Advances in Microbial Physiology Volume 41

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Advances in

MICROBIAL PHYSIOLOGY VOLUME 41

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY Edited by

R. K. POOLE Department of Molecular Biology and Biotechnology The Krebs Institute f o r Biomolecular Research The University of Shefield Firth Court, Western Bank Shefield SIO 2TN, UK

Volume 41

ACADEMIC PRESS A Harcaurt Science and Technology Company

San Diego San Francisco New York Boston London Sydney Tokyo

This book is printed on acid-free paper. Copyright 0 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press 24-28 Oval Road, London NW I 7DX. UK http:Nwww.hbuk.co.uWap/ Academic Press A Harcourt Science and Technology Company

525 B Street, Suite 1900, San Diego, California 92101-4495. USA http://www.apnet.com ISBN 0-12-027741-7 A catalogue for this book is available from the British Library

Typeset by M Rules, London, UK Printed in Great Britain by MPG Books Ltd, Bodmin, Cornwall 990001020304MP98765432 1

Contents

CONTRIBUTORS TO VOLUME 41 .................................

ix

Factors Affecting the Production of L-Phenylacetylcarbinol by Yeast: A Case Study Alison L. Oliver. Bruce N . Anderson and Felicity A. Roddick Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 . Biochemical production of L-phenylacetylcarbinol . . . . . . . . . . . . . . 4 3. Development and optimization of the fermentation process . . . . . . 11 4 . An industrial process for the production of L-phenylacetylcarbinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Fungal Production of Citric and Oxalic Acid: Importance in Metal Speciation. Physiology and Biogeochemical Processes Geoffrey M . Gadd 1. 2. 3. 4.

5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Metal chemistry of oxalic and citric acids . . . . . . . . . . . . . . . . . . . . 50 Fungal biosynthesis of oxalic acid and calcium oxalate formation . 53 Role of metals and oxalate in lignocellulose degradation and plant pathogenesis ...................................... 61 Catabolism of oxalic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Fungal biosynthesis of citric acid . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Fungal organic acid production and metal biogeochemistry . . . . . . 68 Fungal organic acid production and metal biotechnology . . . . . . . . 76

vi

CONTENTS

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

78 79

Bacterial Viability and Culturability Michael R. Barer and Colin R. Harwood 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 The ‘viable but non-culturable’ (VBNC) hypothesis . . . . . . . . . . . . 96 ‘As yet uncultured’ (AYU) bacteria . . . . . . . . . . . . . . . . . . . . . . . . . 99 ‘New’methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Factors influencing the outcome of culturability tests . . . . . . . . . . 1 1 1 Should bacterial viability be assessed at the individual or community level? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 124 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 126 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Histidine Protein Kinase Superfamily Thorsten W. Grebe and Jeffry B . Stock 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histidine protein kinase subfamilies . . . . . . . . . . . . . . . . . . . . . . . Cognate receiver domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Domain shuffling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139 197 206 211 214 214

Bacterial Tactic Responses Judith P. Armitage Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 232 2 . What is meant by ‘bacterial taxis’? ........................ 3 . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 4 . How Bacteria swim and glide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 5. The chemosensory pathway of E . coli ...................... 238 6. Responses to electron acceptors and light . . . . . . . . . . . . . . . . . . .263 7 . What is the role of tactic responses in natural environments? . . . .269 8 . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

vi i

CONTENTS

The Bacterial Flagellar Motor

Richard M . Berry and Judith P. Armitage Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The flagellar structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Flagellar motor function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Models of the flagellar motor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

292 292 296 310 322 328 329 329

Authorindex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

339 363


Contributors to Volume 41

Bruce N. ANDERSON, Department of Chemical and Metallurgical Engineering, Royal Melbourne Institute of Technology University, GPO Box 2476V, Melbourne, Victoria 3001, Australia ([email protected]) Judith P. ARMITAGE, Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK ([email protected]) Michael R. BARER, Department of Microbiology and Immunology, The Medical School, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK ([email protected]) Richard M. BERRY, The Randall Institute, King’s College London, 26-29 Drury Lane, London WC2B 5RL, UK Geoffrey M. GADD,Department of Biological Sciences, University of Dundee, Dundee DD 1 4HN, UK ([email protected]) Thorsten W. GREBE, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA ([email protected]) Colin HARWOOD, Department of Microbiology and Immunology, The Medical School, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK CSL Limited, 45 Popular Road, Parkville 3052, Alison L. OLIVER, Australia

X

CONTRIBUTORS TO VOLUME 41

Felicity A. RODDICK, Department of Chemical and Metallurgical Engineering, Royal Melbourne Institute of Technology University, GPO Box 2476V, Melbourne, Victoria 300 1, Australia ([email protected]) Jeffry B. STOCK, Department of Molecular Biology, Princeton University, Princeton, NJ 08544,USA ([email protected])

Factors Affecting the Production of L-Phenylacetylcarbinol by Yeast: A Case Study Alison L. Oliver, Bruce N. Anderson and Felicity A. Roddick Department of Chemical and Metallurgical Engineering, RMIT University. GPO Box 2476V Melbourne, victoria 3000, Australia

ABSTRACT

L-Phenylacetylcarbinol (L-PAC)is the precursor for L-ephedrine and Dpseudoephedrine, alkaloids possessing a-and 0-adrenergic activity. The most commonly used method for production of L-PAC is a biological method whereby the enzyme pyruvate decarboxylase (PDC) decarboxylates pyruvate and then condenses the product with added benzaldehyde. The process may be undertaken by either whole cells or purified PDC. If whole cells are used, the biomass may be grown and allowed to synthesize endogenous pyruvate, or the cells may be used as a catalyst only, with both pyruvate and benzaldehyde being added. Several yeast species have been investigated with regard to L-PACproducing potential; the most commonly used organisms are strains of Sacchuromyces cerevisiue and Cundidu utilis. It was found that initial high production rates did not necessarily result in the highest final yields. Researchers then examined ways of improving the productivity of the process. The substrate, benzaldehyde, and the product, L-PAC,as well as the by-products, were found to be toxic to the biomass. Methods examined to reduce toxicity include modification of benzaldehyde dosing regimes, immobilization of biomass or purified enzymes, modification of benzaldehyde solubility and the use of twophase reaction systems. Various means of modifying metabolism to enhance enzyme activity, relevant metabolic pathways and yield have been examined. Methods investigated include the use of respiratory quotient to influence pyruvate production and induce fermentative activity, reduced aeration to increase PDC activity, and carbohydrate feeding to modify glycolytic enzyme activity. The effect of temperature on L-PACyield has been examined to identify conditions ADVANCES IN MICROBIAL PHYSIOLOGY VOL 41

ISBN 0-12-027741-7

Copyright 0 1999 Academic Press All rights of reproduction in any form reserved

2

ALISON L. OLIVER, BRUCE N. ANDERSON AND FELICITY A. RODDICK

which provide the optimal balance between L-PAC and benzyl alcohol production, and L-PAC inactivation. However, relatively little work has been undertaken on the effect of medium composition on L-PACyield. Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Biochemical production of L-phenylacetyl carbinol ....................... 2.1. Mechanism of L-PACproduction ...................... 2.2. Pyruvate decarboxylase and provision of substrates . . . . . . . . . . . . . . . . . . 2.3. Alcohol dehydrogenase and reaction by-products .................... 3. Development and optimization of the fermentation process . . . . . . . . . . . . . . . 3.1. Selection of a high-yielding producer organism ...................... 3.2. Physicochemical conditions and their effect on L-PACproduction . . . . . . . 3.3. Physiological condition of cells for optimum L-PAC production . . . . . . . . . 3.4. Nutrient effects in L-PAC production ............................... 3.5. Production of L-PACby batch, fed-batch or continuous fermentation . . . . 3.6. The use of additives to modify metabolic activity .................... 3.7. Reduction of the toxic effects of substrate, product and by-product . . . . . 3.8. Other methods for influencing L-PACproduction ..................... 4. An industrial process for the production of L-phenylacetylcarbinol . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 4 4 6 9 11 12 13 16 18 20 21 23 33 34 39 40

ABBREVIATIONS

ADH BCD CSL 2,4-D DMF Ki kl'a

Km L-PAC NAA PCO, PDC PEG PO, RQ TPP YADH

Alcohol dehydrogenase P-Cy clodextrin Corn steep liquor 2,4-Dichlorophenoxy acetic acid N,N-Dimethylformamide Inhibition constant - the concentration of a compound at which a reaction is inhibited by 50% Volumetric liquid mass transfer coefficient for oxygen Michaelis constant - the substrate concentration at which the reaction velocity is 50% of its maximum L-Phenylacetylcarbinol a-Naphthoxy acetic acid Partial pressure of carbon dioxide (millibar) Pyruvate decarboxylase Polyethylene glycol Partial pressure of oxygen (% saturation) Respiratory quotient Thiamine pyrophosphate Yeast alcohol dehydrogenase

THE PRODUCTION OF L-PHENYLACETYLCARBINOLBY YEAST

3

1. INTRODUCTION

L-Phenylacetylcarbinol (L-PAC)is used as a substrate in the manufacture of Lephedrine and D-pseudoephedrine, alkaloids possessing a-and P-adrenergic activity (Long and Ward, 1989b). Ephedrine is used in the treatment of conditions including hypotension and asthma, while pseudoephedrine, an optical isomer of ephedrine, is used mainly as a nasal decongestant in cold and influenza medications. These alkaloids can be produced by one of two methods: direct extraction from plant material (Ephedra spp.) or synthesis using L-PAC as a precursor. Until early this century, plant material was the sole source of both ephedrine and pseudoephedrine; however, the total alkaloid content in Ephedra spp. is generally low, the highest concentration being approximately 2.5% by weight, and occurs as a mixture of ephedrine and pseudoephedrine. The alkaloid content is also subject to seasonal variation, requiring the collection of large amounts of plant material followed by time- and labour-intensive processing (Morton, 1977). In contrast, the synthesis of ephedrine or pseudoephedrine via L-PACis less labour intensive and independent of climatic conditions, enabling a continuous supply of product of guaranteed quality. While there are several chemical methods for L-PAC synthesis (Culik et al., 1984; Nikolova et al., 1991), the usual method of production is via yeast fermentation. Production via fermentation is preferable to chemical synthesis because of the mild conditions used, and because the pharmacologically active laevorotatory form (L-PAC) is produced whereas chemically synthesized PAC is a racemic mixture (D-PACand L-PAC).D-PACcannot be used to prepare L-ephedrine or D-pseudoephedrine. In addition, the waste products of other processes, e.g. molasses, can be used as raw materials in the L-PAC fermentation medium, after which the spent broth is amenable to biological waste treatment. The final ephedrine and pseudoephedrine concentrations produced by synthetic routes are similar to those present in plant extracts. This review is divided into three sections: first in the specific processes involved in the conversion of benzaldehyde to L-PAC are examined and the background on the enzymes involved in the process is provided; in the second the effects of manipulating fermentation parameters on yeast behaviour are examined; and in the third an industrial process used for the production of LPAC is outlined (Oliver et al., 1997).

