected cell will produce a large number of progeny phages. The average number of viral particles released per infected cell is called the “burst size” and is characteristic of the phage. b. The life cycle of temperate phages can develop in one of two directions: (1)the lytic mode of development, described above, and (2) lysogenization, in which a temperate phage maintains its presence in a host cell without causing death of the cell and producing free infectious particles. Lysogenization is a latent infectious state. During this state, the relationship between the phage and its host is called lysogeny, which is defined as the indefinite persistence of the phage DNA as a prophage in the host cell without the production of phage particles (Kokjohn, 1989). Bacteria having the potential to produce phage particles are called lysogens or lysogenic bacteria. The latent form of the phage genome that remains in the host without destroying it is called a prophage. The prophage is usually integrated into the bacterial genome, but in some cases it exists independently; for example, prophages P1 of E.coli and F116 of l? aerugirzom are maintained as low copy-number plasmids (Miller et al., 1977; Sternberg and Hoess, 1983). Lysogens are immune to superinfection by the same (and sometimes another closely

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related) phage. This immunity usually does not inhibit the entry of superinfecting phage DNA, but it inhibits the transcription of its genome in the cell as the result of a phage repressor protein (e.g., the product of the cI gene for phage lambda (A)) that is provided by the resident prophage. This phage repressor protein also prevents the expression of the prophage genes by binding to the DNA of the phage and maintains the temperate life cycle of phages. Lysogeny is controlled by both the bacterium and the phage (Herskowitz and Hagen, 1980). If the cell is starving, the lysogenic state will be favored. If the cell is active, induction and the lytic cycle will be favored. 4. Lysogenic Induction

Lysogenic induction is the process by which phage reproduction is initiated in a lysogenized culture. Some environmental factors (e.g., ultraviolet light and other mutagens, changes in growth conditions) can cause a prophage to convert from the temperate cycle to the lytic cycle, probably because these factors result in a signal that ultimately leads to the destruction of the repressor protein. For example, in phage A, the lysogenic induction process starts with the interaction of the repressor protein encoded by the cl gene with the RecA protein of the host cell (Clark, 1973). The RecA protein is activated by the inducing agent and promotes cleavage of the repressor protein into two polypeptide fragments (Roberts and Devoret, 1983). The cleaved repressor protein cannot bind to the phage promoters and inhibit the transcription of the genes of the phage, and the vegetative life cycle of the phage is initiated when enough repressor protein is cleaved (Bailone et a]., 1979). Induction can apparently also occur spontaneously. 5. Lysogenic Conversion

In some situations, a change in the phenotype of the host cell (i.e., the lysogen) can be caused by a temperate phage. Such acquisition by a lysogen of new, phage-encoded, phenotypic properties is called lysogenic conversion (or phage conversion) (Prescott et a]., 1996). For example, diphtheria toxin will be produced only when Corynebacteriurn diphtheriae is lysogenized with temperate phage fi, because the toxin gene is carried by the phage and not by the bacterium. Lysogenic conversion may be important in the ecology of both a phage and its host and may have an effect on the potential for the transfer of genes by transduction in the environment. For example, some phenotypic properties encoded by a temperate phage may favor the survival and distribution of the host and, thereby, enhance the survival of the phage and the distribution of its genes.

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6. Archived Prophages

Another kind of prophage, called an archived prophage, is not inducible by ultraviolet radiation [see below) but can be induced by perturbations of purine metabolism in the host. This process is independent of the recA gene, and the host has no immunity to superinfection (Downs and Roth, 1987). Archiving has only been observed in S . typhimurium. The physical condition of the archived prophage DNA and its prevalence in and significance for gene transfer among bacteria in natural environments are not known. B. TYPESOF TRANSDUCTION

During the life cycle of a phage, bacterial genes may be incorporated into a phage capsid and injected into another bacterium, resulting in the transfer [transduction) of bacterial genes. There are essentially two types of transduction: generalized transduction and specialized transduction. Another type, capsduction, may belong to another system of gene transfer. 1. Generalized Transduction

In generalized or unrestricted transduction, any DNA fragment of a bacterial genome or plasmid can be transferred when the fragment is packaged within a phage capsid. Generalized transducing phage particles can be produced either during primary infection of a bacterial cell with a lytic phage or during induction of a prophage. After virulent or temperate phages enter the lytic cycle, phage DNA is replicated, and the DNA of the bacterial chromosome is cleaved into fragments at the pac site by nucleases. During the assembly stage, not only phage DNA can be packaged into the capsid, but random fragments of partially degraded chromosomal and plasmid DNA from the host can also be packaged in the place of phage DNA. Any single or group of bacterial genes may be incorporated into the phage at essentially equal frequency. The resulting phage lysates will contain both normal phages and generalized transducing phages. The transducing phages can inject the DNA from the bacterial chromosome or plasmids into another bacterial cell. However, they cannot initiate a lytic cycle in the recipients, and the transductants will not show immunity to superinfection, as they do not contain phage genes. Cotransduction may occur when two genetic loci are packaged in the same phage and transduced together into the recipient. Cotransduction can be used to measure the relative distance between the two genetic

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loci by the probability of their coinheritance. If the distance between the two genes is short, the probability of their coinheritance is high. 2. Specialized flansduction

In specialized or restricted transduction, a specific set of bacterial genes (adjacent bacterial sequences), as the result of imprecise excision of the prophage during induction of the lytic cycle, is introduced into another bacterium by a temperate phage. After the DNA of a temperate phage is injected into the potential donor cell, it is integrated into the bacterial chromosome at a defined region. The phage DNA then replicates with the host DNA and remains in the progeny cells of the lysogen as a prophage until induction occurs. After being induced to the lytic stage, the phage DNA normally is correctly excised from the bacterial chromosome, and the exact initial viral DNA molecule is reproduced. However, imprecise excision of the prophage from the host genome can occur at low frequencies, resulting in a transducing particle that possesses most of the phage genome as well as a portion of the bacterial genome immediately adjacent to the integration site. The transducing particles are then packaged, released from the donor cell, adsorbed on new recipient bacteria, the DNA is injected, and the bacterial genes may become stably incorporated in the chromosome of the new recipient. Only temperate phages have been shown to be capable of specialized transduction, and only genes close to the integration site of the phage are packaged and transduced, The most extensively studied example of specialized transduction is phage h of E. coli. The genome of this phage inserts into the host chromosome at attachment (aft) sites. The genes for galactose catabolism and biotin synthesis are located on either side of the attachment site on the chromosome of E. coli and can be transduced by phage h. The lysate resulting from the induction of lysogenized E. coli contains normal phage and a few transducing particles. This lysate is often called a low-frequency transducing lysate (LFT lysate), as it only contains a few transducing particles and results in a low frequency of transduction. Because the transducing phage may lose some genes necessary for its integration and reproduction during the excision process, the defective phage will need help from a normal phage (called a helper phage if it is in the same cell) to aid in integration and reproduction. After a defective phage is integrated into the host chromosome with the assistance of a helper phage, both phages are linked together on the same host chromosome, and the transductants are not stable. Eventually, a lysate containing equal numbers of defective phage and normal helper

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phage is generated after induction and excision. This is called a highfrequency transducing lysate (HFT lysate), as this lysate is very effective in transduction. During specialized transduction, a phenomenon called abortive transduction can occur. Transduced DNA fragments from a lysed lysogen that are not integrated into the endogenote of the new recipient can persist in the recipient but cannot replicate, although the genes on the transduced DNA fragment can be expressed (Stent, 1963). In colonies that develop from such transduced cells, only one of the daughter cells receives the transduced DNA in each cell division, resulting in a clone in which only some of the cells contain the transduced DNA fragment. The nonintegrated DNA fragment may be protected from degradation by an associated protein and may persist within the clone for many generations (Ikeda and Tomizawa, 1965). 3. Capsduction A transduction-like system, termed capsduction, has been demonstrated in Rhodobacter capsulatus (Joset and Guespin-Michel, 1993; Solioz et al., 1975). In this system, the structure of the gene transfer agent (GTA) is similar to that of a small virulent phage: the average diameter of the head is 30 nm, and the size of the tail ranges from 5 to 6 nm x 40 ? 10 nm. At least five major and three minor proteins comprise the capsid, which contains linear double-stranded DNA with an M, of 3.6 megadaltons (MDa). GTA particles are synthesized and released in one or two synchronized waves during the growth of GTAproducing cells. Because no phage-like functions (e.g., cell-killing, plaque-formation) and no plasmid-like structures have been found in the various GTA-producing strains, the GTA-encoding genes are probably located on the host chromosome. Although the modes of production and extrusion of this particle are not known, the GTA can mediate transduction through the steps of adsorption, injection, and homologous recombination that are characteristic of a phage. The transductionlike properties of GTA strongly suggest that the particles might originate from remnant genetic information of a putative phage, although it is possible that the information for capsduction could be originally bacterial (Joset and Guespin-Michel, 1993). Chiura and Takagi (1994) also reported that phage-like particles (PLPs) from Agrobacterium kieliense transferred genes coding for the utilization of 2,4-dichlorophenoxyaceticacid, and PLPs from F ~ Q V O ~ C I C terium spp. transferred genes for the synthesis of four amino acids. Their observations suggest that the PLPs produced by certain marine

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bacteria may be important newly discovered elements for generalized transduction in the environment.

c. FACTORS THAT AFFECT TRANSDUCTION IN THE ENVIRONMENT ‘Transduction has been shown to occur under laboratory conditions in over 51 bacterial species representing more than 25 bacterial genera (Kokjohn and Miller, 1992). Consequently, transduction is probably a widespread and common mechanism of gene transfer among bacteria. The packaging of genetic material in phage particles has many advantages under natural conditions, as it not only provides protection for the DNA from nucleases and adverse physicochemical environmental conditions, hut it may also represent an evolutionary survival strategy for bacterial genes (Stotzky et a]., 1990). Therefore, transduction is probably also an important method for gene transfer in natural environments. As transduction is an interactive process of gene transfer between transducing phages and host bacteria (donor and recipient, respectively), high concentrations of bacteria and phages are probably required. The numbers of phages and bacteria that have been detected in different types of soil, water (fresh, marine, coastal, estuarine, and waste), wet sludge solids, and hospital settings are more than sufficient to ensure transduction, indicating that transduction can occur in the environment (Kokjohn and Miller, 1992). Transduction in natural environments probably occurs in the following stages (Stotzky, 1989): (1)induction of a prophage to the lytic stage or successful infection by a lytic phage; (2) incorporation of a bacterial gene(s) into the new virion; (3) release of the virion containing the bacterial gene into the ambient environment; [4) infection of another bacterial cell with this virion; (5) incorporation, propagation, and expression of the original bacterial gene in the infected bacterium; (6) transfer of the new genetic information to the progeny of the infected bacterium; and (7) repeat of the lytic-prophage cycle to spread the new genetic information to other bacteria in the habitat. The biological and physicochemical environmental factors that can influence transduction in the environment are: 1. Phage Host Range

Because the adsorption of a transducing particle requires an interaction with a unique receptor on the surface of the recipient, the entry of transducing DNA is usually limited to only one or a few closely related bacterial species. This requirement for specific receptor sites is the

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major barrier to the broad environmental distribution of genes by transduction. Moreover, some phage-resistant strains have evolved by mutation within a phage-sensitive host population (Stent, 1963). However, numerous phages with host ranges that cross species and genetic boundaries have been reported (Coetzee, 1975; Col6n et al., 1972; DBnarie et a]., 1977; Goldberg et al., 1974; Green and Goldberg, 1985; Kaiser and Dworkin, 1975; Kelln and Warren, 1971; Murooka and Harada, 1979; Ruhfel et al., 1984; Thorne, 1968, 1978; Yu and Baldwin, 1971). In addition, some extraordinarily broad host range phages, such as PRRl and PRD1, can infect any Gram-negative bacterium that contains an IncP-1 plasmid, which encodes a specific receptor for phage adsorption and can be extensively transmitted and maintained in a large number of Gram-negative species (Olson and Shipley, 1973; Olson et al., 1974). Some of these BHR phages have been shown to mediate interspecific and even intergeneric transduction, suggesting that interspecific and intergeneric transduction may also occur in the environment. 2. Restriction-Modification System

This bacterial system acts to protect bacteria from infecting phages. Phage DNA is usually identified as foreign by endonucleases of the host and is degraded. However, some phages have developed anti-restriction mechanisms to avoid the degradation of their DNA by recipient bacteria (Krishnapillai, 1971; Kriiger and Bickle, 1983; Moffatt and Studier, 1988; Spoerel et al., 1979). Holloway and Krishnapillai (1975) showed that, in the case of phage F116 of I? aeruginosa, phage DNA and transduced bacterial DNA can be resistant to restriction by recipient cells. Some environmental factors, such as elevated temperature (Rolfe and Holloway, 1966) and UV radiation (Walker, 1984), can also inactivate the restriction-modification system of bacteria. Moreover, foreign DNA is seldom completely destroyed by the restriction system, and a few molecules of DNA usually escape degradation (Reanney et a]., 1982). 3. Immunity to Superinfection

The phenomenon of immunity to superinfection by temperate phages is similar to that of plasmid incompatibility, that is, both mechanisms can exclude similar or closely related foreign phages or plasmids from the same host cell. In some cases, an immunity to superinfection may be accompanied by phage conversion, which may increase the fitness of the recipient cells to the environment. Some lysogens can inhibit or eliminate adsorption of the superinfecting phage, whereas others, in-

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cluding l? aeruginosa lysogenic to phage F116, do not. Transduction can occur in a lysogen containing phage F l l 6 , but the transduced lysogen will not be killed by subsequent infection with another phage F116. When a lysogen containing phage F l l 6 was used as the recipient, the frequency of transduction obtained in situ was higher than with a nonlysogen (Saye and Miller, 1989; Saye et al., 1987b, 1989, 1990). Consequently, in this system, immunity to superinfection actually increased the frequency of transduction in the environment. 4. Lysogeny

Lysogeny has apparently been adopted by many bacteria as a form of existence and survival in the environment, as many bacteria isolated from nature have been shown to be lysogenic (Freifelder, 1987). Holloway (1969) even estimated that 100% of all natural isolates of P aeruginosa were lysogenic for at least one phage and that many of these bacteria contained several prophages. Lysogeny may be a method by which both phages and bacteria can be protected in the environment. Moreover, lysogens may be an important source of phage genomes in nature, and lysogenic hosts may have some advantages in some natural ecosystems. For example, phages containing determinants for resistance to antibiotics and heavy metals may be advantageous to lysogenic hosts in certain environments [Marsh and Wellington, 1994). However, the distribution and proportion of lysogeny by various phages in different natural environments remain to be established. 5. Molecular Size of Transduced DNA

Transducing particles usually package a single fragment of bacterial DNA that is about the same size as the parental phage genome. Appropriate maximal and minimal sizes of the transduced DNA are needed to package efficiently the DNA fragment into a specific phage capsid. If the molecular size of the transduced DNA is too big, the capsid cannot package it. If the size is too small, the DNA will not be packaged and transduced efficiently. When the molecular size of a plasmid was similar to that of the phage genome, transduction of that plasmid by phage F l l 6 of Pseudomonas aeruginoscl was more efficient than that of snialler plasmids (Saye et a]., 1987a). However, when the small plasmids of Staphylococcus aureus formed linear concatemers of larger molecular size than the individual plasmids within the bacterial cells, these larger plasmids could be transduced efficiently to recipient cells by phage $11 (Novick et d.,1986).

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6. Survival of Phages in the Environment

After phages are released from host cells into the environment, their “lifetimes” are finite, and they will eventually “die” if they cannot infect appropriate hosts. In some phages, inactivation (loss of infectivity) occurs rapidly (Saye et al., 1987b; Wiggins and Alexander, 1985), but the mechanisms involved and how representative these phages are of phages, in general, in natural environments remain unknown. However, free phages can adsorb on clay minerals (important components of soils and sediments), especially on montmorillonite, and on other particles, which can increase their survival and persistence in terrestrial and aquatic habitats (Babich and Stotzky, 1980; Bystricky et al., 1975; Duboise et d., 1979; Lipson and Stotzky, 1984, 1987; Schiffenbauer and Stotzky, 1982, 1983; Stotzky, 1980, 1985, 1986, 1989; Stotzky and Babich, 1984, 1986; Stotzky et al., 1981; Yin and Stotzky, 1997; Yin et al., 1997; Zeph et al., 1988). Such persistence would remain undetected (cryptic) until an appropriate host is infected and the genes carried by the phages are again expressed in the habitat. This needs to be considered in the assessment of the risks related to release of GEMS into the environment, as well as in the evolution of bacteria and phages. Yin and Stotzky (1997) reported that phage F116(Rms149) was detected in nonsterile soil more than 2 1 days after inoculation, as were transductants of I! aeruginosa RM242. Higher numbers of P aeruginom RM242(Rms149) were enumerated in soil amended with montmorillonite than in unamended soil. Moreover, the multiplication of phages is very efficient. For example, a single nondefective phage with a burst size of 100 could produce about lo8 progeny phages after four lytic cycles. These new transducing phages can transfer their genes to many host cells, resulting in large numbers of transductants. Saye et al. (1987b) suggested that lysogens may also continuously replenish the pool of transducing particles by spontaneous induction of prophages. The widespread distribution of lysogens may be important in maintaining the potential for transduction in natural environments. 7. Multiplicity of Infection (MOI) The MOI is the ratio of virus particles to potential host bacterial cells (phage:bacteria ratio). Differences in the MOI may change the frequency of transduction. For example, the frequency of in vitro transduction of E. coli W3110(R702) by phage P1 Cm cts increased as the MOI increased from 0.3 to 9.5 (Zeph et al., 1988). The optimal MOI for transduction may change under different conditions. For example, the optimal MOI

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was different when I? aeruginosa RM242 was transduced by phage F116(Rms149) in vitro and in soils, and when the transductants were selected on different selective media (Luria Agar (LA) and Pseudomonas Isolation Agar (PIA)) (Yin and Stotzky, 1997). Therefore, determination of the optimal MOI for a given condition is very important in studying transduction. Generally, a lower MOI reduces the probability of both transducing and infectious phage particles entering the same recipient cell (Miller, 1992b; Primrose et aJ., 1982), which may decrease the number of transductants killed and increase the frequency of transduction. Different phages have different optimal MOIs for transduction. Even at the optimal MOI, higher concentrations of transducing phages and bacteria should result in higher numbers of transductants (Yin and Stotzky, 1997), which is especially useful when a phage-bacterial system with a low frequency of transduction is studied in natural environments. 8. Concentration uf Phages and Bacteria

Because the frequency of transduction in natural environments is usually low, sufficient concentrations of bacteria and phages are required to assure successful transduction. For example, the number of transductants increased as the concentrations of phage and bacteria increased at the same optimal MOI (Yin and Stotzky, 1997). Numerous studies (see Kokjohn and Miller, 1992) have indicated that bacterial concentrations range from lo4 to lo9 colony-forming units (CFUs) per gram of soil in terrestrial habitats and from l o 3to lo9 CFU/ml in aquatic ecosystems; the concentrations of phage range from lo3 to 10l1 PFU/ml in aquatic ecosystems. Although no direct phage counts in soil by transmission electron microscopy (TEM) are available (the result, in part, of the difficulty in preparing soil samples suitable for TEM), phage concentrations in terrestrial habitats are undoubtedly higher than the lo2 to lo7 PFU/g of soil estimated by plating phages in soils on specific indicator host strains (Marsh and Wellington, 1994), as this estimation is low, based on studies of phage concentrations in aquatic ecosystems determined by TEM and plaquing (Marsh and Wellington, 1994). Nevertheless, the concentrations of both phages and host bacteria appear to be sufficiently high in different environments for transduction to occur. However, the concentrations may vary at specific sites in a natural habitat, and if the numbers of phages and bacteria are present at low levels, transduction may occur at undetectable levels. Therefore, the frequency of transduction may differ significantly from one location to another in the same habitat. Moreover, the physicochemical and biological properties of natural environments are variable and change-

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able, and these changes have an effect on the survival of bacteria and phages (Stotzky, 1986, 1989). However, not only have many bacterial genera adapted to such changes in natural habitats (Chet and Mitchell, 1976), but bacteria may congregate in specific regions of a habitat, especially at solid-liquid interfaces, and form microcolonies (Costerton et al., 1986; Stotzky, 1986), which would increase the probability of interaction between genetically distinct lysogens and nonlysogens and enhance transduction among these concentrated bacterial populations. particles/ml) have been found High concentrations of phages in many aquatic ecosystems (Bergh et al., 1989; B~rsheimet al., 1990; Ewert and Paynter, 1980; Ogunseitan et al., 1990; Proctor and Fuhrman, 1990), suggesting that phages are important not only in regulating bacterial population densities in these environments, but also that they exist in dynamic equilibrium with their host bacteria and, thus, greatly affect the microbial ecology (Kokjohn et al., 1991). As about 90% of all phages isolated from natural environments appear to be temperate (Freifelder, 1987),phages may have a more important and active role in gene transfer in nature than previously assumed. However, numerous questions need to be answered. For example: How high are the numbers of phages in terrestrial ecosystems? Are most phages in different natural ecosystems temperate or virulent? Do they persist in the free state for long or for just short periods but maintain a dynamic equilibrium by continuous lysis of host bacteria? Are free phages or lysogens a major reservoir of bacterial genes carried by phages in different ecosystems? 9. Type of Recipient Cells Even within the same species of bacteria, different strains can have different propensities for transduction, which can result in different frequencies of transduction, even in the same environment. For example, I? aeruginosa PAO1, but not I? aeruginosa PAT2, was transduced by phage F116 carrying plasmid RP4 in sterile soil (Stotzky et ul., 1990). In contrast, other closely related bacterial strains may have almost the same frequency of transduction, such as l? aeruginosa PA01 and I? ueruginosa RM242 transduced by phage F116(Rms149) in sterile and nonsterile soils (Yin and Stotzky, 1997). 10. Metabolic State of the Host

Although phages can bind on metabolically inactive host bacteria as well as on cell fragments, most successful and efficient transduction occurs when the recipient cells are in an active metabolic state (Stent, 1963). For example, significantly more transductants were detected 2 h

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after the addition of I! aeruginosa RM242 to nonsterile soil previously inoculated with phage F116(Rms149) than 2 1 days after addition of the bacteria (Yin and Stotzky, 1997). In many natural ecosystems, bacteria are not very metabolically active, and, therefore, transduction may not occur frequently. However, if the conditions of the ecosystem are changed to stimulate bacterial growth, such as the addition of nutrients and water, and the release of phage particles from lysogens is induced (e.g., by UV radiation, elevated temperature, enhanced nutrition), the active bacteria and the newly produced phages, as well as phages that are surviving as the result of adsorption on particles, may interact and result in enhanced transduction. In addition, some environmental conditions, such as an elevated temperature, will inactivate some restriction systems of the host and favor the stability of the transduced DNA. Consequently, transduction may be enhanced under favorable environmental conditions. 11. Temperature

In general, efficient transduction requires higher temperatures in the laboratory than in situ. However, although environmental temperatures are often lower than those used in the laboratory, they are seldom too low to prevent transduction. Transduction in natural environments has been observed at ambient temperatures in the laboratory, for example, at 25°C for the transduction of E. coli by coliphage P1 (Germida and Khachatourians, 1988; Zeph et a]., 1988) and of F! aeruginosa by phage FllG(Rmsl49) (Yin and Stotzky, 1997) in soil; and at ZOOC, and even 5OC, for the transduction of I! aeruginosa by phage F l l 6 in water (Aniin and Day, 1988; Saye et id., 1987a, 1990). Phages often have an optimal temperature for their adsorption and replication (Seeley and Primrose, 1980). This temperature usually reflects the ecological origin of the phage more than the optimal temperature for growth of the host bacterium (Kokjohn and Miller, 1992).Hence, in the environment, the optimal temperature for the adsorption and replication of the phage may have a greater effect on transduction than the optimal temperature for bacterial growth, suggesting that phages may have a more active and important role in transduction than the bacterial host, which appears to be only a passive recipient. 12. Composition and Concentration of Ions Phages often require adequate concentrations of specific cations, such as MgL+and (:a2+, for optimal adsorption on their host, possibly because the cations reduce repulsive electrostatic forces between the phage and 1982; Stent, the bacterium (Marsh and Wellington, 1994; Primrose et a].,

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1963; Stotzky, 1986). The isoelectric point (PI) of most viruses and bacteria is low (e.g., pH 2.5 to 4.0), and, therefore, both viruses and bacteria are net negatively charged at the pH of most natural environments and laboratory conditions (Lipson and Stotzky, 1984, 1987; Stotzky, 1986; Stotzky et al., 1981). Some phages will not infect their host when suspended in distilled water or at low concentrations of monovalent ions, and many phages are unstable in a minimal salt medium (Miller, 1992b). Although the presence of divalent cations increases the probability of contact between the host and phage and, thus, increases the frequency of transduction, adsorption can be reduced if the concentrations are too low or too high (Tolmach, 1957). Although Mgz+ and Ca2+ appeared to have a positive effect on the adsorption of phage F116 on I! aeruginosa RM242 and on the frequency of transduction (Yin and Stotzky, 1997), Primrose et al. (1982) stated that there are no convincing data to show a relation between the levels of divalent cations in a particular habitat and the divalent cation requirement for adsorption of phages isolated from that habitat. The relation between the levels and types of cations and the frequency of transduction in natural environments needs to be established.

13. p H The pH affects the growth and survival of bacteria and phages in the environment (Stotzky, 1989) and apparently also the frequency of transduction. Transduction of E. coli by phage P1 was higher in a soil with a pH of 7.9 than in a soil with a pH of 6.8 (Germida and Khachatourians, 1988). However, as these soils also differed in texture, organic matter content, and other physicochemical characteristics, differences in the transduction frequencies cannot be attributed solely to these differences in pH. In studies on transduction of I! aeruginosa by phage F116(Rms149)in soil (Yin and Stotzky, 1997),transductants were not detected when the pH of the soils was below 4.5, whereas transductants were detected in soils with a pH above 5.1. The frequency of transduction, as well as bacterial growth and phage survival, was also lower in various soil-clay mixtures that had a pH below 5.6 than in the same mixtures amended with CaC03 to a pH close to 7. Consequently, transduction may not occur easily in an environment in which the pH does not favor the growth of bacteria or the survival of phages, and it does not permit transduction. 14. Duration of Interaction Between Bacteria and Phages

The time of contact for optimal transduction between phages and hosts appears to differ for different bacteria and phages and under

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different conditions. In general, maximal transduction in vitro occurs within less than 30 min for most bacterial strains and phages. However, under environmental conditions, even with the same strains and phages, optimal contact times may he longer. For example, maximal transduction of E. coli by phage P1 in vitro occurred within 30 min, whereas in soil, transductants were not detected until 3 h after inoculation of the phage and the host, and maximal numbers of transductants were detected only after 24 h (Zeph ef a].,1988). Similar results were o5tained with the transduction of E! aeruginosn by phage F116(Rms149) in vitro and in soil (Yin and Stotzky, 1997). 15. Surfaces Surfaces in the environment may have positive or negative effects on transduction. Soil has a high content of solids, and phages may bind on clay and other particles, which can protect the phages from inactivation and prolong their persistence in the absence of their hosts (Stotzky, 1986, 1989). As mentioned earlier, clay minerals, as important components of soil, have a significant effect on the persistence and ecology of viruses in soil (e.g., Lipson and Stotzky, 1984,1987; Stotzky, 1986,1989; Stotzky et al., 1981, 1990). In general, free infective phages can persist in soil for relatively long periods (Williams et al., 1987). Even though free phage particles are in an unprotected state k e . , outside a host) in the environment, the nucleic acids of phages are in a protected state (i.e., inside capsids). Therefore, free transducing phages and their DNA may survive in natural environments under certain circumstances and retain the ability to infect and transduce host bacteria. Clay minerals and other surface-active particles can also act as concentrating surfaces for phages and bacteria, as well as for proteins and other nutrients (Stotzky, 1986), thus promoting the growth of hosts and increasing the probability of transduction. However, the high so1id:liquid ratio of soil also limits the movement of bacteria and phages, which may reduce the probability of contact between them and, hence, of transduction (Stotzky, 1989). The interactions of transducing phages and host bacteria with different surfaces appear to affect transduction. For example, the numbers of transductants enumerated in soil amended with various amounts of montmorillonite or kaolinite and inoculated with l? Qeruginoso and phage F l l 6 differed, and higher numbers of transductants were generally obtained in soil amended with montmorillonite than with kaolinite (Yin and Stotzky, 1997). In addition, different phages appear to have different affinities for and mechanisms of binding on clays. For example, phage F116 of I? aeruginosa, in contrast to phage P1 of E. coli, did

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not bind tightly on montmorillonite and kaolinite, and the adsorption of P1 was greater on montrnorillonite than on kaolinite (Yin et al., 1997); coliphages T1 and T7 had a greater affinity for montmorillonite than for kaolinite, T7 had a greater affinity for both clays than T1, and both coliphages had a higher affinity for both clays than for bacteria, including the host E. coli (Schiffenbauer and Stotzky, 1982, 1983). Similar differences in affinities and mechanisms of binding have been observed with various viruses of mammals (e.g.,Lipson and Stotzky, 1984, 1987; Stotzky, 1986, 1989; Stotzky et al., 1981, 1990). Although the water columns of aquatic habitats have a lower content of surfaces than soil, phages and bacteria also appear to concentrate on the surfaces of suspended particulate matter, where the probability of transduction is increased (O’Morchoe et al., 1988). Ripp and Miller (1995) found that both production of phage F116 and the transduction frequencies of l? aeruginosa in a freshwater environment increased in the presence of suspended particulate matter. 16. Solar Radiation

Because solar radiation may damage the DNA of bacteria and cause the induction of prophages to the lytic stage, it probably affects transduction. Saye and Miller (1989) demonstrated that the induction of prophage by solar UV radiation could occur in situ. More studies on the effects of solar radiation on transduction in the environment are needed.

D. TRANSDUCTION IN SITU Although transducing phages and their host bacteria are found in many different environments (e.g., Goyal et al., 1987; Kokjohn, 1989; Kokjohn and Miller, 1992; Miller, 1992b; Ripp et al., 1994),transduction in situ has been studied only to a limited extent. 1. Animal Systems

Velaudapillai (1960) demonstrated the effect of factors that control the synthesis of flagellar antigens on transduction between strains of Salmonella in live chick embryos and mice. Jarolmen et al. (1965) showed the in vivo transduction of S. aureus to tetracycline resistance by staphylococcal phage 80 in the kidneys of mice that were injected intravenously with a pathogenic strain of S. aureus and 6 days later with the transducing phage. Novick and Morse (1967) demonstrated transduction of erythromycin resistance between strains of S. aureus by phage 411 in the kidneys of mice that were injected intravenously with a

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lysogenic drug-resistant strain and a nonlysogenic strain. Freter et al. (1979) observed the transduction of E. coli to chloramphenicol resistance by phage h::Tn9 in the intestines of gnotobiotic mice. Baross et al. (1974) reported the transduction of the genetic determinant that encodes agarase activity between strains of Vibrio parahaernolyticus in the gills of oysters by phage P4. 2. Plants Kidambi et al. (1994) reported phage-mediated gene transfer in the phylloplane. Transduction was detected among strains of t! aeruginosa on leaves of bean and soybean. 3. Soils

Zeph et al. (1988) showed the transduction of chloramphenicol- and mercury-resistance genes to E. coli by phage P1 in sterile and nonsterile soils. The transductants were confirmed by heat induction of phage P1 Cm cts::Tn501 and with a biotinylated DNA probe (Zeph and Stotzky, 1989; Zeph et al., 1991). Germida and Khachatourians (1988) reported phage PI-mediated transduction to E. coli of genes for amino acid synthesis and tetracycline resistance in two nonsterile soils. Transduction of Streptomyces lividans by phage $C31 KC301 was also demonstrated in sterile and nonsterile soil (Herron and Wellington, 1990; Marsh and Wellington, 1992; Marsh eta]., 1993). Yin and Stotzky (1997) generally detected more transductants of P aeruginosa by phage F116(Rms149) in sterile than in nonsterile soil, in soil amended with montmorillonite than with kaolinite, and in soil in which the pH had been increased by the addition of CaC03. Although transduction was markedly affected by the pH of the soils, the sequence of inoculation of the host bacterium and the phage had only a minor effect. Transductants were detected u p to 2 1 days (the longest time evaluated) after the addition of I? aeruginosa RM242 or phage F116(Rms149) to nonsterile soil originally inoculated with either phage F116(Rms149) or P aeruginosu RM242, respectively. The phage in soil was enumerated directly by plating soil dilutions, without filtration or centrifugation, on lawns of antibiotic-resistant host bacteria on antibiotic-containing agars, which provided a simple and efficient quantitative method for enumerating phages in soil (Yin et al., 19971. Putative transductants were confirmed by growth on media containing various combinations of antibiotics and by restriction-enzyme digestion of isolated plasmid Rms149; that is, the expected antibiotic-resistance phenotypes were expressed on various antibiotic-containing media, and the plasmid was

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demonstrated in the presumed transductants (Yin and Stotzky, 1997). Plasmid Rms149 appeared not to be stable in strains of l? aeruginosa, and some rearrangement of DNA, which may have involved transposons, apparently occurred in this plasmid. 4. Aquatic Systems

Most studies on transduction in natural environments were originally performed in aquatic habitats. Transduction of both chromosomal and plasmid DNA of l? aeruginosa was demonstrated in lake water (Miller et al., 1992; Morrison et al., 1978; Ripp and Miller, 1995; Ripp et al., 1994; Saye and Miller, 1989; Saye et al., 198713, 1989,1990) and in river water (Amin and Day, 1988). Coliphage-mediated transduction of E. coli has also been observed in wastewater treatment facilities (Osmand and Gealt, 1988). Reciprocal transduction of chromosomal genes was ohserved between lysogens and nonlysogens and among lysogens of strains of I? aeruginma in a freshwater habitat; that is, both of the parental l? aeruginosa strains obtained some chromosomal genes from each other by transduction (Saye et al., 1990).