4


2. BIOCHEMICAL PRODUCTION OF LPHENYLACETYLCARBINOL 2.1. Mechanism of L-PACProduction

The biochemical production of L-PAC was first demonstrated in the early 1920s by Neuberg and co-workers who described its formation after the addition of benzaldehyde to a culture of actively fermenting top yeast (McKenzie, 1936). They proposed two possible mechanisms: the first, thought to be the most likely of the two scenarios, was the conversion of pyruvate by carboxylase to an activated acetaldehyde followed by the enzymic combination of benzaldehyde and the activated acetaldehyde by carboligase; the second proposal was the joining of benzaldehyde and pyruvate via a carboligase, followed by decarboxylation of the complex using carboxylase (McKenzie, 1936). Pyruvate decarboxylase (PDC) is now known to be responsible for both nonoxidative decarboxylation and a carboligase reaction, which results in the production of a-hydroxy ketones (Pohl, 1997). The production of L-PACby yeast was considered to be analogous to the production of acetoin (methylacetylcarbinol)which was demonstrated by Green el al. in 1942 using a crude yeast enzyme extract. Weight was added to the hypothesis that acetaldehyde was an intermediate in the production of acetoin when Gross and Werkman ( 1947) demonstrated the incorporation of isotopically labelled acetaldehydeinto acetoin by a dried yeast ‘juice’.Acetoin was not considered to be a normal fermentation product by Happold and Spencer (1952), although it is a known product of wine yeast fermentation (Romano and Suzzi, 1996).Happold and Spencer’s proposal is supported by Hohmann (1997), who suggested that the production of acetoin is ‘probably not important under physiological conditions,’owing to the low concentrations of acetaldehyde present in yeast cytosol. Happold and Spencer found that the presence of additional acetaldehyde resulted in the formation of acetoin. Green et al. (1942) found that the production of significant quantities of acetoin required the presence of both acetaldehyde and pyruvate rather than either in isolation. The role of a single enzyme for the production of acetoin was advanced by Juni (1952) who was unable to separate his brewer’s yeast extract into separate fractions for pyruvate decarboxylation and for acetoin formation. He later introduced the concept of a two-site mechanism for the formation of acetoin (Juni, 1961). Although it was previously assumed that PDC catalysed the condensation of benzaldehyde and pyruvate to L-PAC,confirmation did not occur until BringerMeyer and Sahm (1988) demonstrated the production of L-PACby purified PDC from both Zymomonas mobilis and Saccharomyces carlsbergensis. Their results were confirmed by Cardillo et al. (199 I ) who showed that benzaldehyde was condensed more readily than other substituted aldehydes by

5


Saccharomyces spp. Although Fuganti et al. (1988) questioned whether the enzyme responsible for L-PAC production was known, they reconfirmed the ability of baker's yeast to produce L-PAC and considered the mechanism of LPAC production to be analogous to the production of acetoin, thereby implying that PDC was essentially responsible for L-PACproduction. Crout et al. (199 1) provided definitive proof of the ability of purified yeast PDC to convert a range of aldehydes to ketols, including L-PAC, by following the reactions using 'H-nuclear magnetic resonance (NMR) spectroscopy. Kren et al. (1993) also demonstrated the production of various ketols by purified yeast PDC. PDC, located in the cytosol, catalyses the irreversible conversion of pyruvate to acetaldehyde with the resultant loss of a molecule of CO,. The reaction requires the cofactors thiamine pyrophosphate (TPP) and a magnesium ion, which is thought to act as a link between the apoenzyme and the thiamine pyrophosphate (Pohl, 1997).

CH,CHO + CO, Acetaldehyde

CH,COCOOH PYruvate decarboxY1ase, Pyruvate TTP, Mg2+

(1)

PDC then catalyses the condensation of acetaldehyde and pyruvate, forming acetoin:

,

CH,COCOOH + CH,CHO PDC CH,CHOHCOCH, Pyruvate Acetaldehyde p p , Mg2+ Acetoin

+ CO,

(2)

After the formation of acetaldehyde according to Equation 1, L-PAC is formed (analogous with Equation 2) by the condensation of acetaldehyde with added benzaldehyde:

,

PDC C6H,CHOHCOCH, CH,COCOOH + C,H,CHO PAC Pyruvate Benzaldehyde p p , Mg2+

+ C02

(3)

L-PACis considered to be a product of the stationary phase of growth (Liew et al.,1995). In the process employed by Oliver et al. (1997), the pyruvate consumed in the reaction is generated by the yeast via glycolysis and is allowed to accumulate exogenously during the exponential phase of growth. Commercial processes are generally divided into two stages: a first stage where the yeast is grown and pyruvate is accumulated, followed by a bioconversion stage where benzaldehyde is added and L-PAC produced. For fermentations where pregrown yeast is used as a catalyst only, direct pyruvate supplementation is required in the bioconversion phase.

6


2.2. Pyruvate Decarboxylase and Provision of Substrates

As described above, PDC is the enzyme responsible for the production of LPAC. PDC is usually found to exist as either dimers or tetramers whereby the active PDC holoenzyme generally exists as a tetramer, while the apoenzyme exists as a dimer (Pohl, 1997). The existence of the dimer and tetramer forms is pH dependent. In yeast, PDC has been reported to exist only as a tetramer in the pH range 5.5-6.5, as both tetramers and dimers at pH values up to 9.5, and dimers only at pH > 9.5 (Pohl, 1997). However, Hohmann (1997) has reported the existence of PDC only in the form of dimers at a pH of 8.4. PDC from 2. mobifis has been found to exist only in the form of tetramers (Pohl, 1997). While PDC subunits were thought previously to have different compositions, they are now known to be identical (Hohmann, 1997). A total of six PDC genes has been identified in Saccharomyces cerevisiae, of which three are structural genes (PDC 1, PDC5 and PDC6) and the remaining three are considered to be genes related to expression of PDC (PDC2, PDC3, PDC4) (Flikweert etal., 1996; Hohmann, 1997; Pohl, 1997; ter Schure et al., 1998).A single structural gene coding for PDC has been identified in 2. mobilis (Pohl, 1997). While the majority of work in this area appears to have been performed on S. cerevisiae, genes for PDC have been identified in a number of yeast species, not including Candida utilis. Flikweert and co-workers undertook studies to assess the role of each of the isoenzymes in overall PDC activity, and found that the expression of each of the isoenzymes in S. cerevisiae differed. Using batch culture with either ethanol or glucose as carbon substrate, PDC 1 was expressed constitutively while PDC5 was induced in the presence of glucose. PDC6 was present at insignificant levels. These findings were echoed in a review on pyruvate decarboxylases by Hohmann ( 1 997), who indicated that only PDC 1 and PDC5 played an apparent role in sugar catabolism, with 80-90% and 10-20% of total PDC activity being attributed to these two genes, respectively, for glucose-grown biomass. In addition to the production of acetoin, S. cerevisiae PDC has been found to be involved in the production of fuse1 oils, which are flavour compounds present in alcoholic beverages and bread. Fuse1 oils are produced by the decarboxylation of branched chain 2-0x0 acids, derived from aromatic amino acids. The products are then dehydrogenated by alcohol dehydrogenase (ADH). The activity of PDC with the 2-0x0 acids is significantly lower than for pyruvate (ter Schure et al., 1998). Production of novel aldehyde analogues is discussed later in this chapter. PDC activity can be induced and manipulated both by the degree of aeration supplied to the culture and by the choice of carbohydrate substrate in the medium. PDC activity is required to enable glycolytic flux only under anaerobic conditions; therefore, a reduction in aeration should result in the induction of PDC. Both Sims and co-workers (1991) and Rogers et al. (1997) have

THE PRODUCTION OF L-PHENYLACETYLCARBINOL BY YEAST

7

demonstrated that an increase in C. utilis PDC activity occurs when oxygen concentration is reduced. Such a response is highly beneficial for the production of L-PAC.However, both groups of researchers found that the activity of ADH, responsible for generation of unwanted by-products, exceeds that of PDC under anaerobic or partially anaerobic conditions. The effect of aeration conditions on yeast physiology is discussed later in this chapter. Induction of PDC activity by carbohydrate source is dependent on the yeast species involved and the carbohydrate source supplied. Glucose is one substrate capable of inducing glycolytic enzymes, especially PDC. A number of workers have demonstrated that the addition of glucose to cultures results in an increase in the level of PDC activity. Maitra and Lob0 (1971) found that the addition of glucose as a pulse to Saccharomyces spp. cultures resulted in an increase in the production of glycolytic enzymes, including PDC, following a short lag period. The medium used for growth was carbohydrate-free and acetate was used as the carbon source. Similarly, Schmitt and Zimmerman (1982) demonstrated an 18-fold increase in the PDC activity of S. cerevisiae after glucose was added to a shake flask culture grown on ethanol as the sole carbohydrate source. These results are in agreement with those of Harrison (1972) and Sims et al. (1991). Sims and co-workers also demonstrated the reversible nature of PDC activation. They showed that, under anaerobic conditions, when deprived of glucose (by centrifuging and resuspending biomass in glucose-free medium) the PDC activity of C. utilis was reduced by 50%. PDC activity was restored by the addition of glucose to the medium under anaerobic conditions; however, if the culture was aerated in addition to glucose supplementation, there was no change in PDC activity. Such enzyme activation does not occur for all carbohydrate types. When yeast species are grown anaerobically on glycosides which give the Kluyver effect, PDC activity is reduced compared with glycosides, such as glucose, which can be metabolized anaerobically (Sims and Barnett, 1991). As a result of their research, they found that PDC activity may be rate limiting in anaerobic conditions. This finding is in agreement with that of van Urk et al. (1989). PDC is a substrate-pyruvate-activated enzyme (Hubner et al., 1978; Hohmann, 1997), which is also allosterically inhibited by inorganic phosphate (Boiteux and Hess, 1970). Boiteux and Hess reported an increase in the Michaelis constant (K,) of purified PDC from S. carlsbergensis from 1.3 mM in the absence of inorganic phosphate to approximately 11 m~ in the presence of 100 nm phosphate. The inhibitory effect was determined to be competitive in nature, the variation in K , having no effect on the maximum activity of the enzyme. The sensitivity of PDC to inhibition by phosphate was determined to be of the same order of magnitude as its sensitivity to activation by pyruvate. Saccharomyces spp. and C. utilis are commonly used for the production of L-PAC;however, in early work, little was done to compare enzyme activities directly. The PDCs of S. cerevisiae and C. utilis were compared by van Dijken

8


and Scheffers (1986), and van Urk et al. (1989). They determined that the activity of PDC from S. cerevisiae was approximately eight times that of PDC from C. urilis, although the PDC from the former was determined to be more sensitive to inhibition by phosphate. With respect to phosphate, the PDC from C. utilis displayed similar K , values to that from S. carlsbergensis, namely 3.6 mM in the absence of phosphate, and 11 mM in the presence of 100 mM phosphate. In contrast, PDC from S. cerevisiae exhibited K , values of 3.0 mM in the absence of phosphate and 48 mM in the presence of 100 mM phosphate. Consequently, the availability of phosphate plays an important role in the activity of PDC and thus PDC productivity. A combination of reduction in the cytosolic concentration of phosphate and increased pyruvate concentrations was proposed by van Urk et al. (1989) as contributing factors to increased PDC activity after pulsing the culture with glucose. The process used by Oliver et al. (1 997) involved pulsing with molasses midway through the fermentation, hence a similar increase in PDC activity would most probably have resulted. Significant work has been undertaken by Rogers and co-workers (Chow et al., 1995; Shin and Rogers, 1996a; Rogers et al., 1997) to evaluate the kinetics of PDC from C. utilis in both purified form and in whole cells. They recorded PDC activities of 0.85-0.9 U/mg protein for whole, stationary-phase biomass after growth in batch culture. After partial purification, Chow et al. (1995) recorded an increase in PDC activity to 4.8 U/mg protein, which was comparable with commercially obtained PDC. Pohl (1997) suggests that a specific activity in the range of 45-60 U/mg can be achieved for PDC purified from yeast and plants. Rogers et al. (1997) reported a number of kinetic parameters for purified PDC from C. utilis. The K , values for benzaldehyde and pyruvate were 42 m M (4°C pH 7.0) and 2.2 mM (25°C pH 6.0) respectively, with concentrations in excess of 10 m~ pyruvate required to give saturating conditions. The inhibition for acetaldehyde was approximately 20 m ~while , substrate inhiconstant (Ki) bition was evident at benzaldehyde concentrations in excess of 180 mM ( 19.1 gm Chow et al. (1995) undertook studies to determine the kinetics of deactivation of PDC by benzaldehyde. They determined that the deactivation followed first-order kinetics for benzaldehyde; however, the response was not linearly related to time for benzaldehyde concentrations between 100 and 300 mM. In much of the work undertaken on production of L-PAC, PDC has been found not to be the factor limiting L-PAC production (Vojtisek and Netrval, 1982; Shin and Rogers, 1996a; Tripathi et al., 1997) since some PDC activity remained at the end of the fermentations. Rather, both Tripathi and co-workers ( I 997) and Vojtisek and Netrval(1982) found that low pyruvate concentration in the medium limited yields. Glycolytic enzymes were implicated as potentially rate limiting by both Tripathi and co-workers, and Nikolova and Ward (1991).


9

Based on the information above, the conditions used by Oliver et al. (1997) (see Section 4) appear to be highly conducive to the production of L-PAC. Pyruvate is present in significant quantities and, according to Hohmann ( 1 997), the capacity of pyruvate dehydrogenase is limited compared with that of PDC, thereby restricting metabolism of pyruvate via the only alternative route. Further, the biomass is supplemented with carbohydrate in combination with reduced aeration. The work of Sims and Barnett (199 1) and Sims et al. (199 1) indicates that the combination of these two conditions is conducive to the induction of PDC.

2.3. Alcohol Dehydrogenase and Reaction By-products

The complete conversion of added benzaldehyde to L-PACis not achieved in whole cell systems because of the formation of by-products, namely benzyl alcohol and benzoic acid (Smith and Hendlin, 1953; Ose and Hironaka, 1957; Agarwal et al., 1987; Long and Ward, 1989b; Mahmoud et al., 1990a,b,c; Tripathi et al., 1991). Minor by-products include acetoin, butane-2,3-dione, 1phenylpropan-2,3-dione, 1-phenyl-1,2-propandioland 2-hydroxypropiophenone (Pohl, 1997). Benzaldehyde volatilizes readily, which may lead to some losses, even in fermenters equipped with exhaust air condensers. The main by-product of the L-PACprocess, however, is benzyl alcohol with reports of up to 50% of added benzaldehyde being converted to benzyl alcohol (Ose and Hironaka, 1957). For each gram of added benzaldehyde converted to benzyl alcohol rather than to L-PAC,the potential L-PACyield is reduced by approximately 1.4 g. The benzyl alcohol yields obtained in the study by Oliver et al. (Oliver, 1996; Oliver et al., 1997) were up to 4 g/l, equivalent to lost L-PACproduction of nearly 6 g/l. It is clear that minimizing the formation of benzyl alcohol production during L-PAC fermentations has the potential to increase the final L-PACyield significantly.This is further demonstrated by the work of Shin and Rogers (1996a,b) and Rogers et al. (1997), who performed a mass balance on the process for production of L-PAC using purified PDC. They were able to account for 98% of benzaldehyde consumption, the remaining 2%being attributed to evaporative losses and experimental error. All of the pyruvate added was accounted for as PAC, acetoin, free acetaldehyde or residual pyruvate. It appeared that further yields of L-PAC may have been achieved had additional benzaldehyde been added, as there was residual PDC activity after 6 h. Benzyl alcohol production from benzaldehyde is facilitated by the enzyme alcohol dehydrogenase, as shown in Equation 4. C,H,CHO NADH+H+ Benzaldehyde

ADH

C,H5CH,0H +NAD+ (4) Benzyl alcohol

10


The production of benzyl alcohol, in addition to a range of other aromatic alcohols, by pure ADH has been demonstrated by Long et al. (1989), although Bowen et al. (1 986) had previously investigated the inhibition of yeast ADH by aldehydes and found that their enzyme preparation displayed no reactivity with benzaldehyde. The reaction is reversible, although equilibrium constants ( K , ) of lo4 at pH 7.0 (Conn et al., 1987) and 7 x 10-9-1.8 x lo-'' (Jones, 198d) have been reported. In view of the reported equilibrium constants, the formation of ethanol, or benzyl alcohol, would be highly favoured under the fermentation conditions used for L-PACproduction if the equilibrium constants were similar for all ADH isoenzymes. The inhibition of both fermentation and growth by ethanol have been demonstrated to be completely independent and non-competitive (Aiba et al., 1968; Brown et al., 1981; Pascual et al., 1988). There are differences in ADH activities between Crabtree-positive and Crabtree-negative yeasts, with the ADH activity of Crabtree-negative yeasts, e.g. C. utilis, being significantly lower than that of Crabtree-positive yeasts, e.g. S. cerevisiae. van Urk et al. (1990) measured activities of 1.61-2.91 U/mg protein for C. utilis and 5.7-7.0 U/mg protein for S. cerevisiae. Accordingly, with respect to reported ADH activities, it would appear that Crabtree-negative yeasts such as C. utilis would be better suited to L-PAC production than Crabtree-positive yeasts. However, the activity of PDC from C. utilis ( 0 . 0 8 4 1 1 Wing protein) was much lower than that of S. cerevisiue (0.58-1.12 U/mg protein). Further, in contrast to S. cerevisiae, the PDC activity of C. urilis did not increase when the culture was pulsed with glucose. The latter finding is in contrast to that of Sims et al. (1991) who found that PDC was synthesized de novo in response to glucose pulsing. Verduyn et al. (1988) compared the substrate specificities of ADH purified from freshly grown Hansenula polymorpha and C. utilis with commercially obtained ADH purified from S. cerevisiae. They found that the ADHs from both H. polymorpha and C. utilis were capable of reducing methylglyoxal and displayed greater activity than the ADH from S. cerevisiae when butanol was the substrate. The activity of the enzyme from C. utilis was actually higher for butanol than for ethanol under optimal pH conditions, unlike the enzyme from Succharornyces. A further dissimilarity to the S. cerevisiae enzyme was the maintenance of the degree of activity with increasing chain length of substrate: ADH from C. utilis was found to be reactive with 2-propanol and 2-butanol. While the ADH from C. utilis appears to have several distinct advantages over the enzyme from S. cerevisiae, the affinity of the former for ethanol is considerably lower than that of the latter, although this is counterbalanced by its greater stability. Verduyn and co-workers noted that three isoenzymes existed in S. cerevisiae, two being cytoplasmic and one mitochondrial. However, work undertaken by Nikolova and Ward (1991) using mutants lacking all three of these isoenzymes showed that some ADH activity remained. ADH-IV activity


11

has since been demonstrated in S. cerevisiue and is thought to have potential for the production of benzyl alcohol (Rogers et ul., 1997). Other aldehydes for which whole baker’s yeast has been found to be reactive include a,a,a-trifluorotolualdehyde, nitrobenzaldehyde, anisaldehyde, fluorobenzaldehyde, tolualdehyde and chlorobenzaldehyde. All of these aldehydes were used as substrates and converted to the corresponding alcohols when substituted in either the ortho-, metu- or puru-positions (Nikolova and Ward, 1994b), although increasing substrate hydrophobicity reduced yeast catalytic activity. These substrates were tested in a two-phase medium of hexane containing 2% (v/v) water. While activity was demonstrated for all of these substrates, the conversion of benzaldehyde to benzyl alcohol proceeded at almost the highest rate. Only the rate of conversion of p-nitrobenzaldehyde was higher, while the lowest rate was for a,a,a-trifluoro-o-tolualdehyde. Other studies by Leskovac et ul. (1997) found that aliphatic aldehydes were the most efficiently reduced by yeast ADH (YADH), followed by branchedchain aldehydes and lastly by aromatic aldehydes. Leskovac and co-workers used 4-dimethylamino-truns-cinnamaldehydeand chloroacetaldehyde as substrates; the YADH was of unspecified origin. The conversion of chloroacetaldehyde was essentially irreversible, the first known instance of a near-irreversible reaction involving YADH. The reduction of acetaldehyde to ethanol by ADH is an important metabolic mechanism in yeasts because of its role in the regeneration of NAD+, an electron acceptor in the phosphorylation of glyceraldehyde-3-phosphate, an intermediate in the glycolytic pathway. The regeneration of NAD+ assumes particular importance when in situ accumulation of pyruvate is desired. To date, there has been no success in completely preventing the production of benzyl alcohol in whole cell cultures, despite many reported attempts. There has, however, been some success in reducing the amount of benzaldehyde converted to benzyl alcohol. Methods which have been used include the random induction of mutant strains, the use of isoenzyme mutants, benzaldehyde dosing protocols and the use of additives to variously enhance PDC activity and reduce ADH activity. As briefly mentioned above, work is also taking place on developing purified enzyme systems. Studies which were undertaken specifically with the aim of reducing the benzyl alcohol yield, rather than increasing PDC activity, are discussed in greater detail later in this review.

3. DEVELOPMENT AND OPTIMIZATION OF THE FERMENTATION PROCESS

The development and optimization of a fermentation process is dependent on an array of factors, including the type of organism used, its physiological

12


condition, the physical conditions for growth/production and the medium composition, all of which when correctly combined create conditions conducive to high product yields. Studies into the optimization of L-PAC production by these various processes are reviewed below. 3.1. Selection of a High-yielding Producer Organism

L-PAC yields can be significantly affected by the choice of the production strain. Desirable qualities include the presence of a high level of PDC activity, low ADH activity, and high tolerance levels for benzaldehyde, benzyl alcohol and PAC. High affinity for benzaldehyde by PDC is beneficial but rarely measured. The importance of some of these criteria was demonstrated by Bringer-Meyer and Sahm (1988) when they compared purified enzymes from whole cell cultures of S. carlsbergensis and 2. mobilis. Although the PDC activity of 2. mobilis was five times that of the enzyme from S. carlsbergensis, a lower affinity for benzaldehyde resulted in a L-PACyield in 2. rnobilis, which was four to five times lower. Most early studies compared strains on an holistic basis, measuring their ability to produce PAC, and productivity over a set period, rather than identifying the biochemical basis for their performance. No single species of organism has been accepted universally as the strain most suited to L-PAC production, although Saccharomyces spp. have been identified as the best L-PACproducers in comparative studies by a number of groups (Becvarova and Hanc, 1963; Gupta et al., 1979; Netrval and Vojtisek, 1982; Mahmoud et al., 1990a), and are known to be producers of acetoin (Romano and Suzzi, 1996). Importantly, Netrval and Vojtisek demonstrated that the yeast with the highest initial productivity was not necessarily the most productive over an extended period (24 h), in contrast to Becvarova and Hanc’s suggestion (based on 6-h transformations) that the highest yields were produced by the yeast with the highest decarboxylase activity. Netrval and Vojtisek’s findings have been supported by those of Shin and Rogers (1996a,b) and Rogers et al. (1997) who found that, while initial rates of L-PAC production were higher at higher PDC activities, this did not necessarily result in higher final yields. These findings have also been supported by the work of Tripathi et al. (1997) and Bringer-Meyer and Sahm (1988). Prior to Becvarova and Hanc’s (1963) comparison of six brewing and food yeasts, screening of organisms was limited. Both brewer’s (Smith and Hendlin, 1953, 1954; Netrval and Vojtisek, 1982) and baker’s (Becvarova et al., 1963; Voets et al., 1973; Long et al., 1989; Long and Ward, 1989a; Nikolova and Ward, 1992a) yeasts were, and have continued to be, common choices owing to their ready availability and low cost. Both possess good PDC activity, although brewer’s yeast also has high ADH activity. Although some ADH activity is desirable for the regeneration of NAD+, particularly if pyruvate is