E. CONCLUSIONS FROM TRANSDUCTION STUDIES Transduction occurs in the environment. It may be as important, if not more so, than conjugation and transformation as a means of gene transfer in some environments, especially in structured ones with a high so1id:liquid ratio, such as soil, where cell-to-cell contact may not occur often and free DNA may not survive for long periods. The survival of phages is enhanced by their adsorption on clay minerals and other particles in soil and other environments, and the cryptic genes carried by the phages could be expressed after they infect appropriate hosts. V. Concluding Remarks

Some overall conclusions and implications can be derived from studies on the three major mechanisms of gene transfer (i.e., transformation, conjugation, and transduction) in natural environments. 1. Numerous data indicate that gene transfer occurs in the environment by all three mechanisms. 2. Gene transfer in two directions has been demonstrated by: (1)cellto-cell contact transformation; (2) retrotransfer in conjugation; and (3) reciprocal chromosomal transduction. Consequently, gene transfer by all these mechanisms could occur bidirectionally in the environment.

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3. There may be additional mechanisms for gene transfer among bacteria in nature. For example, protoplast fusion could be considered a form of gene transfer among bacteria in the environment; for example, streptomycetes can fuse their hyphae (anastomosis), and this can result in gene exchange (Rafii and Crawford, 1989); some Gram-negative bacteria produce vesicles derived from the bacterial outer membrane that can contain DNA, and such nuclease-resistant vesicles may have the ability to transfer the DNA by membrane fusion (Dorward and Garon, 1990). Transposition, in which specific mobile molecules of DNA change position within or among a bacterial genome and plasmids, may also be important in gene transfer in the environment, as indicated by reports of transposon-mediated conjugation (see Clewell, 1993: Salyers et d., 1995). Consequently, transposition should not be considered only as a mechanism that mediates intracellular gene transfer, as some environmental conditions may stimulate transposon movement and result in both intracellular and intercellular gene transfer. Although electroporation is usually considered to be a laboratory method for inserting genes into both prokaryotic and eukaryotic cells, lightning in natural environments may electroporate indigenous cells. 4. New definitions of the three major mechanisms of gene transfer may be needed in the future. Even now, the traditional definitions of these three mechanisms are gradually becoming more and more ambiguous as the result of such discoveries as cell-to-cell transformation, capsduction, transposon-mediated conjugation, and retrotransfer. Considering the rapid developments in the area of gene transfer, new explanations, mechanisms, phenomena, and even discoveries can be expected. 5. Interactions and synergy among and between the mechanisms of gene transfer are possible in the environment. Each mechanism is not entirely independent but may interact with others and may be used alternatively by the same bacteria or phages, depending on the environmental conditions. Bacteria in the environment live as mixtures and in a community rather than as single species or strains, as on an agar plate or in a test tube in the laboratory. Therefore, several mechanisms of gene transfer may be acting simultaneously in a mixed bacterial community, and ambient environmental conditions may favor one or another mechanism. When the environmental conditions change, the mechanisms of gene transfer may also change. For example, a bacterium may receive a novel gene via one of the mechanisms under one environmental condition and then use another mechanism to transfer the gene to other bacteria under other conditions. In this way, GEMS could transfer their genes to indigenous microorganisms, which could retain

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them for some time and subsequently transfer them to other indigenous bacteria in the same or a different environment. The GEMs could also receive genes from indigenous microorganisms that may help the GEMs to adapt to and survive in the environment into which they have been introduced. Hence, a bacterium capable of transferring its genes by more than one mechanism might be able to use the most favorable one under a specific environmental condition. Gene transfer in nature is probably much more complex than originally assumed, which increases the difficulty and complexity of assessing the consequences of the release of GEMS but which may increase the possibilities for microbial diversity and evolution. Gene transfer among bacteria has been studied for over half a century in many laboratories around the world, but its importance and the mechanisms involved in natural environments still need further investigation. The following suggestions may be helpful for the further study of gene transfer in these environments. 1. Most studies in this area have been carried out under enclosed, anthropocentric, and controlled conditions in microcosms or mesocosms containing material sampled from the environment. Studies now need to be conducted in situ in open environments and under natural conditions. However, the risks associated with the release of GEMS to the environment must be considered. 2. In most experiments, specific donors (whether cells, phages, or DNA) and recipients have been added to the environment in which gene transfer is being studied. More studies should focus on gene transfer among indigenous bacteria and between introduced and indigenous bacteria. 3. The number of model systems used in studies on the mechanisms of gene transfer in the environment has been limited, More model systems, especially with organisms relevant to specific habitats, should be intensively studied. 4. The physicochemical and biological factors of specific environments that regulate the different mechanisms should be determined and related to the mechanisms. 5. The interactions between the mechanisms and their relation to specific environments need to be studied and elucidated. 6. The actual frequencies of gene transfer by the various mechanisms in the environment need to be determined. 7. The importance of horizontal gene transfer to the evolution of microorganisms in nature needs to be determined. 8. The primary mechanisms of gene transfer in different environments need to be determined.

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Salyers, A. A,, and Shoemaker, N. B. (1994). Broad host range gene transfer: Plasmids and conjugative transposons. FEMS Microbiol. Ecol. 15, 15-22. Salyers, A. A,, Shoemaker, N. B., Stevens, A. M., and Li, L.-Y. (1995). Conjugative transposons: An iiniisual and diverse set of integrated gene transfer elements. Microbial. Rev. 59, 579-590. Sandaa, R.-A., and Enger, 0. (1994). Transfer in marine sediments of the naturally occurring plasmid pRAS1 encoding multiple antibiotic resistance. Appl. Environ. Microhiol. 60, 4234-4238. Saunders, C. W., and Guild, W. R. (1981). Monomer plasmid DNA transforms Streptococc u s pneumonine. Mol. Gen. Genet. 181, 57-62. Saye, D. J., and Miller, R. V. (1989).The aquatic environment: Consideration of horizontal gene transmission in a diversified habitat. In “Gene Transfer in the Environment” (S. B. Levy and K.V. Miller, eds.), pp. 223-259. McGraw-Hill, New York. Saye, D. J., Kokjohn. T. A., and Miller, R. V. (1987a). Pseudomonos aeruginosa phage US1 suppresses the Les- phenotype. 87th Genl. M f g . Am. SOC.Microbiol., M-11, p. 238.

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Saye, D. J., Ogunseitan, O., Sayler, G. S., and Miller, R. V. (1987b). Potential for transduction of plasmids in a natural freshwater environment: Effect of plasmid donor concentration and a natural microbial community on transduction i n Pseudomonas aeruginosa. Appl. Environ. Microbiol. 53, 987-995. Saye, D. J,, Replicon, J., and Miller, R. V. (1989). F116L-mediated transduction of the Pseudomonas aeruginosa chromosome in a freshwater environment. 89th Genl. Mtg. Am. SOC.Microbiol., Q-149, p. 354. Saye, D. J., Ogunseitan, O., Sayler, G. S., and Miller, R. V. (1990). Transduction of linked chromosomal genes between Pseudomonas aeruginosa during incubation in situ i n a freshwater habitat. Appl. Environ. Microbiol. 56, 140-145. Schiffenbauer, M., and Stotzky, G. (1982). Adsorption of coliphages T I and T7 to clay minerals. Appl. Environ. Microbiol. 43, 590-596. Schiffenbauer, M., and Stotzky, G. (1983). Adsorption of coliphages T I and T7 to host and non-host microbes and to clay minerals. Curr. Microbiol. 8, 245-259. Schilf, W., and Klingmuller, W. (1983). Experiments with Escherichia coli on the dispersal of plasmids in environmental samples. Recomb. DNA Tech. Bull. 6, 101-102. Schlegel, H. G. (1986). “General Microbiology,” 6th ed. Cambridge Univ. Press, Cambridge. Scolnik, P. A,, and Marrs, B. L. (1987). Genetic research with photosynthetic bacteria. Annu. Rev. Microbiol. 41, 703-726. Scott, J. R. (1992). Sex and the single circle: Conjugative transposon. J. Bacteriol. 174, 6005-6010. Scott, J. R. (1993). Conjugative transposons. In “Bucillussubtilis and other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics” (A. L. Sonenshein, J. A. Hoch, and R. Losick, eds.), pp. 597-614. American Society for Microbiology, Washington, DC. Seeley, N. D., and Primrose, S. B. (1980). The effect of temperature on the ecology of aquatic bacteriophages. J. Gen. Virol. 46, 87-95. Seto, H., and Tomasz, A. (1976). Calcium-requiring step in the uptake of deoxyribonucleic acid molecules through the surface of competent pneumococci. J. Bacteriol. 126, 1113-1118. Shoemaker, N. B., Smith, M. D., and Guild, W. R. (1980). DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in pneumococcus. Plasmid 3, 80-87. Smit, E., and van Elsas, J. D. (1992). Methods for studying conjugative plasmid transfer in soil. In “Genetic Interactions among Microorganisms in the Natural Environment” (E. M. H. Wellington and J. D. van Elsas, eds.), pp. 113-126. Pergamon, Oxford. Smit, E., van Elsas, J. D., van Veen, J. A., and de Vos, W. M. (1991). Detection of plasmid transfer from Pseudomonas fluorescens to indigenous bacteria in soil by using bacteriophage R2F for donor counterselection. Appl. Environ. Microbiol. 57, 3482-3488. Smith, H. O., Danner, D. B., and Deitch, R. A. (1981). Genetic transformation. Annu. Rev. Biochem. 50,41-68. Solioz, M., Yen, H. C., and Marrs, B. (1975). Release and uptake of gene transfer agent by Rhodopseudomonas capsulatu. J. Bacteriol. 123, 651-656. Sparling, P. F. (1966). Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92, 1364-1371. Spencer, H. T., and Herriott, R. M. (1965). Development of competence of Haemophilus influenzae. J. Bacteriol. 90, 911-920. Spoerel, N., Herrlich, P., and Bickle, T. A. (1979). A novel bacteriophage defence mechanism: The anti-restriction protein. Nature 276, 30-34.

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Breathing Manganese and Iron: Solid-state Respiration KENNETHH. NEALSON Center for Great Lakes Studies University of Wisconsin at Milwaukee Milwaukee, Wisconsin 53204

BRENDALITTLE Naval Research Laboratory Stennis Space Center Stennis, Mississippi 39529

I. Introduction 11. Respiration: Organismal and Environmental A. Aerobic Respiration B. Anaerobic Respiration C. Environmental Respiration 111. Metal-Reducing Bacteria in Captivity IV. Reduction of Metals by Iron and Manganese Reducers A. General Features B. Biochemistry of Metal Reduction C. Regulation of Metal Reduction D. Products of Metal Reduction V. Electron Transport In Metal Reducers A. Cytochromes B. Quinones VI. Metal-Reducing Bacteria in Natural Environments A. Corrosion B. Release of Adsorbed Pollutants C. Clay Reduction D. Bioremediation VII. Summary References

I. Introduction

This review concerns a relatively new concept in microbial metabolism: the use of solid metal substrates by bacteria that grow anaerobically, using iron and manganese oxides as oxygen substitutes for respi2 13 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 45 Copyright D 1997 by Academic Press All rights of repruduction in any form reserved. OOfi5-2164/97 $25.00

214

KENNETH H. NEALSON AND BRENDA LITTLE

ration. Recognition of the organisms and their metabolism is new, but the knowledge that such respiration occurs in the environment has been taught for many years in environmental science and oceanography. Measurements of carbon turnover and porewater chemistry have demonstrated that iron and manganese are potent oxidants of organic matter in sediments (Froelich et a]., 1979) and anoxic water columns (Nealson et al., 1991). Data from these environments [Fig. 1) indicative of some kind of anaerobic respiration led several groups to attempt isolation of metal-reducing bacteria (MRB). In this review the concept of respiration as an organismal and environmental process is discussed, with attention to how geochemical (environmental) data suggest the presence of MRB. There follows a discussion of the current state of knowledge concerning MRB, the problems they pose, and the potential uses they may represent.

II. Respiration: Organismal and Environmental A. AEROBIC RESPIRATION

Aerobic respiration is the process whereby electron transport occurs from a fuel (reduced compound) to oxygen, and in the process protons are pumped across a membrane to create a chemiosmotic gradient, or proton motive force (PMF). The PMF is then used to generate biologically useful energy in the form of adenosine triphosphate (ATP), or to run other cellular processes directly. For eukaryotes the oxidant is molecular oxygen, and respiration is usually assessed by direct measurement of oxygen consumption. Most readers should be comfortable with this concept of respiration. In general, the higher forms of life on earth use organic carbon as an energy source and molecular oxygen as the oxidant to burn the organic fuel. With a few notable exceptions, eukaryotes cannot survive for long periods in the absence of molecular oxygen; their metabolism is geared to aerobic respiration, and in its absence the organisms suffocate. There are some exceptions: a few fungi exist anaerobically via fermentation, and some anaerobic protists (ciliates and flagellates) have anaerobic bacteria as symbionts and are capable of exploiting anaerobic niches via these partners. The concept of breathing is, of course, intimately connected to this view of respiration, as oxygen is a gas, and many eukaryotes have special organs specifically for the exchange of gas and the associated breathing process.

LAKE MICHIGAN (FreshwaterSediment)

BLACK SEA (Marine Basin) % max

Yomax

I 3-4

50

100

I

I

1

02

sod'

.. MnTT II

-

-t

5

k

10

Fett

E I

I

k W

W 0

a

15

20 NOZ-

10.3pM; NH4+ = 120 pM;SO4' = 300 pM; CHI = 300 pM

NO2-= 10.1 pM; NH~+=30pM;SO~==25rnM;HiS=100pM

FJG.1. Gradients of electron donors and/or acceptors in anoxic stratified environments. This diagram shaws porewater nutrients from freshwater (Lake Michigan) sediment on the left, and water column measurements of the same nutrients horn a stratified marine environment [Black Sea). The numbers are as a percentage of the maximum value seen for such environments, with the 100% values shown at the bottom.

216

KENNETH H. NEALSON AND BRENDA LITTLE TABLE I CLASSICAL ELECTRONACCEPTORS FOR B ACXKRIAL RESPIRA.I.ION

Chemical statc

ProkaryProduct

otic

Oxygen

co,

Water (H20) Methane (CH,)

+ +

Nitrate (NO,) Nitrite (NO,)

Nitrogen (N,) Nitrogen (N,)

+ +

Sulfate (SO:-) Polysulfide (So) sulfite SO,^-)

Sulfide [H,S) Sulfide (H,S) Sulfide (H2S)

+ + +

TrimethylamineN-oxide (TMAO)

Trimethyl nniine

b .

Dimethylsulfoxide (DMSO]

Dimethylsulfide

+

Fumarate

Succinate

+

Glycine

Acetate

+

Oxidant

Gases

Solutions

Eukaryotic

+

B. ANAEROBIC RESPIRATION

In marked contrast to the eukaryotes, prokaryotes are notorious for survival under anoxic conditions, with a major mode of metabolism being that of anaerobic respiration. In this metabolic mode, any of a variety of inorganic and organic compounds may be used as “oxygen substitutes” for the oxidation of organic carbon: some are dissolved gases, while others are dissolved salts of solids (Table I). The concept of breathing is somewhat altered in these situations, as the prokaryotes have no special organs or organelles for gas exchange. However, the concept of respiration with dissolved electron acceptors other than oxygen has been known for many years and has offered no particular conceptual problems to biologists or biochemists. Thus, both the substrates shown in Table I and the organisms that use these substrates for anaerobic respiration are well known (see Zehnder, 1988).

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

217

C. ENVIRONMENTAL RESPIRATION

In many physically stabilized environments, where mixing and convection are minimal phenomena, gradients of chemicals form due to the activity of microorganisms, and quite often the gradients are the direct result of respiratory activities. Concentration profiles show oxygen depletion for both a Lake Michigan sediment and the Black Sea stratified water column (Fig. 1).In the freshwater sediment, anoxia occurs about 2 cm below the aerobic sediment-water interface. From that point downward, breakdown of organic carbon is accomplished either via fermentation or anaerobic respiration, and environmental gradients reflect these processes. For example, after oxygen depletion, a zone of nitrate and nitrite depletion develops, followed by zones of manganese and iron reduction, and finally a zone of methane production (COz reduction). In the Black Sea water column, oxygen depletion occurs at approximately 50 m, followed by zones of nitrate, nitrite, manganese, and iron reduction. In marine systems, however, sulfate reduction, rather than methanogenesis, predominates, leading to the appearance of sulfide in the deep zones. In lake systems, where sulfate concentration is low, sulfate is rapidly consumed, and the remaining organic carbon is disposed of via COz reduction to methane. Thus, the high concentration of sulfate in seawater (Table 11) leads to sulfur-dominated marine systems as compared to their methanedominated freshwater counterparts. Figure 1 demonstrates a fact known by marine chemists for many years and discussed in detail by Froelich et al. (1979); namely, that respiration observed in the environment involves some components not normally considered by bacteriologists-that is, the role of metals as oxidants of organic carbon. In both systems shown in Fig. 1,there is an appearance of MnZ+and Fez+, and for most stratified environments throughout the world, similar profiles can be constructed (Davison, 1993; Nealson and Saffarini, 1994). The profiles are a measure of the respiratory activity of the environment-oxidizing equivalents that are being reduced at the expense of the oxidation of organic carbon in nature. It was observations like these that led to the interest by microbiologists in organisms that could respire solid metal substrates. Arguing from general principles, one might expect that manganese or iron could act as an electron acceptor for the oxidation of organic matter for two reasons. First, both have redox potentials that should allow them to be utilized in preference to many other electron acceptors; second, both are often found in abundance in sedimentary environments (Table 11).The redox potential of manganese is near that of nitrate,

218

KENNETH H. NEALSON AND BRENDA LITTLE TABLE I1 COMI'AKISON OF

Electron acceptor

ELECTRON ACCEPTORS AVAILABLE IN THE ENVIRONMENT

p") [W]"

Natural ahundanre (pM)

Electron acc/cm;

Free energy (kJ/M gliicosc)

0 2

+13.75

300'

4.3

-3190

NO; Mn4+ Fe

+12.65

10-20'

n0.85

-3030

+8.9

<1 to >1000d

e2.6

-3090h

-0.80


4

-1410'

so;-

-3.5

112

-380

1

-350

'+

>inoo"

250 (FW) 28000 (Mar)P

CO,

-4.13

Variahld

"This is the electron activity for unit activities of oxidant and reductant at neutral pH, as calculated by Zchndcr and Stumm (1966). "'l'he solubility of oxygcn is tcmpcraturc sensitive. but this is a rcasonable number for natural walers 'Nitrate is usually quite low, with these numbers representing high values, except in polluted areas. dBoth Mn a i d Fe form iusoluble oxidized tornis, so thal in water coluinns they tend to be very low (less than 1 pM), hut in sediments they can reach 111714values or Iiigher. "Sulfate ~:~inr:entration varies widely in trashwater e n v i n i n ~ ~ ~ el,ul ~ i l susually does not exceed 200-300 pM, while in marine environments i t lypic:ally is at 28 mM. /COz varies widely, depending 011 alkalinity, nrgmic carlmn inpul, eti: ,?Thisnumber is based on the oxitlalion ol"'standard" organic carbon, as discussed by Froelich et a / . (1979).

l q . 1 11s ' iitiinIx:r can wry, ~lnp~:iiding on the Mn oxide (Nnnlson and Saffarini, 1994). Wiis numher can vary, dopcnding on thc Fc oxide (Nealson and Saffarini, 1994).

while that of iron is lower, hut still significantly above that of sulfate. Thus, in studies of environmental respiration, King (1990) demonstrated that addition of iron or manganese oxides inhibited the respiration of sulfate. While indirect effects could have led to the same results, direct interaction of these oxidants with bacteria could not he dismissed. Using different approaches, several workers have attempted to assess the importance of iron and/or manganese in environmental respiration, and in some cases these metals are major redox components. For example, Aller (1990) and Aller et al. (1991) showed that oxidized manganese was the major electron acceptor in sediments fiom the Amazon shelf, often accounting for 70% or more of the organic carbon that was respired. Similarly, Canfield ef al. (1993) have shown that Mn recycles many times in offshore sediments and can account for

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

219

virtually all of the organic carbon oxidized in some sediments. Aguilar and Nealson (1994) measured the flux of reduced Mn2+from sediments of Oneida Lake (NY) and calculated the rate of oxidation of carbon oxidation, using a stoichiometry of approximately 2.5:l (Mn2+produced per COz produced, as shown in Table 11). The authors calculated that a few percentage points of the annual primary productivity of this eutrophic lake was remineralized via Mn4+oxidation or environmental respiration of this metal. In summary, the study of “environmental respiration” led to the discrepancy between the known “classical” oxidants for respiration (Table 1) with those observed to be active in the environment (Fig. 1). While Mn4+and Fe3+reduction were major environmental processes, there were no known bacteria that could live by respiration of iron or manganese. Even now, the concept of dissimilatory metal reduction is not common in microbiology textbooks. The reason for this discrepancy can be traced to the beliefs of bacterial physiologists that oxides and oxyhydroxides of iron and manganese are solids, and that solids are unavailable for direct reduction by bacteria. Thus, any interactions that occurred were assumed to be via indirect reduction by soluble reductants: the reduction of the metal oxide by an indirect, nonenzymatic process. There is little doubt that such indirect reactions can and do occur (Stone, 1987a,b),as shown for the reduction of Mn(1V) by sulfate reducers (Burdige and Nealson, 1986) or iron reducers (Myers and Nealson, 1988b), but there is also no doubt that dissimilatory reduction is a process common to several groups of bacteria and of possible importance in a variety of environments and processes. It is these organisms, the dissimilatory metal-reducing bacteria (MRB or DMRB), and their metabolism that will be discussed in this review-organisms that have found a niche on solid metal oxide surfaces, where they rapidly reduce the metal oxides and grow at the expense of organic carbon oxidation.

Ill. Metal-Reducing Bacteria in Captivity

In 1988, three publications reported dissimilatory reduction of iron and/or manganese by pure cultures of bacteria. The growth of the facultative anaerobe Shewanella putrefaciens (a.k.a. Alteromonas p u trefaciens; MacDonell and Colwell, 1985) on iron (DiChristina et a]., 1988) and manganese (Myers and Nealson, 1988a) was reported, and some aspects of its anaerobic versatility were presented. Earlier reports (Obueckwe et al., 1981; Semple and Westlake, 1987) suggested that a

220

KENNETH H. NEALSON AND BRENDA LITTLE TABLE 111

PHYLOCENETIC AFFILIATIONS OF METAL REDUCERS Archaea

No known representatives

Bacteria Gram-positives

Bacillus spp. Bacillus infernus Bacillus spp. SG-1

R u s h et a]., 1994 Boone et al., 1995 DeVrind eta]., 1986

Shewanella putrefaciens Shewanclla alga Ferrirnonas baleurica Aerornonas spp.

Myers and Nealson, 1988a Cnccavo ef a].,1997 Rosello-Mora et al., 1995 Nealson, unpublished

Geobacter rnetallireducens Geobacter acefoxidans Desulfurornonas acetoxidans Pelobncter carbinolicus Desulfurornusa spp.

Lovley eta]., 1993a,b Lonergan et al., 1996 Roden and Lovley, 1993 Lonergan et a/., 1996 Lonergan et a[., 1996

Geospirillum barnesi

Lonergan et al., 19%

Geothrix fermentens

Coatesh

Geovibrio ferrireducens

Caccavo et a]., 1997

Gram-negatives gamma'

delta"

epsilon' novelh novel"

'Among the domain bacteria, three groups of the proteobacteria have members that are metal reducers. "IJnpublished results cited in Lonergan et ol., 1896.

strain of this same species was a dissimilatory iron reducer, but no demonstration of growth at the expense of metal reduction was made. Lovley and Phillips (1988) reported the isolation of Geobacter metallireducens (then referred to as GS-15), an obligate anaerobic iron reducer that grew with Fe3+ as the sole electron acceptor and actively reduced Mn4+. For several years, these two organisms were the only two dissimilatory metal reducers studied, but the situation has changed markedly in the past few years, with the isolation of many new strains and species. A summary of known metal reducers (Table 111) shows their diversity and their phylogenetic affiliations. This field is in its infancy, and one can expect that this already broad group will grow substantially.

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221

The largest group of metal reducers known to date are obligate anaerobes that can be placed in the Geobacter group, as defined by Lonergan and colleagues (1996). This group includes a number of bacteria, most of which share the ability to reduce elemental sulfur to sulfide. The group is in the delta group of the Proteobacteria, and is aligned with sulfate-reducing bacteria (SRB) such as Desulfovibrio spp., some of which have also been reported to be iron reducers (Coleman et al., 1993), although not proven to grow at the expense of iron reduction. The major work on biochemistry and physiology has been limited to G. metallireducens (Lovley et al., 1993a). G. metallireducens grows with lactate, acetate, and a variety of other carbon sources, requires cell contact with solid iron oxides to effect reduction, and can reduce a number of other inorganic electron acceptors, including nitrate and U6+ (Lovley et al., 1991). S. putrefaciens, and a closely related species Shewanella alga (Caccavo et al., 1992), are facultative organisms that grow not only on oxygen and metals as electron acceptors, but display a remarkable versatility-some strains are able to reduce and grow on 10 or more different electron acceptors. Many MRB strains can also reduce elemental sulfur (polysulfide), an unusual ability for an aerobe (Perry et al., 1993; Moser and Nealson, 1996), and produce sulfide from the anaerobic reduction of thiosulfate. These organisms require surface contact for the reduction of solid metal oxides (Arnold et al., 1988), colonize the surfaces during reduction (Figs. 2 and 3; Little et al., 1997a), and have markedly different rates of metal dissolution on different metal oxides (Burdige et a]., 1992; Little et al., 1997a). Unlike G. metallireducens, S. putrefaciens is unable to grow on acetate anaerobically, but it does grow with formate as the sole source of carbon and energy (Scott and Nealson, 1994), and utilizes H2 as an energy source for metal reduction (Lovley et a]., 1989a). IV. Reduction of Metals by Iron and Manganese Reducers

A. GENERALFEATURES

Although the study of dissimilatory microbial metal reduction is a new field, some general features are emerging. Several metals reportedly reduced by MRB are shown in Table IV, and some of these may be environmentally significant for both biodegradation and bioremediation (Fredrickson and Gorby, 1996). Whether catalysis reactions are specific and/or independent is, in general, not known, although for S. putrefaciens mutants have been obtained that are deficient in

222

KENNETH H. NEALSON AND BRENDA LITTLE

F I G . 2. Imagns of cells of Shewonella putrefaciens growing on manganite (MnOOH). Top panel (a) shows uninoculated MnOOH imaged by environmental scanning electron microscopy (ESEM].Middle pariel (b) shows ESEM image of MnOOH (after 90 h of growth with S. putrefaaens MR-4) with bacteria that are obscured by the presence of extracellular polymer. Bottom panel (c) shows the same field as in the middle panel, but after sample drying (polymer dehydration) and imaging by standard scanning electron microscopy (SEM).

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22 3

224

KENNETH H. NEALSON AND BRENDA LITTLE TABLE IV METALS REDIJCED BY IRON/MAN~;ANESE-~DUCINC BACTERIA

Metal Fe3+

Form or mineral name Soluble chelate (EDTA, NTA)" Fe(OH)? (ferrihydrite)

Reference

FeaO4 (magnetite) Fe-rich clays (smectite)

Arnold et al. (1986) Arnold et 01. (1986) Roden and Zachara (1996) Little e t a ] . (1997a,b) Roden and Zachara (1996) Little eta]. (1997a,b) Roden and Zachara (1996) Kostka and Nealson (1995) Kostka et al. (1996)

Mn3+

Soluble chelate (pyrophosphate) MnOOH (manganitc)

Kostka et al. (1995) Larsen et al. (in review)

Mn4'

MnOz (amorphous) MnOz (birnessite) MnOz (6-MnOz) MnOz (pyrolusite)

Burdige et al. (1992) Burdige et al. (1992) Burdige et 01. (1992) Burdige st a]. (1992)

u6+

Soluble UO$

Lovley eta!. (1991)

Ih+

Soluble 10;'

Ferrenkopf e i al. (in press)

Crb+

Solrible CrO;'

Lovley el al. (1993b)

co3+

co '+-EDTA

Lovley eta]. (1993b)

FeOOH (goethite) Fez03 (hematite)

"EI)TA = ethylenntliarnine tctramxtic acid: N'I'A = nitrilo triacetic acid

Fe but not Mn reduction, and vice versa, thus implying that the terminal reductases for the two processes are different (see Nealson and Saffarini, 1994). Similar mutants have not been reported for other metals, or for other MRB. Growth coupled to reduction of metals has been demonstrated only for Fe and Mn, with one report of growth of G. metallireducens on UG+ (Lovley et al., 1993b). In general, the abundance of the other metals is very low, on the order of ppm or less, so that it is not easy to imagine the evolution of specific systems to harvest such minuscule amounts of energy. While dissimilatory reduction of selenium is known to be a bacterial process (Oremland et a]., 1994), to our knowledge, none of the h4RB have been shown to reduce selenium.

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225

Burdige et al. (1992) showed that a culture of S . putrefaciens growing on a series of manganese oxides with different mineralogies gave very different rates of reduction. In general, the rates of reduction were proportional to metal oxide surface area, so that a highly crystalline oxide like pyrolusite (with a very low surface-to-volume ratio) was reduced very poorly: almost not at all. Roden and Zachara (1996) studied the relationship between iron oxide reduction and surface area, concluding that the rate and extent of iron oxide reduction were controlled by the surface area and site concentration of the solid phase. From studies such as these, it seems likely that the major controlling factor in solid metal reduction is surface area, with such other factors as crystal structure, morphology, free energy, and particle aggregation participating to lesser degrees (Roden and Zachara, 1996). Yield studies of bacteria on solid substrates are difficult because of the attachment of the bacteria to the oxides (Figs. 2 and 3). When growing on manganese oxides (Fig. 21, S. putrefaciens forms a layer of extracellular polymer that obscures individual cells when viewed by environmental scanning electron microscopy (ESEM). The nature of this polymer is not yet elucidated, but it is likely to be a polysaccharide based on the fact that it is difficult to visualize when samples are dehydrated and viewed by standard scanning electron microscopy (SEM) (Fig. 2). Interestingly, when the same cells are grown on iron oxides, no extracellular polymers are conspicuous (Fig. 3) when examined by ESEM.

B. BIOCHEMISTRYOF METAL REDUCTION As of this writing, iron or manganese reductases have not been purified or characterized, although high levels of both activities have been observed in whole cells as well as in cell-free extracts. Myers and Myers (1992, 1993) reported that cytochromes and the iron reductase activity of S . putrefaciens are located in the outer membrane, consistent with the observations that metal oxides are solids and that cell contact is required for metal reduction. Tsapin et al. (1996) purified a small (12 kDa) tetraheme cytochrome cg of very low potential (-233 mV) with a high sequence similarity to the cytochrome cg of Desulfovibrio desulfuricans, and showed that this cytochrome in its reduced state could reduce Fe3+. Pleiotropic mutants missing this cytochrome are unable to reduce iron and several other electron acceptors (Lies and Nealson, unpublished). Despite this circumstantial evidence, the proof that this cytochrome is actually the iron reductase has not been presented. However, the gene is now cloned and sequenced

226

KENNETH H. NEALSON AND BRENDA LITTLE

1 ('**

(SRB)

$2032-

(CH20)n

5042-

Frc:. 4. Direct and indirect reduction of Mn4+.'This diagram shows the ways in which S . putrefaciens can reduce manganese. The solid lines indicate reactions that are catalyzed primarily by bacteria, while the squiggly lines indicate those reactions that occur by inorganic chemical transformations. MRB = metal-reducing bacteria. SRB = sulfur(sntfate, thiosulfate, or elemental sulfur) reducing bacteria.