13

being generated in situ, high ADH activity can result in high levels of benzyl alcohol, which potentially reduces the L-PACyield by reducing the amount of available benzaldehyde for conversion to L-PAC. Baker’s yeast may be preferable because of its generally lower ADH activity. Other strains used include locally isolated strains of S. cerevisiae (Agarwal et al., 1987;Tripathi et al., 1988; Agarwal and Basu, 1989) and various yeast strains from culture collections (Becvarova and Hanc, 1963). The L-PAC yields from local isolates were comparable to yields from yeasts obtained from culture collections. One way of seeking further increases in L-PACyield is via the use of genotypically and/or phenotypically modified biomass, although success is not guaranteed. For example, Nikolova and Ward ( 1 99 1) tested eight S. cerevisiae ADH I, II and I11 isoenzyme mutants, and achieved similar benzyl alcohol concentrations for all the strains tested regardless of their isoenzyme configurations. Also, a benzaldehyde-tolerant strain of C. utilis with increased productivity and benzaldehyde consumption rate isolated by Dissara and Rogers (1995) had a lower final L-PACyield than the parent strain because of a lower specific growth rate. The use of chemical (ethylmethane sulphonate, N-methyl-N’-nitro-Nnitrosoguanidine, nitrous acid) and physical (ultraviolet and gamma rays) agents to generate mutant strains has met with some success, with increases in L-PACyield ranging from 10% using S. cerevisiae (Sambamurthy et al., 1984; Ellaiah and Krishna, 1987) to 100%using S. cerevisiae and C.Jlareri (Seely et al., 1989b). The types of changes induced have included acetaldehyde and ephedrine resistance (Seely et al., 1989b), increased productivity, benzaldehyde tolerance and benzaldehyde consumption rate (Sambamurthyeta!., 1984; Ellaiah and Krishna, 1987; Dissara and Rogers, 1995). Benzaldehyde, L-PAC and sodium cyanide resistance has been induced by exposure of Saccharomyces spp. cultures to each of the three chemical agents (Gupta et al., 1979; Ellaiah and Krishna, 1987), although only benzaldehyde-tolerant strains had improved rates of conversion of benzaldehyde to L-PAC. Another approach has been the use of site-directed mutagenesis to increase the carboligase activity of PDC. When applied to PDC extracted from Z. mobilis, the resultant mutant enzyme gave a three-to four-fold increase in LPAC production when coupled with an enzymatic system to remove acetaldehyde [Bruhn et al. 1995, 1996 (as cited by Pohl, 1997)l. 3.2. Physicochemical Conditions and their Effect on L-PAC Production

The physical environment of a fermentation affects cell metabolism and structure, and product stability. Product stability is increasingly likely to be directly

14


affected if the product is excreted into the fermentation medium, as is L-PAC. The three variables over which process control is most precise are dissolved oxygen, pH and temperature. 3.2.1. Effect of Dissolved Oxygen Concentration on Metabolism Both Voets et al. (1973) and Ellaiah and Krishna (1988) demonstrated the benefit of limited aeration on L-PAC production by S. cerevisiae. Aeration of medium at a rate of 0.3 Vl.min enhanced L-PAC production compared with non-aerated cultures, although at 0.5 Vl.min, L-PAC production was reduced (Voets et al., 1973). Ellaiah and Krishna (1988) demonstrated that L-PACyield varied with the mass transfer coefficient (kLa), the maximum L-PAC yield being obtained when the kLa was 2.35 mm/l.h. Culik et al. (1984) deliberately reduce3 aeration to induce fermentative activity in S. coreanus and hence to stimulate L-PACproduction. They found that increasing the rate of aeration when L-PAC production began to slow increased the final L-PACyield, although the specific reasons for enhanced LPAC production were not detailed. Rogers and co-workers (1997) also employed a strategy of reduced aeration to stimulate the activity of PDC in their fed-batch and three-stage continuous culture systems with resulting LPAC yields of 22 g/l (in defined medium) and 10.6 g/l, respectively. Mahmoud et al. (1990a,b,c)induced anaerobiosis in S. cerevisiae by sparging the culture with nitrogen gas, which may have improved the L-PAC yield by ensuring the regeneration of NAD+, essential for the production of pyruvate from sucrose. However, the benzyl alcohol yields may have also been increased in the nitrogen-sparged cultures, compared with the aerated cultures, owing to the absence of molecular oxygen, which is known to exert a repressive effect on ADH activity (Nagodawithana et al., 1974; Jones et al., 1981; Ward and Young, 1990). In their study of the role of PDC on the Kluyver effect on C. utilis, Sims et al. (1991) found that PDC was synthesized de novo under anaerobic conditions in the presence of D-glucose. Under aerobic conditions the PDC was partially deactivated. Anaerobic conditions in these experiments were achieved by sparging with nitrogen gas; however, Sims and co-workers also demonstrated that depletion of oxygen by actively growing biomass was also sufficient to enable synthesis of PDC. Concomitant with the increase in PDC activity under anaerobic conditions was an increase in ADH activity, which in the case of the process used for L-PAC production could lead to higher concentrations of benzyl alcohol, i.e. reduced L-PAC yields. It is likely that reduction in aeration (Culik et al., 1984). and even sparging of cultures with nitrogen as practised by Mahmoud et al. (1990a,b,c), was intended to increase PDC activity. It is also possible that the reductions in L-PAC yields observed by Voets et al. (1973) and


15

Ellaiah and Krishna (1988) under conditions of increased aeration were due to deactivation of PDC as a result of the cultures becoming aerobic. The findings of van Dijken and Scheffers (1986) suggest that increased glycolytic flux results from glucose pulsing under anaerobic conditions for C. utilis, thus increasing the levels of pyruvate. This is important in that the L-PAC yield is potentially enhanced owing to increased pyruvate availability. In contrast to the findings of Rogers and co-workers, Tripathi et al. (1997) found that L-PACyields were higher if the biomass was grown anaerobically, with the biotransformation taking place under aerobic conditions. The greater yields were attributed to the induction of PDC under the anaerobic growth conditions accompanied by increased pyruvate concentrations. However, with aerobic growth, it is likely that pyruvate would be diverted from the production of L-PAC by consumption through aerobic metabolic pathways. Variation in the levels of enzymes owing to differences in aeration is not peculiar to PDC. In a paper published in 1972, Harrison noted that growth of microorganisms or cultured cells under either aerobic or anaerobic conditions affected the levels of enzymes present. Anaerobic conditions were likely to enhance the levels of enzymes related to fermentation pathways, and low dissolved oxygen concentrations resulted in increases in glycolytic enzyme concentrations. Aerobic conditions enhanced the levels of Krebs cycle enzymes. Oliver et al. (1997) (see Section 4)used a system where, after the growth of biomass, the level of aeration was reduced in conjunction with pulsing of molasses. Based on the discussion in preceding paragraphs, an increase in PDC activity would have occurred. Pyruvate production continued for some time into the bioconversion phase of the fermentation process of Oliver and coworkers even under conditions of increasing benzaldehyde concentration. 3.2.2. Eflect of pH on Cellular Metabolism

When using S. cerevisiae for L-PAC fermentations, a pH range of 4-6 has generally been employed (Smith and Hendlin, 1953; Voets et al., 1973; Gupta et al., 1979; Long and Ward, 1989a,b), while Rogers and co-workers (1997) employed a pH of 6.2 for the bioconversion stage of their three-stage process using C. utilis. Rogers et al. (1997) also found that L-PACproduction was more sensitive to pH than was benzyl alcohol production. 3.2.3. Effect of Temperature on Metabolism

According to Rogers (1990), the rate of L-PAC production by C. utilis was approximately 1.5 times higher at 30 "C than at 20 "C; however, benzyl alcohol

16


yield was also increased at the higher temperature. The use of a lower temperature improved L-PAC yield with respect to benzyl alcohol yield and increased L-PAC stability, although the growth rate declined at temperatures below 25 "C. The use of a lower temperature may have also helped to reduce alcohol toxicity. Temperatures as high as 35 "C were found to have little effect on L-PACproduction by S. cerevisiae (Ellaiah and Krishna, 1988), although temperatures ranging from 25 "C (Vojtisek and Netrval, 1982; Nikolova and Ward, 1991) to 28-30 "C are used more routinely (Smith and Hendlin, 1953, 1954; Voets et al., 1973; Netrval and Vojtisek, 1982; Nikolova and Ward, 1991, 1994a). The manipulation of fermentation temperatures to improve yields has generally not been a key component of laboratory studies. 3.3. Physiological Condition of Cells for Optimum L-PAC Production

The biotransformation of a chemical is largely dependent on the physiological condition of the biomass, which, in turn, is influenced by the medium composition and physicochemical conditions used throughout the process. This applies to both the growth phase and the bioconversion phase of the L-PACfermentation. While a popular choice for laboratory studies, the use of purchased commercial baker's (Long and Ward, 1989a,b;Voets e f al., 1973; Nikolova and Ward, 1992a,c, 1994a) and brewer's yeast (Smith and Hendlin, 1953;Vojtisek and Netrval, 1982) as added biocatalyst reduces the ability for control and/or optimization of biomass preparation. The absence of control over biomass preparation may be of particular concern where purchased biocatalysts are used after very short acclimatization periods. Although baker's yeast is considered to have good PDC activity, as described above, the reduction in aeration results in enhanced production of PDC, provided that the acclimatization period is sufficient to allow for further induction of PDC production. Insufficient time for induction of PDC may result in reduced L-PACyields. No studies were undertaken to determine enzyme activities or the effect of the acclimatization period on those activities. The effect of cell age has been considered for S. cerevisiae (Agarwal et al., 1987), with the greatest L-PAC production observed with biomass that was 15-24 h old. The L-PACyield was considered to be dependent on a combination of enzyme activity and benzaldehyde tolerance, with reduced yields from older or younger biomass owing to lower activity or tolerance. Freshly cultivated inoculum ranged from 16 h (Ellaiah and Krishna, 1987) to 24 h (Netrval and Vojtisek, 1982; Mahmoud et al., 1990a,b,c; Nikolova and Ward, 1991, 1992b) and even to 28 h old (Becvarova and Hanc, 1963). The age of commercially obtained brewer's and baker's yeast was never stated, and as little as


17

1 h was allowed for adaptation to transformation conditions prior to commencement of the benzaldehyde feed (Smith and Hendlin, 1954; Voets et al., 1973; Long and Ward, 1989b).Yeast freshness was considered to be important by Becvarova et al. (1963) and Voets et al. (1973). The cessation of L-PAC production has been directly linked with reduced viability by Long and Ward (1989b); however, the loss of viability coincided with exposure to high benzaldehyde concentrations.Although PDC activity is resistant to benzaldehyde concentrations as high as 7 g/l, sucrose metabolism, cell growth and viability, which may in turn reduce the L-PAC production capacity, are affected by concentrations as low as 2-3 g/l. Indeed, recent reports (Rogers et al., 1997) placed the minimum concentration required for inhibition of growth of C. utilis at 1 gA, with a growth inhibition constant of just 0.30 g/l. They found that the specific rate of L-PAC production was strongly affected at benzaldehyde concentrations of 3 g/l. Biomass rendered non-viable by other means can still catalyse the transformation of benzaldehyde to L-PAC provided that pyruvate is supplied, as demonstrated with non-viable immobilized S. cerevisiae biomass (Seely et al., 1989a). Other authors have noted differences in metabolic activity whilst using acetone powders of yeast. Increased residual benzaldehyde and acetyl benzoyl concentrations were detected, but benzyl alcohol was not detected when acetone-dried powders were used by Voets et al. (1973). transcinnamaldehyde, a compound not normally detected in fermentations using freshly grown biomass, was detected in S. cerevisiae acetone powder fermentations (Voets et al., 1973). In contrast, Ose and Hironaka (1957) found that benzyl alcohol was produced by acetone dried yeast powders. When both benzaldehyde and pyruvate are present in sufficient concentration, PDC activity is reportedly the rate-limiting factor in L-PAC production by S. carlsbergensis (Vojtisek and Netrval, 1982), and low PDC concentrations were identified as a cause of low L-PACproduction capacity by S. cerevisiae (Mahmoud et af.,1990a). Continuous culture, particularly at high dilution rates, has been shown to be an effective method for increasing S. cerevisiae PDC activity, with four- and seven-fold increases in specific productivity for 23- and five-fold increases in flow rate during the growth and transformation phases of the fermentation, respectively (Tripathi et al., 1988, 1991). Culik et aE. (1984) adapted biomass for L-PAC production by preventing diauxic growth during the growth phase. This was achieved by ensuring sufficient oxygen supply. Fermentative metabolism was then induced in the Saccharomyces sp. by reducing aeration and regulating the feeding of sucrose. Using such methods, L-PAC yields of 10 g/l or more could be achieved. Alternatively, Seely et al. (1989b) allowed the cultures of S. cerevisiae to become oxygen-limited or anaerobic for up to 16 h prior to commencement of biotransformation. By following these guidelines, cells from any stage of