(Tsapin et al., 1997), so it should be possible to generate insertional mutants and specify a function for this cytochrome. In addition, five other c-type cytochromes have been purified from S. putrefaciens, two of which have low redox potentials consistent with metal reductases, and one of which is membrane-bound (Tsapin et a].,1997). Lovley et ul. (1993b) partially purified a cytochrome c3 from Desulfovibrio vulgaris that could reduce U", and concluded that it was the U reductase. In many cases of metal reduction, the possibility that indirect reactions occur must be considered. Manganese is a particular problem because it is so easily reduced by other reductants (Stone, 1987a,b) as outlined in Fig. 4 (Myers and Nealson, 1988b). Fez+and HS- are both produced by S. putrefaciens and can reduce Mn4+.Thus, any sulfur- or iron-reducing bacterium can appear to be an Mn reducer via indirect reactions, and, in fact, with catalytic amounts of iron or So, a biogeochemical cycle resulting in Mn reduction can be established. For these reasons, the identification of microbes as Mn4+ reducers should be regarded with suspicion until mutants are produced that show the separation of direct and indirect reduction.

c.

REGULATION OF

METAT. REIXJCTJON

Studies by King (1990) showed that addition of Fe3+or Mn4+resulted in inhibition of sulfate reduction or methanogenesis in sediments, suggesting that these substrates were preferred electron acceptors for sediment communities. Earlier work by Burdige and Nealson (1986)

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

227

demonstrated that pure cultures of sulfate reducers continued to produce sulfide in the presence of Mn4+,reducing the Mn4+via sulfide production. Thus, the relationship between metal respiration and respiration of sulfate or sulfur is probably not simply one of the physiological response of single organisms, but may represent a complex community response, perhaps even a competition for substrates. In fact, if sulfate reduction is assessed by measurement of sulfide, then the effect of adding Mn4+could be totally directed at the product of the reaction rather than the process itself, that is, sulfate reduction would be apparently inhibited (because of the lack of sulfide production), while the process could be proceeding at the same rate (see Fig. 4). To our knowledge, work with pure cultures to address such questions has not been initiated. Several studies of regulation of metal reduction by S. putrefaciens indicate that regulation occurs at several levels. Arnold et al. (1990) and DiChristina (1992) concluded that a physiological competition exists between iron and other electron acceptors, with oxygen or nitrate capable of inhibiting iron reduction. Similarly, Myers and Nealson (1988b) showed that oxygen or nitrate inhibited reduction of Mn4+by S. putrefaciens, but that less electropositive electron acceptors like fumarate or sulfite had no such inhibitory effect. It appears that metal reduction follows thermodynamic rules for a respiratory substrate. In the presence of oxygen or nitrate, no metals are “breathed,” but in the absence of other good electron acceptors bacteria use iron or manganese for respiration. Interestingly, some substrates like iron (which is converted to Fez+)or sulfur (which is converted to HzS) result in apparent increases in Mn4+respiration via indirect reactions, discussed above (Fig. 4). Anaerobic respiration in bacteria is controlled by a variety of regulatory systems (fnr, arcBA, etc.) that sense oxygen, or some product of oxygen, and control metabolic pathways (like the tricarboxylic acid cycle) and specific reductases at the level of biosynthesis (Spiro and Guest, 1990; Lin and Iuchi, 1991; Unden et al., 1995). Mutants in these control systems characteristically result in pleiotropic phenotypes, with a number of different anaerobic processes being simultaneously affected. Several such pleiotropic mutants have been isolated from strains of S. putrefaciens (DiChristina and DeLong, 1994; Saffarini and Nealson, 1993; Saffarini et a]., 1994), and in one case the gene controlling the mutation was isolated, sequenced, and found to be an analogue of the fnr gene from E. coli (Saffarini and Nealson, 1993), suggesting that similar control mechanisms operate to regulate the anaerobic respiration of this metal reducer. Similar studies of regulation at the molecular level have not been reported for other metal reducers.

228

KENNETH H. NEALSON AND BRENDA LITTLE

pyrite FeS, (greigite FeC03 (siderite) Fe (OH)3 (ferrihydrite) Fe,Os (hematite) FeOOH (goethite)

Mn'2

Mn02

4 Uk Mn'+

(CH20)n birnessite manganates MnOOH(manganite)

Mn4+

CO2

(MRB)

(Mn3+)

MnC03 (rhodochrosite)

Soluble Mn"

FIG.5. Fates of soluble Mn and Fe produced under anoxic conditions. Manganese docs not easily form insoluble sulfides or mixed phase oxides. If COZ production is high, rhodochrosite may form: otherwise, MnZ+ remains as the soluble salt and difhises upwards to the oxygen interface. Depending on the chemistry of the anoxic environment, Fez+may have any of a variety of "fates."

D. PRODUCTS OF METALREDUCTION For the most part, the products of metal reduction are soluble forms of the metals, leading to an increase of Fez+or Mn2+in the anoxic zones, but in laboratory studies, a variety of other end-products are seen, depending on experimental conditions (Fig. 5). MnZt tends to be fairly unreactive with regard to end-products, and unless experiments are conducted in closed tubes with high levels of organics (which raises the level of carbon dioxide in the water), the product is primarily dissolved manganous ion. In closed tubes, a pinkish-white mineral phase called rhodochrosite (MnCO,) is formed. In many rhodochrosite samples in nature, the pink rhodochrosite strata are interlayered with white carbonates, suggesting episodic introduction of MnZt. The solubility of manganese sulfide is such that this mineral does not form in laboratory experiments, and is rarely found in nature. Ferrous (Fez+)iron is a much more reactive sedimentary component, rapidly forming a variety of insoluble sulfides, including pyrite. In marine systems, insoluble iron sulfide formation leads to scavenging of the iron from the system, while in freshwater systems with very low sulfate concentrations iron often remains in the system as a major redox

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

229

species. In addition to iron sulfides, siderite or iron carbonate (FeC03) can form, especially in closed-tube laboratory experiments where COz increases as a result of respiration. As shown in Fig. 5, reduced iron can interact with Mn4+ to reduce it, thus being reoxidized to Fe3+.This reaction in part explains why the zone of iron reduction is always found below the zone of manganese reduction. Magnetite is one of the more poorly understood mineral phases in terms of the role of dissimilatory iron reducers. Several authors have noted that magnetite is an extracellular product of iron reducers (Bell et a]., 1987; Lovley et a]., 1987; Roden and Lovley, 19931, routinely formed in experiments where chelated iron (as iron citrate) is used as the source of Fe3+.However, if the pH is kept at 7.0 or below and the concentration of magnetite is kept low, it is possible, in the presence of organic matter, for S. putrefaciens to catalyze the further reduction of magnetite to Fez+(Kostka and Nealson, 1995). It is not known whether such reactions are important in the sedimentary budget of magnetite: its formation, dissolution, or both. V. Electron Transport In Metal Reducers A. CYTOCHROMES

Only a limited amount of work has been reported with components of the electron transport systems of the anaerobic metal reducers. Studies of cell-free extracts have shown that G. metallireducens and Desulfuromonas acetoxidans contain c-type cytochromes that can be reoxidized by a number of different substrates. The cytochromes c from G. metallireducens could be oxidized by Fe3+,U6+,NO;, Hg, Au, Ag, or Cr6+(Lovley et al., 1993b), while those of D. acetoxidans were oxidized by So, malate, Fe3+,or Mn4+(Roden and Lovley, 1993). Some strains of D. desulfuricans are known to reduce Fe3+and U6+ (Coleman et al., 1993),although they do not couple this reduction to cell growth. Desulfovibrio vulgaris cells can reduce uranium, apparently via a cytochrome c3,which can catalyze the reduction of Fe3+,UG+, or Cr6+(Lovley et al., 1993b3).Naik et al. (1993)isolated three c-type cytochromes (28,46, and 68 kDa) in the partially purified preparation of nitrate reductase from G. metallireducens. In S. putrefaciens, spectral analysis of whole cells and extracts (Obueckwe and Westlake, 1982) showed that c-type cytochromes were present, and that the concentration increased with addition of iron to the growth medium. Arnold et al. (1986) also reported the presence of c-type cytochromes, and noted low oxygen tension-stimulated cyto-

230

KENNETH H. NEALSON AND BRENDA LITTLE

chrome synthesis. Myers and Myers (1992) concluded that many c-type cytoclrromes were present and that they were primarily localized in the outer membrane fractions. Morris et al. (1990) resolved nine different c-type cytochrome bands by DEAE-Sepharose chromatography. In the above studies, cytochromes were not purified, nor were they characterized in detail. In more recent definitive work, a large (63.8 kDa) soluble tetraheme flavocytochrome c was purified and identified as a fumarate reductase (Morris et al., 1994; Pealing et al., 1995). Heme c replaces iron sulfur centers characteristic of the membrane-bound funiarate reductases of other organisms. The heme midpoint redox potentials are -220 and -320 mV. The gene coding for this protein has been isolated and sequenced (Pealing et al., 1992). Tsapin et al. (1996) recently purified and sequenced (see Tsapin et al., 1997) a small (12.8 kDa) c3-type cytochrome from S . putrefaciens. This molecule, like the flavocytochrome of Morris et al. (1994), has a very low redox potential of -233 mV and is a tetraheme molecule with structural similarities to the cytochromes c3 from D. acetoxidans and the cytochrome portion of the flavocytochrome reported by Morris et al. (1994). The gene for this protein has been cloned and sequenced, revealing a leader sequence for transport to the periplasm (Tsapin and Nealson, unpublished data).

B. QUINONES No detailed quinone analyses have been presented from G. metallireducens, but on the basis of difference spectra of lipophilic extracts it was concluded that the quinones are menaquinones and that the types and levels are similar to those found in sulfate and sulfur reducers (Lovley et a]., 1993a). Not surprisingly, the facultative S. putrefaciens has a wide variety of quinone types, and these vary extensively between aerobically and anaerobically grown cells. The aerobic cells contain two ubiquinones, (1-7 and Q-8, and one menaquinone, MK-7, along with a small level of a methylated menaquinone, MMK-7. Under anaerobic conditions, irrespective of electron acceptor, the major quinone was MMK-7, with Q-7, Q-8, and MK-7 being minor types. Several other quinones appeared under anaerobic conditions, including several MMK types (Lies et al., 1996; Sano eta]., 1997). Under microaerophilic conditions, some strains produced 15 or more different quinones.

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231

VI. Metal-Reducing Bacteria in Natural Environments

MRB have been isolated from a variety of environments, including marine and lake sediments, and oil-field injection waters (Nealson and Saffarini, 1994; Lonergan et al., 1996). In addition to their role in the biogeochemical cycling of carbon, the ability of MRB to reduce a variety of different substrates can lead to environmental consequences and problems, including corrosion, release of bound metals or radionuclides, and a change in the physical properties of clays. Conversely, reactions related to metal reduction have potential for exploitation in areas of bioremediation. A. CORROSION

While it has been established that the most devastating microbiologically influenced corrosion (MIC) takes place in the presence of microbial consortia in which many physiological types of bacteria interact in complex ways, dissimilatory iron and/or manganese reducers represent one of the important components of the complex corrosion community (Fig. 6). Obueckwe et al. (1981a,b) used polarization studies to demonstrate that a Pseudomonas (Shewanella)isolate from an oil field caused anodic polarization of mild steel coupons accompanied by conversion of ferric to ferrous compounds. There was a loss of passivity and an intense depolarization of the anode in the presence of the organism. Electron micrographs demonstrated that a dense crystalline surface deposit covering the uninoculated metal was removed when the organism was introduced. Furthermore, the presence of the organism prevented formation of a protective ferric layer. Obueckwe et al. (1987) recognized that Shewanella influenced corrosion by the reduction of Fe3+to Fez+and S20,2-to S2-. In experiments designed to inhibit one or both of these reduction reactions, both the production of S2- and Fez+ simultaneously and the production of Fez+ alone by bacteria were responsible for anodic dissolution of the carbon steel. When only S2was produced, the initial increase in anodic reaction was due to reactions of the S2- with the metal. The resultant FeS eventually protected the metal, followed by a decrease in the anodic reaction. Corrosivity of soils has been associated with soluble iron, and soils with an Fez+ content above 333 pg-* are very corrosive (Booth et al., 1967). Little et al. (1997a) used electrochemical noise to demonstrate the aggressive attack of carbon steel by pure cultures of S. putrefaciens and to further differentiate mechanisms of iron and thiosulfate reduction.

232

g-

I

N

s"

I &

g

KENNETH H. NEALSON AND BRENDA LITTLE

f

-

.-d

a

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

23 3

Little et al. (1997b) used synthetic iron oxides (goethite, FeOOH; hematite, Fe,O,; and ferrihydrite, Fe(OH),) as model compounds to simulate passivating films on carbon steel to demonstrate that corrosion of carbon steel by S. putrefaciens was related to surface mineralogy. Dissolution of these oxides exposed to pure cultures of the metal-reducing bacterium was followed by direct measurement of ferrous iron, using atomic absorption spectroscopy (AAS), coupled with microscopic analyses. Bacteria colonized all mineral surfaces and formed biofilms within 48 h. After 190 h, confocal laser scanning microscopy (CLSM) images (Fig. 3) have been used to show that bacteria penetrate both goethite and ferrihydrite but are restricted to the surface of hematite even after 200-h exposure (Little et al., 1997a).

B. RELEASE OF ADSORBED POLLUTANTS Iron and manganese oxides are regarded as the “scavengers of the sea” (Goldberg, 1954)because of their ability to adsorb other metals and trace components. Interaction of metal oxides with trace metals is considered to be of major importance in sediment and water column chemistry (Balistrieri and Murray, 1982, 1984; Tessier et al., 1996). Manganese oxide fibers have been used to collect radium from seawater (Moore, 1975), and metal oxides have been proposed as a means for disposal of radionuclide waste (Mott et al., 1993).As a consequence, when iron or manganese oxide reduction occurs in municipal water systems, not only is the water fouled by excess soluble manganese and/or iron, but trace components bound to the metal oxides may also be released (Francis and Dodge, 1990). Such reactions may account for the distribution of trace metals (Francis and Dodge, 1990; Rose et al., 1993; Tessier et ~ l . , 1996) and radionuclides like uranium in sediments (McKee eta]., 1987) and anoxic water columns (McKee and Todd, 1993). C. CLAYREDUCTION

Kostka et QI. (1996) reported reduction of structural iron from smectite clays by strains of S . putrefaciens. Reduction of the clay is accompanied by changes in color, swellability, and other properties (J. Stucki, personal communication), suggesting a major impact in anaerobic soils or sediments where it occurs. The environmental importance of such reactions is not known, but the knowledge that bacteria can couple organic carbon oxidation to reduction of clays should be considered in studies of anaerobic sediments.

234

KENNETH H. NEALSON AND BRENDA LITTLE

D. BIOREMEUIATION Metal-reducing bacteria may offer potential applications in bioremediation, including degradation of toxic organic pollutants. Because of the reasonably high redox potentials of Feat and Mn4+,organisms that use them are relatively efficient at converting organic carbon pollutants into harmless COz and organic byproducts that can be metabolized by other anaerobic bacteria. G. rnetaffireducenscan oxidize certain aromatics under anaerobic conditions using iron as the oxidant (Lovley et nf., 1989b, 1990; Kazumi et al., 1995). The direct approach to bioremediation has the following advantages over using more standard respiring bacteria (e.g., aerobes, nitrate reducers, or sulfate reducers): (1) iron oxides are solids and can be delivered to a contaminated site without the possibility of their diffusing away; (2) iron oxides are rather specific substrates, as far as is known, so that competition from other bacteria for the electron acceptor should be minimal; and (3) in stratified aqueous environments reduced iron should diffuse upwards, be reoxidized by molecular oxygen in the overlying oxic zone, and returned to the anoxic zone via gravity, thus acting as a “pump” for oxidizing equivalents, as proposed by Nealson and Myers (1992). The introduction of MRB and their potential in pollutant removal for both the short and long term might be very high, especially if iron reducers not inhibited by oxygen were available. Two reports suggest that Shewnneffa spp. can donate electrons to chlorinated hydrocarbons, thus reductively dechlorinating toxic compounds by converting tetrachloromethane to trichloromethane (Picardel et al., 1993; Petrovskis et a]., 1994). No other chlorine transformations were observed, and the reaction was inhibited by oxygen, but not by other electron acceptors. These results are consistent with a fortuitous reduction by reduced c-type cytochromes under anaerobic conditions (Picardel et af., 1993). Ferrous iron, especially in the presence of surfaces, can catalyze the reduction of nitro groups on substituted nitrobenzenes (Klausen et al., 1995) and other nitroaromatic compounds (Heijman et nl., 1995). Thus, any organism that can produce Fez+can potentially catalyze the reduction of toxic nitrates. In reduction of the pollutant, iron is reoxidized, so that a catalytic amount of iron could be used to cycle between the oxidized pollutants and respiring bacteria (Fig. 7). Metals can be removed from solution by indirect reactions via the production of sulfide and resulting production of insoluble metal sulfides. Many metal-reducing bacteria are also capable of generating hydrogen sulfide through the reduction of sulfate (Desuffovibrio),sulfur

xFe2

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

6

2 35

“bound

“bound Fe3+

FIG.7. Degradation of nitroaromatics by Fe-reducing bacteria. This figure, modeled after that presented by Heijman et al. (1995), shows the inorganic reduction of nitroaromatics. The process is strongly enhanced if surfaces such as minerals or clays are present, indicated by the “bound” iron.

(Desulfuromonas, Shewanella), or thiosulfate (Shewanella). Toxic and trace metals can be removed via precipitation as insoluble sulfides, which have very low solubility products (Stumm and Morgan, 1981). Fude et al. (1994) demonstrated Cr6+reduction/detoxification via H2S by a consortium of SRB. Metals can also be removed via direct reduction by the MRB. While iron and manganese are solubilized, other metals are converted to insoluble forms upon reduction. Of note are chromium (Cr6+)and uranium (U6+),which are soluble in oxidized form, but insoluble as the respective Cr3+and U4+ reduced species. Reduction of U6+ has been demonstrated for both G. metallireducefis and S. putrefaciens (Lovley et al., 1991), and has been proposed as a mechanism for concentrating and thus removing radionuclide waste. The cytochrome c3 from G. metallireducens can reduce U6+ (Lovley et d.,1993b), presenting the possibility of immobilized enzyme treatment of waste materials. In this regard, the recent cloning of cytochrome cgfrom S. putrefaciens (Tsapin et al., 1997) could offer a ready supply of protein for such work. Chromium reduction has also been reported for D. vulgaris, and the cytochrome c3 partially purified from this organism is capable of Cr6+ reduction. S. putrefaciens can reduce Cr6+,but no detailed studies have been presented (Nealson, unpublished). As with uranium, the removal of toxic chromium should be possible using either intact cells or cellfree systems of MRB. VII. Summary

Respiration of solid compounds was, until a few years ago, thought to be very unlikely, and, although metal reduction was known to occur, organisms that coupled growth and respiration to reduction of oxidized

236

KENNETH H. NEALSON AND BRENDA LITTLE

iron or manganese were not known. In recent times, this view has changed, and several groups of microbes have been described that live via dissimilatory reduction of metal oxides. MRB represent a fairly wide range of phylogenetic types, although to date no archaea are included among them. They often share the property of being able to reduce elemental sulfur, a trait that was previously unknown in organisms able to grow aerobically. The ability to “breathe” metal oxides adds a new, not previously considered dimension to bacterial respiration. How can an organism transfer electrons to solid substrates? While this question is not yet answered, many recent advances have been made in both the biochemistry and molecular biology of some metal reducers. Of particular note are reports of reductases and cytochromes localized to the outer membranes of the MRB, and isolation of c-type cytochromes with low potentials. Since iron and manganese oxides are solids, their reduction has environmental consequences of both decreasing particulate load and increasing levels of dissolved iron and manganese. In addition, if other components adsorb to the metal oxides, as often occurs in nature, they may be released by the metal reduction. Microscopic studies of metal oxides during microbial reduction show contact of the organisms with surfaces, and distinct environmental responses between organisms grown on manganese versus iron oxides. Finally, the unique abilities of these organisms present both environmental problems and opportunities. Problems resulting from metal oxide reduction include corrosion, alteration of sediment and soil properties, release of toxic adsorbed compounds in sediments, and fouling of public water supplies with MnZ+and Fez+during periods of anoxia. Opportunities involve new approaches to bioremediation: anaerobic consumption of organic pollutants, removal of toxic metals either by sulfide precipitation or by conversion to insoluble reduced forms, and addition of Fez+to environments where it may be active in reduction of nitroaromatics. ACKNOWLEDCMENTS

KHN wishes to thank NASA (exobiology), the NSF (chemical oceanography), and the Wisconsin Sea Grant Program for support for his research on this topic. He also gratefully acknowledges the Office of Naval Research for the Distinguished Visiting Researcher Award during 1996, contract no, NO00149 96-J-06. BJL acknowledges support by the Office of Naval Research, contract no, NO001497 WX30031. We thank the following NRL personnel: Richard Ray for ESEM and CLSM micrographs; and Darlene Jorns, Maria Banker, and Mary Ellen Turncotte for help with the figures. This document is NRL Contribution Number NRLIBAI 73 33-97-0004.

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

23 7

REFERENCES Aguilar, C., and Nealson, K. H. (1994). Can. J. Fish. Aquat. Sci. 51,185. Aller, R. C. (1990). Philos. Trans. R. SOC. London, Ser. A 331,51. Aller, R.C., Aller, J., Blain, N., Mackin, J., and Rude, P. (1991). Oceanography,April 27. Arnold, R. G., Olson, T. M., and Hoffmann, M. R. (1986). Biotechnol. Bioeng. 28, 1657. Arnold, R. G., DiChristina, T. J., and Hoffmann, M. R. (1988). Biotechnol. Bioeng. 32, 1081.

Arnold, R. G., Hoffmann, M. R., DiChristina, T. J., Picardel, F. W. (1990). Appl. Environ. Microbio].56, 2811. Balistrieri, L. S., and Murray, J. W. (1982). Geochim. Cosmochim. Acta 46,1041. Balistrieri, L. S., and Murray, J. W. (1984). Geochim. Cosmochim. Acta 48,921. Bell, P., Mills, A., and Herman, J. (1987). Appl. Environ. Microbiol. 53, 2610. Boone, D., Liu, Y., Zhao, Z.-J., Balkwill, D., Drake, G., Stevens, T., and Aldrich, H. (1995). Int. J. Syst. Bacteriol. 45,441. Booth, G. H., Robh, J. A., and Wakerly, D. W. (1967). Int. Con@. Metal. Corr., 3rd 3,544. Burdige, D., and Nealson, K. H. (1986). Geomicrobiol.1.4,361. Burdige, D., Dhakar, S. P., and Nealson, K. H. (1992). Geomicrobiol.I. 10,27. Caccavo, F., Blakemore, R. P., and Lovley, D. (1992). Appl. Environ. Microbiol. 58, 3211. Caccavo Jr., F., Coates, J. D., Rosello-Mora, R. A,, Ludwig, W., Schleifer, K.-H., Lovley, D. R., and McInerney, M. J. (1997). Arch. Microbiol. In press. Canfield, D. E., Thamdrup, B., and Hansen, J. W. (1993). Geochim. Cosmochim. Acta 57, 3867. Coleman, M., Hedrick, D. B., Lovley, D., White, D. C., and Pye, K. (1993). Nature 361, 436. Davison, W.(1993). Earth Sci. Rev. 34, 119. DeVrind, F. C., Boogerd, J. P. M., and DeJong, E. W. (1986). J. Bacteriol. 167,30. DiChristina, T.J. (1992). J. Eacteriol. 174, 1891. DiChristina, T. J., and DeLong, E. F. (1994). J. Bacteriol. 176,1468. DiChristina, T.J., Arnold, R. G., Lidstrom, M. E., and Hoffmann, M. R. (1988). Water Sci. Technol. 20, 69. Ferrenkopf, A. M., Dollhopf, M. E., Chadhain, S . N., Luther 111, G. W., and Nealson, K. H. (1997). Marine Chem. In press. Francis, A. J., and Dodge, C. J. (1990). Environ. Sci. Techno].24, 373. Fredrickson, J. K., and Gorhy, Y. A. (1996). Cum Opin. Eiotechnol. 7, 287. Froelich, P., Klinkhammer, G., Bender, M. Luedtke, N., Heath, G . R., Cullen, D., Dauphin, P., Hammond, D., Hartman, B., and Maynard, V. (1979). Geochim. Cosmochim. Acta 43,1075. Fude, L., Harris, B., Urrutia, M., and Beveridge, T. J. (1994). Appl. Environ. Microbiol. 60, 5. Goldberg, E. D. (1954). /. Geol. 62, 249. Heijman, C. G., Grieder, E., Holliger, C., and Schwarzenback, R. P. (1995). Environ. Sci. Technol. 29, 775. Kazumi, J., Hagghlom, M., and Young, L. (1995). Appl. Environ. Microbiol. 61,4069. King, G. M. (1990). FEMS Microbiol. Ecol. 73,131. Klausen, J., Troeber, S . P., Haderlein, S. B., and Schwarzenback, R..'F (1995). Environ. Sci. Technol. 29, 2396. Kostka, J. E., and Nealson, K. H. (1995). Environ. Sci. Technol. 29, 2535. Kostka, J. E., Luther, G. W., and Nealson, K. H. (1995). Geochim. Cosmochim. Acta 59, 885. Kostka, J. E., Stucki, J. W., Nealson, K. H., and Wu, J. (1996). Clays Cluy Min. 44,522.

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Larsen, I., Little, B., Nealson, K., Ray, R., Stone, A., and Tian, J. (1997). Envjron. Sci. Techno/. In review. Lies, D., Moser, D., Sano, H., Nishijima, M., and Sakai, M. (1996). Absfr. Amer. Soc. Microbial. Ann. Mfg. K-72,547. Lin, E. C. C., and Iuchi, S. (1991). Annu. Rev Genetics 25,150. Little, B., Wagner, P., Hart, K., Ray, R., Lavoie, D., Nealson, K., and Aguilar, C. (1997a). Corrosion '97, NACE International, Houston, TX, Paper #215. Little, B., Wagner, P., Ray, R., Hart, K., Lavoie, D., Nealson, K., and Aguilar, C. (1997b). Biodegradation. In review. Lonergan, D. J., Tenter, H., Coates, J., Phillips, E., Schmidt, T., and Lovley, D. (1996). J. Bacteriol. 178,2402. Tmvley, D., and Lonergan, D. (1990). Appl. Environ. Microbiol. 5 6 , 1858. Lovley, D., and Phillips, E. (1988). Appl. Environ. Microbiol. 51,683. Lovley, D., Stolz, J., Nord Jr., G. L., and Phillips, E. (1987). Nature 330,252. Lovley, D.,Phillips, E., and Lonergan, D. J. (1989a). Appl. Environ. Microbiol. 55, 700. T.ovley, D., Baodecker, M. J., Lonergan, D., Cozzarelli, I., Phillips, E., and Siegel, D. (l%gb). Nature 339,298. Lovley, D., Phillips, E., Gorby, Y,and Landa, E. R. (1991). Nature 350,413. Lovley, D., Giovannoni, S. J., White, D. C., Champine, J. E., and Phillips, E. (1993a).Arch. Microbiol. 159,336. Lovley, D., Widman, P., Woodward, J., and Phillips, E. (1993b). Appl. Environ. Microbiol. 59, 3572. MacDonell, M. T., and Colwell, R. R. (1985). Syst. Appl. Microbiol. 6,171. McKee, B., and Todd, J. F. (1993). Limnol. Oceanog. 38, 408. McKec, B., DeMaster, D., and Nittrouer, C. (1987). Geochim. Cosmochirn. Acta 51,2779. Moore, W. S. (1975). Nature 253,262. Morris, C. J.. Gibson, D. M., and Ward, F. B. (1990). FEMS Microbiol. Lett. 69,259. Morris, C.J., Black, A. C., Pealing, S. L., Manson, F., Chapman, S., Reid, G., Gibson, D., and Ward, F. (1994). Biochem. J. 302,587. Moser, D. P., and Nealson, K. H. (1996). Appl. Environ. Microbiol. 62,2100. Mott, H. V., Singh, S., and Kondapally, V. R. (1993). Res. Technol., October, 114. Myers, C. R., and Myers, J, M. (1992). 1.Bacteriol. 174,3429. Myers, C.R., and Myers, J. M. (1993). FEMS Microbiol. Lett. 108, 15. Myers, C. R., and Nealson, K. H (1988a). Science 240,1319. Myers, C. R., and Nealson, K. H. (1988b). Georhim. Cosrnochini. Acta 52,2727. Naik, R. R., Francisco, M. M., and Stolz, J. F. (1993). FEMS Microbiol. Lett. 106,53. Nealson, K.H., and Myers, C. R. (1992). Appl. Environ. Microbiol. 58, 439. Nealson, K.H., and Saffarini, D. (1994). Annu. Rev. Microbiol. 48,311. Nealson, K. H., Myers, C. R., and Wirnpee, B. (1991). Deep Sea Res. 38, S907. Ohueckwe, C., and Westlake, W. (1982). Can. I. Microhiol. 28, 989. Obueckwe, C.,Westlake, W., and Cook, F. (19fl1). Can. I. Microbiol. 27, 692 Ohueckwe, C. 0..Westlake, D. W. S., Plambeck, J., and Cook, F. D. (1981a). Corrosion 37, 461. Obueckwe, C. O., Westlake, D. W. S., Plambeck, J., and Cook, F. D. (1981b). Corrosion 37, 632. Ohueckwe, C. O.,Westlake, D.W. S., and Planibeck, J. (1987). Appf. Microbiol. Biofechno]. 26, 294. Oremland, R. S., Switzer Blum, J., Culbertson, C., Visscher, P., Miller, L., Dowdle, P., and Strohmaier, F. (1994). Appl. Environ. Microhiol. 60,3011.

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2 39

Pealing, S. L., Black, A., Manson, F., Ward, F., Chapman, S., and Reid, G. (1992). Biochemistry 31,12132. Pealing, S . L., Myles, C., Reid, G., Thomson, A., Ward, F., and Chapman, S. (1995). Biochemistry 34,6153. Perry, K., Kostka, J., Luther, G., and Nealson, K. (1993). Science 259, 801. Petrovskis, E. A., Vogel, T. M., and Adriaens, P. (1994). FEMS Microbiol. Lett. 121,357. Picardel, F., Arnold, R., Couch, H., Little, A., and Smith, M. (1993). Appl. Environ. Microbiol. 59, 3763. Roden, E. E., and Lovley, D. (1993). Appl. Environ. Microbiol. 59, 734. Roden, E. E., and Zachara, J. (1996). Environ. Sci. Technol. 30, 1618. Rose, A. W., and Bianchi-Mosquera, G. C. (1993). Econ. Geol. 88,1226. Rosello-Mora, R., Ludwig, W., Kempfer, P., Amann, R., and Schleifer, K. H. (1995). Syst. Appl. Microbiol. 18,196. Rusin, P. A., Quintana, L., Brainard, J. R., Strietelmeier, B. A., Tait, C. D., Ekburg, S. A., Palmer, P. D., Newton, T. W., and Clark, D. L. (1994). Environ. Sci. Technol. 28,1686. Saffarini, D. A,, and Nealson, K. H. (1993). J. Bacteriol. 175,7938. Saffarini, D. A., DiChristina, T. J., Bermudes, D., and Nealson, K. H. (1994). FEMS Microbiol. Lett. 119,271. Sano, H., Sakai, M., Goto, M., Nishijima, M., Lies, D., Tsapin, A., Moser, D., and Nealson, K. (1997). Appl. Environ. Microbiol. In press. Scott, J. H., and Nealson, K. H. (1994). J. Bacteriol. 176,3408. Semple, K., and Westlake, W. (1987). Can. J. Microbiol. 33, 366. Spiro, S.,and Guest, J. R. (1990). FEMS Microbiol. Rev. 75,399. Stone, A. T. (1987a). Geochim. Cosmochim. Acta 51,919. Stone, A. T. (1987b). Env. Sci. Technol. 21,979. Stumm, W., and Morgan, J. J. (1981). “Aquatic Chemistry,” 2nd ed. Wiley, New York. Tessier, A., Fortin, D., Belzile, N., DeVitre, R., and Leppard, G. (1996). Geochim. Cosmochim. Acta 60, 387. Tsapin, A. I., Nealson, K. H., Meyer, T., Cusanovich, M. A., van Beeumen, J., Crosby, L. D., Feinberg, B. A., and Zhang, C. (1996). J. Bacteriol. 178,6386. Tsapin, A. I., Nealson, K. H., Meyer, T., and van Beeumen, J. (1997). Abstr. Amer. Sac. Microbiol. Ann. Mtg. K-70,353. Unden, G., Becker, S., Bongaerts, J., Holighaus, G., Schirawski, and Six, S. (1995). Arch. Microbiol. 164,81-90. Zehnder, A. J. B., ed. (1988). “Biology of Anaerobic Organisms.” Wiley, New York. Zehnder, A. J. B., and Stumm, W. (1988). In “Biology of Anaerobic Organisms” (A. J. B. Zehnder, ed.). Wiley, New York.