18

ALISON

L. OLjVER, BRUCE N. ANDERSON AND FELICITY A. RODDICK

growth up to late log phase could be successfully used for L-PACproduction with little variation in yield. 3.4. Nutrient Effects in L-PACProduction

The role of nutrition in the production of L-PAChas received limited attention to date. Manipulation of fermentation media, in particular, complex industrial media, to enhance the production of L-PAC requires an understanding of the metabolic roles of macronutrients and micronutrients.The work undertaken on yeast nutrient requirements in general and their effects on metabolism is extensive, and it is neither practical nor possible to review all aspects of the work here. Some aspects of yeast nutrition relevant to L-PAC production will therefore be discussed briefly. Vojtisek and Netrval(l982) investigated the use of corn steep liquor (CSL) and yeast hydrolysate, and found them both to be beneficial for the production of L-PAC by S. carlsbergensis when added to an otherwise simple medium. Noronha and Moreira (1993) developed a L-PAC production medium (details not published) with a glucose concentration of approximately 100 g/l (S. Noronha, 1996, personal communication) to stimulate the fermentative activity of S. cerevisiae. Others, all employing S. cerevisiae, have used media ranging from fully defined media (Agarwal ef al., 1987; Tripathi et al., 1991) to very simple media containing nothing more than molasses and urea and/or phosphate (Ellaiah and Krishna, 1987, 1988). Glucose, sucrose and pyruvate are frequently used substrates for the L-PAC production process with the sucrose supplied either as pure sucrose or as molasses. Glucose or sucrose is generally used when biomass is grown in situ prior to the initiation of the bioconversion phase (Netrval and Vojtisek, 1982; Ellaiah and Krishna, 1988; Nikolova and Ward, 1991). Sucrose has also been used as a substrate for the generation of pyruvate during a short acclimatization period prior to benzaldehyde dosing when commercially obtained biomass was used (Long and Ward, 1989a,b;Nikolova and Ward, 1991).When the biomass is used essentially as a catalyst, pyruvate is generally used and has been found to produce higher and more reproducible L-PAC yields because the regeneration of NADH is limited, and hence the production of benzyl alcohol is also limited (Nikolova and Ward, 1991). It is not uncommon for industrial fermentation media to contain one or more complex materials. Generally inexpensive and readily available complex carbon and nitrogen sources may also be used as a source of vitamins, trace elements and growth factors (Zabriskie et al., 1980), in some cases fulfilling all of the vitamin requirements of the organism (Stanbury and Whitaker, 1984). However, various compounds may be present in inhibitory concentrations (Oura, 1983; Jones and Greenfield, 1984; Cejka, 1985), may be


19

non-metabolizable (Reed and Peppler, 1973; Meyrath and Bayer, 1979), or may be complex ionic nutrients (Jones and Greenfield, 1984; Berry and Brown, 1987), functions which may be considered as either beneficial or detrimental. Optimization of individual nutrients is difficult because a change in the concentration of the complex material alters the concentrations of all individual components. Increased yields may be obtained by using defined medium, although the use of a defined medium on an industrial scale may limit the commercial viability of the process (Zabriskie et ul., 1980, Stanbury and Whitaker, 1984). Among the benefits of using defined medium are constant composition, and the exclusion of toxic or inhibitory compounds; however, the cost of the medium may be disproportionate to the value of the product and the overall yields may be no better. Defined media are frequently used on a laboratory scale to determine nutrient requirements. Dissara and Rogers (1995) achieved L-PACyields of up to 22 g/l using a defined medium, on a 5-1 scale, compared with 13 g/l in undefined medium. On the other hand, Vojtisek and Netrval (1982) achieved higher L-PAC yields (4.7-5.6 g/l) when yeast hydrolysate and/or CSL was included in the medium compared with yeast hydrolysate alone (2.5 gA). However, the basal medium used by Vojtisek and Netrval was much simpler than the defined medium used by Dissara and Rogers. The work undertaken on production of L-PACto date has been quite limited in the extent to which the effects of nutrients on metabolism have been closely examined. An investigation undertaken by Derrick and Large (1993) on the growth of C. utilis in continuous culture using valine and ammonium as nitrogen sources found that, under nitrogen limitation, increases in pyruvate yield of up to 100-foldwere achieved. Thus growth under nitrogen limitation may in turn lead to enhanced L-PACyields. One of the drawbacks from the use of complex industrial fermentation media is the presence of an array of different forms of the same macronutrients leading to diauxic growth patterns. Diauxic growth is more frequently seen in batch cultures; in continuous culture the biomass can adapt and eventually use all of the nutrients simultaneously. Culik et al. (1984) experienced scale-up problems owing to diauxic growth; ammonium and sucrose were used preferentially, followed by the utilization of carbon and organic nitrogen from corn extract, and finally ethanol produced initially from sucrose was used. Diauxic growth is not exhibited by all organisms; C. utilis, for example, is capable of the simultaneous use of metabolically produced ethanol in conjunction with added glucose (Ghoul et al., 1991), whereas the addition of glucose to S. cerevisiue cells will cause an immediate return to net ethanol generation rather than consumption (van Urk et al., 1988, 1990). Oliver (1996) et al. and Oliver (1 997) undertook a study aimed at simplifying a complex medium used industrially for the production of L-PAC by C. utilis. Medium components included molasses, CSL and whey in addition to

20


pure sources of glucose, phosphate, nitrogen, magnesium and thiamine. Although C. utilis is known to be prototrophic for vitamins, the thiamine was provided to ensure that the cofactor demand exerted by PDC was met. They found that up to 60% of the original molasses content of the medium could be substituted by the equivalent amount of sucrose in the form of raw sugar, with no loss in either pyruvate or L-PACyield. Furthermore, both CSL and whey could be omitted from the medium leading to slight increases in pyruvate yield but no change in L-PACyield. These results are consistent with the findings of Derrick and Large (1993) discussed above. Other benefits arising from these modifications to the medium included reduced inorganic content and chemical oxygen demand of the spent medium, the latter largely due to lower residual reducing sugar levels. The protocol used by Oliver (1996) and Oliver et al. ( 1997) included pulse feeding of carbohydrates (either molasses or sucrose). Rogers et al. (1997) also employed pulsed carbohydrate feeding in some of their studies. The benefits of using pulse feeding of carbohydrate in relation to the induction of PDC activity are outlined in Section 2.2. 3.5. Production of L-PAC by Batch, Fed-batch or Continuous Fermentation

The majority of studies on the production of L-PAChave been undertaken on a batch basis for comparison of yeast strains and to optimize production; the development of fed-batch or continuous production of L-PAC is relatively recent. The advantages of fed-batch or continuous production lie in the use of the same culture of organisms for production over an extended period, so reducing downtime, reducing of substrate and product toxicity by continuous dilution of medium, leading to higher final yields. Agarwal and Basu (1989) compared batch and fed-batch fermentationsusing free cells of S. cerevisiue and found that fed-batch fermentationscould be run for up to 14 times longer than batch fermentations. ‘Semi-continuous’may be a more appropriate term than ‘fed-batch’because aliquots of medium were periodically replaced rather than additional medium being added to the total volume. The fed-batch protocol was most beneficial up to the third cycle when the total LPAC yield was up to 120% higher than the total yield for an equivalent number of batch fermentations,i.e. based on an equivalent medium volume. Thereafter,the productivity decreased but was relatively constant, with approximately 50% more L-PACat any given time than for an equivalent number of batch fermentations. Mahmoud et al. (1990b) compared batch and ‘semi-continuous’fermentation protocols using S. cerevisiue immobilized in alginate. Unlike Agarwal and Basu (1989) who also used a strain of S. cerevisiue, Mahmoud et ul. (1990b) replaced the entire volume of medium at the end of each 24-h cycle rather than


21

a proportion thereof but similarly recorded a decrease (approximately 50%)in L-PACproduction after the first two cycles, which could not be restored by the regeneration or re-immobilization of the biomass. L-PACproduction remained relatively constant for the remaining cycles. Over seven cycles a fivefold increase in total L-PACyield was measured relative to a single batch fermentation, although it was unclear whether this referred to batch fermentations conducted in shake flasks (Mahmoud et al., 1990a) or a single cycle in a column reactor (Mahmoud et ul., 1990b). When transformations were run continuously, flow rate was determined to influence productivity (Tripathi et al., 1991) with a sevenfold increase in productivity, through a fivefold increase in flow rate for S. cerevisiue. The continuous system developed by Rogers and co-workers is fundamentally different from the systems developed by Agarwal and Basu (1989) and Mahmoud et al. (1990b).While the systems established by the last two groups embodied the concept of periodical replacement of part or all of the medium, the fermentation conditions were kept constant. In the case of Rogers et al. (1997), conditions were modified to exploit particular aspects of yeast physiology and metabolism. They found that a single-stage continuous system was unsuitable for use because of the toxicity of the benzaldehyde. Continuous culture may also affect enzyme activities, since the PDC and ADH activities of C. utilis were lower than those of S. cerevisiue when grown under the same continuous culture conditions (Derrick and Large, 1993). A well-controlled fed-batch system was considered by Rogers et al. (1997) to be preferable to a continuous system. Two- and three-stage systems were developed of which the three-stage system was the best. The separate stages were designed to: (1) optimize biomass yield; (2) induce semi-fermentativeactivity and enhance PDC activity; and (3) provide a biotransformation phase where benzaldehyde was added and converted to L-PAC.Respiratory quotient (RQ) was the major control variable for each stage. Using a continuous process, LPAC yields of up to 10.6 g/l were achieved, while yields of up to 22 g/l have been achieved for a three-stage fed-batch system. Significant reductions in PDC activity occurred. Similar to Sims et al. (1991), Rogers and co-workers reported an approximate fivefold increase in PDC activity, induced by controlling the RQ at 4-5 to induce partially fermentative conditions. Shin and Rogers (1995b) found that an RQ of 5-7 was most favourable for L-PACproduction with minimum benzyl alcohol formation. While pyruvate yields were enhanced at higher RQ levels, higher benzyl alcohol yields resulted.

3.6. The Use of Additives to Modify Metabolic Activity As previously mentioned, the complete conversion of benzaldehyde to L-PAC has not been achieved with any yeast strains as some benzyl alcohol has always

22


been produced. Even the use of ADH isoenzyme mutants (Nikolova and Ward, 1991) failed to prevent benzyl alcohol production. In the absence of an ADHfree yeast, reduced benzyl alcohol production and increased L-PAC yields have been sought by the use of additives, e.g. inhibitors or alternative hydrogen acceptors, which are not required for normal metabolic activity. The use of additives to reduce benzyl alcohol yield by dried brewer’s yeast was originally investigated by Smith and Hendlin (1954) who proposed three different methods: the use of alternative H+ acceptors, the addition of sulphydryl inhibitors (to inhibit ADH), and the use of nicotinic acid analogues to compete with NADH for enzyme active sites. Colloidal sulphur was the only H+ acceptor which successfully reduced the benzyl alcohol yield; however, owing to its toxicity, the L-PAC yield was also reduced. The use of sulphydryl inhibitors was similarly unsuccessful owing to the inhibition of other sulphide-containing glycolytic enzymes. Of the nicotinic acid analogues, only the amides were successful in reducing benzyl alcohol yields. Using concentrations of analogues up to 50 mM, reductions in benzyl alcohol of up to 20% were achieved with an equivalent concomitant increase in the yield of L-PAC. Ose and Hironaka (1957), also using dried brewer’s yeast, confirmed the findings of Smith and Hendlin and tested an additional hydrogen acceptor, acetaldehyde. They found that 70% conversion of benzaldehyde to L-PAC was achieved when a mixture of acetaldehyde and benzaldehyde was added to the medium compared with 40% conversion without added acetaldehyde. The increase in L-PAC yield matched the decrease in benzyl alcohol. By using radiolabelled acetaldehyde, Ose and Hironaka were able to exclude the use of acetaldehyde as a substrate in the production of L-PAC and it was believed to be an alternative hydrogen acceptor to benzaldehyde, thus preventing the production of benzyl alcohol. Acetaldehyde dosing has since been used successfully by Becvarova et al. (1963) and Becvarova and Hanc (1963), who used a mixture of equal parts of benzaldehyde and a 50% aqueous solution of acetaldehyde, or equal parts of benzaldehyde and acetaldehyde, respectively, to achieve increased L-PACyields compared with controls where no acetaldehyde was added. Netrval and Vojtisek (1982), and Vojtisek and Netrval(1982), routinely added a mixture of acetaldehyde and benzaldehyde to fermentations, although no comparison was given for a fermentation without added acetaldehyde. Increases of up to 30% in L-PACproduction by C. utilis were obtained by Oliver (1996) when acetaldehyde was supplied at two different concentrations and under varying dosing regimes. Acetaldehyde was supplied at levels equimolar and twice equimolar to the anticipated maximum benzyl alcohol yield, and, in contrast to the findings of Ose and Hironaka (1 957), led to a marginal (less than 10%) increase in benzyl alcohol production,. While the use of acetaldehyde may result in reduced benzyl alcohol yields, it is important to balance the benefits with the known disadvantages.


23

Acetaldehyde is known to be more toxic than ethanol on a weight basis (Stanley, 1993). Inhibition of growth by concentrations as low as 0.3 g/l have been observed (Stanley et al., 1993), although the acetaldehyde concentration calculated by Stanley to inhibit growth of S. cerevisiae by 50% was 0.5 g/l. This concentration is similar to that reported by Carlsen et al. (1991) as inhibitory to the respiration of S. cerevisiae (0.55 gA) grown on glucose. Some individual enzymes are even more sensitive to the effects of acetaldehyde: PDC has a Ki of 4-7 m~ (0.2 - 0.3 g/l) while ADH has a Ki of 120-160 p~ (5-7 mg/l) (Jones, 1989). Rogers et al. (1997) determined an inhibition constant for acetaldehyde for C. utilis-derived PDC of 20 m~ (equivalent to 0.882 gA). These quantities are much lower than the amounts added to the fermentation medium by Oliver (1996) (up to 1.5 gA), suggesting that some inhibition of PDC and hence reduced L-PAC yields may have occurred. The physiological role of acetaldehyde is believed to be as a competitor with benzaldehyde for active sites on ADH. The miscibility of acetaldehyde with aqueous solutions is greater than for benzaldehyde and so it should be more readily available to ADH than benzaldehyde. Dosing with acetaldehyde allows the reaction to proceed in the direction favoured under fermentation conditions, in addition to enabling the regeneration of NAD+ for use in glycolysis. The addition of acetaldehyde to medium has been reported by Stanley (1993) to stimulate glycolytic flux in anaerobic fermentations through increased generation of NAD+. Ellaiah and Krishna (1987) tested the additives a-naphthoxy acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), ethylenediaminetetraacetic acid (EDTA) and niacinamide on a strain of S. cerevisiae. None were successful in enhancing L-PAC yield; reductions of up' to 25% were observed. On addition of 20 p g / d niacinamide, there was a negligible decrease in L-PAC yield ( 6 5 % ) in contrast to the findings of Smith and Hendlin (1954). Other respiratory inhibitors which have been tested for their ability to improve L-PAC yield by C. utilis include sodium azide, sodium cyanide and salicyl hydroxamic acid (Rogers, 1990). Of these, only the addition of sodium azide resulted in an increase (10%)in L-PAC yield and was accompanied by a similar decrease in benzyl alcohol yield. 3.7. Reduction of the Toxic Effects of Substrate, Product and By-product

Production of L-PAC is self-limitingowing to the toxic nature of benzaldehyde, benzyl alcohol and L-PAC. The potential effects of benzaldehyde on biomass and fermentation outcome include retardation or inhibition of growth, reduction of viability and alteration of cell permeability to substrate and product (Long and Ward, 1989b). Liew et al. (1995), who studied the use of a

24


continuous membrane bioreactor, attributed reduced flux across the membrane to lysis of biomass on addition of benzaldehyde. Benzyl alcohol is substantially more toxic than ethanol, with benzyl alcohol concentrations as low as 5 g/l causing up to 80% inhibition of both L-PAC and benzyl alcohol production rates for C. utilis (Rogers et al., 1997) in addition to inhibition of growth. Ethanol concentrations of up to 21 g/l caused only minor inhibition of initial benzyl alcohol production, and the Kifor growth of C. utilis was 39 g/l ethanol (Rogers et al., 1997). The same group has reported a Ki for growth of 4.1 gA L-PAC,which is therefore also much more toxic than ethanol. In terms of the sensitivity of individual enzymes, purified yeast ADH is more sensitive to benzaldehyde (with inactivation at concentrations as low as 0.2 g/l) and PAC than purified yeast PDC which is most sensitive to benzyl alcohol (Long and Ward, 1989b). Owing to the highly toxic nature of benzaldehyde, the ability of yeast to adapt is limited. Pelleting of free cells in fermentations when exposed to benzaldehyde was noted by Mahmoud et d.(1990a) and was thought to be a mechanism for reduction of the toxic effect of benzaldehyde. Because of the limited tolerance of yeast to benzaldehyde, benzyl alcohol and L-PAC,various physical methods have been developed to reduce the exposure of the yeast. The techniques used include benzaldehyde dosing regimes, immobilizationof biomass, biphasic fermentations and the use of additives. The role of nutrientshffering agents in reducing the toxic effects of benzaldehyde also merits consideration, although the number of studies in the area is extremely limited. The addition of 20% yeast extract to enzyme suspensions was found by Long and Ward (1989b) to be helpful in slowing the inactivation of ADH with 61% residual activity after 6 h in the presence of 7 g/l benzaldehyde. 3.7.1. Substrate Dosing

One of the simplest and least costly methods of reducing the biomass exposure to inhibitory concentrations of benzaldehyde is to alter the benzaldehyde dosing regime and thus limit the maximum benzaldehyde concentration in the medium at any time. Much of the early experimental work undertaken on the production of L-PACwas conducted on a small scale in shake flasks, leaving little scope for variation in the benzaldehyde dosing regime. Long and Ward (1989b) compared the L-PAC yields obtained from S. cerevisiae using shake flasks, where a total of 12 gA of benzaldehyde was added to each flask, being added as either two aliquots each of 6 g/l.h or six aliquots each of 2 g/l.h. Markedly different behaviour was demonstrated by the yeast for the two conditions. When six small aliquots of benzaldehyde were added to the broth, the yeast viability was maintained at close to the initial level for the


25

first 4 h of the biotransformation,while the cell viability in the culture dosed with 6 g/l.h benzaldehyde dropped to approximately 30% of the initial value within the same period. Sucrose consumption continued in the medium with the small benzaldehyde doses and the residual benzaldehyde concentration was lower than in the medium receiving the two large doses. Higher L-PACtitres were also recorded in the medium with the six benzaldehyde doses - approximately 4 4 . 5 g/l compared with 3.3 g/1 for the medium with two doses although the maximum L-PACconcentration occurred earlier in the fermentation for the condition with two doses. The benefits of protecting biomass from large initial concentrations of benzaldehyde were clearly demonstrated. During their development of a semi-continuous process for L-PACproduction using S. cerevisiae, Mahmoud et al. (1990b) compared intermittent and continuous benzaldehyde dosing protocols. They found that, by adding benzaldehyde continuously over 6 h, up to double the amount (12 g/l) could be added compared with the amount previously added intermittently (6 g/l) without inhibition of L-PACproduction, resulting in 2.5 times the L-PAC yield (10 g/l rather than 4 g/l). The advantages offered by continuous benzaldehyde dosing over intermittent feeding were also demonstrated when a wild-type strain and a strain of S. cerevisiae adapted to benzaldehyde were exposed to intermittent and continuous dosing of benzaldehyde to the same final benzaldehyde concentration (8 g/l). For the adapted cells, 70% of the added benzaldehyde was converted to L-PAC under continuous dosing, while only 26% was converted in the intermittently dosed medium. For the wild-type cells, the conversion efficiencies were 57% and 41%, respectively. The use of a continuous feeding protocol appeared to reduce the inhibitory effect of benzaldehyde and led to more efficient conversion of benzaldehyde to L-PAC. Studies by Agarwal et al. (1987) found that, by maintaining medium benzaldehyde concentrations greater than 4 m, PDC activity remained higher than ADH activity in S. cerevisiae. The reduced rate of benzyl alcohol production at higher concentrations was likely to be the result of inactivation of ADH by benzaldehyde. Long and Ward (1989b) found that residual activity of purified ADH exposed to 0.2 g/l benzaldehyde had dropped to 82% after 6 h compared with a residual activity of 90.6%for ADH incubated in the absence of benzaldehyde, and had dropped to 33% for ADH incubated in the presence of 3 g/l benzaldehyde. In contrast, the PDC activity remained constant at 89% and 87% in the presence of 0.2 g/l and 7 g/l benzaldehyde, respectively. Thus, benzaldehyde concentration and dosing regime may be used to help control the formation of by-products. While the potential use of high benzaldehyde concentrations to lower benzyl alcohol yields was recognized by Long and Ward (1989b), they also noted that the inhibition or inactivation of ADH by benzaldehyde could restrict the regeneration of NAD+.This may be beneficial in whole cell systems where the biomass is used as a catalyst and exogenous pyruvate is added to the

26


medium. However, when endogenous production of pyruvate is required, as in the system used by Oliver et al. (1997), the addition of benzaldehyde resulting in inhibition of ADH may also result in reduced glycolytic flux and hence reduced L-PACyields. Work undertaken by Rogers et al. (1997) expanded on that of Agarwal et al. (1987) by demonstrating that higher yields of by-products were formed at benzaldehyde concentrations of less than 150 m and greater than 200 m, and when the molar ratio of benzaldehyde to pyruvate was between 0.5 and 1.O. Shin and Rogers (1995b) found that benzyl alcohol was preferentially produced by both free and immobilized biomass at concentrations of less than 30 m benzaldehyde, while L-PAC was the main product when benzaldehyde concentrations were in excess of 40 m. A two-phase protocol was utilized by Oliver et al. (1997) comprising batch generation of the C. utilis biomass followed by fed-batch L-PACproduction. In the production phase, conversion of the accumulated pyruvate to L-PAC was maximized by the addition of a large initial aliquot of benzaldehyde, this was accompanied by the addition of nutrients and carbohydrate to promote further pyruvate production prior to the commencement of continuous benzaldehyde dosing and a second addition of carbohydrate. 3.7.2. Immobilization of Enzymes or Biomass

Benzaldehyde concentrations as low as 1-2 g/l are capable of inhibiting the growth of C. utilis (Dissara and Rogers, 1995) and S. cerevisiue (Long and Ward, 1989b) and, although higher initial concentrations of benzaldehyde (6 g/l) can result in higher L-PACproduction, there is an almost immediate effect on cell viability (Long and Ward, 1989b). Some protection from the toxic effects of substrates and/or products can be conferred by immobilizing the biomass, a process also used successfully for enzymes. The potential benefits of immobilization include modification of metabolism, stabilizing and extending the life of the catalyst, broadening the range of reaction conditions, simplifying product recovery and the separation of biomass from spent broth, and simplifying biomass recycling (Navarro and Durand, 1977; Ward and Young, 1990; Shacar-Nishri and Freeman, 1993). One of the negative aspects of immobilization of biomass, as observed by Rogers et al. (1997), is the more limited control over metabolism manifested as reduced L-PAC yields, although enhanced resistance to benzaldehyde was noted. The reduced L-PAC yields were attributed to lower levels of accumulated pyruvate (by a factor of 2-3 times). A further effect of immobilization is limitation of mass transfer owing to reduced diffusion. Both Shin and Rogers (1996b) and Rogers et al. (1997) reported an increased K,,, for pyruvate with immobilized purified PDC.