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Enzymatic Deinking PRATIMA BAJPAI Chemical Engineering Division Thapar Corporate Research and Development Centre Patiala 147 001,India

I. 11. 111. IV. V. VI. VII. VIII.

Introduction Enzymes Used in Deinking Performance of Enzymes in Deinking Effect of Enzyme on Pulp Yield and the Quality of Fiber and Paper Possible Mechanisms of Enzymatic Deinking Factors Affecting Enzymatic Deinking Benefits of Enzymatic Deinking Current Research Needs IX. Conclusions References

I. Introduction

Due to the rapid depletion of worldwide forest resources and its impact on ecological balance, the paper industry has turned to fastgrowing wood species, alternative nonwood fibers, and the use of secondary fibers. This trend is expected to continue globally. The use of secondary fibers in newsprint, tissue paper, and in higher grades of paper has increased greatly over the last two decades, particularly as a result of successful deinking operations. Worldwide, recovered paper comprised 37% of the raw material supply in 1991 (Stork et al., 1994). The American Forest and Paper Association has projected that overall wastepaper recovery in the United States will rise from approximately 40% in 1993 to 50% or more by 2001 (Anon., 1994a). Profitable conversion of this relatively abundant and inexpensive raw material into quality products demands effective and efficient means for removal of contaminants, with inks being a significant problem. One hundred and forty-five deinking facilities are now either operating, under construction, or planned for construction in the United States (Anon., 1994b). According to a Jaakko Poyry study, worldwide deinking capacity will rise to 31 million tons by 2001, with particularly large expansion in newsprint, printing, and tissue grades (Uutela, 1991). Meeting these ambitious goals will require intensified investment in research and development of improved deinking processes. 241 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 45 Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved. 0065-2164/97 $25.00

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PRATIMA BAJPAI

Some paper grades (e.g., newspapers printed with oil-based inks) can be deinked with relative ease. Nonimpact printed papers are more difficult to deink (Vidotti et al., 1992), and the quantity of such papers continues to increase as a proportion of total recovered paper volume. Similarly, color printing via offset lithography is expanding in the United States at an annual rate of 25% (Prasad et a]., 1993), and the crosslinking inks used in this process are also difficult to remove. Thus, removal of ink remains a major technical obstacle to greater use of recycled paper. In addition, deinking processes are sources of substantial amounts of solid and liquid waste. Disposal is a problem, and deinking plants would benefit from more effective and pollution-free processes. Enzymatic deinking represents a new approach to converting secondary fibers into quality products (Franks and Munk, 1995; Heise et d., 1995,1996: Jeffries et al., 1992, 1993, 1994,1995; Joyce et al., 1995; Ow and Eom, 1990,1991: Ow et al., 1995; Prasad, 1993; Prasad et al., 1992a, 19%?b,1993; Puls et nl., 1993). On the laboratory and industrial scales, it has proven to be an effective and economical method for deinking wastepaper. Drainage enhancement is known to be of secondary benefit in enzymatic deinking. Many enzyme preparations have been investigated on different papers and inks. A number of pilot and mill-scale trials have been conducted, and promising results have been obtained.

II. Enzymes Used in Deinking

Deinking of secondary fibers involves dislodging ink particles from fiber surfaces and separating dispersed ink from fiber suspensions by washing or flotation. Enzymatic approaches involve attacking either the ink or the fiber surfaces. Different enzymes have been used for deinking, including lipases, esterases, pectinases, hemicellulases, cellulases, and ligninol ytic enzymes. Lipases and esterases degrade vegetable-oil-based inks. Pectinascs, hemicellulases, cellulases, and ligninolytic enzymes alter the fiber surface or bonds in the vicinity of the ink particles, thereby freeing ink for removal by washing or flotation. Many patent applications have been filed or granted concerning the use of enzymes in deinking (Table I). Several patents specify the use of (particularly alkaline) cellulases for deinking. A few patents claim that esterases can be used, whereas others specify the use of lipases or pectinases. One patent application employs laccase from white rot fungi. Most of the published literature on deinking deals with cellulases and hemicellulases.

243

ENZYMATIC DEINKING TABLE I PATENTS FOR ENZYMATIC DEINKLNG Title Enzymatic deinking process Elimination of ink from reclaimed paper Elimination of ink from reclaimed paper Deinking waste-printed paper using enzymes Process for removing printing ink from wastepaper Biological ink elimination from reclaimed paper Elimination of ink from reclaimed paper Process for wastepaper preparation with enzymatic printing ink removal Deinking wastepaper with the incorporation of lipase Removal of ink from recycled paper Elimination of ink from reclaimed paper Chemicals for deinking Deinking chemicals

Enzyme used

Reference

Alkaline cellulase Alkaline cellulase Cellulase

Baret eta]., 1991

Cellulases

Eom and Ow,1989

Cellulase or pectinase

Eom and Ow, 1990

Cellulase and/ or pectinase Esterase

Gen. Y. and Go, 1991

Ligninolytic enzyme (laccase)

Call, 1991

Lipase

S h q o and Sakaguchi, 1990

Lipase

Guy Vare et al., 1990

Nomura and Shoji, 1988 Fukunaga and Kita, 1990

Sugi and Nakamura, 1991

Esterase Not specified Not specified

Urushibata, 1984 Hagiwara, 1988

Cellulases encompass a collection of enzymes whose primary function is to hydrolyze p-1,4-glucosidic linkages in cellulose. Microorganisms that produce cellulases are almost invariably able to hydrolyze xylan as well. There are few examples of organisms only able to hydrolyze one of these two structural polysaccharides. Cellulases from a wide range of microorganisms have been purified, analyzed, and categorized according to their substrate specificities. The breakdown of cellulase requires at least three basic activities: 1)endo-j3-glucanase, which randomly cleaves glucosidic bonds within an unbroken glucan chain, 2) cellobiohydrolase, which cleaves cellobiose dimers from the nonreduc-

244

PRATIMA BAJPAI

ing ends created by activity 1, and 3) P-glucosidase, which splits cellobiose into glucose (Eriksson et al., 1990; Wood, 1989). Total combined activity is greater than the sum of individual activities (Din et al., 1991). The rationale for this synergy is as follows. Although cellulose is chemically homogenous, it is structurally diverse and comprises amorphous regions where the cellulose chains are not closely linked and crystalline areas where inter- and intrachain hydrogen bonding results in an ordered array of cellulose microfibrils. It is this latter form of polysaccharide that is particularly recalcitrant to enzyme attack. It is generally agreed that the endoacting enzymes hydrolyze bonds in the amorphous regions, creating many nonreducing ends in the cellulose molecules. The exoacting cellulase (cellobiohydrolase) initiates cellulose hydrolysis at these newly created termini and continues releasing cellobiose from the crystalline regions of the polymer, By leaving cellobiose, the P-glucosidase prevents the disaccharide from primarily inhibiting the cellobiohydrolases. The synergistic effects are high with crystalline cellulose, low with amorphous and extensively hydrated cellulose, and nil with cellulose derivatives. Such observations suggest that directed mixtures of cellulases might be more effective for deinking than single enzymes or natural mixtures. Some cellulases share certain common features, including a structural and apparently bifunctional organization characterized by a central core containing a catalytic domain with a tail that has a glycosylated region and a cellulose-binding domain (Elliston et al., 1990; Esterbauer et al., 1990). For exoglucanases, the glycosylated region serves as a hinge, linking the cellulose-binding domain to the catalytic domain. The cellulose-binding domain either functions as an anchor or serves to relax the surface and inner structure of cellulosic fibers (Puls et al., 1993). Research on celluloses is intense, and the rapidly changing definitions of structure and function will continue to change. Separating the catalytic and binding domains produces differing effects (Din et al., 1991). Though lacking hydrolytic activity, the binding domain adheres to these areas and disrupts fiber structure. Further penetration exfoliates fibers, exposing cellulase chain ends and roughening surfaces, Despite a low affinity for cellulose, the catalytic domain cleaves glycosidic linkages and smooths fiber surfaces. These differing activities may generate contrasting results, with roughening leading to improved fiber bonding and smoothing to increased freeness (Jeffries et al., 1992). Further research on enzyme structure and enzyme domains, as well as on differential activities, is needed. Using the two domains separately or, when recombined, in various proportions could improve the effectiveness of a variety of processes. Perhaps the reduced

ENZYMATIC DEINKING

245

size and lower molecular weight of the catalytic domain would permit diffusion into and activity within the fiber wall. Hemicelluloses are much more variable in composition than cellulose. Due to their complex nature, several different enzymes are needed for their enzymatic degradation and modification (Eriksson, 1990; Eriksson et al., 1990). The two main gIycanases that depolymerize the hemicellulose backbone are endo-l,4-P-xylanase and endo-l,4-P-mannanase. Small oligosaccharides are further hydrolyzed by 1,4-~-D-glucosidase. The sidegroups are split off by a-L-arabinosidase, a-D-glucuronidase, and a-D-galactosidase. Esterified sidegroups are liberated by acetyl xylan esterase and acetyl galactoglucomannan esterases. Endoxylanases, perhaps the best known xylanolytic enzymes, are noted for initiating endwise attacks on the xylan backbones of common hemicelluloses. P-xylosidases, on the other hand, convert water-soluble dimers and oligomers to xylose.

Ill. Performance of Enzymes in Deinking

Work by research teams in Germany, Korea, and the United States has examined the application of cellulases to deinking of old newspapers (ONPs) (Kim et al., 1991; Prasad et al., 1993, 1992b; Putz et al., 1994). Realizing that much of wood-containing paper production occurs at lower pH values, there should be some advantages to be gained from the use of the acidic class of cellulases from Trichoderma spp. in this application. The lower pH optimum (-5-5.5) of these cellulases should prevent alkaline yellowing of wood-containing papers during the deinking process. This in turn could eliminate the need for the normal deinking chemicals used in the pulping stage and improve the efficiency of the post-deinking brightening steps. Kim et al. (1991) and Ow and Eom (1990,1991) reported that increasing the pH from 4.7 to 8.0 decreased enzyme activity and reduced the brightness of deinked pulp. Presoaking with enzymes before pulping appeared beneficial: a 10-min presoak gave brighter and stronger pulp. Longer presoaking times decreased brightness, presumably due to reduced ink particle size (Kim et al., 1991; Ow and Eom, 1990). It was speculated that longer presoaking allowed finely dispersed ink particles to readhere to fiber surfaces or to penetrate into porous parts of fibers, thereby limiting the effectiveness of flotation. Soaking after pulping but before flotation adversely affected deinking. This result was also attributed to readherence of ink particles to fibers. This trial was among the

246

PRATIMA BAJPAI TABLE IIA

DEINKING BKICIITNESS IMPROVEMENT DURING ENZYMATIC Old newsprint printed by

Enzyme used

Black and white letterpress Black and white flexo Colored flexo

Cellulase + hemicellulase Cellulase Cellulase

Brightness" (% ISO) 58 (53)

55 (51)

51 (46)

~~

Based on Prasad et ol (1992b).

*Values i n parentheses dcnotc control withnut e n ~ y m e

first to demonstrate that the brightness of enzymatically deinked pulp was similar to that of conventionally deinked pulp. Prasad et nl. (1992b, 1993) evaluated low-pH cellulase and hemicellulase mixtures for deinking of letterpress- and color offset-printed newsprint. All operations were performed at pH 5.5. The highest increase in brightness for letterpress paper was obtained with a hemicellulase preparation that was primarily xylanase (Table IIA), while the lowest residual ink areas as measured by image analysis were achieved using a cellulase preparation (Table IIB). For color-offset papers, the best brightness values were obtained with a mixture of cellulases and hemicellulases (Table IIB). This same preparation, however, yielded the highest residual ink area. Heitmann et al. (1992) and Prasad et al. (1992a) also used similar enzymes to deink flexographic-printed newspaper. Enzymatic treatment and flotation removed the water-based ink with ease, resulting in brightness levels well above those obtained with conventional inking (Table IIA). However, inks of this type are so finely divided that flotation is impaired (Chabot et al., 1993). Such results suggest that enzyme treatment under acidic conditions are best for deinking this raw material and emphasize that deinking methods must be tailored to paper, ink, and printing type. Paik and Park (1993) tested the effectiveness of hemicellulases from Aspergillus niger and cellulases from fiicboderma viride. Brightness was found to increase with increasing enzyme dosage and with increasing reaction time at constant enzyme dosage. Soaking with enzyme before pulping was beneficial but prolonged soaking, reduced ink particle size, lowered flotation effectiveness, and reduced brightness. Higher brightness gains were obtained with cellulase and hemicellulase

247

ENZYMATIC DEINKING TABLE IIB

OPTICAI. PROPERTIES OF ENZYMATICALLY DEINKED COLORED OFFSETNEWSPRINT Deinked pulp

Enzyme preparation

bright-

Residual ink

Scatt.

ness

area

coeff.

ml

(%I

(%I

(mz/kgI

41 49

93

34.5

96

50.2

70.5 78.0 75.8 69.9

Enzyme dose/

IS0

g PU'P LJ

Blank"

-

-

Control"

-

-

I

0.2"

0.033

I1

0.2"

0.033

52 52

77 77

111 IV

lOOd 19d

0.500

53

65

0.033

54

79

Based on Prasad e t a / . (1993) Enzyme preparation I contained fi IJ/ml of CMCase, 10.0 U/ml of xylanase, and 42 U/ml of filter paper activity. Enzyme preparation I1 contained fi U/ml of CMCase, 6 U/ml of xylanase, and 1 U/ml of filter paper activity. Enzyme preparation 111contained 0.20 U/ml of CMCase, 200 U/ml ofxylanase, and 0.005 U/ml of filter paper activity. Enzyme preparation IV contained 26 U/ml of CMCase, 580 U/ml of xylanase, and 50 U/ml of filter paper activity. "Reslushed pulp without enzyme treatment and no flotation "Reslushed pulp without enzyme treatment but with flotation ?)Osage based on CMC. "Dosage based on xylan.

mixtures; the optimal blend gave higher brightness gains than conventional deinking. The phenomenon of ink particle size reduction merits further investigation. Regardless of ink type or printing process, enzymatic treatment tends to reduce ink particle size. Kim et al. (1991) reported that reductions in particle size varied with pulping time in the presence of cellulases for standard newsprint, and that overall reductions were greater than those noted with conventional deinking. Prasad et al. (1992b) and Rushing et al. (1993) reported reductions varying from 16 to 37% depending on ink type. Credible explanations have not appeared. Quantifying this effect, verifying its causes, and learning to control it remain major research objectives. The bleach chemical requirement is usually lower for enzymatic deinking than for chemical deinking processes. Kim et al. (1991) have reported that newspaper pulps bleached after being deinked by enzy-

PRATIMA BAJPAI TABLE Ill DEINKING OF WOOD-FREE SHAVINGS: PILOTRESULTS Brightness (% ISO) Deinking treatment

Chemical Chemical Based

+ cellulase

011

Postflotatioii

Postwash

75 80

84

88

Baret et a1. (1991).

matic and conventional means had similar brightness values. In case of conventional deinking, hydrogen peroxide is used in the pulping as well as in the bleaching step, but in the case of enzymatic deinking, hydrogen peroxide is used only in the bleaching process. Enzymatically deinked pulps were thus easier to bleach and required half the hydrogen peroxide. A similar study (Rushing et al., 1993) with letterpress newspaper produced enzymatically deinked pulps with lower initial brightness values than those for conventionally deinked pulps. Subsequent bleaching with hydrogen peroxide, however, produced similar brightness values, with peroxide usage lowest for the enzymatic process. Putz et al. (1994) reported that brightness levels obtained from bleaching offset-printed newspaper pulps after enzymatic deinking slightly exceeded those of pulps produced by conventional deinking, with the same quantity of hydrogen peroxide applied during pulping. Mixed office waste (MOW) offers a special challenge. Increasing amounts of the wood-free paper that comprises this waste stream are made under alkaline conditions using calcium carbonate as the filler of choice. The benefits of neutral cellulase for this deinking application were exploited by a French group (Baret et a]., 2991). They used a neutral cellulase as posttreatment after standard alkaline chemical treatment. An additional brightnesdink removal benefit was observed (Table 111). Baret et al. (1991) evidently did not consider using neutral cellulase without any other chemical pretreatment. This alternative was investigated by researchers at the USDA Forest Products Laboratories (FPL) (Jeffries et a]., 1994). They claimed enhanced deinking of wood-free noncontact printed waste with cellulases. This work has been expanded upon in a series of publications and conference reports (Jeffries et al., 1994; Rutledge-Cropsey et al., 1994; Sykes et al., 1995).

249

ENZYMATIC DEINKING TABLE IV EITECTOF ENZYMEDOSEON DEINKNC.* Enzyme dose (ECU/kg pulp)

Residual ink

0

118

(PPm)

200

10

400

20

600

34

800

39

Based on Jeffries st 01. (1994). *Repulping with a neutral/alkaline cellulase tion.

+ flota-

The work published by the FPL group compared the relative benefits of acidic and neutral cellulases to deink mixed office waste. Not surprisingly, the neutral cellulases demonstrated a benefit over the acidic cellulases, even when the pH of the waste furnish was adjusted to the proper initial pH region with sulfuric acid. (A furnish is composed of the materials in a pulp stock mixture, such as the various pulps, dyes, additives, and other chemicals, blended together in the stock preparation and fed to the wet end of the paper machine to make the paper or paper board.) Table IV shows the types of responses they observed with a neutral cellulase. These data were adapted from one of the group’s publications (Jeffries et d., 1994) and were combined with an assumed endocellulase activity value: endocellulase units per gram (ECU/g). This endocellulase assay is performed using soluble carboxymethyl cellulose (CMC) and measures viscosity change as a function of time and amount of enzyme. The viscosity change observed in this assay relates to chain cleavage or the “endo” action of the cellulose. By any measure, the deinking response observed by the FPL group required relatively small amounts of enzyme to achieve the optimum ink removal response. The dose-response curve reported by this group is unusual and has not been satisfactorily explained to date. The FPL group also conducted pilot trials on enzymatic deinking 1994). The pilot results agreed with laboratory experi(Jeffries et d., ments for two enzymes. With one of the enzymes, the ink removal efficiency was 94% in pilot compared to 96% in laboratory trials.

250

PRATIMA BAPAI TABLE V DEINKINC OF

MULTICOMPONENT CELLULASE" Brightness (YOISO)

Enzyme dose (ECU/kg pulp)

Feed

Postflotation

Post-

wash

0

78.5

80

80.8

180

79.6

80.6

81.8

360

78.8

m i

81.2

Based on Franks and Munk (199.5). *Repulping at 5% consistency and neutral pH.

Jeffries et al. (1995) observed that, in continuous processing of 2300-kg batches of 100% toner-printed office wastepaper, cellulases greatly reduced the residual particle count while increasing brightness and freeness. The strength properties and fiber length were essentially unchanged. To verify the effects observed by the FPL group, researchers from Novo Nordisk, Denmark, performed several laboratory flotation runs using a commercially available neutral cellulase, Novozym 342, hereinafter called Multicomponent A (Franks and Munk, 1995). The furnish employed in these experiments was an ideal furnish that used homogeneous paper printed on a Kodak copier. Rather than using residual ink measurements, they used brightness response as a measure of the benefit of cellulase treatment (Table V). By this measure, the Novo results paralleled the response seen by the FPL investigators. Prasad (1993) reported that treatment with a pure alkaline cellulase significantly improved brightness levels of photocopied and laserprinted papers relative to pulping in water without enzymes (Table VI). Brightness improvement of 4 IS0 units was observed, and residual ink area was reduced by 94%. Enzymatic treatment affected fiber length distributions. Such results might be expected since such papers typically contain bleached softwood chemical pulp and cellulases are more likely to affect the fiber distribution of chemical pulps. Enzymetreated pulps showed a similar increase in freeness and strength properties, such as breaking length and burst index relative to control pulps (Tables VII and VIII).

251

ENZYMATIC DEINKING TABLE VI 01: ENZYMATIC TREATMENT ON BRIGHTNESS EFFECT AND DIRTCOUNT

Treatment Blank Control Enzyme

Brightness (Yo ISO)

Dirt count (mm2/M2)

82.1

4200

84.2

1200

88.3

70

Based on Prasad (1993).

TABLE VII OF ENZYMATIC TREATMENT ON FREENESS EFFECT

Treatment Blank Control Enzyme

Freeness (CSF, ml) 400 440

490

Based on Prasad (1993).

Heise et al. (1996) carried out industrial-scale investigations on enzymatic deinking of nonimpact printed toners. Three trials were run at the Voith Sulzer pilot plant in Appleton, Wisconsin. Increased ink removal was achieved at a low level of a commercially available enzyme preparation in combination with a surfactant (Table IX). The brightness of enzymatically deinked pulp was two points higher than that of control pulp (Table X). The enzyme trials also displayed improved drainage and comparable strength when compared with a control (Table XI). No significant differences in the quality and treatability of the process water was noted. Effluent samples from these trials were lower in oxygen demand and toxicity than effluents from the control. Yang et al. (1995) carried out successful deinking of mixed wastepapers (laser/xerographic-printed and ultraviolet-coated wastepapers) and old newspapers/old magazines (ONPs/OMGs) using specially formu-

252

PRATIMA BAJFAI

TABLE VIII EWXT OF ENZYMATIC TREATMENT ON STRENGTH PROPERTIESOF PAPER Breaking length Treatment

(km)

Tear index (mN . m’g-’)

Blank Control

6.5 6.2 6.8

8.5 7.92 8.0

Enzyme

Burst index (KPa . ~n’g-’) 4.55 4.38 4.75

Based on Prdsad (1993)

TABLE IX RESILIUAL I N K IN CONTROL AND ENZYME TRIALS Dirt count

>225 pm (Tappi) >I60 pm 80-160 pm 10-80 prn Based on Hcise

st

Reference

Enzyme 1

Enzyme 2

258 417 167 310

173 239 99 110

26 47 24 200

01. (19Yti).

TABLE X DKIGIITNESS OF PULP FRflM

CONTROL AND ENZYMR RUNS Brightness (% IS01

Dcinking trial

Control Enzyme 1

Enzyme

2

Flotation feed

Final Pulp

74.3 75.8 77.2

82.2 85.7 86.2

Based on Heise el a]. (1996).

lated mixtures of enzymes followed by flotation. The enzymatic deinking process was found to eliminate or substantially reduce the use of chemicals in deinking. The quality of enzymatically deinked pulp was found to be superior to that of pulp obtained by conventional chemical

253

ENZYMATIC DEINKING TABLE XI STRENGTH PROPERTIES OF PULPFOLLUWING DEINKING Parameter CSF (ml) Kajanni (mm) Tensile index (kN mig) Burst index (k Pa m2/g) Tear index (mN m2/g) Viscosity (mPa. S)

Control

Enzyme 1

Enzyme 2

510

5 70

565

1.88

2.01

1.87

0.0410

0.0431

0.0412

2.20

2.42

2.34

4.28

4.39

4.25

17.2

17.4

16.5

Based on Heise et ol. (1996).

deinking. The brightness of enzymatically deinked MOW papers containing 90% laser copies, 3% colored paper, and 7% of other papers was significantly greater than the chemical method. Enzymatic deinking achieved a 94% lower dirt count (i.e., visible dirt) as well as an 82% lower total dirt count. Korean researchers (Ow et d.,1995) conducted enzymatic deinking trials in a newsprint mill. The treatment with blended cellulase resulted in about a two-point improvement in the brightness of deinked American old newspaper pulp (AONP). An enzyme deinking mill trial was also run with white ledger grade paper, the results of which showed a reduction in residual ink count at the mill of one of the biggest Korean tissue makers for several days during August 1994. Ink removal efficiency in terms of ink particle number was found to be increased from 93.9 to 97.7% by using the enzymatic deinking process with a blended cellulose compared to the conventional method. Ink removal efficiency measured by total ink area was also found to increase from 93.9 to 98.3Yo by using blended cellulose deinking. Zeyer et d. (1995) studied the performance of enzymes in deinking of old newsprint. Their results demonstrated that the arrangement of unit operations is an important factor. No deactivation of enzymes by shear stress was observed. Statistical investigation of particles on handsheets demonstrated that it is very likely that many ink particles are still in their original location. The majority of published reports dealing with enzymatic deinking, whether with ONPs or MOW, have used multicomponent cellulase systems that are produced by a specific microorganism. The Novo researchers used monocomponent cellulases SP476 and SP613 (herein-

254

PRATIMA BAJPAI TABLE XI1 DEINKINC WITH MONUCOMPONENT CELLULASE

Enzyme dose (ECU/kg Pulp1

Brightness (Yo ISO)

count

Ink

[PPml

n

80.6

son

400

82.8

400

400

83.5

400

400

84.0

400

300

84.0

300

~~

~

Based on Franks and Munk (1995).

after called Mono A and Mono B) for deinking of MOW (Franks and Munk, 1995). Mono A contains three discrete regions: a cellulose binding domain (CBD), a spacer or linker area, which ties the CBD to the active region, and a core protein. Cellulases like Mono A, which contain the CBD, have the ability to attack the crystalline region of cellulose structures. Mono B differs from Mono A in that it consists exclusively of the core protein region with no CBD or spacer included in its structure. This cellulase is active exclusively on amorphous regions of the cellulase structure and causes less structural change within the cellulose structure. The response obtained using Mono A was found to be similar to that observed for the Multicomponent A cellulase preparation. Mono B, on the other hand, demonstrated a dose response closer to what would be expected for more typical enzymatic systems (Table XII). Both brightness response and ink count data are included in these data. The concomitant increase in brightness and decrease in ink count values helped confirm that the use of brightness as an assessment method could provide a rough measure of response in these systems. However, these studies did not proceed far enough to lead to firm conclusions. Nevertheless, they show that the monocomponent portion of the multicomponent cellulase systems plays a major role in the enhancement of deinking of wood-free noncontact printed waste. Direct physical contact between enzyme and substrate is a prerequisite to activity (Cowling and Brown, 1968). Paper sizing and other additives may prevent or limit contact. Many researchers have studied the implications of sizing effects (Rutledge-Cropsey et a]., 1994; Zeyer

ENZYMATIC DEINKING

255

et al., 1993, 1994a, 1995). It has been suggested that sizing physically shields fibers from enzymes. Earlier work with textiles showed that starch sizing must be removed via a-amylase or other treatment before cotton fabrics can be altered by cellulase treatment (Tyndall, 1990, 1992). Paper sizings differ in mode of action and can therefore limit contact by various means. Sizing agents can limit enzyme activity by increasing fiber hydrophobicity, physically shielding fiber surfaces from enzyme attachment, or preventing access via covalent bonds with cellulose. For example, alkyl succinic anhydride increases hydrophobicity but also forms covalent bonds with cellulose. The literature shows that paper sizing reduces enzymatic deinking efficiency and that reduction can vary with sizing agent. For nonimpact-printed papers, deinking efficiency was lowest for papers sized with rosin/alum (Rutledge-Cropseyet al., 1994). Such papers demonstrated the greatest resistance to wetting and the highest fiber hydrophobicity. Paper sized with alkyl succinic anhydride was less resistant to wetting but almost as difficult to deink. These findings confirm the need for additional research, but future investigations must not be limited to sizing effects, since numerous additives are used in papermaking and effects may vary dramatically. Coatings, dyes, metals, and other additives may denature or inhibit enzymes. Several patents claim that alkaline lipase facilitates removal of oilbased inks. Nakano (1993) reported that an alkaline lipase efficiently removed offset printing inks. The effect was attributed to enzymatic hydrolysis of the drying oil or thermosetting resin in the inks. Lipases should be effective with inks carried in such material as vegetable oils, and the approach merits additional research, especially if the trend towards greater utilization of such inks accelerates. The high proportion of lignin-rich mechanical pulps in newsprint suggests that enzymes catalyzing removal of surface lignin may hold promise for deinking. This approach has been evaluated using the white rot fungus Phanerochaete chrysosporium and with lignin-degrading enzymes (Ander, 1993; Call and Strittmatter, 1992; Call et al., 1990). Lignin release was observed in all cases. Ink removal by a laccase preparation proved comparable to conventional chemical deinking. The pulps deinked with laccase showed high brightness and were easier to bleach. Further research on such enzymes, used alone or in combination with cellulose and/or hemicellulases, would lead to an better understanding of the interactions between fiber surfaces and ink, if not improved deinking effectiveness. Recent literature describes a novel deinking process that couples separation technology and cellulase treatment (Woodward et a]., 1994a,

256

PKATIMA BAJPAI

1994b). Ink particles dislodged from newsprint, presumably by cellulose activity, readhered to smaller fibers originally present or created by enzymatic action. The shorter fibers and adhered ink were then separated from longer deinked fibers. The latter were considered usable without further treatment. Since ink adhered to the shorter fibers, conventional washing or flotation was unnecessary and ink was not released into effluents. The reason for the strong association between ink and short fibers was not determined. It has been reported that separation of such fibers is technically feasible (Eul et a]., 1990; Floccia, 1988). IV. Effect of Enzyme on Pulp Yield and the Quality of Fiber and Paper

The results in terms of yield are inconclusive. Some yield reduction appears to result fro= losses of fines and other small particles due to enzymatic action. More precise control over enzyme dosages and reaction time are expected to minimize such losses. Kim et al. (1991) reported that reducing sugars were released during enzymatic deinking of old newspaper but that yield losses were immaterial. Relatively short reaction times were thought to have restricted enzyme attack of the fibrils on fiber surfaces. In another study with old newspaper, sugar release increased with enzyme dosage and reaction time (Paik and Park, 1993). Yield was reduced by 5%, but freed sugars did not explain all the loss. Microfibrils freed from fibers by enzymatic activity were said to have been lost during flotation. Even so, yields from enzymatic deinking were higher than those obtained with conventional deinking. Early workers were concerned that cellulases would reduce fiber length and adversely affect paper strength. However, Bauer-McNett classification of enzymatically deinked newspaper pulps yielded a short-fiber fraction smaller than that from conventionally deinked pulps (Kim et a]., 1991). Other investigators found similar trends-reduced fines content and improved drainage-when comparing pulps deinked with and without enzymes (Heitmann et a]., 1992; Prasad et al., 1992a, 1992b, 1993; Rushing et al., 1993). Putz et al. (1994) found no significant enzymatic effects on fiber fraction length or mass. Limiting enzyme action to removal of microfibrils is thought to remove sufficient hydrophilic material to improve drainage (Pommier et al., 1989). Freeness may also be improved by enzymatic action on small colloidal particles as well as on fines (Grant, 1990; Jackson et al., 1993; Putz eta].,1990; Young, 1989). Low enzyme concentrations can destroy fines but are not likely to harm intact fibers. Instead, fines and other

ENZYMATIC DEINKING

257

small suspended particles, with their large surface area, are attacked preferentially. This was confirmed by silver-enhanced colloidal gold labeling and visualization via light microscopy (Jackson et d., 1994). Enzyme binding to fines and suspended particles may also improve freeness (Jackson et al., 1993). Binding of cellulases or hemicellulases could aggregate small particles, much as occurs when polymers are used as retention aids. Hemicellulase treatment has been observed to reduce fines content without causing measurable hydrolysis. Yang et al. (1995) reported that freeness of enzymatically deinked MOW paper pulp was 32% higher than that of control pulp. Heise et ~ l . (1995, 1996) and Prasad (1993) found that enzymatic treatment significantly increased pulp freeness from 510 to 570 CSF and from 440 to 490 CSF, respectively. Prasad et al. (1993) observed that freeness increased in all enzyme-treated samples compared to a control (Table XIIIA). The freeness increase varied from 50% for a cellulase-treated colored flexoprinted newsprint to 14% for black-and-white printed newsprint treated with a hemicellulase preparation. Rutledge-Cropsey and Abubakr (1995) investigated the effects of enzymatic deinking on paper machine runnability, specifically drainage and wet web strength enhancement. Three deinking trials were conducted on industrial-scale equipment. Two trials used the enzymatic deinking method developed at FPL, and one trial was a surfactant control. The pulp produced in each of the three trials was used in a pilot paper machine run. During each run, overall runnability, drainage pressing response, and wet web strength were evaluated. In general, the enzyme-treated pulp ran better than the control pulp. Mainly, it was the enhanced drainage and wet web strength that contributed to improved runnability. One of the first reports on enzymatic deinking of old newspaper showed increases in strength properties relative to conventional deinking (Kim et al., 1991). Other trials (Heitmann et al., 1992; Prasad et al., 1992b, 1993) comparing enzymatically deinked pulps from a variety of papers to those deinked in water yielded similar results (Tables XIIIA and XIIIB). In the most recently reported FPL pilot deinking study (Jeffries et al., 1994), the physical properties of deinked pulp were comparable to those of control pulp produced using a thermally inactivated enzyme. Baret et d.(1991) claimed to observe a physical property enhancement even when applying as much as 4 litershon of Multicomponent A to the pulp. A hemicellulase preparation demonstrated the largest strength increase with the least improvement in freeness (Tables XIIIA and XIIIB). The strength improvement was attributed to changes in hemicellulose composition and degradation of lignin-hemicellulose linkages. Lignin release following such treatment has been reported

258

PRATIMA BAJPAI TABLE XIIIA

STRENGTH IMPROVEMENT DURING ENZYMKIX: DEINKINC Old nowsprint printed by

zyme used

Rlack-andwhitc

Hemic:ellulase

Black-andwhite flexo Colored flexo

Tensile index* “m/gl

Burst index* (Wa. mZ/g

Tear index“ (mN m2/g

420 (370)

30.6 (26.2)

1.6 (1.3)

10.4 (8.7)

Cellulase

290 (210)

37 (29)

2.4 (1.7)

9.7 (9.0)

Hemicellulase

430 (300)

38 (31)

1.5 (1.1)

10.4 (H.9)

En-

Freeness* (CSF, mll

letterpress

Based on Prasad et 01. (1992b). *Values in parentheses arc control without enzyme.

(Prasad et a]., 1992b). On the other hand, the mechanisms by which cellulases affect strength properties are not clear. Cellulases at high dosages clearly reduce strength properties, and chemically pulped fibers are more severely damaged (Jurasek et al., 1987; Oltus et al., 1987; Pommier et a]., 1989; Stork and Puls, 1994). Severe fiber damage can also increase fines and reduce freeness. A low enzyme dosage may only affect fiber surfaces. Partial digestion of fiber surfaces could promote fibrillation, resulting in improved bonding and stronger paper (Jurasek et al., 1987). Overall, the considerable strength improvements observed in deinking trials are meaningful and consistent with those produced by enzymes used in secondary fiber formation (Jackson et a]., 1993; Pommier et a]., 1989, 1990). V. Possible Mechanisms of Enzymatic Deinking

Various mechanisms have been proposed for removal of ink by enzymes. Although the role of lipases and esterases in deinking is understood, there is a fundamental lack of understanding of how mixtures of cellulases and hemicellulases can enhance deinking of repulped paper products. To economically justify the use of relatively expensive cellulases and hemicellulases, a performance improvement has to be demonstrated. Ultimately, this benefit has to be attributed to the specific

259

ENZYMATIC DEINKING TABLE XIIIB MECHAMCAL PROPERTIES OF ENZYMATICALLY DEINKED COLOROFFSETNEWSPRINT Enzyme" preparation Con-

Parameters

Blank'

trol'

I

I1

III

IV ~

CSF freeness (ml) Fiber length distribution +2a +4a +65 +150 -150

Tensile index (Nm/g) Burst index (m2/g) Tear index Imz/gl

265

250

300

300

315

320

30 25 7 7 30

30 26 7 5 32

31 28 7 6 27

30 29 7 6 27

30 29 7 6 27

30 27

32.7

31.6

36

37

39.2

35.6

1.6

1.5

1.7

1.7

1.9

1.6

7.8

7.5

7.9

8.3

8.7

7.8

a 7 28

Based on Prasad el al. (1993). Enzyme preparation I contained 6 U/ml of CMCase, 10.0 U/ml of xylanase, and 42 U/ml of filter paper activity. Enzyme preparation I1 contained 6 U/ml of CMCase, 6 U/ml of xylanase, and 1 U/ml of filter paper activity. Enzyme preparation I11 contained 0.20 U/ml of CMCase, 200 IJlml of xylanase, and 0.005 U/ml of filter paper activity. Enzyme preparation IV contained 26 U/ml of CMCase, 580 U/ml of xylanase, and 50 IJlml of filter paper activity. 'Reslushed pulp without enzyme treatment and no flotation. bReslushed pulp without enzyme treatment hut with flotation 'Mesh retained.

activity of these enzymes. Since the main function of an enzyme is to cleave specific bonds between sugar units, an explanation as to how this activity can enhance deinking must be provided. Most researches have advanced at least general explanations, but few have provided supporting data. Woodward et al. (1994a) suggested that catalytic hydrolysis may not be essential, that enzymes can remove ink under nonoptimal conditions. Mere cellulose binding may disrupt fiber surfaces in a manner and to an extent sufficient to release ink during pulping. Jeffries et al. (1995) proposed that deinking may be caused not by enzymes but by additives used to enhance enzyme stability. Residual ink areas obtained with heat-deactivated commercial enzyme preparations, however, did not exceed those observed after pulping in water (Jeffries et

2 60

PRATIMA BAJPAI

al., 1994). In addition, ink removal varies inversely with enzyme inhibition (Zeyer eta].,l993,1994a, 1994b). Korean researchers (Kim eta]., 1991) pointed out that enzymes partially hydrolyze and depolymerize cellulose molecules at fiber surfaces, thereby weakening bonds between fibers and freeing them from one another. Ink particles simply are dislodged as fibers separate during pulping. Another mechanism proposed by these researchers is that enzymatic treatment weakens the bonds, perhaps by increasing fibrillation or removing surface layers of individual fibers (Eom and Ow, 1990). However, the suggestion that enzymatic activity could be sufficient to remove whole surface layers at the low dosages and short reaction times commonly employed is questionablc. Zeyer et al. (1993, 1994a, 1994b) reported that mechanical action is critical and a prerequisite to enzymatic activity. Experiments involving mechanical and enzymatic treatment of printed cotton and rayon fabric showed that deinking efficiency increases linearly with applied friction. These results agreed with those obtained with combined enzymatic treatment and stone washing of textiles. Mechanical action was said to distort cellulose chains at or near fiber surfaces, thereby increasing vulnerability to enzymatic attack. Assuming that fiber-fiber friction increases with pulp consistency, such an explanation seems consistent with earlier findings that enzymatic deinking is more effective at medium consistency as opposed to low consistency (Jeffries et al., 1994). However, research conducted by Putz et nl. (1994) disputes the importance of mechanical action. Applying greater shear forces via pulping at higher consistency or for an extended time did not improve brightness. Also, high shear forces can denature enzymes (Kaya et a/., 1994; Reese and Mandels, 1980). Cellulases peel fibrils from fiber surfaces, thereby freeing ink particles for dispersal in suspension (Eom and Ow, 1990). This peeling mecha1983) has also been nism (Chanzy and Henrissat, 1985; Lee et d., implicated in pulp freeness increases after enzymatic treatment of secondary fiber (Ow and Eom, 1990; Pommier et a]., 1989). Enzyme dosages and reaction times, however, seem too low to cause measurable cellulose degradation. Hemicellulases facilitate deinking by breaking lignin-carbohydrate complexes and releasing lignin from fiber surfaces (Paik and Park, 1993). Ink particles are dispersed with the lignin. Hemicellulase treatment facilitated ink removal from newsprint and was accompanied by lignin release (Heitmann et al., 1992; Prasad et a]., 1992b). Jeffries et nl. (1994) reported that enzymatic effects may be indirect, that is, they removed microfibrils and fines, thereby improving freeness

ENZYMATIC DEINKING

261

and facilitating washing or flotation. Fines content is not always reduced during enzymatic deinking (Putz et al., 1994), however. Enzymatic treatment of nonimpact-printed paper removed fibrous material from ink particles, thereby increasing particle hydrophobicity and facilitating separation during flotation (Jeffries et al., 1994). This promising hypothesis should be tested with a wide array of enzymes, paper grades, and inks. Zeyer et al. (1994a) provided evidence that only easily accessible cellulose chains are subject to enzymatic cleavage. Their data also suggest that such mechanical action as surface friction on the fiber is able to alleviate this restriction by opening the outermost layers of the fiber to fully expose the cellulose chains. Only then is removal of a significant amount of ink possible. It is likely that a particular deinking system would involve several of these mechanisms but that the relative importance of each mechanism would be dependent on fiber substrate ink composition and enzyme mixture. It should also be noted that none of the studies used pure enzyme components (endoglucanases, exoglucanases, and cellobiohydrolases) of cellulase or hemicellulase to determine how the different components function in deinking. The study of pure enzyme components needs to be pursued to better understand the role of individual components and the importance of synergy in enzyme mixtures. VI. Factors Affecting Enzymatic Deinking

Operating environments are critical to success, and many variables must be optimized. These include, among others, temperature, pH, enzyme activity/dosage, reaction time, pulp consistency, and mechanical action (Daneault et al., 1994; Kochavi et al., 1990). Effectiveness is mostly limited by pH. Cellulases and hemicellulases vary in sensitivity to pH, with some having optimal activity in alkaline environments, others in neutral conditions, and still others in acidic conditions. Choosing an appropriate enzyme and maintaining proper pH during treatment will determine success or failure. As an example, matching pulping environment pH to enzyme requirements yielded brightness levels above those obtained via conventional alkaline deinking (Paik and Park, 1993). Deinking standard and colored newsprint with enzymes having optimal pH requirements between 4 and 5 worked well (Prasad et al., 1992b, 1993). The initial pH slurry had a pH of 5.5, and no adjustment was necessary. In contrast, an initial slurry of alkaline sized nonimpact printed paper had a pH of 8.5 (Jeffiies et al., 1994) compared to a pH optimum of 5.5-7.5 for enzymatic activity. Adjust-

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ment of pH levels via acid addition was necessary for effective deinking. This could raise operating costs and limit commercialization. However, using enzymes active in acidic environments might confer added benefits: acidic conditions reduce yellowing of derived products (Kim et al., 1991). Proper enzyme dosage and reaction time vary with enzyme, paper, and ink type. Too much enzyme or overly long reaction times can damage fibers. Cellulases aid hemicellulases presumably evolved to degrade wood, and their exploitation amounts to walking a fine line between desirable and undesirable effects. To date, enzyme dosage and treatment time have been determined by trial and error for the enzyme and environment in question. More research is needed in this critical area, as well as on the means for stopping reactions. Adding basic reagents to stop reactions may be counterproductive, since reagents like sodium hydroxide contribute to product yellowing. The timing of enzyme addition is a significant concern, as past treatments seem to have been decided largely subjectively. Enzymes have been added before pulp disintegration (Kim et al., 1991; Paik and Park, 1993), after disintegration and during mixing (Prasad et a]., 1992b, 19931, and during pulping (Jeffries et al., 1994). Despite the diversity of approaches, objective comparisons cannot be made because many factors varied among the experiments. A more recent report (Putz et a]., 1994) comparing several procedures clearly demonstrated that enzyme addition during initial mixing of paper and reaction medium was most effective. Enzymatic deinking would he especially attractive if surfactants and alkaline chemicals were not needed. Operating costs and environmental impact would be lowered. The literature shows mixed results. In an early newsprint trial, acceptable results were optimized with cellulose treatment and flotation in the absence of other deinking chemicals (Kim et a]., 1991). When computer printouts were used as the furnish, however, the same enzyme and environment were inadequate for ink collection; foaming and collecting agents were required. In another trial (Prasad, 1993), nonimpact-printed paper was deinked successfully without conventional deinking chemicals. The furnish was alkaline sized and contained calcium carbonate, and adequate froth was generated during flotation. Regardless of furnish or enzyme preparation, most investigators have included calcium chloride and a surfactant as flotation aids. A later report indicates that enzymatic treatment produced half the chemical oxygen demand (COD) loads produced by conventional deinking (Putz et d., 1994).

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VII. Benefits of Enzymatic Deinking

Enzymatically deinked pulp possesses superior physical properties, higher brightness, and lower residual ink compared to chemically deinked recycled pulps. More importantly, the size distribution and shape of removed ink could be effectively controlled using the enzymatic process to maximize the efficiency of the size-based flotation process. This can be accomplished by selectively varying enzyme composition, charge, and residence time, as well as by variation of other additives and pH in the system to effectively dislodge the normally large, flat, and rigid ink particles into much finer and nonplatelet forms. The enzymatic deinking process also improves freeness compared to chemically deinked pulps. The feasibility of enzymatic deinking in acidic environments has been confirmed. Applied commercially, this should reduce overall chemical requirements and minimize yellowing of reclaimed papers after alkaline deinking. Reduced chemical usage means lower waste treatment costs and reduced environmental impacts. Lower bleaching costs can also be anticipated. Enzymatically deinked pulps have proven to be easier to bleach and require fewer bleaching chemicals than pulps deinked by conventional means. Few cost comparisons between enzymatic and conventional deinking have been published. In analyses based on small-scale laboratory tests, the costs of the two methods were considered similar. Larger-scale trials are needed for thorough evaluation (Jeffries et al., 1994; Zeyer et al., 1993). Future economic analyses should also consider other potential benefits associated with enzyme usage: lower energy consumption, reduced need for chemicals in ink removal and flotation, and lower bleaching requirements. Improved drainage and faster machine speeds resulting from increased freeness may also yield significant cost savings. VIII. Current Research Needs

It is obvious that there are many areas of research that need to be addressed to develop enzymatic deinking further. There is a need to carefully evaluate the fiber furnish used. The amount of mechanical and chemical pulps, as well as the hardwood/softwood composition, in the mixture probably have some effect on the activity and efficiency of enzymes in this system. In this respect, some preliminary work by Jackson (unpublished data) at North Carolina State University (NCSU) has shown that enzyme binding is quite different for hardwoods and

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softwoods. This would imply different enzymes activities on the various components of deinking furnishes. There is also a concern as to whether lignin on mechanical fiber surfaces, such as is encountered in ONPs, can invariably absorb enzymes, provide a barrier to enzyme attack, or leach from the fiber and deactivate enzymes. Another important variable is the ink composition and the printing process. For example, soy or water-based inks, different types of xerographic and laser printing toners, and letterpress, offset, or flexo printing processes can all be expected to react differently in enzyme deinking. This has been investigated to some extent for ONPs, but a single comprehensive study that compares several different types of printing processes has not been performed. This would shed some light on the process changes required to effectively deink papers printed with different inks. In the area of MOW deinking, some studies that compare the different printing processes and toner types have been done with conventional, sonic, and agglomeration methods, but not in connection with enzymes. Work done on ONP deinking has demonstrated that the addition of enzymes in the pretreatment stage is the most beneficial addition point, but very little has been done to isolate the best enzyme addition point for MOW deinking. This is an important aspect of improvement of enzymatic deinking technology. The effects of mechanical action, investigated by Zeyer et al. (1993) and Kaya (1994) also need to be examined further to resolve the conflicts in the literature concerning the importance of mechanical action and the possibility of enzyme denaturation by shear forces. It is also of interest to study the effects of enzyme treatment on ink redeposition. Enzymes may retard redeposition of ink particles onto fibers. This could be another benefit of deinking with enzymes. Several methods have been proposed for the study of ink redeposition (Joyce et a]., 1995). These methods include collecting ink particles from rejects and reintroducing them into the deinking system, where a clean paper supply is used, and measuring redeposition by analyzing the pulps and rejects with image analysis. Another possible method would be to introduce an ink that is labeled by color or fluorescence into the normal deinking process and analyzing the rejects and pulps using image analysis. It has been difficult to measure this effect, so that the development of a measurement technique would be helpful to all in the field of deinking. Additives found in MOW furnishes could be very important. Ionic strength, type of ions present, and pH may all effect enzyme activity (Buchert et al., 1993). Kaya (1994) observed different effects depending

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on the surfactant used. Generally, cationic or nonionic surfactants were most compatible with enzyme activity, but anionic surfactants seemed to inhibit enzyme activity. Sizing and fillers found in the furnishes and whether the sheet is alkaline or acid would greatly affect the activity of the enzyme being used. To better understand the interactions between enzymes, fiber, and ink, individual components of cellulase and hemicellulase complexes should be studied. Pure components of enzyme complexes should be studied in deinking trials alone and in combination to determine which components or combinations provide the greatest benefit or result in impediments to the process. Several investigators at NCSU are pursuing various elements of this problem. Some research will deal with pure components of hemicellulases and some with cellulases. One area is the study of the adsorption isotherms of cellulose and xylanase components for various fiber substrates (e.g., bleached chemical hardwoods and softwoods, and mechanical pulp fibers). A related topic of interest is the possible recovery of active enzyme components in order to recycle enzymes and thus reduce enzyme costs. This would allow determination of recycling potential for various enzymes and whether there is a preferential loss of selected enzyme components by irreversible binding. There are several interactions between lignin and xylanase components. Studies should be performed to determine the binding energy between lignin and xylanase and how to recover xylanases after deinking. There is also a need to determine which enzyme components have the most effect on ink detachment, redeposition, final brightness, the size and nature of the ink particles produced, and the yield and strength properties of the final pulp. Enzymatic deinking is likely to produce fewer and simpler waste disposal problems. Some capital savings could also accrue as a result of more efficient dispersion and flotation. These benefits will be important only if the deinking efficiencies described in the literature are realized. Finally, enzyme costs can be expected to fall in the future as demand rises. Genetic engineering can be expected to simplify production and lessen purification costs. IX. Conclusions

The feasibility of enzymatic deinking on a commercial scale is extremely high. However, introducing this technology in a mill-scale operation will necessitate extensive customization of enzyme formulation and process variables to achieve optimal effectiveness. Rather

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extensive experience with mill-based trials has shown that specific formulations vary widely based on furnish, process water, equipment configuration in various mills, and desired deinked pulp specifications. The enzymatic deinking process will naturally lead to a new chemical balance throughout the mill water system. To effectively introduce enzymatic deinking into the pulp and paper industry, the costs and risks of conversion must be minimized. There are several research areas that must be more fully investigated to realize the potential of enzyme deinking. It is apparent that enzymes can facilitate deinking, but the exact process conditions by which this will occur have yet to be established. The research areas that require further investigation include 1) the interaction of enzymes and fiber surfaces, 2) the synergistic effect between various enzyme components, 3) the effect of fillers and additives, ionic strength, cationic species, and pH, 4) the effect of ink composition and the printing process, 5) methods to evaluate redeposition and factors that affect redeposition, and 6) the point of enzyme addition, consistency, and the effects of shear forces.

REFERENCES Ander, P. (1993). 1. Korean Tappi 25(2), 70-76. Anon. (1994a). Popar Recycler 5(1), 1, 7-8. Anon. (1994h). Recycled Paper News 4(5), 10. Baret, J. L., Leclerc, M., and Lamort, J. P. (1991). Int. Appl. No. PC‘T DK91/00090. Buchert, J., Tenkanen, M., Pitkanen, M., and Viikari, L. (1993). TappiJ. 76(11), 131-135. Call, H. P. (1991). Ger. Pat. 4,008,894. Call, H. P., and Stritlmatter, G. (1992). Das Papiar 46(10A), V32-V37. Call, 13. P., Vun Kaven, A., and Leyerer, H. (1990). Das Papier 44(10A), V33-V41. Chabot, B., Daneault, C., Lapointe, M., and Marchildon, L. (1993). Prog. Paper Recycl. 2(4),21-29. Chanzy, H., and Henrissat, B. (1985). FEBS Lett. 184(2),285-288. Cowling, E. B., and Brown, W. (1968). Adv. G e m . Ser. 95,52-187. Daneault, C., Leduce, C., and Valade, J. L. (1994). Toppi J. 77(6), 125-131. Din, N., Gilkes, N. R., Tekant, B., Miller, R. C., Warren, A. J., and Kilburn, D. G. (1991). Biotechno/oRy 9(11), 1096-1099. Elliston, K. O., Yablonsky, M. D., and Eveleigh, D. E. (1990). In “Enzymes in Biomass Conversion” (G. F. Leatham and M. E. Hinimel, eds.), ACS Symp. Ser., No. 460, pp. 290-300. American Chamical Society, Washington, DC. Eom, T. 7.. and Ow, S. S. K. (1989). Brit. Pat. GB2,231,595A. Eom, T.J., and Ow, S. S. K. (1990). Ger. Pat. GB3,934,772. Eriksson, K. E. L. (1990). Wood Sci. Tachnol. 24, 79-101. Eriksson, K. E. L., Blanchctte, R. A., and Ander, P. (1990). “Microbial and Enzymatic Degradation of Wood and Wood Components,” pp. 89-1 77. Springer-Verlag, Berlin.

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Esterbauer, H., Hayn, M., Abuja, P. M., and Claeyssens, M. (1990).In “Enzymes in Biomass Conversion” (G. F. Leatham and M. E. Himmel, eds.), ACS Symp. Ser., No. 460, pp. 301-312. American Chemical Society, Washington, DC. Eul, W., Meier, J., Arnold, G., Bereger, M., and Suess, H. U. (1990).In ”Proc. Tappi Pulping Conf.,” pp. 757-765. Tappi Press, Atlanta, GA. Floccia, L. (1988). In “Proc. Tappi Int. Pulp Bleaching Conf.,” pp. 181-197. Tappi Press, Atlanta, GA. Franks, N. E., and Munk, N. (1995). In “Proc. Tappi Pulping Conf.,” pp. 343-347. Tappi Press, Atlanta, GA. Fukuda, S., Hayashi, S., Ochiai, H., Ihizumi, T., and Nakamura, K. (1990). Jpn. Pat. 229,290/90. Fukunaga, N., and Kita, Y. (1990). Jpn. Pat. 2-80,683. Gen, Y., and Go, S. (1991). Jpn. Pat. 882/91. Grant, R. (1990). Pulp Paper Int. 32(5), 118-119. Guy Vare, J. A,, Lucrelk, M., Sharyo, M., and Sakaguchi, H. (1990). Jpn. Pat. 150,984/90. Hagiwara, M. (1988). Jpn. Pat. 2-80684. Heise, 0. U., Unwin, J. P., Klungness, J. H., Finneran Jr., W. G., Sykes, M., and Abubakr, S. (1995). In “Proc. Tappi Pulping Conf.,” pp. 349-354. Tappi Press, Atlanta, GA. Heise, 0. U., Unwin, J. P., Klungness, J. H., Finneran Jr., W. G., Sykes, M., and Abubakr, S . (1996). Tappi 1. 79(3), 207-212. Heitmann, J. A., Joyce, T. W., and Prasad, D. Y. (1992). In “Proc. 5th Int. Conf. Biotechnol. Pulp and Paper Industry,” Kyoto, 27-30 May, pp. 175-180. Jackson, L. S., Heitmann, J. A., and Joyce, T. W. (1993). TappiJ. 76(3), 147-154. Jackson, L. S., Heitmann, J. A., and Joyce, T. W. (1994). Prog. Paper Recycl. 3(2), 32-41. Jeffries, T., Patel, R. N., Sykes, M. S., and Klungness, J. H. (1992). In “Proc. Mat. Res. SOC. Symp.,” pp. 277-287. Jeffries, T., Klungness, J. H., Sykes, M. S., and Rutledge-Cropsey, K. (1993). In “Proc. Tappi Recycl. Symp.,” pp. 183-188. Tappi Press, Atlanta, GA. Jeffries, T. W., Klungness, J. H., Sykes, M. S., and Rutledge-Cropsey, K. R. (1994). Tappi J. 77(4), 173-179. Jeffries, T. W., Sykes, M. S., Cropsey, K. R., Klungness, J. H., and Abubakr, S. (1995). In “Proc. 6th Int. Conf. Biotechnol. Pulp and Paper Industry,” pp. 141-144. Joyce, T. W., Heitmann, J. A., and Eriksson, L. A. (1995). Proc. Wastepaper ‘95,4th Int. Wastepaper Technology Conf., 13-15 June, pp. 399-407. Jurasek, L., Paice, M. G., Yaguchi, M., and O’Leary, S. (1987). In “Biomass Conversion Technology: Principles and Practice” (M. Moo-Young, J. Lamptey, B. Click, and H. Bungay, eds.), pp. 131-137. Pergamon, New York. Kaya, F. (1994). Ph.D. Thesis, North Carolina State University at Raleigh. Kaya, F., Heitmann, J. A., and Joyce, T. W. (1994). 1.Biotechnol. 36(7), 1-10, Kim, T.-J., Ow, S., and Eom, T. J. (1991). In “Proc. Tappi Pulping Conf.,” Vol. 2, pp. 1023-1030. Tappi Press, Atlanta, GA. Kochavi, D., Videback, T., and Cedroni, D. (1990). Am. DyestuffReporter 79(9), 24-28. Lee, S., Kim, K. H., Ryu, J. D., and Taguchi, H. (1983). Biotechnol. Bioeng. 25(1), 33-52. Nakano, J. (1993). J. Korean Tappi 25111, 85-91.

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Noniura, Y., and Shoji, S. (1988). Jpn. Pat. 59,494, Honshu Paper Mfg. Co. Ltd. Oltus, E., Mato, J., Bauer, S., and Farakas, V. (1987). Cellulose Chem. Techno]. 21, 663-672. Ow, S., and Eom, T.-J. (1990). In “Proc. EUCEPA Symp. Additives, Pigments and Fillers in the Pulp and Paper Industry,” Barcelona, pp. 85-94. Ow, S., and Eom, T.-J. (1991). Jpn. Tappi J. 45(12), 1377-1382. Ow, S. K., Park, J.-M., and Han, S.-H. (1995). In “Proc. 6th Int. Conf. Biotechnol. Pulp and Paper Industry,” pp. 163-168. Paice, M. G., Bernier, R., and Jurasek, L. (1988). Biotechnol, Bioeng. 32, 235-239. Paik, K. H., and Park, J. Y. (1993). 1.Korean Tappi 25(3),42-52. Pommier, J. C., Fuentes, J. L., and Goma, G. (1989). Tappi]. 72(6), 187-191. Pommier, J. C., Fuentes, J. L., and Goma, G. (1990). Tappi]. 73(12), 197-202. Prasad, D. (1993). Appita 46(4), 289-292. Prasad, D. Y., Heitmann, J., and Joyce, T. (1992a). Papiripnr 36(4), 122-130, Prasad, D. Y., Henmann, J. A., and Joyce, 1.W. (1992b). Prog. PaperRecycf. 1(3),21-30. Prasad, D. Y., Heitrnann, J. A., and Joyce, T. W. (1943). Nordic Pulp Paper Res. 1. 8(2), 284-286. Puls, J., Stork, G., and Schuseil, J. (1093). Das Papier 47(12) 719-728. Putz, H.-J., WLI,S., and Gottsching, L. (1990). Dus Papier 44(10A), V42-V48. Putz, H.-J., Renner, K., Gottsching, L., and Jokinen, 0. (1494). In “Proc. Tappi Pulping Conf.,” pp. 877-884. Tappi Press, Atlanta, GA. Reese, E. T., and Mandels, M. (1980). Biotechnol. Bioeng. 22, 323-335. Rushing, W., Joyce, T. W., and Heitmann, J. A. (1993),In “Proc. 7th Int. Symp. Wood and Pulping Chemistry,” Beijing, pp. 233-238. Rutledge-Cropsey, K.,and Abubakr, S. M. (1995). In “Proc. Tappi Pulping Conf.,” pp. 639-643. Tappi Press, Atlanta, GA. Rutledge-Cropsey, K., Jeffries, T., Klungness, J. H., and Sykes, M. (1994). In “Proc. Tappi Recycl. Symp.,” pp. 103-105. Tappi Press, Atlanta, GA. Sharyo, M., and Sakaguchi, H. (1990). Jpn. Pat. 2,160,984. Stork, G., and Puls, J. (1994). Das Papier 48(6), 310-319. Stork, G., Pereira, H., Wood, T. M., Dusterhoft, E. M., Toft, A., and Puls, J. (1994). In “Proc. Tappi Recyc. Conf.,” pp. 107-117. Tappi Press, Atlanta, GA. Sugi, T., and Nakamura, K. (1991). Jpn. Pat. 249,291/91. Sykes, M., Klungness, J . , Abubakr, S., and Cropsey, K. (1995). In “Proc. Tappi Recyc. Symp.,” p. 61.Tappi Press, Atlanta, GA. Tyndall, R. (1990). Am. Dyestuff Reporter 79(5), 22-30. Iyndall, R. M. (1992). Textile Chemist Colorist 24(6),23-26. Urushibata, H. (1984). Jpn. Pat. 59-9299. [Jutela, E. (1991). Poper Techno/. 32(10),44-49. Vidotti, R. M., Johnson, D. A., and Thompson, E. V. (1992). In “Proc. Tappi Pulping Conf.,” pp. 643-6.52. Tappi Pross, Atlanta, GA. Wood, ’I:M. (1989). In “Enzyme Systems for Lignocellulose Degradation” (M. P. CoughInn, 4 . 1 , pp. 17-36. Elsevier, New York. Woodward, I., Stephan, L. M., Koran, L. I., Wong, K. K. Y., and Saddler, J. N. (1994a). Bintechnolology 12(9),90.5-908. Woodward, J., Stephai, L. M., Koran, L. J., Wong, K. K. Y., and Saddler, J. N. (1994b). Cham. Rng. News 72(31), Yang, J. L., Ma, J . , atid Eriksson, K. E.L. (1995). In “Proc. 6th hit. Conf. Biotechnol. Pulp and Paper Industry,” pp. 257-162.

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Zeyer, Chr., Rucker, J. W., Joyce, T. W., and Heitmann, J. A. (1994b). Textile Chemist Colorist 26(3), 26-31. Zeyer, C., Heitmann, J. A., Joyce, T. W., and Rucker, J. W. (1995). In “Proc. 6th Int. Conf. Biotechnol. Pulp and Paper Industry,” pp. 169-172.

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Microbial Production of Docosahexaenoic Acid (DHA, C22:6) AJAYSINGH AND OWEN P. WARD* Department of Biology Microbial Biotechnology Laboratory University of Waterloo Waterloo, Ontario NZL 3G1, Canada

I. Introduction A. Nutritional arid Clinical Importance of DHA B. Sources of DHA 11. Biosynthesis of Polyunsaturated Fatty Acids A. Carbon Metabolism B. Fatty Acid Biosynthesis 111. Potential Microorganisms for DHA Production A. Lower Fungi B. Microalgae C. Psychrophilic Bacteria W. Kinetics of Microbial Growth and DHA Production A. Incubation Time B. Fermentor Cultivation V. Factors Affecting Microbial DHA Production A. Media Components B. Inoculum Development C. pH D. Temperature E. Light F. Aeration VI. Downstream Processing VII. Epilogue References

I. Introduction Docosahexaenoic acid (DHA, 22:6), a long-chain polyunsaturated fatty acid of the omega-3 family, is widely recognized to be an important dietary constituent (Fig. 1).It is the primary structural fatty acid of the highly active neural tissues and makes up about 60% of the structural lipid in the gray matter of the brain (O’Brian and Sampson, 1965). Since *To whom all correspondence should be addressed. TEL: (519) 885-1211; FAX: (519) 746-4989. 271 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 4 5 Copyright 0.1997 by Academic Press All rights of reproduction in any form reserved. 0065-2164/97525.00

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COOH

all-cis-9,12,15-octadecatrienoic acid (a-linolenicacid, 18:3)

COOH

all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA, 205)

al1-cis-4,7,10,13,16,19-do~0sahexaenoic acid (DHA,22:6) FIG.1. Chemical structure of major 0-3 polyunsaturated fatty acids.

the retina, cerebral cortex, testes, and sperms of mammals are particularly rich in DHA (Anderson, 1970; O’Brian and Sampson, 1965; Poulos et d.,1975),it has perceived functions in the nervous and reproductive systems (Dratz and Deese, 1986; Neuringer ef d.,1984; Salem et al., 1986). Polyunsaturated fatty acids (PUFAs) consist of two families ( 0 - 3 and 0-6) of fatty acids containing 18 to 22 carbons. 0-6 or n-6 fatty acids are the predominant highly unsaturated fatty acids found in plants and animals, whereas the 0 - 3 or n-3 fatty acids are commonly found in marine animals and phytoplankton. Linoleic acid (18:2, 0-6) and a-linolenic acid (18:3,0-3) are essential dietary fatty acids, and are precursors for a number of long-chain PUFAs such as arachidonic acid (ARA, 20:4, 0-6), eicosapentaenoic acid (EPA, 20:5, w-3), docosapentaenoic acid (DPA, 22:5, 0-31, and docosahexaenoic acid (DHA, 22:6, 0 - 3 ) . As

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID LINOLEIC ACID

1

EICOSAPENTAENOIC ACID

273

DOCOSAHEXAENOIC ACID

Marine food

Docosapentaenoic acid

(Prostaglandin]

pgmmpJ FIG.2. Mammalian metabolism of polyunsaturated fatty acids.

well as being important as structural lipids, these long-chain PUFAs are precursors of ecosanoids (Weber et al., 1986) such as prostaglandins, thromboxanes, and leukotrienes (Fig. 2). The roles of 0-3 PUFAs as important dietary compounds for preventing arteriosclerosis and coronary heart diseases, for alleviating inflammatory conditions, and for retarding the growth of tumor cells have been recognized during the last decade (Krukonis, 1990; Shikano et a]., 1993). These beneficial effects are a result both of 0 - 3 fatty acids causing competitive inhibition of compounds produced from 0-6 fatty acids and of beneficial compounds produced directly from the 0-3 PUFAs themselves (Samuelssen, 1983). A. NUTRITIONAL A M CLINICAL IMPORTANCE OF DHA DHA is considered an essential fatty acid because it cannot be synthesized de nova by humans. Although a-linolenic acid is elongated and desaturated to DHA in mammals, it is believed that this conversion is very slow, particularly in the presence of large amounts of linoleic acid, as the two substrates compete for the same enzyme system (Dyerberg, 1986), and that DHA found in human tissues originates from dietary DHA (Salem et al., 1986). The most abundant 0 - 3 PUFA in human milk is DHA, which is at about 30 times the level observed in cow's milk (Jensen, 1989). DHA

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AJAY SINGH AND OWEN P. WARD

concentration is consistent among well-fed mothers from different countries and ethnic backgrounds (Crawford et al., 1976). A deficiency of DHA in the human infant diet, either as a result of use of formula milk or because of mothers adhering to a strict vegetarian diet, remarkably reduces (up to 50%) DHA content in erythrocyte lipid, phosphatidylcholine, and phosphatidylethanolamine compared to normal breast-fed infants (Putnam et al., 1982; Sanders et al., 1978: Simopoulos, 1989). A decrease in both 0 - 3 and 0-6 PUFAs in serum triacylglycerols of infants fed cow’s milk and a 23-fold increase in the blood triene:tetrane ratio are reported indications of essential fatty acid deficiency compared to breast-fed infants (Putnam et al., 1982). Carlson et al. (1990) reported that formula-fed infants compared to breast-fed or fish-oil-supplemented formula-fed infants exhibited poorer performance in some of the parameters of the Fagan Test of Infant Intelligence (FTII). Similarly, studies on the development of intelligence in human infants and children have indicated significantly higher IQs in children (18 months to 15 years) who were breast-fed (Taylor and Wadsworth, 1984; Morrow-Tlucak et al., 1988; Morley et al., 1988). Also, DHA-supplemented (fish oil source) formula-fed infants exhibited a more rapid rate of development of visual acuity compared to control formula-fed babies (Uauy, 1990; Uauy et a]., 1990). However, fish supplementation has disadvantages in terms of taste and odor; in addition, fish oil is very sensitive to oxidation because of high levels of unsaturation. The ratio of ARA to EPA to DHA in fish oils (0.2:2.1:1.0) is completely different than from that in human milk (2.0:0.2:1.0). The high levels of EPA i n fish oil may act as an antagonist or an inhibitor of the infant’s own endogenous ARA biosynthesis (Kinsella, 1990). Therefore, fish oil supplementation of infant formula may require an ARA co-supplernentation to overcome the detrimental effect of EPA (Kinsella, 1990). The preferential association of DHA with neural tissue and the modification of the membrane properties imparted by this fatty acid have suggested a role in membrane excitation (Chacko et al., 1977). It is generally associated with stearic acid in phosphatidylserine and phosphatidylethanolamine of synaptosomal membranes and vesicles (Baker, 1979; Breckenridge et al., 19721, where it may produce an optimal acyl-chain packing array for the function of transmembrane proteins involved in the excitatory response (Fliesler and Anderson, 1983; Applegate and Glomset, 1986; Gibson and Brown, 1993). The liver is the major site where dietary a-3fatty acids are converted to DHA (Nouvelot et a]., 1986). The resulting DHA can then be transported through the plasma to the central nervous system (Yorek et a]., 1984). This pathway is considered to be the main source of DHA for the central nervous system (Scott and Bazan, 1989).