27

Immobilization may be by entrapment within a matrix, adsorption to a support or cross-linking to either a support or to adjacent cells. In studies on L-PACproduction, entrapment in alginate has been the most common method of immobilization, although Seely et al. (1989a) investigated the use of polyazetidine for the immobilization of whole cells, Nikolova and Ward (1994a) tested a variety of polymers using lyophilized yeast, and Shin and Rogers (1995a,b) used polyacrylamide and calcium alginate for the immobilization of PDC. Rogers et al. (1995, 1997) compared the performance of free and immobilized biomass and PDC from various biotransformation processes on the basis of productivity and molar conversion yield (Table 1). Both are important criteria for comparing industrial-scaleprocesses, although the use of productivity, rather than specific productivity as a performance indicator limits the ability to compare different strains or conditions directly. The L-PACyields of the immobilized and free cell processes were similar; however, owing to the reduced productivity of the immobilized biomass additional reaction time was required. Increased L-PACyields and productivities were recorded for the purified and immobilized PDC transformations compared with the whole cell methods. Modified enzyme activity was observed by Shin and Rogers (1995a) when they compared the performance of PDC from C. utilis, both free and immobilized in polyacrylamide beads. The immobilized PDC was reported to have displayed higher activity ( K , = 72 m)and greater tolerance to benzaldehyde (Ki = 161 m) than free PDC, although figures were not given for the free PDC. A Ki for L-PACof 240 r m was reported for immobilized PDC. The modifications in activity and tolerance were attributed to the presence of a benzaldehyde concentration gradient in the beads. The optimum pH differed for free and immobilized PDC, while the reaction with immobilized PDC was more sensitive to temperature. Among the disadvantages of immobilization noted were the lower conversion efficiency and longer time required for biotransformation. Immobilized PDC used on a continuous basis generated an average L-PACconcentration of 4.5 g/l and exhibited a half-life of 29 days, its stability apparently affected by both denaturation of the enzyme and its leakage from the matrix. The average L-PAC concentration contrasted with the maximum of 17.1 g/l achieved using immobilized PDC in a 16-h batch biotransformation. Tripathi et al. (1997) reported free cells to be more efticient at biotransforming benzaldehyde than immobilized cells; they noted that this contradicted the findings of other groups. However, it appears that the benzaldehyde concentration used in their work was within the range where ADH activity exceeded PDC activity. Nikolova and Ward (1994a) investigated the effect of the physicochemical properties of a selection of polymers on the production of L-PACand benzyl alcohol by baker’s yeast. Freshly lyophilized biomass was used as a catalyst for

Table I Comparison of kinetic evaluations for various methods of L-phenylacetylcarbinol (L-PAC)production. Reproduced with permission from Rogers er al. (1997) Process

L-PAC(gll)

Batch and fed-batch proeesse~ Free cells 12.4 22.4 Free cells (cyclodextrins) 12 Immobilized cells 9.9 10 15 Free PDC 28.6 27.1 Immobilized P d c

continoous pl-omses Immobilized cells Immobilized cells (semicontinuous) Three-stage system (free cells) PDC = pyruvate karboxylase.

Reaction time (h) productivity (g/l.h)

Molar conversion yield (96)

Reference

17 14

57 65

Culik et al. (1984) Wang et al. (1994)

0.73 1.6

3 24 22 8 12

3.3 0.42 0.7 3.6 2.3

59 58 95 93

Mahmoud er ul. (1990~) Seely er al. (1989a) Mahmoud et ul. (199Ob) Shin and Rogers (199%) Shin and Rogers (196a) Shin and Rogers (1996b)

4

-

0.6

45

Shin and Rogers (1995b)

4.5

-

0.4-0.8

57

Mahmoud et al. (199Ob)

10.6

-

0.44

56

Wang (1993)

60

rn W

c

8 z

?


29

the transformation of benzaldehyde to L-PAC. The highest L-PAC yield (0.98 g/l) was produced by the free cell suspension followed by the hydrophilic, polyethylene glycol-containing polymers (ENT-4000 and PU-6), the polypropylene glycol-containing gels (PU-3 and ENTP-2000), and lastly the alginate and silicone gels. The hydrophobic gels, PU-3 and ENTP-2000, produced the highest and lowest L-PAC:benzyl alcohol ratios, respectively (1:0.08 and 1:1.80); the free cell suspension was second only to the PU-3 gel in the ratio of product to by-product obtained. The results of the biotransformation were attributed to the physicochemical properties of the gels including hydrophilicity,hydrophobicity and porosity, although little detail as to the relative properties of the polymers was given. The high porosity of alginate beads enables easy and relatively unrestricted diffusion of substrates and products, an important factor in preventing toxicity (Smidsrod and Skjak-Braek, 1990).Mahmoud et al. (1990a,b) investigated the use of S. cerevisiae ATCC 834 immobilized in calcium alginate for both batch and semi-continuous fermentations. The immobilized cells could withstand benzaldehyde concentrations up to 0.6% w/v, while the growth of free cells was inhibited in 0.4% benzaldehyde - 2 . 5 4 times higher than reported by Gupta et al. (1979), who found that 0.15% benzaldehyde was sufficient to inhibit the growth of S. cerevisiae CBS 1171. Increased benzaldehyde tolerance, attributed to the presence of a benzaldehyde concentration gradient, as for Shin and Rogers (1995b), enabled the production of up to 7.5 times more L-PACthan for free cells. The benzaldehyde uptake rate by immobilized cells remained constant at varying initial benzaldehyde concentrations up to 0.6% w/v benzaldehyde. At higher initial benzaldehyde concentrations, the benzaldehyde uptake rate decreased. In contrast, the benzaldehyde uptake rate decreased beyond an initial benzaldehyde concentrationof only 0.4% with free cells. Variation in the L-PAC yield with variation in the bead cell mass was attributed to the development of concentration gradients within the beads and therefore a lower concentration of benzaldehyde being available to the internal biomass. In semi-continuous fermentations undertaken by Mahmoud et al. (1990b), the same batch of biomass beads was used for up to seven cycles with some bead damage and biomass loss observed after three cycles. The observed bead damage coincided with an approximate 50% drop in L-PACproduction, which was possibly due to the increased exposure of the yeast to the benzaldehyde and transformation products, and hence increased toxic effects. Reactivation of the beads after the third cycle made no difference to the L-PACyield per cycle, with similar yields per cycle (2.5-3 g/l) for the third through to the seventh biotransformationcycle. The total amount of L-PACproduced in seven cycles was five times the amount produced in just one cycle. Bead cell mass is an important parameter, with 5 g of beads per 50 ml of medium determined to be the optimal concentration by Tripathi et al. (1991)

30


for their strain of S. cerevisiae. Higher concentrations of beads resulted in a rapid decline in productivity which was attributed to oxygen limitation. The beads were resuspended in fresh medium that did not contain any pyruvate prior to the biotransformation. Supplementationof the medium with pyruvate may have enabled the use of higher bead concentrations without a loss of productivity. The creation of cross-links between cells using polyazetidine was patented by Seely et al. (1989a) as an alternative method of immobilization. Although the cross-linking of cells to solid substrates including glass beads, sand and ion- exchange resins was described, the cross-linking of cells directly to other cells was preferred, owing to the cost advantages compared with the additional cost of using a physical support. Freshly grown biomass was rendered nonviable, and the cell-wall structure modified to enhance permeability to substrates and products during the preparation process. An added advantage of this method is the ability to store the immobilized biomass for extended periods after preparation. The advantages of immobilizing yeast using polyazetidine on the basis of L-PAC yield could not be determined because the immobilized system was not compared with a free cell system; yields of up to 12 g/l were achieved. While the immobilization matrix appears to have a definite effect, the preparation of the biomass prior to immobilization also has a significant effect on L-PACproduction. Shin and Rogers (1995b) enhanced PDC activity prior to harvesting and immobilization of the biomass by reducing the aeration and stirring rate. They noted that enzyme activity decreased initially after immobilization but was restored to near pre-immobilizationlevels once a glucose feed had been introduced. Liew et al. (1995) used a method dissimilar to the other continuous methods described above, namely a continuous membrane bioreactor where biomass was recovered by membrane filtration. There appeared to be a number of distinct disadvantages with this method; the flux was affected by high biomass concentrations and, as mentioned previously, by lysis of biomass after the addition of benzaldehyde. 3.7.3. Modi$cation of Benzaldehyde Solubility

The effect of benzaldehyde solubility on L-PAC yields has been tested by incorporating co-solvents to increase the concentration of dissolved benzaldehyde in the medium beyond the solubility limit of benzaldehyde in water (0.03 g/lOO ml). Ideally, neither L-PACyield nor L-PACformation should be directly nor adversely affected by the co-solvent used. The effect of increasing benzaldehyde solubility on L-PAC production by S. cerevisiae was tested by Mahmoud et al. (1990a), who dissolved 0.6 ml of benzaldehyde in 6 ml of

THE PROOUCTION OF L-PHENYLACETYLCARBINOL BY YEAST

31

N,N-dimethylformamide (DMF) prior to adding the benzaldehyde to the medium. They found that increasing the solubility of the benzaldehyde in this manner had no significant effect on L-PAC yield, and therefore the benzaldehyde was neither more toxic nor more readily converted to L-PACwhen it was more soluble. The results presented by Mahmoud et al. (1990a), however, did not preclude the possibility of DMF toxicity as a counterbalance to potentially higher L-PACyields in the presence of higher benzaldehyde concentrations. The lack of improvement in yield using DMF as a co-solvent contrasts with the results of Seely et al. (1989a) who, using a strain of S. cerevisiae, described increased conversion rates and final yields when co-solvents were incorporated in the medium to increase the concentration of dissolved benzaldehyde. The co-solvents used included methanol, ethanol, propanol and butanol as well as ethylene glycol, glycerol and polyethylene glycol (PEG) of varying molecular weights. The inclusion of 20% by weight of ethanol as a cosolvent for benzaldehyde in the reaction medium was reported to have increased both the rate of reaction and the final yield of L-PAC (from 0.6 g/l in the absence of ethanol with 25 m~ benzaldehyde, to up to five times in the presence of 25 m~ benzaldehyde and to up to 10 times (5.5 g/l) in the presence of 100 m~ benzaldehyde). L-PAC yields of 10.5 g/l were reported in the presence of glycerol compared with 6 g/l in the absence of the co-solvent. The use of PEG as a co-solvent was considered particularly advantageous because,unlike short-chain alcohols it has no adverse effect on the PDC, . Growth has been found to be more sensitive than fermentation to ethanol inhibition, with concentrations as low as 4 8 % (w/v) being sufficient to reduce the growth rate of a laboratory strain of S. cerevisiae (Brown et al., 1981).An ethanol concentration of 12% (w/v) was required to inhibit growth completely, similar to the ethanol concentration found to inhibit the growth of a commercial S. cerevisiue strain (Brown et al., 1981); 9-10% (w/v) ethanol is commonly reported as being required for complete inhibition of growth (Casey and Ingledew, 1986). The growth rate inhibition constant (Ki) reported for the S. cerevisiae strain used by Brown and co-workers was 2.01% (w/v); the Ki for fermentation was 4.46% (w/v) with a slightly higher fermentation Ki (6.1% (w/v) for the commercial yeast strain. A fermentation Ki of 3.7% was reported by Pascual et al. (1988) for S. cerevisiae. These ethanol concentrations are all significantly lower than the 20% (w/v) ethanol added to the reaction medium by Seely et al. (1989a). However, fermentativeactivity has been reported in the presence of ethanol concentrations as high as 20% (Aiba et al., 1968) and 30% (Casey and Ingledew, 1986), and a lesser degree of toxicity has been reported for ethanol that has been added to yeast cultures compared with endogenously produced ethanol (Nagodawithana and Steinkraus, 1976; Novak et al., 1981; Casey and Ingledew, 1986). However, Pamment and coworkers (Dasari et al., 1990) indicated that the apparent inhibition due to endogenously produced ethanol also included that due to the by-products of the fermentation. On the