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TABLE I

EFFECTSOF DHA DEFICIENCY IN THE DIET POSSIBLE PHARMACOPHYSIOLOGICAL Reduction of DHA levels in erythrocyte lipids Increase in platelet and smooth muscle reactivity Reduction of DHA level in brain and retina Poor brain development in infants Visual dysfunction Impairment in learning abilities Neurological disorders such as kinky hair disease and Alzheimer’s disease Multiple sclerosis and polydipsia

Some pharmacophysiological effects of DHA deficiency in diet are shown in Table I. Dietary manipulation of the PUFAs induces modifications of membrane lipid composition in all the tissues, including brain and retina (Neuringer et a]., 1986; Bourre et a]., 1990; Connor et al., 1990).Deficiency of DHA and 0-3 fatty acids in the diets of animals resulted in impairments in retinal functions, reflected in decreased visual acuity and abnormal electroretinograms (Bourre et al., 1989; Neuringer et al., 1991), and in learning abilities (Lamptey and Walker, 1976; Yamamoto et al., 1988). Visual dysfunction was observed in humans with an inadequate dietary intake of 0 - 3 fatty acids (Uauy et al., 1990). Although it is not possible to undertake PUFA-deficiency studies in humans, there are reports suggesting an important function in the development of the brain tissues of infants. A congenital defect known as “kinky hair disease” has been described in infants where the lipid classes and fatty acid distribution in the brain were normal in most ways except for a marked depression of DHA (O’Brian and Sampson, 1966),which may indicate that neuronal degeneration occurs when this fatty acid is depleted under certain circumstances. A selective loss of DHA as well as of 20:4 and 2 2 4 fatty acids in the brain tissues of Alzheimer’s disease patients has been reported (Soderberg et al., 1991). Based on epidemiological data, multiple sclerosis may also be related to a dietary deficiency of 0 - 3 fatty acids during a crucial stage of brain development (McAlpine et al., 1963). B. SOURCES OF DHA

The current commercial source of DHA is marine fish and its oil. Levels of DHA in fish oils are somewhat variable and can range from

2 76

AJAY SINGH AND OWEN P. WARD

TABLE I1 SOURCES OF DHA

Source

Fish

Total lipids (% in biomass]

DHA of total lipids)

(Yo

6-14

5-15

Shellfish

1-3

8-1 5

Fungi

5-20

20-50

Algae

20-50

10-35

5-10

5-20

Bacteria

to 20% (Table 11). Fishes such as salmon, sardine, mackerel, menhaden, anchovy, and tuna, which usually contain a high proportion of fat tissue, are used for fish oil production. The composition and content of 0 - 3 fatty acids in fish oils from each source varies depending upon species of fish, season, and geographical location of catching sites (Bimbo, 1987). High levels of DHA have been reported in certain fish tissues such as the orbital sockets of tuna (Yazawa et al., 1991). Antarctic fish generally have higher levels of DHA than more common marine fishes (Kinsella, 1990). On a global basis, about two million tons of fish oil are produced annually (Ward, 1995). A comparison fatty acid profile of different fish oils is presented in Table 111. Most fish oils are produced by a wet reduction process under inert gas or in closed containers to reduce chances of quality deterioration due to oxidation (Ackman, 1982). Refined fish oils with increased levels of w-3 fatty acids are produced by a process known as winterization. High-grade fish oils are deodorized to eliminate undesirable fish flavor, and antioxidants such as tocopherol, tertiary butylhydroxyquinone (TBHQ), or octyl gallate is added to prolong shelf-life. However, due to relatively a low proportion of DHA in fish oil and the difficulties encountered in extraction and purification of 0 - 3 fatty acids (Contreras et al., 1971; Haagsma et al., 1982; Fujita and Makuta, 1983; Aveldano et al., 1983; Nilsson ef al., 19881, large-scale production of DHA is difficult. Furthermore, other sources of w-3 fatty acids are being explored because of the limited availability of fish oil (Yongmanitchai and Ward, 1989). 8

277

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID TABLE I11

FATTY ACIDSPROFILES OF SELECTED FISHOILS % of total fatty acids Fatty acid

Cod

Seal

Sardine

Menhaden

(1)

(2)

(3)

(4)

11.0

14:O

6.9

7.0

6.6

16:O

18.9

9.0

15.5

19.9

16:l

6.2

13.0

9.5

13.7

18:O

3.4

1.0

3.7

3.3

18:l

29.6

17.5

17.3

10.9

18:2

3.3

-

2.5

1.1

18:3 (n-6)

1.8

-

18:3 (n-3)

2.9

1.1

-

1.1

20:l

-

12.8

8.1

1.3

-

-

20:4

0.4

2.5

0.6

205

11.9

7.5

9.6

14.6

22:l

-

5.6

7.8

225

3.2

-

-

22:6

9.0

9.5

8.5

7.5

(1) = Li and Ward (1993); (2) = Myrnes st al. (1995); (3) = Seto et al. (1992);(4) = Ackrnan et 01. (1988).

Although fish represent the most important source of DHA in the human diet, in many cases DHA is also an essential fatty acid for the fish. The fish receive their dietary DHA from phytoplankton (Tinoco, 1982). Therefore, the natural source of w-3 fatty acids is algae, which contain these PUFAs as important components of their photosynthetic membranes. Thus, PUFAs accumulate in the food chain and are eventually incorporated into fish oils. DHA has been found in many species of phytoplankton and seaweed in levels ranging from 12 to 35% of total fatty acids (Ackman et al., 1968; Sicko-Goad et al., 1988; Joseph, 1975; Cohen, 1986). Studies have shown that the contents of 0-3 fatty acids in fish and phytoplankton are similar to those found in some marine algae from the same areas (Ackman, 1982). Several laboratories have confirmed that most fish species can be depleted of DHA simply by restriction of 0 - 3 fatty acids in their diet. Unlike fish, these primary producers contain the genetic

2 78

AJAY SINGH AND OWEN P. WARD

and enzymatic systems for DHA biosynthesis. Microalgae, like higher plants, contain only those fatty acids that they have genetic competence to synthesize (Kyle et al., 1992). Aside from marine algae, some other microorganisms contain considerable amounts of 01-3 fatty acids and are possible sources of commercial production of DHA. Microorganisms cultured in controlled bioreactors offer an alternative and unlimited source of desired fatty acids. The diversity of species can facilitate the selection of microbial strains producing a large proportion of their lipid material as a single predominant fatty acid form (Ward, 1995). Environmental conditions can be manipulated to optimize microbial growth and product formation in order to achieve maximum productivity of the desired product. The fatty acid compositions and profile of prokaryotic and eukaryotic microorganisms are different. Among prokaryotes, blue-green algae contain some PUFAs, but they produce C12 to C18 fatty acids with maximum of four double bonds. Certain bacteria have been found to contain a substantial amount of DHA (Yano et a]., 1994; Hamamoto et a]., 1995). Among eukaryotic microorganisms, PUFAs are usually found in the polar lipid components. They vary widely in terms of their structure, that is, the carbon chain lengths, the degree of unsaturation, and the location of double bonds. However, they are typically all-cis in configuration and can be assigned into one of the two families: w-3 and W-6. Several genera of yeast are good sources of oil, but they contain mainly saturated and monounsaturated fatty acids. PUFAs found in yeast are limited to 18:2 and 18:3 acids. The eukaryotes that have been studied extensively for their long-chain PUFAs are mainly classes of lower fungi. Fungal fatty acids consist of a homologous series of saturated and unsaturated aliphatic acids ranging from 10 to 24 carbons in chain length. Even-numbered carbon chains with all-cis configuration predominate. Fatty acids with 16 or 18 carbons are most abundant, with palmitic acid (16:O) being the principal saturated and 18:l and 18:2 the major unsaturated fatty acids. II. Biosynthesis of Polyunsaturated Fatty Acids A. CARBON METABOLISM

Acetyl-CoA is the principal building block for de novo synthesis of fatty acids (Weete, 1980). However, an additional series of metabolic events occurs prior to the formation of acetyl-CoA. A summary of carbon metabolism in oleaginous fungi is shown in Fig. 3. The two key enzymes, ATP:citrate lyase and malic enzyme, are involved in lipid

2 79

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID Acetyl-CoA

Pyruvate -+

T

Oxaloacetate

\ Malate

MITOCHONDRIA

t

6

Triacylglycerols

P

I

Malonyl-CoA

FIG.3. Carbon metabolism in oleaginous fungi. Key enzymes: 1,aconitase; 2, isocitrate dehydrogenase; 3 , ATP:citrate lyase; 4, malate dehydrogenase; 5, malic enzyme; 6, fatty acid synthetase.

accumulation in oleaginous fungi (Ratledge, 1981). A correlation has been observed between the activity of the ATP:citrate lyase and the ability of yeast (Boulton and Ratledge, 1981) and fungi (Kendrick and Ratledge, 1992a) to accumulate more than 20% of their biomass as lipid. ATP:citrate lyase is located in the cytosol fraction of the oleaginous organisms and provides acetyl-CoA from citrate for fatty acid biosynthesis: Citrate

+ ATP + CoA + Acetyl-CoA + Oxaloacetate + ADP + Pi.

Another key enzyme, malic enzyme, generates the NADPH by which the acetyl units can be reduced and used as the backbone of the fatty acids (Boulton and Ratledge, 1985). On the basis of a study of three oleaginous microorganisms, two yeast and one fungi, it has been postulated that lipid accumulation is a result of the concerted action of at least two separate metabolic events (Botham and Ratledge, 1979; Ratledge, 1981). First, the NAD-dependent isocitrate dehydrogenase of mitochondria has an absolute requirement for AMP, so that when AMP

280

AJAY SINGH AND OWEN P. WARD

concentration is low, as occurs during nitrogen deprivation, citric acid will accumulate. Second, ATP:citrate lyase cleaves citrate to acetyl-CoA and oxaloacetate, so that fatty acid synthesis is constantly primed with substrate. The sites of biosynthesis of fatty acids are mainly in the cytoplasm outside the mitochondria. However, most of the acetyl-CoA is derived from the oxidation of pyruvate in mitochondria, and the mitochondria1 membrane is relatively impermeable to acetyl-CoA (Gurr and Harwood, 1991). Therefore, oleaginous eukaryotic microorganisms accumulate citrate in the mitochondria, which is then transported into the cytoplasm and cleaved there by the ATP:citrate lyase. Since nonoleaginous organisms do not possess the citrate-cleaving enzyme and most rely on the less effective carnitine-mediated system for production of acetyl-CoA in the cytoplasm (Kohlow and Tan-Wilson, 1977), desaturation of fatty acids occurs with the fatty acyl groups attached to phospholipids. In fungi, the desaturation occurs with fatty acyl groups specifically attached to the sn-2 position of phosphatidylinositol (Ratledge, 1992). On the other hand, desaturation of fatty acyl groups attached to phosphatidylcholine has been reported in plants (Stumpf, 1987).

B. FATTYACIDBIOSYNTHESIS Fatty acids are synthesized from acetyl-CoA by the concerted action of the two complex enzyme systems, acetyl-CoA carboxylase and fatty acid synthetase. Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis, that is, the ATP- and Mnz+-dependent carboxylation of acetyl-CoA to malonyl-CoA (Gurr and Harwood, 1991). The second step is conversion of malonyl-CoA to fatty acids, which is catalyzed by fatty acid synthetase and requires acetyl-CoA and NADPH. This involves a series of condensation-reduction-dehydration-reduction reactions that result in lengthening of the acyl by two carbons (Turner and Aldridge, 1983), and the formation of even-numbered fatty acids. Malonyl-CoA supplies all the carbon atoms of the long-chain fatty, acids with the exception of the two methyl terminal carbons, which are supplied normally by acetyl-CoA. Substitution of propionylor isopropionyl-CoA for acetyl-CoA results in the formation of an oddnumbered or branched-chain fatty acid (Bressler and Wakil, 1961). Fungi are known to be active in de novo biosynthesis of palmitic and stearic acids through reductive polymerization of acetate (Radwan, 1991), resulting in an abundance of fatty acids with 16 and 18 carbons acids, with palmitic acid as the major saturated fatty acid (Weete, 1980).

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

281

Unsaturated fatty acids are synthesized by at least two mechanisms. One pathway is anaerobic, whereby a double bond is introduced into a medium-chain-length fatty acid, followed by chain elongation. This pathway is only limited to certain bacteria (Gurr and Harwood, 1991). The other pathway involves an aerobic route in which direct desaturation of a preformed fatty acid is conducted in the presence of molecular oxygen and reduced compounds such as NADH or NADPH. This aerobic pathway is the most common, being found in protozoa, fungi, algae, some bacteria, and mammals (Boulton and Ratledge, 1985). The first double bond is invariably introduced into the 9 position of the saturated fatty acids, Thus, palmitoleic (16:1, 0-9) and oleic (18:1, 0-9) acids are the most common monoenes in fungi. PUFAs in fungi are usually synthesized from 18:l monounsaturated fatty acids by a series of chain elongation and desaturation steps (Fig. 4). Almost all fungi can produce 18:l from stearic acid (18:O) by aerobic desaturation. The subsequent desaturation of 18:l results in the formation of a-linolenic (18:3, 0-3) and y-linolenic (18:3, 0-6) acids through linoleic acid (18:2, 0-6). In some fungi, desaturation and elongation proceed to form long-chain PUFAs such as ARA (20:4, 0-6), EPA (20:5, 0-3),DPA (22:5, 0-3), and DHA (22:6, 0 - 3 ) . The fungal species particularly active in the synthesis of these PUFAs belong to a lower class of fungi, Phycomycetes. Both desaturation and chain elongation are enzyme-catalyzed steps (Ackman and Cunnane, 1992). Chain elongation involves the same mechanism in all pathways, introducing two carbon atoms from the donor (either acetyl- or malonyl-CoA)to the acyl chain. Desaturation is a relatively slow step in relation to elongation and is considered rate-limiting (Ackman and Cunnane, 1992). The 0-6 family of PUFAs is synthesized from linoleic acid (182, 0-6) via desaturation and chain elongation, but the additional double bonds are exclusively introduced toward the carboxy-terminal of the molecule. ARA (20:4) may be synthesized in this pathway via either of two routes (Wassef, 1977): (1) desaturation of linoleic (18:2, 0-6) to y-linolenic (18:3, 0-6) acid followed by chain elongation, or (2) chain elongation of 18:2 (0-6) to Il,l4-eicosadienoic acid (20:Z) followed by carboxyl-directed desaturation. The first route occurs in the lower fungus Saprolegnia parasitica (Gellerman and Schlenk, 1979) and the red alga Porphyridium cruentum (Nichols and Appleby, 1969),whereas the second route is the major one in the slime mold Physarum polycephalum (Korn et al., 1965) and certain Protozoa (Erwin, 1973). The 0-3 type of desaturation usually produces linoleic (18:2,0-6)and a-linolenic (18:3, 0-3) acids from oleic acid (18:1, 0-9) by introducing

282

AJAY SINGH AND OWEN P. WARD

1

I

A9

1 . . A 15

Octadeeadienoicacid

1EL

1

1

A6

A6

Eicosadienoic acid

I

A5

Eicosatrienoic acid

I I

EL

I

A5

temperature

Docosapentaenoic acid

0-9 route

I

I

0 - 6 route

w-3 route

A4

FIG.4. Biosynthesis of PUFAs in fungi. An, fatty acyl desaturase acting at nth atom of fatty acid; EL, elongase.

double bonds at positions 12, 13, 15, and 16 of the acyl molecule (Wassef, 1977).Further chain elongation and desaturation reactions will yield EPA (20:5, w-3), DPA (22:5, 0 - 3 ) , and DHA (22:6, w-3). Thus, 0 - 3 desaturation is the main pathway for DHA biosynthesis in most organisms. A number of marine and other microorganisms, such as Porphyridium yezoevensis (Kayama et al., 1986), Nannochloropsis salina (Ben-Amotz et al., 1987), Chlorella spp. (Chu and Dupuy, 1980), and Crypthecodinium cohnii (Henderson et al., 1988) seem to follow this pattern.

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

283

Henderson et al. (1988) investigated the biosynthesis of oleic acid and DHA by a heterotrophic marine dinoflagellate, Crypthecodiniurn cohnii strain WH-d. Radioactivity incorporated into lipid by the dinoflagellate grown in the presence of I4C-acetatewas recovered largely in phosphatidylcholine and triacylglycerols. Saturated fatty acids contained most of the radioactivity incorporated into both polar lipids and triacylglycerols. Thus, the observed pattern of labeling is indicated that saturated fatty acids were the products of fatty acid synthetase and reflected desaturation of saturates to monoenes, which in turn further desaturated to a PUFA. The synthesis of PUFA was indicated by the gradual incorporation of radioactivity into fatty acids having four or more double bonds including DHA. Interestingly, when C. cohnii was grown with [1-14C]acetate or with [I-14C]-octanoate,all of its fatty acids were labeled (Beach et a]., 1974). Distribution of 14C in 18:l and 22:6 indicated that octanoate was used as a source of C2 units and was not chain-elongated. However, when C. cohnii was grown with 14C-labeled C10-C18 fatty acids that were anticipated as precursors 18:l and 22:6, only 18:l was labeled. The precursor-product relationships, inferred from the patterns of distribution of radioactivity, suggested that the biosynthesis of 18:l was compartmentalized from that of 22:6, and that known pathways to the a-3PUFAs were either inaccessible to exogenous intermediates or did not operate in C. cohnii. Ill. Potential Microorganisms for DHA Production A. LOWERFUNGI

In the past decade, many studies on the production of DHA using fungi have been carried out. Phycomycetes, a group of diverse organisms among the lower fungi, is the only class of fungi that appears to be a promising source of PUFAs. Some species within the orders Saprolegniales, Peromosporales, and Etomophthorales produce significant amounts of DHA (Haskins et al., 1964; Shaw, 1965; Tyrell, 1967). Higher fungi (e.g., Ascomycetes, Basidiomycetes, and Deuteromycetes) do not produce any significant amount of DHA. Some species of the genus Thraustochytrium, a marine fungus, synthesize an unusually high proportion of DHA and have potential for commercial exploitation. Genus Thraustochytrium has been designated to the order Thraustochytriales and is characterized by monocentric thalli that are attached to their substrate by branched, radiating epi- or endobiotic rhizoid-like extensions of the thallus (the ectoplasmic net). Asexual reproduction in species of Thraustochytrium is normally conducted by conversion of the vegetative thallus to a zoosporangium

284

AJAY SINGH AND OWEN P. WARD

within which many laterally biflagellate zoospores are formed (Moss, 1986). Zoospores are liberated as angular and nonflagellate bodies and, after a period of rest, become transformed into pyriform biflagellate bodies. Release of zoospores occurs upon the bursting and partial or complete disintegration of the distal part of the sporangial wall. Following a swarm period, zoospores settle, encyst, and form daughter thalli (Bessey, 1961). Recently, Iida et al. (1996) studied reproduction in cultures of Thraustochytrium aureum ATCC 34304. Release of zoospores from a zoosporangium (diameter 40 pm) was observed during the early growth phase, that is, 1 day subsequent to inoculation and at the beginning of the exponential growth phase. Zoospores, oval in shape and about 5 pm in diameter with heterokont flagella, swam rapidly but stopped after some time and became larger trophic cells, maturing into sporangia. Zoospores were not observed microscopically in any other growth phase of ?: aureum. The lipid classes and component fatty acids of three marine fungi, 7: aureum, 7: roseum, and Schizochytrium aggregatum, were examined by Kendrick and Ratledge (1992a). The neutral lipid fraction contained predominantly triacylglycerols and small amounts (
MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

285

PUFAs, which has important implications for downstream processing. A fatty acid profile of different DHA-producing fungal species is given in Table IV. The DHA content of different strains of tested Thraustochytrium spp. and Schizochytrium spp. ranged from 1.5 to 35% of total fatty acids (Singh et al., 1996). The predominant fatty acids produced by Schizochytrium strains ATCC 2088 and 20889 were 16:l (40% of total fatty acids) and 18:l (65% of total fatty acids), respectively. Thraustochytrium sp. ATCC 20892 produced the highest DHA yield (67.6 mg/liter, or 35% of total fatty acids), relatively low amounts of 16:O (29%) and 18:l (13%), and insignificant amounts of other PUFAs. Although T roseum exhibited a similar fatty acid profile to that of Thraustochytrium sp. 20892, T aureum produced 18:O as another major fatty acid (Li and Ward, 1994).

B. MICROALGAE Microalgae are considered a potential source of a wide spectrum of chemicals, including PUFAs, carotenoids, and antioxidants. High levels of DHA have been detected in many species of microalgae and is particularly abundant in algal classes Dinophyceae, Haptophyceae, and Cryptophyceae (Sargent et al., 1987). Lipids in actively growing and dividing algae are mainly glycolipids located in thylakoid membranes (Harwood and Russel, 1984). These glycolipids are characteristically rich in 0-3 PUFAs. Algal triacylglycerols, present as membrane-bound oil droplets in their cytoplasm, generally contain lower concentrations of 0-3 PUFAs and higher concentrations of saturated and monounsaturated fatty acids than the corresponding glycolipids (Sargent et al., 1987). The fatty acid composition of lipids can vary quite markedly throughout the growth cycle, and biosynthesis of lipid can be significantly influenced by the physiological state of the organism and environmental conditions. The PUFAs of dinoflagellates (Dinophyceae) mainly consist of 18:4, 20:5, and 22:6. DHA constitutes from 1 2 to 34% of total fatty acids in these microalgae (Table V). Four particular dinoflagellate species, Crypthecodinium cohnii (Beach and Holz, 1973), Gonyauhx catenella, Gymnodinium simplex (Joseph, 1975), and Gyrodinium cohnii (SickoGoad et al., 1988) are good sources containing 30% or more of DHA in total lipids. The diatoms (Bacillariophyceae) are widely distributed in both fieshwater and marine habitats. However, EPA (15-30% of total fatty acids) is the major PUFA in this class of microalgae. Pheodactylum

TABLE N

-%

A COMPARISON OF FATTYACID PROFILES OF DHA-PRODUCING FUNGI

k

56 of total fatty acids

v)

Organism

Conidiobolus nanodes 92299 Entomophthom exitalis 3742 Schizochytrium sp. 20888 S . aggregatum 28209 Thraustochytrium sp. 20892 I: aureum 34304 I: roseum 28210 I: stn'atum 24473 IT: visurgence 28208

14:O

16:O

16:l

18:O

18:l

182

18:3

1.1 6.2 2.0 3.2 2.9

22.7 23.3 20.9 33.4 12.5

1.6 8.0 40.4

0.4 2.1 4.1 3.0

-

-

-

20.8

3.2

7.8 33.9 20.2

-

24.8 35.4 9.7 2.7 11.6 18.7 16.0 48.2 15.7

1.2 2.1 4.1 3.0

2.0 0.9

15.0 4.7 11.3 0.8 3.1 4.9 4.2 8.0 3.4

3.7 2.2 3.3 0.2

3.7 2.2 3.3 0.2

1.5

-

20:4

205

3.9 1.1

-

-

10.9 0.8 2.3 3.0 0.3 3.8

3.9

1.8

0.2 2.7

22:6 2.3 0.9 4.7 22.9 53.1 42.8 52.3 0.7 16.1

Reference

Kendrick and Ratledge (1992a) Kendrick and Ratledge (1992b) Singh et al. (1996) Singh and Ward (unpubl.) Singh et al. (1996) Bajpai et al. (199lb) Singh and Ward (1996) Li and Ward (1994) Singh and Ward (unpubl.]

z

z

z0

2 3

TABLE V

MICROALGAE A COMPARISON OF FAIT Acm P R O F U S OF DHA-PRODUCING % of total fatty acids Organism Amphidinium carteri Chaetoceros calcitrans CCAL1315 Cricosphaera carterae LB 1014 Crypthecodinium cohnii WH-d Cryptomonas ovafa Euglena gracilis 2 Gonyaulav polyedra Gymnodinium nelsoni

G. w'lczeki DINO-EX Gyrodinium cohnii Isochrysis galbana AL41 I. galbana 11-4 I. galbana LB 2307 Microalgae MK8805 Nitzschia frustulum Pavlova luthen CS-182 R lutheri LB 1293 Phaeodactylum tricornutum UTEX640 Prorocentrum cordatum NI-539 Rhodomonas lens Thalassiosira pseudonana CS-173

14:O

16:O

16:l

18:O

18:l

18:2

18:3

2.0 24.1 6.3 46.9 1.0 11.8 2.0 6.0 3.4 19.0 10.0 11.8 17.1 14.5

12.0 17.5 26.4 18.6 10.0 10.3 36.0 35.0 35.4 20.0 14.0 10.1 14.4 27.8

1.0 28.1 19.0

2.0 4.3 6.5 1.5

1.0 -

8.9 8.1 6.6

16.5 22.8 9.7 13.3 9.5 13.0 6.9

2.0 3.2 31.7 4.9 2.0 7.9 3.0 16.0 3.2 14.0 2.9 2.5 16.0 10.5 1.2 0.8 7.7 2.4 1.1 2.0 0.1

-

25.7 18.0 3.2

1.1

4.0 1.1

-

1.0

-

-

5.0 0.3 1.0 13.5

1.0 2.6 1.0

10.6 8.1

0.7 4.5

0.1

-

17.1 20.9 16.9 23.1

0.8 0.3 0.8 0.3 2.2 1.0 0.4

1.2

5.0 12.6

-

9.2

-

2.9 2.0 1.0 3.0

1.6 3.8

2.1 0.5 3.5 2.4 0.8 16.0 0.6

20:4

20:5

22:6

3.0

-

-

0.1

20.0 4.3 0.4

22.0 0.4 1.5 8.8 7.0 1.0 23.0 18.0 18.5 30.0 9.2 11.1 15.7 41.3

Harringtonetal. (1970) Burgess etal. (1993) Burgess et al. (1993) Henderson et al. (1988) Beach etal. (1970) Hayashietd(1993) Patton etal. (1966) Harringtonetal. (1970) Nichols etal. (1984) Harrington and Holz (1968) Alonso et a!. (1992) Grimaetal. (I9931 Burgess el al. (1993) Kyle ef a]. (1992)

1.7 11.2 2.7 8.9 14.5 5.0 6.2

RenaudandParry (1994) Dunstanetal. (1993) Burgess eta]. (1993) Yongmanitchai &Ward (1992a) Nichols etal. (1984) Beach etal. (1970) Brown et al. (1996)

12.0 1.9 3.0 1.0 0.8

-

-

0.3 0.6 0.1

0.9 0.8

-

0.2

-

23.9

-

-

12.0 3.7 14.0 5.0 12.1

-

0.4

24.6 25.1

-

-

0.7 0.5 0.1 0.4

-

26.7 17.7 3.0 29.9 4.4

13.0 0.2

32.7

-

Reference

288

AJAY SINGH AND OWEN P. WARD

tricornutuin, a freshwater diatom) has been found to contain low levels of DHA (about 9%) in lipid (Yongmanitchai and Ward, 1992a). Emiliana huxleyi (Volkman et a]., 1981),Isochrysis galbana (Alonso et al., 1992), and Cryptomonas spp. (Beach et al., 1970) accumulate up to 15% of DHA in total lipids and are good examples of microalgal classes Haptophyceae, Chrysophyceae, and Cryptophyceae, respectively. A wide variation in fatty acid composition (including w-3) among 4 2 different isolates of a single strain of Isochrysis galbana Parke isolate AL41 was reported by Alonso et al. (1992). For example, EPA content ranged from 16.8 to 29.4% and DHA content from 4.9 to 13.8% of total fatty acids. Since all isolates were manipulated, cultivated, and harvested under identical conditions, and all were analyzed with the same apparatus and method, environmental variations are ruled out. The probable explanation is that the parent strain of I . galbana was genetically heterogeneous, the fatty acid variation among isolates being an expression of the underlying genetic variation. This also explains the discrepancies noted in fatty acid composition between different studies on I . galbana (Helm and Laing, 1987; Volkman et al., 1989). C. PSYCHROPHILIC BACTERIA

Long-chain PUFAs, previously thought to be absent in prokaryotes, with the exception of a single marine Flexibacter sp. (Johns and Perry, 1977), are now known to be produced by some other species of marine bacteria. The existence of pressure-adapted psychrophilic deep-sea bacteria has raised interesting questions as to the specific types of physiological and biochemical adaptations necessary for growth and survival in the abyssal environment (Marquis, 1976; Jannasch and Taylor, 1984). One of the most interesting characteristics of low-temperature and high-pressure adaptation in the psychrophiles is the presence of longchain PUFAs such as DHA and EPA. In order to gain insight into lipid-based pressure and temperature adaptations, DeLong and Yayanos (1986) determined the fatty acid composition of membrane phospholipids in a collection of deep-sea bacteria. The fatty acids observed were typical of marine vibrios except for the presence of long-chain PUFAs, EPA, and DHA. Large amounts of EPA (12.3-36.7%) in three isolates and DHA (10.5-24.7%) in six isolates were detected. A general increase in the relative amount of PUFAs as a function of decreasing temperature and increasing pressure suggested the involvement of longchain PUFAs in the maintenance of optimal membrane fluidity and function over environmentally relevant temperatures and pressures.

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

289

Yano et al. (1994) have also demonstrated the presence of DHA at levels of 6.4 to 11.6% of total fatty acids in five bacterial strains isolated from the intestines of deep-sea fish. Recently, Hamamoto et al. (1995) analyzed the cellular fatty acid composition of 22 psychrophilic and psychrotrophic vibrios isolated from deep-sea sediments and demonstrated the presence of DHA (7.4-22.7% of total fatty acids) in 1 2 isolates and EPA (14.2-19.7% of total fatty acids) in 5 isolates. All the isolates contained 16:l as a major fatty acid (24.1-53.4%) together with 14:0, 14:1, 16:0, 17:0, and 1 8 3 acids. The profiles of fatty acids of DHA-producing psychrophilic bacteria is shown in Table VI. IV. Kinetics of Microbial Growth and DHA Production A. INCUBATION TIME

Oleaginous microorganisms tend to store their energy source in the form of lipid as the culture ages (Weete, 1980). The cellular PUFA content of microorganisms follows the sigmoid curve, that is, the concentration of PUFAs increases until it reaches maximum at the late logarithmic or early stationary growth phase and then gradually reduces at the late stationary and death phases (Yongmanitchaiand Ward, 1989). Bajpai et al. (1991b) studied the pattern of growth and lipid and DHA production by Thraustochytrium aureum ATCC 34304 with incubation time. Biomass and cellular lipid content increases to a maximum at 6 days, after which biomass plateaus and lipid content declines. DHA content in lipids remains more or less constant at around 50% during the cultivation period. On the other hand, the maximum biomass yield (9.8 g/liter) of T roseum ATCC 28210 was after 5 days of incubation, with the amount of DHA increasing in parallel with the biomass reaching maximum (85.9 mg/g dry cells) on the 5th day (Li and Ward, 1994). Given the close correlation between biomass and DHA production in the cultures of T roseum, the potential exists to significantly enhance volumetric DHA titers by focusing on optimization of biomass production. Keeping this in view, Singh and Ward (1996) optimized media components and fermentation strategy to improve biomass and volumetric DHA yields of I: roseurn ATCC 28210. A typical profile of substrate utilization and lipid and DHA formation by T roseum ATCC 28210 in a shake-flask culture is exhibited in Fig. 5. In a batch shakeflask culture and using an optimized medium, about 95% of the total carbohydrates present in the medium was utilized by I: roseum within 5 days of incubation, producing maximum biomass (10.4 g/liter) and total lipid (19.6%, w/w) contents. DHA content in biomass and volu-

t3

c n 0

TABLE Vl A COMPARISON OF FATTYAcm PROFILES OF SOME DHA-PRODUCING PSYCHROPHILI~ BACTERIA h

?

YO of total fatty acids Organism Vibrio sp. PE36 Vibrio sp. T3615 Vibrio sp. T4714 Vibrio sp. 5705 Vibrio sp. 814-4 Vibrio sp. 5710 !I marinus ATCC 15381

16:l

18:O

18:l

182

20.9 9.1 17.8 10.7

29.3 45.3

1.4

3.6

-

-

-

-

-

-

-

6.9 16.8 6.0 12.5

53.4 35.5

0.5 5.6

1.2 5.0

-

-

0.6 0.8

1.1 2.7

-

-

0.8 6.9

-

14.0

16:O

5.2 12.0 12.1 34.7 16.4 12.9

37.9 36.5 34.7

3.0

-

18:3

1.2

0.8

1.1 2.1

-

-

20:4

-

-

20:5

22% 24.7 9.4 11.6 21.5 17.9 22.6 17.9

Reference

zv, 8

DeLong and Yayanos (1986) Yano eta]. (1994)

Yano eta]. (1994) Hamamoto et al. (1995) Hamamoto et al. (1995) Hamamoto et al. (1994) DeLong and Yayanos (1986)

0

2z yci

5

291

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

30

1

+Carbohydrate

+Biomass - 100

-

8og -

- 60 .Ip,

n

- 40; fl

- 20 to

0 0

1

2

3

4

5

6

7

8

Time (days)

FIG. 5. A typical profile of biomass, lipid, and DHA production in a shake-flask culture by Thruustochytrium roseurn ATCC 28210 [Singh and Ward, 1996). The culture was incubated on a rotary shaker (200 rpm) at 25°C.

metric DHA yield also increased with cultivation time, showing a positive correlation with biomass yield and reaching maximum values of 102.2 mg/g and 1.01 g/liter, respectively, after 5 days. However, DHA content in the lipid remained more or less constant (50-54%) for up to 5 days and decreased slightly during postexponential lipid depletion. Also, there were no significant changes in the amount of total saturated or total unsaturated fatty acids. A fed-batch culture system of ?: roseum ATCC 28210, in which additional doses (40% of initial composition) of medium components were added to the culture, was also developed that gave the highest yields of biomass (17.1 g/liter) and DHA (2.0 g/liter) achieved to date (Fig. 6). In Thruustochytrium sp. ATCC 20892, biomass, lipid, and DHA contents increased in parallel, reaching maximum after 4 days (Singh et d., 1996). Biomass content of the culture remained almost constant between days 3 and 7,but lipid content decreased after 4 days. Yields of unsaturated fatty acids, including DHA, increased up to day 4 and then decreased. A general decrease in unsaturated fatty acid content has been reported to occur on aging in many microorganisms (Ratledge, 1989). However, in some marine microalgae the content of PUFAs significantly increases as the culture grows older (Yongmanitchai and Ward, 1989).

292

AJAY SINGH AND OWEN P. WARD 225 200 175

p

150

to 125 s - 0

fig

1005

.9 +Biomass +Lipid +DHA in bioinarr

2 *E

50

t D H A yield

25 0

0

2

4

6

8

1

0

1

2

1

4

Time (days)

FIG.6. Production of high yields of DHA by Thrnustochytriurn roseurn ATCC 28210 in a fed-batch culture (Singh and Ward, 1996). Culture was initiated in a medium containing 25 g starch/liter. Additional starch and nutrients (at 40% of initial level) were supplied after 4 days at 2-day intervals.

Stationary-phase cells of Crypthecodinium cohnii, a marine dinoflagellate, were found to be somewhat richer in neutral lipids and poorer in polar lipids than log-phase cells, though the fatty acid composition of triacylglycerols and phosphatidylcholine fractions were not different. The lipid content of lsochrysis galbana increases with an increase in culture age (Utting, 1985). In a chemostat culture, the lipid production of 1. galbana increased as dilution rate increased up to 0.0295 h-l, remained constant until 0.0355 h-l, and then decreased (Grima et al., 1993). Although DHA content in lipid remained more or less constant over a range of dilution rates (0.006 to 0.0436 h-l), maximum DHA productivity was obtained at dilution rates between 0.1295 and 0.0355 h-l. At D = 0.0048 h-l, cell growth of I. galbana isolate 11-4 was strongly limited by light (Grima et al., 1994). The proportion of neutral lipids in biomass grown at 0.0048 h-l was 67.5%, while in biomass grown at 0.029 h-l the neutral lipid proportion decreased to half of that obtained at 0.0048 h-l, Interestingly, at 0.029 h-l unsaturation was somewhat higher than at 0.0048 h-l, with a significant increase in DHA content from 3 to 10% of total fatty acids.

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

293

A marine bacterial strain, T3615, isolated from the intestine of deepsea fish, produced maximum biomass (122 mg/liter) and DHA yield (0.8 mg/liter) after 6 days of incubation at 5°C and under atmospheric pressure (Yano et al., 1994). Similar biomass yields of this strain were obtained in 3 days when culture was grown at higher pressure (41.4 MPa). Psychrophilic Vibrio sp. strain 5710 exhibited differences in fatty acid composition of cultures at mid-log and early stationary phases (Hamamoto et al., 1994). The proportion of 16:0, 16:1, and 18:l increased from mid-log phase cells to cells at early stationary phase, while 18:2, 18:3, and 22:6 decreased proportionally as cultivation proceeded.

B . FERMENTOR CULTIVATION

Microorganisms grown in a fermentor under controlled environmental conditions (e.g., pH, temperature, aeration) are expected to produce higher yields of the desired products. However, this does not hold true with DHA production by Thraustochytriurn. When IT: aureum ATCC 34304 was cultivated in a laboratory fermentor (temperature 25OC, aeration 2.5 litedmin, impeller speed 300 rpm), lower biomass and DHA yields were obtained as compared to shake flasks (Bajpai et al., 1991b), probably due to an inhibitory effect of mechanical stirring on growth. IT: aureurn cultures grown in a fermentor showed a coagulation tendency compared with ones grown in shake flasks, where they exhibited disperse growth (Iida et al., 1996). On the other hand, IT: roseurn ATCC 28210 cultivated in a 10-liter fermentor (temperature 25OC, aeration 0.5 litedmin, impeller speed 200 rpm) produced DHA yields similar to those obtained in shake-flask cultures of this strain (Singh and Ward, unpublished results). Kyle et al. (1992) compared cell growth of a microalgal strain, MK8805, in shake flask, airlift, and stirred-tank reactors. Cell growth in a rotated shake flask was generally found to be oxygen-limited at biomass densities greater than 10 g/liter. The stirred-tank reactor was better than the airlift reactor. Production batches have been produced up to a 350-liter working volume in conventional stirred-tank reactors, and cultures containing 25 g dry weight of biomass could be harvested in an 84-h batch run. A high volume-to-surface-area ratio is required for efficient light energy utilization by marine microalgal cultures (Burgess et al., 1993). Matsunaga et al. (1991) developed a biosolar reactor to achieve efficient photosynthetic biomass production using sunlight. The light is supplied to the growing cells from light-diffusing optical fibers and allows efficient use of light energy and high cell densities (Takano et ~ l .1992). ,

2 94

AJAY SINGH AND OWEN P. WARLl

As compared to the conventional culture method in flat glass bottles, Isochrysis gafbana T-Is0 (UTEX LB 2307) produced twofold higher DHA yields in a closed photobioreactor in which efficient light distribution was achieved using light-diffusing optical fibers (Burgess et af., 1993). A comparison of data for optimal yields of DHA by selected fungi, microalgae, and bacteria is presented in Table VII. As can be seen, the DHA yields and productivity of fungi are much higher than that of other organisms. V. Factors Affecting Microbial DHA Production

DHA production by microorganisms is influenced by several environmental and nutritional factors, including carbon and nitrogen sources, salts and trace metals, pH, temperature, light intensity, and oxygen supply. A. MEDIACOMPONENTS 1. Carbon Source Glucose is generally a preferred carbon source for microorganisms. It is the most commonly used sugar in fermentation media and is efficiently converted into lipids by a number of yeast and fungi (Ratledge, 1989). Mono-, di-, and polysaccharides of glucose promote good growth of different species of Thraustochyfriurn and produce a high lipid content in biomass. Although similar yields of biomass and lipid contents were obtained with glucose and starch, T. aureurn ATCC 34304 (Bajpai et af., 1991b) and ?: roseurn ATCC 28210 (Li and Ward, 1994) produced maximum DHA yield with starch, while Thraustochytriurn sp. ATCC 20892 (Singh et a!., 1996) produced maximum DHA yield with glucose. In contrast, the growth and content of cellular lipid of these cultures were poor on fructose, sucrose, and lactose. This suggests that, where carbon source substantially limited cell growth, minimum conversion of carbohydrates to lipid occurred in these organisms. In general, the DHA content of lipid in these cultures was higher (65-75% of total fatty acids) when cellular lipid content was low (1-2%) and lower (40-50% of total fatty acids) when biomass contained high lipid content (14-18%). Although vegetable oils such as linseed, corn, and canola oils as sole carbon source have been found to support high biomass and lipid yields in the cultures of Thraustochytriurn, DHA content of biomass was generally low (Bajpai et af., 1991b; Li and Ward, 1994).

h

COMPARISON OF LIPIDAND DHA YIELDS OF DIFFERENT ORGANISMS

Lipid yield Organism

Thraustochytrium aureum ATCC 34304 II: roseum ATCC 28210 Batch culture Fed-batch culture Thraustochytrium sp. ATCC 20892 Schizochytrium aggregatum ATCC 28209 Isochrysis galbano 11-4 I. golbana LB 207 Microalgae MK8805 Vibrio sp. T3615

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

f5

TABLE VII

DHA in

DHA

(g/liter)

biomass (mg/gl

yield (mglliter)

DHA

0.9

104

511

85

2.0 4.1

102 117

1061 2000

212 167

177

productivity (mgllitedd)

Reference Bajpai et al. (1991b) Singh and Ward (1996) Singh and Ward (1996) Singh et al. (1996)

1.2

100

707

0.2

20

38

6

Sin& and Ward (unpubl.)

0.3

33

34

-

0.2

16

17

4

Grima et ol. (1994) Burgess et al. (1993)

1.3

90

560

-

0.01

9

0.8

0.13

Kyle et 01. (1992) Yano e t a ] . (1994)

295

296

AJAY SINGH AND OWEN P. WARD

A high C:N ratio is generally good for lipid accumulation, usually being triggered by nitrogen depletion (Ratledge, 1989). Substrates present in excess in the medium were shown to support good growth and substantial synthesis of lipid in different species of Thraustochytrium. The major effect of glucose concentration was on the lipid content of T aureum ATCC 34304 (Bajpai et al., 1991b) and Thraustochytrium sp. ATCC 20892 (Singh et al., 1996). While the proportion of DHA in lipids did not vary with glucose or starch concentration, a dramatic increase in the DHA content of biomass and volumetric DHA yield was observed in T aureum ATCC 34304 by increasing glucose concentration to 20 g/liter (Bajpai et al., 1991a) or starch concentration to 25 g/liter (Bajpai et al., 1991b) in the medium. 2. Nitrogen Source The nitrogen content of the medium affects the proportion of saturated to unsaturated fatty acids in microorganisms (Erwin, 1973; Yongmanitchai and Ward, 1989). In fungi, aside from concentration, source of nitrogen also plays an important role. Inorganic nitrogen sources are generally not well utilized by the species of Thraustochytrium (Goldstein, 1963a). These cultures cannot metabolize K N 0 3 and urea, and manifest poor assimilation of nitrogen from (NH4)S04 (Goldstein, 1963b). In contrast, organic nitrogen sources are efficiently utilized. Sodium glutamate is generally a preferred nitrogen source for these fungi (Bajpai et al., 1991b; Singh et al., 1996; Iida et al., 1996). Although aspartate and glutamine are energetically more favorable than glutamate, addition of these amino acids yielded reduced growth of 7: aureum ATCC 34304 (Iida et al., 1996). Yeast extract, peptone, tryptone, and cas-amino acid promote high biomass but with decreased DHA content in biomass of T nureum ATCC 34304 (Bajpai et al., 1991b) and T. roseurn ATCC 28210 (Singh et al., 1996). Yeast extract represents an important source of growth factors, in addition to being a nitrogen source. Supplementation of yeast extract (2 g/liter) to the basal medium containing sodium glutamate was found to increase biomass and DHA yield by about 30% of a T. roseum ATCC 28210 culture (Li and Ward, 1994). A microalgal strain, MK8805, produced high biomass and low lipid and DHA content at high nitrogen concentrations and low biomass and high lipid content at low nitrogen concentrations, a typical observation with single-cell oleaginous organisms (Kyle eta]., 1992). The highest biomass density of this culture was obtained with media containing 1% tryptone and 1% yeast extract, while DHA and lipid contents were maximum with 0.1% tryptone and 0.1% yeast extract.

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

297

3. Supplementation of Exogenous Unsaturated Fatty Acids

Although it is known that microorganisms and cultured cells are able to incorporate exogenous fatty acids into phospholipids or neutral lipids (Clejan et al., 1988; Williams et al., 1990), media containing free fatty acids usually suppress biosynthesis of other fatty acids in microorganisms (Silbert et al., 1968; Yongmanitchai and Ward, 1989; Bajpai and Bajpai, 1993). In yeast and bacteria, supplementation of triacylglycerols such as cuttlefish liver oil to a growth medium has been reported to increase the cellular content of w-3 PUFAs (Imada et a]., 1979; Ando et al., 1992). Zooplankton such as Brachionus plicatilis and Artemia salina (Watanabe et al., 1982) and photosynthetic bacteria such as Rhodopseudomonas capsulatus (Kobayashi et al., 1967) are used commercially as feed in mass cultivation of fish larvae. However, these organisms have little or no PUFAs that are essential fatty acids for marine fish (Yone and Fuji, 1975; Kaufman et al., 1982). Therefore, attempts have been made to enrich fish feed with 0-3 PUFAs to enhance fish production, Euglena gracilis contains about 8% (w/w) of total fatty acids, comprising about 1% (w/w) DHA and 3.6% (w/w) EPA (Hayashi et al., 1993). Supplementation into the growth medium of DHA or EPA (0.5%) resulted in accumulation of 76.05% DHA and 75.68% EPA, respectively. Supplementation of their ethyl esters also increased corresponding fatty acids, but not to the extent of free fatty acids. The total lipid content of the Euglena gracilis cells supplemented by DHA and EPA was 31.3 and 25.3% (8.9% w/w of total lipid in nonsupplemented media), respectively. Watanabe et al. (1994) investigated the incorporation of exogenous DHA into bacterial lipids as a method for DHA-linked phospholipid production. The cultivation of 23 bacterial strains in a medium with DHA showed that EPA-producing bacterium Shewanella sp. SCRC2738, Bacillus subtilis W23, B. cereus, Antarctic marine bacterium strain S-7, Rhodopseudomonas capsulatus (utilized for mass production of fish larva), Pseudomonas aerugenosa, Staphylococcus uureus, Serratia marcescens, and Escherichia coli K12 all incorporated DHA into their phospholipids. Strain SCRC-2738 incorporated the largest amount of DHA among the bacterial strains tested, DHA being 16% (w/w) of total fatty acids in phosphatidylethanolamine and 29% in the phosphatidylglycerol. 4. Salinity

Microorganisms isolated from a marine environment are frequently characterized by salinity optima that approximate the conditions of

298

AJAY SINGH AND OWEN P. WARD

their natural habitats (Vishiniac, 1960). Thus, natural sea water is necessary in cultivation of marine organisms, unless the basal medium contains sufficient amounts of metal salts. Vishiniac (1960) studied the salt requirement of 49 marine Phycomycetes isolates (members of Thraustochytriaceae and Ectogellaceae). When inoculated into media containing 1 to 5% NaC1, 27 isolates made no growth with 1% NaCl and 4 others grew less well with 1% than with 2.5% NaC1. At 5% NaC1, 11 failed to grow, while 7 grew only poorly. The optimum concentration of NaCl for growth of Phycomycetes isolates was 2.5%, and was not affected by the temperature. Similar results were obtained in another study with T. motivum and i? multirudirnentale, where neither culture developed in the absence of NaCl or at concentrations above 5%; they exhibited optimal growth at 2.5-3.0% NaCl concentrations (Goldstein, 196313).Attempts to replace NaCl with KC1 failed in the above experiments. Iida et al. (1996) compared artificial sea water (Tropic Marin, Germany] with aged sea water and synthetic sea water at a salt concentration half that of sea water in the medium (12.5 g/liter NaC1, 2.5 g/liter MgS04, 0.5 g/liter KCl, 0.1 g/liter CaCO,, and 0.05 g/liter NaHCO,), and found no differences in the biomass yield of T aurezzrn ATCC 34304. They also examined the effect of salt concentration on growth of T aureum 34304 in the range from zero to twice that of sea water. The highest glucose consumption rate and biomass were obtained at a salinity of 0.5 (ratio to sea water). The culture did not grow in zero salinity and growth was completely inhibited at a salinity of 2.0, showing a narrow dependence of growth on salt concentration. There were no significant differences in the biomass and DHA yields of ?: roseurn ATCC 28210 grown at NaCl concentrations of 10 to 25 g/liter (Singh and Ward, 1996). The growth of a DHA-producing marine dinoflagellate, Crypthecodinium cohnii, was inhibited at 0.3 and 5% NaC1, and failed in the absence of NaCl (Beach and Holz, 1973). The neutral lipid content was higher and polar lipid content lower at the extremes of salinity than at 1.8% NaC1. While the fatty acid composition of the phosphatidylcholine was relatively stable at all salinity levels, there was an NaC1-dependent inverse relationship between 1 8 : l and 22:6 in the triacylglycerol, that is, 18:l was high at high salinity and DHA was high at low salinity. 5. Trace Metals

Several metal ions, such as Mn2+,Co2+,Zn2+,and Mg2+,promote synthesis of lipids and PUFAs (DHA, ARA, GLA) in microorganisms

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

299

(Ratledge, 1992; Ward, 1995). Acetyl-CoA carboxylase catalyzes the initial step of fatty acid synthesis and requires bivalent metal ions (Mg2+ or Co2+)as cofactors (Naganuma et al., 1987). Supplementation to the basal medium of a trace metal mixture consisting of Mn2+,Co2+,Fez+, Zn2+,and Cu2+significantly enhanced DHA productivity in Thraustochytrium sp. ATCC 20892 (Singh et al., 1996) and T roseum ATCC 28210 (Singh and Ward, 1996). A positive effect of trace metals has also been found in organisms producing other PUFAs such as A M . While a deficiency of Mn2+decreased the PUFA and ARA contents of Mortierella sp. S-17 (Sajbidor et al., 1992), more lipid and GLA accumulated by M. ramanniana when the medium was supplemented with Cu2+and Zn2+(Hansson and Dostalek, 1988). However, no significant effect of trace metal supplementation was found in cultures of Thraustochytrium sp. ATCC 20891 (Barclay, 1992). Deficiency of MnZ+was shown to reduce the content of 01-3 fatty acids in Euglena gracilis (Constantopoulos, 1970). Silicate deprivation in a medium of the diatom Cyclotella crypfica yielded an increase in the cellular lipid content but suppressed biosynthesis of PUFAs, particularly DHA (Shifrin and Chisholm, 1981; Enright et al., 1986). Silica concentration in the range of 0 to 100 mglliter had little effect on growth of Pheodactylum tricornutum, but high concentrations were found to be toxic, resulting in reduced growth (Yongmanitchai and Ward, 1989). 6. Vitamins

Although vitamins were not found to be essential for growth, thiamine-HCl(10 pglliter) and cyanocobalamine (1pglliter) have been used in the medium for studies of the growth and development of different species of Thraustochytrium such as T motivum sp. nov., T multirudimentale sp. nov., and ?: roseum Goldstein sp. nov. (Goldstein, 1963a,b), and DHA production by T aureum ATCC 34304 (Bajpai et al., 1991a,b), T roseum ATCC 28210 (Singh and Ward, 1996), and Thraustochytrium sp. ATCC 20892 (Singh et al., 1996). The effect of different vitamins using a defined medium containing glucose and sodium glutamate was studied on the growth of T aureum ATCC 34304 (Iida et al., 1996). Among the various vitamins tested, a combination of thiamine, vitamin BIZ,and pentothenic acid produced maximum biomass, while folic acid and pyridoxine produced the least. B . INOCULUM DEVELOPMENT

Cultures of Thraustochytriurn spp. are generally maintained on 3.0% agar slants containing basal medium (glucose, glutamate, mineral salts)

300

AJAY SINGH AND OWEN P. WARD

supplemented with yeast extract and subcultured every 2 months (Bajpai et al., 1991a,b; Singh et a]., 1996). Vegetative inocula were developed in 250-ml Erlenmeyer flasks containing 50 ml basal medium with glucose (Bajpai et a]., 1991a,b; Singh et al., 1996) or starch (Li and Ward, 1994). T aureum ATCC 34304 inoculum was grown at 25°C for 40 h with orbital shaking at 300 rpm (Bajpai et al., 1991b), and T roseum ATCC 28210 inoculum was grown at 25°C for 48 h with orbital shaking at 250 rpm (Li and Ward, 1994). A 5Yo inoculum from these flasks was generally used to inoculate a 50-ml production medium in 250-ml Erlenmeyer flasks. An inoculum size of 5% (v/v) was found to be optimum for the production of DHA by T aureum ATCC 34304 (Bajpai et al., 1991b). While inoculum age had little effect on culture biomass of T aureum, lipid content of biomass and DHA yield dramatically declined when 48or 72-h inocula were used instead of a 24-h inoculum (Bajpai et al., 1991b). However, inoculum age did not significantly affect the proportion of DHA in lipid. C. pH The best pH for lipid production varies between pH 5.0 and 8.0 depending on the microbial species (Weete, 1980).An initial culture pH of 6.0-7.0 has been found to be optimal for DHA production in fungi (Bajpai et al., 1991b; Singh et al., 1996; Li and Ward, 1994). A significant decrease in DHA yields is observed at an initial culture pH below 5.0 and above 8.0. A culture of T aureum 34304 in the basal medium resulted in an increase in pH in accordance with consumption of sodium glutamate; when the pH exceeded 8, no increase in growth was observed (Iida et al., 1996). However, the culture pH scarcely affected the growth rate of the strain as long as the pH of the culture medium was within the range from 5.5 to 8.0. DHA-producing microalgal species such as lsochrysisgalbana (Grima et al., 1992,1993) are generally grown at media pH of 8.0, and the pH is maintained by injecting COz. However, no attempt has been made to optimize the initial pH in these cultures.

D. TEMPERATURE Increased synthesis of unsaturated fatty acids at lower temperatures has been observed in many microbial species (Neidelman, 1987; Yongmanitchai and Ward, 1989). It has been postulated that, because of increased solubility of oxygen at low temperatures, a greater amount of intracellular molecular oxygen is available that would be required by

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oxygen-dependent enzymes catalyzing the desaturation of PUFAs (Brown and Rose, 1969). It has also been suggested that lowering the growth temperature tends to increase the proportion of unsaturated fatty acids and the membrane fluidity of the organism, as an adaptation to cold environments (Suutari and Laasko, 1994). The optimum temperature for growth and lipid formation by species of Thraustochytrium is 25-28OC. The fatty acid profiles of 'I: aureum ATCC 34304 observed at different temperatures indicate an increase in 16:O and 18:l acids and a concomitant decrease in 20:5 and 22:6 acids with an increase in cultivation temperature (Bajpai et al., 1991b). In Thraustochytrium sp. ATCC 20892, maximum DHA (63.8% oftotal fatty acids) was observed at 15"C, whereas DHA only amounted to 17.9% of total fatty acids at 35°C (Singh et al., 1996). A decrease in DHA at higher temperatures was accompanied by an increase in 16:0, 18:0, and 18:l acids. A beneficial effect of temperature shifting on DHA production was observed when Thraustochytrium sp. ATCC 20892 was first grown at the optimum temperature (25OC) and then at a lower one (15°C). Examination of the subsequent fatty acid profile indicates an increase in DHA and total unsaturated fatty acids. Although biomass yields were similar under various culture conditions, maximum DHA yield (707 mg/liter) and volumetric productivity (177 mg/liter/d) were obtained when the culture was grown at 15OC for 1 day after initial growth at 25°C for 3 days. A high starting temperature (28OC) followed by a shift to a lower one (12OC) has also been shown to induce EPA formation in several species of Mortierella (Shimizu et al., 1988). Since low temperatures are disadvantageous for practical purposes because of a low growth rate of organisms and a high energy cost for cooling, a temperature shift after optimal growth may be an effective way of obtaining enough cells with high PUFA and DHA content in a relatively short cultivation period. Temperature was the principal regulation factor for the degree of unsaturation in lipids of Entomophthora exitalis, an oleaginous fungus (Kendrick and Ratledge, 1992b). As the growth temperature was decreased from 30 to ZOOC, the PUFA content increased from 18 to 27% (w/w) of total fatty acids. While the level of DHA remained more or less constant in the different lipid fractions, the increase in unsaturation was the result of an increased proportion of ARA in the phospholipids and the sphingo- plus glycolipid fraction. Marine microalga Zsochrysis galbana T-Is0 (UTEX LB 2307) exhibited optimum growth at 25°C (Burgess et al., 1993). On reducing the temperature to 17OC, a decrease in total fatty acids and an increase in DHA

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content has been observed. In contrast, in a different strain of the same species, I. gulabana Parke, a decrease in total fatty acids as well as in DHA content with decreasing temperature was observed (Grima et al., 1992). Microalgal strain MK8805 was capable of growing at temperatures from 10 to 30°C (Kyle et al., 1992). The best growth of this strain was observed at 26"C, though the highest DHA productivity was seen at 20°C due to the high oil content of cells. In contrast to the general observations in other oleaginous microorganisms, the level of unsaturation and DHA content was found to increase with temperature. In response to decreasing growth temperature from 20 to 2"C, Vibrio marinus, a deep-sea bacterial isolate, had decreased proportions of 16:O and increased 22:6 (DeLong and Yayanos, 1986). The fatty acid composition of other isolates (PE36 and MT41) also changed as a function of pressure. An increase in growth pressure was accompanied by an increase in the relative proportions of 1 4 : O and 22:6. Another psychrophilic strain, Vibrio sp. 5710, showed no growth above 13OC and had its maximum growth at 4 to 8OC (Hamamoto et al., 1994). Growth of this strain was observed even at -5OC, with the maximum amount of DHA (16.4% w/w of total fatty acids) produced at this temperature, which suggests the role of PUFAs, particularly DHA, in the adaptation of the membrane to low temperatures and high pressures in psychrophilic deep-sea bacteria.

E. LIGHT The occurrence of light-stimulated growth in fungi is unusual and has been only seen in Blastocladiella ernersonii (Cantino and Horenstein, 1956), T roseum Goldstein sp. nov. (Goldstein, 1963b), and T aureum ATCC 34304 (Bajpai et a]., 1991b). In 'I: roseum, the effect of light on growth was studied in shake cultures on a rotary shaker. Illumination was provided by a 450-fc fluorescent bulb, and the red portion of the spectrum was provided by a tungsten bulb (350 fc). The stimulatory effect of light upon growth was most pronounced during the early stages of the logarithmic phase, and 150% greater Klett readings (no. 42 filter) were observed from the light cultures as compared to the dark cultures (Goldstein, 196313). Exposure of T aureum ATCC 34304 to light (Lab-line incubator shaker, Model 3525, fitted with two 24-inch 33-watt fluorescent tubes in the lid at a distance of 1 4 inches from the shaker platform) had the dramatic effect of increasing lipid content of biomass and DHA yields by 40-50%, while biomass was increased by 13% (Bajpai et al., 1991b). In contrast, Iida et al. (1996) found no differences in biomass yield between light- and dark-grown cultures of the same

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

303

strain ?: aureum ATCC 34304, though light cultures were brownish and darker than the dark cultures, which were pale yellow. Similarly, minor quantitative differences were noted among the lipid fraction and their fatty acids of Crypthecodiniurn cohnii,a marine dinoflagellate grown in the light or dark (Beach and Holz, 1973). Light is an important environmental parameter that has been reported to affect fatty acid metabolism in marine microalgae. In general, low light intensity enhances the formation of unsaturated fatty acids in many species of microalgae (Thompson et al., 1990; Renaud et d., 1991), though in many photosynthetic algae, the absence of light enhanced 0-6 fatty acids and decreased 0-3 fatty acids (Yongmanitchai and Ward, 1989). Burgess et al. (1993) studied the effect of light intensity in the range of 1.4-9.5 pE m-2 s-l on growth and DHA production of Isochrysis galbana T-Is0 (UTEX LB 2307) in a light-diffusing optical fiber bioreactor. At a light intensity of 4.5 pE m-2 s-l, threefold higher biomass productivity was obtained as compared to the culture grown using a conventional culture method (Grima et al., 1992). The DHA decreased with increasing light intensity. The reduction of unsaturated fatty acid content with increasing light intensity was also observed in 1. galbana Parke, although DHA content remained unaffected by light intensity (Grima et al., 1992). When light limits growth, cells have to synthesize chloroplast membrane in order to collect scarce light efficiently (Grima et a]., 1993). These membranes are composed of highly unsaturated polar lipids (Forder and Steer, 1976).

F. AERATION Availability of oxygen often determines the degree of unsaturation of fatty acids obtained in a particular culture (Ward, 1995). Molecular oxygen is involved in fatty acyl desaturation mechanisms and could conceivably be the rate-determining factor for PUFA biosynthesis. Despite its importance, little information is available with regard to the oxygen requirements of DHA-producing strains. Oxygen tension had striking effects on population growth and lipid composition in Crypthecodiniurn cohnii (Beach and Holz, 1973). Cells with an enhanced supply of oxygen from flask (100 cm3/min) and fermentor cultures (200 cm3/min) multiplied more rapidly, contained less neutral lipids and more polar lipids, and their lipids were more unsaturated than cells grown with a restricted supply of oxygen. In fermentor cultures, the major neutral fraction, triacylglycerol (44% of total neutral lipids), and the major polar lipid fraction, phosphatidyl-

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AJAY SINGH AND OWEN P. WARD

choline (79% of total polar lipids), contained 25 and 66% DHA, respectively. Restriction of oxygen supply caused a rapid depression of DHA in triacylglycerols, while DHA level in phosphatidylcholine remained relatively unchanged. In contrast, oxygen depletion at constant temperature and growth rate caused no significant changes in lipid composition and DHA levels in culture of Entomophthora exitalis (Kendrick and Ratledge, 1992b). VI. Downstream Processing

The focus of biotechnology on highly valuable fine chemicals such as PUFAs requires not only mass cultivation techniques but also development of downstream processing or recovery methods. At this point, the commercialization of purified biomolecule (DHA, ARA, or EPA) or enriched lipid fractions (phospho- or glycolipids) improves the added value of the productivity system. DHA can be recovered from microbial biomass either in the form of a lipid mixture or fatty acid esters. Lipids can be extracted by a number of organic solvents. The procedure employed by Bligh and Dyer (1959), involving the use of a chloroform and methanol (2:1) mixture in the presence of water, is one of the most extensively used methods for lipids from microbial cells. Conventional Soxhlet extraction for 2 h using hexane has also been found to efficiently extract 95% of the oil from freeze-dried microalgal biomass (Kyle et al., 1992). Lipids can be further fractionated into neutral lipid and polar lipid class using a column of silicic acid or a silica gel cartridge by passing through different organic solvent systems with increasing polarity (Yongmanitchai and Ward, 1992b; Kendrick and Ratledge, 1992a). Several methods have been proposed to produce concentrated PUFAs particularly high in DHA and EPA contents. These methods are generally based upon a combination of techniques such as saponification, solvent extraction, urea inclusion, molecular distillation, fractionation distillation, liquid chromatography, and supercritical COz (Adolf and 1982; Aveldano et ol., 1983;Nilsson et al., Emken, 1985; Haagsma et d., 1988). The process of urea crystallization allows separation to be made on the basis of fatty acid saturation. Urea complexes preferentially with saturated and monounsaturated fatty acids, facilitating removal of solid complexes and resulting in an enhanced concentration of polyunsaturated components. For highly purified preparations of DHA or EPA, preparative high-performance liquid chromatography (HPLC) may be employed to produce gram-scale product with over 90% purity (Grant, 1988).

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

305

Barclay (1992) developed a recovery process of producing a fatty acid mixture containing >90% 0 - 3 fatty acids (65-70% DHA) from the cellular biomass of Schizochytriurn sp. ATCC 20888. The process involved extraction of lipids by chloroform and methanol mixture, and saponification of lipids by methanolic NaOH, extracting free fatty acids and crystallizing fatty acids in different solvents such as petroleum ether, hexane, or acetone at -72 to -74%. The complete absence of any PUFAs other than DHA in the lipid of microalga strain MK8805 made the isolation and purification of DHA a relatively straightforward process (Kyle et al., 1992). Their procedure involved saponification of freeze-dried biomass with ethanolic KOH, removal of nonsaponifiable fraction by hexane, recovery and crystallization of free fatty acids in diethyl ether at -78"C, and urea complexation in the presence of methanol followed by filtration to remove urea adducts to obtain 82% DHA content in the final mixture. Grima et al. (1995) developed a purification method of separating EPA and DHA from a mixture of fatty acids produced by Isochrysis galbana (isolate 11-4).Reverse-phase HPLC on an octadecylsilyl semipreparative column was used to separate stearidonic acid (SA, 18:4),EPA, and DHA in PUFA concentrate obtained by direct saponification followed by urea complexation of lyophilized I. galbana biomass. Isolated DHA, EPA, and SA fraction purity was 94.9, 96.0, and 94.8%, respectively, with yields of 94.0, 99.6, and 100%. Purity is generally susceptible to improvement only at the expense of a decrease in yield, which depends on the collection cutoff point of fractions. The above study indicated a potential for using the preparative-scale HPLC method to obtain high yields of individual PUFAs with high purity. Some results of recovery and purification of DHA from microalgal lipids are shown in Table VIII. VII. Epilogue

Recognition of the effects of 0 - 3 fatty acids, DHA, and EPA in human health resulting from the pioneering work of Dyerberg et al. (1978) created the momentum for extensive nutritional and pharmacological studies on the effects of these PUFAs in human physiology (Dyerberg, 1986). PUFAs play an important role in human health, particularly in treatment or prevention of heart and circulatory diseases, inflammatory disorders, cancer, and brain development in infants. The commercial source of DHA is marine fish oil, though it is believed that fish do not synthesize these fatty acids themselves; rather, they acquire them from the phytoplankton that they eat.

w

0

cr,

TABLE VIII

RECOVERY AND PURIFICATION OF DHA FROM MICROALGAL LIFJJDS

Organism

Fractionation step

%. w/w, of total fatty acids 14:O

16:O

18:0

18:l

18:2

18:3

Starting material Urea concentrate HPLC - EPA fraction HFLC - DHA fraction

10.1 0.3

20.3 0.2

1.4 0.2

0.9 0.2

1.2

0.8

-

2.0

-

-

0.7 1.5

1.4

-

Starting material

18.1

14.1

6.5 5.3

2.6 4.0

-

-

20:4

20:5

225

0.7 1.1

8.4 23.4

-

22.6 39.4 96.0

3.7

-

94.9

-

-

30.2 60.6 82.3

Reference

9

rsochrysis galbana 11-4

-

Microalgae MK8805 Saponifiable fraction Urea concentrate

-

-

-

11.3 23.8 4.9

-

Grima et 01. (1995)

-

Kyle et al. (1992)

Z U

MICROBIAL PRODUCTION OF DOCOSAHEXAENOIC ACID

307

Since the demand for 0 - 3 PUFAs is increasing, supply of these PUFAs from fish is unlikely to meet future requirements. Also, separation of EPA from DHA in fish oil is difficult to achieve on a processing scale. Therefore, alternative sources are being investigated. During the last decade, several species of fungi, microalgae, and bacteria have been proposed as an alternative source of DHA and EPA to fish oil. Microorganisms cultured in reactors under controlled environmental conditions can be manipulated in a manner that optimizes microbial growth and product formation in order to achieve maximum productivity. The potential also exists to select microbial strains producing a large proportion of their lipid material as a single predominant fatty acid form. Thus, “designer oils,” such as microbial oils containing DHA as the only PUFA, would provide the needed and an economically viable source of DHA for nutritional and pharmacological supplementation. Some marine fungal species, particularly those belonging to the genus Thraustochytrium, produce significant quantities of DHA (up to 50% w/w of total fatty acids). A high proportion of DHA in the total lipids of Thraustochytrium and relatively lower levels or an absence of structurally related PUFAs would simplify downstream processing of DHA (Singh et a]., 1996; Singh and Ward, 1996). Microalgal strain MK8805 is another promising source containing DHA (30% of total fatty acids) as the only 0 - 3 PUFA (Kyle et al., 1992). In general, fungi were found to be better sources of DHA than that microalgae or bacteria. In microbial production of DHA, several factors affecting the growth kinetics and biochemical composition of cells must be balanced. Choice of species, growth conditions, nutrients, and age of culture determines DHA yield. DHA production is promoted by increased oxygen tension and reduced temperature and, in the case of photosynthetic organisms, by light intensity. It is expected that a greater understanding of the factors affecting DHA production by fermentation would lead to development of a viable commercial process. ACKNOWLEDGMENTS

Support for research on PUFAs in the authors’ laboratory by the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. REFERENCES Ackman, R. G. (1982). In “Advances in Fish Science and Technology” (J. J. Connell, ed.), pp. 86-103. Fishing News Books, Surrey. Ackman, R. G., and Cunnane, S. C. (1992). Adv. Appl. Lipid Res. 1, 161-215.

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Index

identification, 3 soy sauce production, 109 A spergillus nid ulan s genetics, 8-9 norsolorinic acid resemblance, 4 Aspergillus oryzae, 108-111 Aspergillus parasiticus genetics, 7-10 identification, 2 Aspergillus sojae, 108-111 ATP:citrate lyase, 278-280 Azotobacter vinelandii. 157

A

Acetate, 27 Acetic acid in cheese, 60-61 in soy sauce, 97,127,129-130 Acetophenone, 65 Acetyl-CoA carboxylase, 280-283,299 Acinetobacter calcoaceticus, 164,166 Activated sludge, 176 Aeration, 303-304 Aerobic respiration, 214 Aflatoxins biosynthetic pathway, 3-5 description, 2 genetics, 7-10 Agrobacterium kieliense, 183 Agrobacterium tumefaciens, 170 Alcohols, 124-125,132 Aldehydes in cheese, 53-54,65 in soy sauce, 132 Algae, 277-278 Alkaline lipase, 255 Alkan-2-ones, 30-31 Alkyl succinic anhydride, 255 Amines, see also specific types cheese flavor and, 65 cheese production, 45-49 decarboxylation, 45-49 Amino acids, see also specific types cheese flavors, 65 free amino acids, 39-45 interconversion, 53-54 nonprotein, 70 reactions in cheese, 65-68 in soy sauce, 92-96 sulfur, 54,56-62 Ammonia deamination, 49,51-52 Anaerobic respiration concept, 216 regulation, 227 Aroma, soy sauce, 100-101 Aspergillus f l a w s genetics, 8-9

B Baceman production, 116 Bacillus subtilis competence development, 157 DNA binding, 157 transformation in situ, 162-164 Bacteria, see also specific species competence development, 157 conjugation description, 166-16 7 in situ, 173-177 mechanisms, 167-172 DHA production by, 288-289 DNA binding, 157-158 recombination, 159 transfection, 161 uptake, 158-159 lactic acid, 31-32 metal reduction, 219-221,224-229 metal substrates, 213-214 oxygen effects, 125-126 phage interaction, 191-192 pH factors, 191 plasmid transformation, 159-160 respiration, 213-219,214-219 transduction description, 177-178 environmental factors, 184-193 in situ, 193-195 types, 181-184 313

314

INDEX

transformation by cell contact, 160-161 description, 154-155 in situ, 161-166 mechanisms, 155-156 Bacteriophages, see Phages Benzaldehyde, 65 Benzothiazole, methionine-related, 61 Bgl 11-Sph-2 gene, 8 Bioremediation, 234-235 Biosynthesis, 278-283, 280-283 Bis(methylthio)methane, 61 Bitterness, 70-72 Blastocladiella emersonii, 302 Bleach, 247-248 Brevibacterium linens in cheese, 56 deamination by, 51-52 lipolysis by, 29 phenylalanine catabolism, 56 proteolysis, 32 Brevibacterium spp., 23 Brewer’s yeast, see Saccharomyces cervisiae

c Calcium cheese flavor and, 65-66 in soy sauce, 135-136, 139 Candida etchellsii, 117 Candido versatilis fermentation processes, 117, 119 organic acid interaction, 129-130 water effects, 1 2 1 Capsduction, 183-184 Carbohydrates, 103, 116 Carbon dioxide, 127-128 Carbon sources, 294, 296 Carbonyl sulfide, 61-62 Caseins flavor effects, 70, 72-73 hydrolysis and, 33-38 Catabolism free amino acids, 45 free fatty acids (FFAs), 30-31 phenylahine, 62-64 sulfiir amino acids, 56-62 tryptophan, 64 tyrosine, 64

Cellulases, deinking by description, 256 mechanisms, 258-261 performance, 245-246, 248-250 properties, 243-245 PUIP effects, 256-245 systems, 253-255 Cheese aldehyde production, 53-54 amines, 45-49 amino acids in, 56-52, 65 bitterness in, 70-72 deamination in, 49, 51-52 fat contents, 27-28 flavor impact, 17-20 free amino acids in, 39-45, 65-68, 7‘0 free fatty acid catabolism, 30-31 fruitiness, 74 glycolysis, 20-27 lactic acid metabolism, 21-23 lactose levels, 23-26 lipases in, 28-30 lipolysis, 27-28 nonprotein amino acids in, 70 pH, 25, 54 phenylacetaldehyde in, 63-64 phenylalanine catabolism, 62-64 protoolysis, 31-32, 33, 37-39 rancidity, 73-74 sulfur amino acids in, 54 transamination, 53-54 tyrosine in, 64 Chitopearl system, 119 Chymosin, 33, 70-73 Citrate, 26-27 Citric acid, 1 2 7 Clay reduction, 233 Competence, 157 Component balance theory, 18 Conjugation description, 166-167 environmental factors, 172-173 in situ, 173-177 mechanisms, 167-172 Conjugative transposons, 170-171 Conversion, lysogenic, 180-181 Copper, 139 Corrosion, 231-235 Corynebacterium diphtheriae, 180 Coryneform bacteria, 56-5 7

315

INDEX p-Cresol, 64, 74 Crypthecodiniurn cohnii DHA production by growth kinetics, 292 light, 303 oleic acid synthesis in, 283 Cryptophyceae , 2 85-2 88 Cytochromes, 225-226, 229-230

D Deacidification, 23 Deamination, 49, 51-52 Decarboxylation, 45-49 Deinking, see Enzymatic deinking Demethiolase, 56 Desulfovibrio spp., 225-226 Desulfuromonas acetoxidans, 229-230 DHA, see Docosahexaenoic acid Diacetyl, 27 Dimethyldisulfide, 59-60 23-Dimethylpyrazine, 65 Dimethylsulfide, 59-60 Dimethyltrisulfide, 59-60 Dinophyceae, 285-288 DMDS, see Dimethyldisulfide DMS, see Dimethylsulfide DMTS, see Dimethyltrisulfide DNA binding, 157-158 conjugation, 167-177 dispersal, 156 persistence, 156-15 7 recombination, 159 release, 155-156 transduction description, 177-178, 177-181 environmental factors, 184-193 types, 181-184 transfection, 161 transformation in situ, 161-166 uptake, 158-159 DNase, 162 Docosahexaenoic acid description, 271-273 microbial production aeration, 303-304 downstream processing, 304-305 fermentor cultivation, 293-294

growth kinetics, 289, 291-294 inoculum development, 299-300 light factors, 302-303 lower fungi, 283-285 media components, 293-294, 294 microalgae, 285-288 pH, 300 salinity, 297-298 supplementation, 297 temperature, 300-302 trace metals, 298-299 vitamins, 299 nutritional importance, 273-275 sources, 275-278

E Electron transport, 229-230 Entomophthora exitalis, 301 Enzymatic deinking benefits, 263 factors, 261-262 industry, 241-242 lignin release, 257-258 mechanisms, 258-261 performance, 245-256 pulp quality and, 256-258 research needs, 263-265 types used, 242-245 Epilithon, 166, 177 Escherichia coli transduction, 182 transformation in situ, 165-166 wastewater treatment and, 176-177 Ethanol, 2 1 Ethyl acetate, 1 3 2 Ethyl esters, 74 Etomophthorales, 283

F fas-IA gene, 9 Fats, in cheese, 27-28 Fatty acid biosynthesis, 280-283 Fatty acid synthase, 9 Fermentation lactose, 57-58 soy sauce, 112,118-119

316

INDEX

FFAs, see Free fatty acids Filamentous fungi, 10 Fish oils DHA in, 275-276 PUFAs in, 274 Flexibacter spp., 288-289 Free amino acids catabolism, 45 in cheese, 39-45 deamination, 49, 51-52 degradation, 41 reactions in cheese, 65-68, 70 Free fatty acids, 29-31 Fruitiness, 74 Fungal enzymes, 130

G

GEMS,see Genetically engineered microorganisms Genetically engineered microorganisms, 154 Gene transfer bacteria, 153-155 Geobacter metallireduceiis bioremediation, 234-235 electron transport, 229 growth, 224 isolation, 220-221 Glutaminase, 111 Glycerol, 131 Glycolysis, 20-27 Gram-negative bacteria, see also specific species broad-host-range transfer, 169-1 70 conjugation, 174-1 77 conjugative transposons, 170-171 DNA binding, 157-158 DNA uptake, 159 plasmid mobilization, 169 plasmid transformation, 160 self-transfer, 167-168 Gram-positive bacteria, see also specific species conjugation broad-host-range transfer, 169-1 70 in situ, 172 self-transfer, 168 transposons, 170-171

DNA uptake, 159 plasmid mobilization, 169 plasmid transformation, 160 Guaiacols, 131

H Haernophilus influenzne competence development, 157 DNA binding, 158 DNA uptake, 159

Haemophilus porninfluenme, 159 Haptophyceae, 285-288 Hemicellulases, deinking by description, 256 mechanisms, 258-261 performance, 245-246, 248-250 properties, 243-245 pulp effects, 256-245 systems, 2 53-25 5 Histamine, 46-47 Host metabolic state, 189-190 phage range, 184-185 range transfer, 170 Hydrogen peroxide, 248 Hydrogen sulfide, 59 Hydroxyacids, 30-31 Hydroxyphenylacetic acid, 64 4-Hydroxy-2,5-dimethyl-3[2H)-furanone, 131 4-Hydroxy-Z(or 5)-ethyl-5(or 21-methyl3(2H)-furaiione, 131 4-Hydroxy-5-methyl-3(2H)-furanone, 131

I Immunity, superinfection, 185-186 Induction, lysogenic, 180 Iron bacterial rcduction, 219-221, 224-229 bacterial respiration, 217-219 bioremediation, 234 corrosion, 231-235 in soy sauce, 139 Isobutanal, 65 Isobutanol, 65

317

INDEX Isochrysis gulbuna fermentor cultivation, 294 growth kinetics, 292 isolation, 288 light, 303 pH, 300-301 temperature, 301-302

Lysogenic induction, 180 Lysogeny, 186 Lytic cycle description, 179 induction, 182

M K KO$, 103-111

Ku, see Koji

L Lactic acid bacteria, 31-32 lactose metabolism by, 20-21 metabolism, in cheese, 21-23 in soy sauce, 96-97, 114-115, 127 Lactobacilli in cheese, 29, 39 free amino acid production, 47 Lactobacillus delbrueckii, 125 Loctobucilfus spp. cheese flavoring, 20-21 proteolysis, 32 Lactococcals, 33-39 Loctococcus spp. lipolysis by, 29 proteolysis, 32 Lactone formation, 30-31 Lactose cheese quality and, 23-26 fermentation, 57-58 metabolism, 20-21 Leuconostoc spp., 26 Light, 302-303 Linoleic acid, 281 Lipase-pregastric esterase, 28-29 Lipases in cheese, 28-30 deinking by, 255 Lipolysis, 27-28 Lipoprotein lipase, 28 Lysogenic conversion, 180-181

Macerating enzymes, 111 Magnesium cheese flavor and, 65-66 in soy sauce, 139 L-Malate, 1 2 8 Malic enzyme, 278-280 Manganese bacterial reduction, 219-221, 224-229 bacterial respiration and, 217-219 bioremediation, 234 pollutant adsorption, 233 Menaquinones, 230 Metal ions, 133-137 Metal reduction bacterial, 221, 224-229 biochemistry, 225-226 electron transport, 229-230 products, 2 28-229 regulation, 226-228 Methanethiol, 56-57 Methional, 60 Methionine, 56-57, 61 n-Methyl ketones, 30-31 Micrococcus spp, lipolysis by, 29 in soy brine stage, 117 Microfibrils, 256, 260 Milk citrate contents, 26 lipoprotein lipase in, 28 psychrotrophs, 30 PUFAs in, 273-274 thiol-producing enzymes in, 58-59 MOI, see Multiplicity of infection Mold enzymes, 111 Molds, see specific species Moromj, 111-118,126 Multiplicity of infection, 187-188 Mycotoxins, see also specific species description, 1-2

318

INDEX

N nar-1 gene, 8 Neisseria gonorrhoeae competence development, 157 DNA binding, 158 Neurospora crassa, 7-10 niaD gene, 7 Nitrogen sources, 296 soy beans, 105-106 Nonprotein amino acids, 70 Nonstarter lactic acid bacteria cheese ripening, 39 L-p-lactate isomerization by, 21 lactose substrate, 26 norA gene, 11-12 nor-2 gene, 9 Norsolorinic acid in aflatoxins, 2-3 enzymology, 6-7 reverse genetics, 11-12 stain degeneration, 10-11 Norsolorinic acid dehydrogenase, 6-7 NSLAB. see Nonstarter lactic acid bacteria

0

Oils DHA in, 275-276 PUFAs in, 274 soy sauce, 103 Oleic acid, 283 Organic acids, see also specific types in soy sauce, 96-98, 126, 128 Oxygen bacteria and, 125-126 soy sauce, 115-116 yeasts and, 123-124 Oxygen depletion, 217

P Pediococcus halophilus, see Tetragenococcus halophilus Penicilliurn camemberti by, 30 PeniciIlium roqueforti, 30 Penicilliurn spp., 30-31

Peromosporales, 283 PGE, see Lipase-pregastric esterase

PH bacteria and, 191 cheese, 25, 54 DHA production, 300 enzymatic deinking, 261-262 Phages bacteria interaction, 191-192 concentration, 188-189 description, 178-181 host range, 184-185 ions, 190-191 metabolic state, 189-190 pH factors, 191 survival, 187 tamperature, 190 transduction types, 181-184 Phnnerochaete chrysosporiurn, 255 Phenethanol, 74 Phenols, 64, 74, 131-132 Phenylacetaldehyde, 63-64 Phenylalanine, 62-64 Phenylethanolacetate, 64 Phosphorus, 139 Phycomycetes, 283 pksA gene, 9 Plants conjugation in, 174 transduction in, 194 Plasmids broad-host-range, 169-1 70 mobilization, 169 retrotransfer, 171-172 self-transfer, 167-169 transformation, 159-160 Plasmin, 33-39 Pollutants, 233 Polyunsaturated fatty acids (PUFAs) biosynthesis, 278-283 description, 272-273 microbial production aeration, 303-304 fermentor cultivation, 293-294 growth kinetics, 289, 291-294 inoculum development, 299-300 media components, 294, 296 pH, 300 salinity, 297-298 supplementation, 297

319

INDEX temperature, 300-302 trace metals, 298-299 vitamins, 299 microorganism production lower fungi, 283-285 microalgae, 285-288 psychrophilic bacteria, 285-288 in milk, 273-274 in oils, 274 Potassium, 136-137, 139 Propionibacterium shermanii lipolysis by, 29 proteolysis, 32 Propionic acid, 60-61 Proteinases, 108 Proteolysis, 31-32 cheese, 33, 37-39 Pseudomonas putida, 172 Pseudomonas spp. cell contact transformation, 160-161 corrosion by, 231 Psychrotrophs, 30 PUFAs, see Polyunsaturated fatty acids Pulp deinking cehlases, 253-254 enzyme effects, 256-258 mixed office waste additions in, 264 cellulase, 253-254 hemicellulase, 257 special problems, 248 old newspaper, 251-253,264 pyr-4 gene, 7 pyrG gene, 7 Pyroglutamic acid, 126

9 Quinones, 230

R

Rancidity, 73-74 RecA protein, 180 Recipient cells, 189 Respiration concept, 2 14-2 19 regulation, 227

Retrotransfer, 171-1 72 Rhizosphere, 175 Rhodobacter capsulatus, 183

S Saccharomyces cerevisiae broad-host-range transfer, 170 in cheese, 64 Salinity, 297-298 Schizoch ytrium aggregaturn, 2 84-2 8 5 sec- genes, 11 Shewanella putrefaciens bioremediation, 235 clay reduction, 233 corrosion by, 231, 233 metal reduction by, 225-227, 229-230 Shoyu, 90-92 Shui, see Koji Sludge, activated, 176 Sodium, 136 Sodium chloride in cheese, 72-73 in soy sauce, 115,119-123, 130 Soils conjugation in, 174-175 transduction effects, 192-193, 194-195 transformation in, 162-164 Solar radiation, 193 Soy beans carbohydrate content, 103 nitrogen content, 105-106 oil content, 103 Soy sauce alcohol in, 124-129 amino acids in, 92-96 aroma, 100-101 bacteria in, 125-126 brine stage aeration, 115-116 microorganisms, 116-118 temperature, 113-115 description, 87-88 fermentation continuous, 118-119 moromi stage, 1 1 2 flavor, 100-101 history, 88-89 koji, 103-111

320

INDEX

manufacture, 103-104 metal ions in, 133-137 moromi, 111-118, 126 organic acids in, 96-98 production substances in, 130-133 traditional, 101 water effects, 119-123 raw materials adequacy, 137-140 composition, 102-103 production factors, 140-143 sugar in, 98-99 types, 89-92 yeasts in, 122-124 Stain degeneration, 10-11 stcE gene, 9 stcj gene, 9 stcK gene, 9 Strecker degradation cheese, 53-54 soy sauce, 1 3 2 Streptococcus pneurnoniae DNA binding, 157 transformation in situ, 161 Streptococcus spp., 170 Succinic acid, 97 Sugars, see also specific types as fermentation media, 294 in soy sauce, 98-99 Sulfur amino acids, 54, 56-62 Superinfection, 185-186

T Temperature bacterial transduction, 190 DHA production, 300-301 soy sauce production, 106-107, 113-115

Tetragenococcus tialophilus, soy sauce production by flavor effects, 117-119 organic acid interaction, 129-130 oxygen effects, 125 role, 126-129 water effects, 1 2 1 Thraustochytriurn roseurn, 289, 291-294

Thraustochytrium spp., DHA production by carbon source, 294, 296 contents in, 291-294 fermentor cultivation, 293-294 growth, 289 inoculum development, 299-300 light, 302-303 nitrogen source, 294 pH, 300 salinity, 2 9 7-2 98 as source, 283-285 temperature, 300-301 trace metals, 298-299 vitamins, 299 'Race metals, see also specific metals DHA production, 298-299 Transamination, 53-54 'Iransduction description, 177-181 environmental factors, 184-1 93 types, 181-184 Transfection, 161 Transformation cell contact, 160-161 description, 154-155 in situ, 161-166 mechanisms, 155-156 plasmid, 159-160 Transposons, conjugative, 170-171 Tryptophan, 64 "yramine, 46-47 Tyrosine, 64 V

Vibrio spp. temperature, 302 transformation in situ, 165-166 W Wastewater treatment, 176-177 Water conjugation in, 175-177 DNA transformation in, 164-166 oxygen depletion, 217 soy sauce production and, 119-123 transduction in, 195

321

INDEX Wheat in soy sauce, 102-103 starch, 108

Y Yeasts, see also specific species oxygen effects, 123-124 in soy sauce, 122-123

1

Zinc, 139 Zygosaccharomyces rouxii, in soy sauce alcohol production, 124-125 fermentation, 119 glycerol production, 129-130 organic acid interaction, 129-130 as source, 117 water effects, 120

This Page Intentionally Left Blank

CONTENTS OF PREVIOUS VOLUMES

Volume 35

Production of Bacterial Thermostable a-Amylase by Solid-state Fermentation: A Potential Tool for Achieving Economy in Enzyme Production and Starch Hydrolysis B. K.Lonsane and M. K Ramesh Methods for Studying Bacterial Gene Transfer in Soil by Conjugation and Transduction G. Stotzky, Monica A . Devanas, and Lawrence R. Zeph Microbial Levan Ibun W Han Review and Evaluation of the Effects of Xenobiotic Chemicals on Microorganisms i n Soil R. J. Hicks, G. Stotzky, and I? Van Voris Disclosure Requirements for Biological Materials in Patent Law Shung-Chang Jong and Jeannette M. Birmingham INDEX

Volume 36

Microbial Cytochromes P-450 and Xenobiotic Metabolism F: Sima Sariaslani Foodborne Yeasts 7:Dea'k High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins Erik l? Lillehoj and Vedpal S . Malik INDEX

Volume 37

Microbial Degradation of the Nitroaromatic Compounds Frank K. Higson An Evaluation of Bacterial Standards and Disinfection Practices Used for the Assessment and Treatment of Stormwater Marie L. O'Shea and Richard Field Haloperoxidases: Their Properties and Their Use in Organic Synthesis M. C. R. Franssen and H. C. van der Plas

Microbial Transformations of Herbicides and Pesticides Douglas J. Cork and James I? Krueger

Medicinal Benefits of the Mushroom Ganoderma S. C. Jong and J. M. Birmingham

An Environmental Assessment of Biotechnological Processes M. S. Thakur, M. J. Kennedy, and N . G. Karanth

Microbial Degradation of Biphenyl and Its Derivatives Frank K. Higson

Fate of Recombinant Escherichia coli K-12 Strains in the Environment G r e g Bogosian and James E Kane

The Sensitivities of Biocatalysts to Hydrodynamic Shear Stress AleS Prokop and Rakesh K. Bajpai 323

324

CONTENTS OF PREVIOUS VOLUMES

Biopotentialities of the Basidiomacromycetes Somasundarum Rajarathnnm, Mysore Nanjarajurs Shash irekh n, and Zakia Ban0

Microbial Pentose Utilization Prashnnt Mishra and Ajny Singh Medicinal and Therapeutic Value of the Shiitake Mushroom S. C. Jong and J. M. Birmingham

INDEX

Yeast Lipid Biotechnology Z. Jacob Volume 38

Selected Methods for the Detection and Assessment of Ecological Effects Resulting from the Release of Genetically Engineered Microorganisms to the Terrestrial Environment G. Stotzky. M. W Broder, J. D. Doyle, and H. A. Jones Biochemical Engineering Aspects of Solid-state Fermentation M. V Ramana Murthy, N. G,Kuranfh, and K. S. M. S. Raghnva Rao The New Antibody Technologies Erik I? Lillehoj and Vedpal S. Malik Anoxygenic Phototrophic Bacteria: Physiology and Advances in Hydrogen Production Technology K. Sasiknln, Ch. R Ramann, I? Rahuveer Hao, and K. L. Kovacs INDEX

Volume 39

Pectin, Pectinase, and Protopectinase: Production, Properties, and Applications Takuo Sakui, Tatsuji Sukamoto, Johan Hollaert, and Erick J. Vandamme Physicochemical and Biological Tkeatments for Enzymatic/Microbial Conversion of Lignocellulosic Biomass Purnendu Ghosh and Ajay Singh INDEX

Volume 40

Microbial Cellulases: Protein Architecture, Molecular Properties, and Biosynthesis Ajay Singh and Kiyoshi Hayashi Factors Inhibiting and Stimulating Bacterial Growth in Milk: An Historical Perspective D. K. O’Tools

Asepsis in Bioreactnrs M. C. Sharma und A. K. Gurtri

Challenges in Conlmercial Biotechnology. Part I. Product, Process, and Market Discovery Ale3 Prokop

Lipids of n-Alkane-Utilizing Microorganisms and Their Application Potential Sumir S. Radwnn nnd Naser A . Sorkhoh

Challenges in Commercial Biotechnology. Part 11. Product, Process, and Market Development Ale6 Prokop

CONTENTS OF PREVIOUS VOLUMES Effects of Genetically Engineered Microorganisms on Microbial Populations and Processes in Natural Habitats Jack D. Doyle, Guenther Stotzky, Gwendolyn McClung, and Charles W Hendricks Detection, Isolation, and Stability of Megaplasmic-Encoded Chloroaromatic Herbicide-Degrading Genes within Pseudomonos Species Douglas J. Cork and Amjad Khalil

325

Biotechnological Potentials of Anoxygenic Phototrophic Bacteria. Part 11. Biopolyesters, Biopesticide, Biofuel, and Biofertilizer Ch. Sasikala and Ch. V Ramana INDEX

Volume 42 The Insecticidal Proteins of Bacillus thuringiensis P Ananda Kumar, R. F! Sharma, and K S. Malik

INDEX

Microbiological Production of Lactic Acid John H. Litchfield Volume 41 Microbial Oxidation of Unsaturated Fatty Acids Ching T. Hou Improving Productivity of Heterologous Proteins in Recombinant Saccharomyces cerevisiae Fermentations Amit Vasavada Manipulations of Catabolic Genes for the Degradation and Detoxification of Xenobiotics Rup Lal, Sukanya Lal, I? S. Dhanaraj, and D. M. Saxena Aqueous Two-Phase Extraction for Downstream Processing of Enzymes/Proteins K. S. M. S. Raghava Rao, N. K. Rastogi, M. K. Gowthaman, and N. G. Karanth Biotechnological Potentials of Anoxygenic Phototrophic Bacteria Part I. Production of Single Cell Protein, Vitamins, Ubiquinones, Hormones, and Enzymes and Use in Waste Treatment Ch. Sasikala and Ch. V Ramana

Biodegradable Polyesters Ch. Sasikala The Utility of Strains of Morphological Group I1 Bacillus Samuel Singer Phytase Rudy J. Wodzinski and A . H. J. Ullah INDEX

Volume 43 Production of Acetic Acid by Clostridium thermoaceticum Munir Cheryan, Sarad Parekh, Minish Shah, and Kusuma Witjitra Contact Lenses, Disinfectants, and Acanthamoeba Keratitis Donald G. Ahearn and Manal M. Gabriel Marine Microorganisms as a Source of New Natural Products V S. Bernan, M. Greenstein, and W M. Maiese

326

CONTENTS OF PREVIOUS VOLUMES

Stereoselective Biotransformations in Synthesis of Some Pharmaceutical Intermediates Ramesh N . Patel

Microbial Synthesis of D-Ribose: Metabolic Deregulation and Fermentation Process I? de Wulf and E. J. Vandamme

Microbial Xylanolytic Enzyme System: Propcrtics and Applications Pratima Bajpai

Production and Application of Tannin Acyl Hydrolase: State of the Art I? K. Lekha and B. K. Lonsane

Oleaginous Microorganisms: An Assessment of the Potential Jacek Lemon

Ethanol Production from Agricultural Biomass Substrates Rodney J. Bothast and Badal C. Saha

INDEX

Volume 44

Biologically Active Fungal Metabolites Cedric Pearce

Old and New Synthetic Capacities of Baker’s Yeast l? D’Arrigo, G. Pedrocchi-Fantoni, and S. Servi Investigation of the Carbon- and SulfurOxidizing Capabilities of Microorganisms b y Active-Site Modeling Herbert L. Holland

Thermal Processing of Foods, A Retrospective, Part I: Uncertainties in Thermal Processing and Statistical Analysis M. N. Ramesh, S. G. Prapulla, M. A. Kumur, and M . Mahadevainh Thermal Processing of Foods, A Retrospective, Part 11: On-Line Methods for Ensuring Commercial Sterility M. N. Ramesh, M. A . Kumar, S. G. Prapulla, and M. Mahadevuiuh INDEX