32


other hand, studies by Rogers et al. (1997) indicated a potential positive contribution from added ethanol in that up to 2-3 M ethanol produced an initial rate increase of 3040% in PDC activity. When benzaldehyde was supplied to C. utilis either neat or diluted with ethanol (1:2, v/v) at two dosage rates by Oliver et al. (1997), there was little effect on L-PACproduction, with only slight increases recorded at the higher benzaldehyde dose rate in the presence of ethanol. Carbohydrate metabolism was enhanced and benzaldehyde conversion was reduced with the addition of ethanol and with increased benzaldehyde flow rate, although the benzyl alcohol concentration was 50% less under the latter condition. The reduction in benzyl alcohol yield was probably due to more rapid inactivation of the ADH, as suggested by Agarwal et al. (1987) following their studies with S. cerevisiae. The creation of benzaldehyde inclusion compounds using P-cyclodextrin (BCD) was used effectively by Mahmoud et al. (1990~) to both increase the availability of benzaldehyde and to reduce the exposure of biomass to the benzaldehyde. In the presence of BCD, a total cumulative amount of 12-14 g/l benzaldehyde could be added to the medium, a significantly higher amount than previously reported (5-6 g/l), without causing serious damage to cells. Using such high concentrations of benzaldehyde, L-PAC yields of up to 12 g/l were achieved in the presence of 1.5% BCD compared to a yield of approximately 5.5 g/l L-PAC for the control experiment. Mahmoud and co-workers proposed that improved L-PACyields in the presence of BCD were the result of lower concentrations of free benzaldehyde, owing to its incorporation into BCD inclusion complexes, which in turn reduced the toxic effects of benzaldehyde but possibly also reduced its rate of conversion. A reduction in the rate of benzaldehyde conversion in the presence of BCD was discounted by the observation that maximum L-PACtitres occurred earlier in the presence of the BCD than in the control fermentations and benzaldehyde consumption was more rapid in the former. The role of cyclodextrins as a metabolic stimulant was confirmed by increased rates of glucose consumption with increasing BCD concentration and a trend, although inconclusive, towards increased cell growth rates. 3.7.4. livo-phase Fermentation Medium

Biphasic systems are particularly useful when either the substrate or product is poorly water soluble, as is the case with the L-PAC production process. The biomass or enzymes are suspended in a partially hydrated, water-immiscible solvent and the substrate or products partition into the solvent, As for immobilized cell systems, the advantages of biphasic systems include easy separation of substrate and product from the catalyst and, in some instances


33

greater stability of either the enzymes or whole cells (Antonini et al., 1981), although substrate and product inhibition may still occur. Nikolova and Ward have investigated the use of whole cells, cell-free extracts and pure enzymes in biphasic systems for the production of L-PACand benzyl alcohol (Nikolova and Ward, 1992a,b,c). Furthermore, the effect of these solvents on the activity of whole biomass immobilized in silicon-alginate was investigated (Nikolova and Ward, 1993). Hexane, hexadecane, toluene, decane, ethylacetate, butylacetate, toluene and chloroform were evaluated for their ability to support and/or improve the bioconversion potential of yeast in the systems mentioned above. The best yields of both L-PACand benzyl alcohol, with the least cell damage, were achieved with the non-polar solvents hexane and hexadecane using moisture levels of 2% for enzyme systems and 10% for whole cell systems (Nikolova and Ward, 1992a,b,c). When immobilized biomass was used in a non-aqueous system (2% moisture), the solvents for which the highest yields for benzyl alcohol were produced were hexane and decane (Nikolova and Ward, 1993). Below 2% moisture, PDC and ADH activity decreased. The use of whole cell systems obviated the need for cofactors and catalyst extraction, offering instead ease of biomass recycling and improved enzyme stability (Nikolova and Ward, 1992b,c). The activity of yeast ADH and ADH isoenzyme mutants of S.cerevisiae in hexane with 2% moisture was one-half to one-third the activity of the same systems in aqueous medium for benzyl alcohol production; PDC activity was not directly compared (Nikolova and Ward, 1992a,b,c). Yields were generally lower in the biphasic medium, with the exception of the ADH isoenzyme mutant containing ADH I, II and LII which had a similar reaction rate in both media (Nikolova and Ward, 1992a,b,c). Non-polar solvents are perceived to be effective in twophase systems because they are believed to prevent the complete removal of water which is present in the enzyme microenvironment. This rationale is likely to apply also in whole cell systems. 3.8. Other Methods for Influencing L-PACProduction

Other methods used to influence the L-PACfermentation process include the application of low-voltage alternating current to the medium during growth and biotransformation (Ellaiah and Krishna, 1988) and the control of the respiratory quotient (Rogers et al., 1997). The application of an alternating current had no effect on the final L-PACyield and, apart from stating that growth was stimulated, no rationale was put forward for its use by Ellaiah and Krishna. The use of respiratory quotient (the ratio of the rate of CO, production to 0, consumption) to control fermentations is based on the manipulation of fermentative activity. Fully respiratory growth (RQ = 1) resulted in increased benzyl alcohol production and correspondingly poor L-PAC yields. The

34


optimal RQ for the production of L-PACby C. utilis was determined to be approximately 4-5 (Rogers ef al., 1997). Neither of these methods are currently used for the production of L-PAC.

4. AN INDUSTRIAL PROCESS FOR THE PRODUCTION OF

L-PHENYLACENLCARBINOL

Work conducted by the authors on the effect of nutrients on L-PACproduction was based on a process used on a commercial scale. As described previously, a two-phase fed-batch fermentation procedure was used. The first phase comprised the growth phase where the conditions were conducive to the growth of biomass and accumulation of an exogenous store of pyruvate, while in the second, the bioconversion phase, PDC catalysed the conversion of the accumulated pyruvate and added benzaldehyde to form L-PAC.The growth-phase medium used originally comprised (in g/l): molasses 104, CSL 5.32, glucose syrup 10.6, urea 3.76 and KH,PO, 0.52. A typical 'standard' fermentation is described below; a time line illustrates the variations (Fig. 1). The physical variables of the fermentation were set at pH 5.2, 24.8 "C and 1.2 volumes per volume per minute (vvm) air. The medium was saturated with oxygen prior to inoculation. For an initial period of 2.5-3 h post-inoculation the PO, was stable after which oxygen consumption increased rapidly. Consumption of oxygen exceeded the rate of replenishment and resulted in a PO, of zero within 10 h of inoculation (Fig. 2). Measurement of the partial pressure of dissolved CO, showed that pC0, increased concomitantly with the decrease in PO,, a further indication of increased metabolic activity. During the growth phase, biomass grew actively and the available carbohydrate supply was metabolized until it was exhausted (approximately 16 h after inoculation). A minimum viable cell count of lo9 cells/ml (dry weight of approximately 19 gA) was achieved at the end of the growth phase. Acidification of the medium, as a result of pyruvate production, occurred continuously during the growth phase, requiring the addition of alkali to maintain the pH at the set point of 5.2. Typical exogenous pyruvate concentrations of 4-5 g/l were achieved by the end of the growth phase using the original medium formulation. The bioconversion phase was initiated by the alteration of the independent variables (pH 6.2, 18.8 "C, 0.6 vvm air), the addition of extra medium components [molasses 54 g/l and 16 mV1 nutrient solution containing (in gA): urea 0.89, whey 0.98, KH,PO, 0.59, MgSO, 0.28, and thiamine HC10.0012] plus a dose of benzaldehyde (0.8 mv1). These changes resulted in a sudden increase in p C 0 , followed by a decrease, suggesting that the cells became stressed. Tivo

35


Start fermentation 10 L Growth Medium

0.4% (v/v) aliquot

(GP)

16 h. 24.8"C, 1.2 vvm. p H 5.2

Addition of carbohydrate (54 g/L GP medium) Bioconversion mixture (16 mUL GP medium) Benzaldehyde aliquot (0.8 mUL GP medium)

Initiate Bioconversion Phase (BPI

2 h, 18.8" C,0.6 vvm. pH 6.2

Addition of carbohydrate (74 GP medium) Continuous benzaldehyde flow commenced ( I .5 mUL GP medium.h)

I

.

I

Initiate Pump Start (PSI I

22 h, 18.8" C, 0.6 wm,p H 6.2

Acidification of medium ceases, continuous addition of acid commences (AR) (approx. 5 h after BP initiated)

A

PdinpCQ (approx. 12 h after BP initiated)

1 0 1 1 Fermentation terminated after

Figure I Schematic diagram of the procedure followed for a standard fermentation by Oliver et nl. (1997).

36

s"

I

8 7

I

7

I

l-

3

I

s

I

8

I

I 9

0

I I

I 0 rn

I

I

I

I

8I

8

I

2I

I

:

I

2I

2 7

8

2

z I z


z :


37

hours later, further molasses was added (74 gA) and a pump started to add benzaldehyde continuously (total 15 m u ) at 1.5 ml/l.h. Acidification of the medium due to the production of pyruvate continued for approximately 5 h after the initiation of the bioconversion phase, albeit at a reduced rate. After this time, the rate of alkali dosing slowed and the pH gradually rose above the bioconversion phase set point of 6.2 (Fig. 2) and acid addition commenced. The increase in pH and the resulting acid dosing to maintain the pH suggested that pyruvate was being metabolized (resulting in L-PACor other less acidic metabolites). The period between the cessation of alkali dosing and the commencement of acid dosing was designated as the acid rollover. The acid rollover coincided with the commencement of an increase in the PO, and a decrease in the p C 0 , The biomass concentration contmued to increase for a few hours after the initiation of the bioconversion phase and then began to decline. Cell viability remained higher than 90%, after which a decline in both biomass concentration and viability commenced. Viability was less than 5% at the end of the fermentation. Approximately 12 h after the initiation of the bioconversion phase, there was a surge in the pCO,, which then subsided over a period of approximately 4 h. The peak was indicative of an increase in PDC activity, CO, being a product of the PDC-catalysed reaction. A small dip in the PO, occurred concomitantly with the increase in CO, concentration. The fermentation was continued for a total of 40 h before it was terminated, based on the protocol for full-scale commercial L-PAC production. The protocol followed was intended to maximize the L-PAC yields achieved, based on the physiological behaviour of yeast as described earlier in this review. Biomass was allowed to grow under good aeration conditions, producing an endogenous supply of pyruvate, until, by the end of the growth phase, the conditions had become fermentative because of the high rate of oxygen consumption. The reduction of aeration at the start of the bioconversion phase helped to ensure that the conditions remained partially fermentative, and the medium was pulsed with carbohydrate to induce PDC activity. A pulse of benzaldehyde was also added to stimulate the PDC activity without causing excessive benzyl alcohol production. The slow benzaldehyde feed started later was intended to maintain PDC activity at higher rates than ADH activity. The main aim of the authors’ study was the simplification of the fermentation medium without detriment to the L-PAC yield. The potential benefits included reduced production costs, and reduced nutrient load and chemical oxygen demand (COD) in the spent broth. They successfully demonstrated that up to 60% of the molasses added to the medium could be replaced by sucrose (as raw sugar), to give an increase in L-PAC (to 14 g/l) and pyruvate (6 g/l) yields. No effect was observed on the benzyl alcohol yield (1.1 g/l). The effect

38


of such a change is an anticipated savings in storage costs owing to the proportionately higher sucrose concentration in raw sugar compared with molasses. Later studies by the authors also demonstrated that the total concentration of carbohydrate in the growth-phase medium could be reduced by up to 20% without significant losses in L-PACyield. A guaranteed minimum of 10 g/l of L-PACcould not be achieved if the bioconversion phase carbohydrate content was reduced by more than 25%. Both CSL and whey powder were added in relatively small quantities and the exact role of either component was unknown. CSL is high in nitrogenous compounds and this, combined with the fact that a considerable quantity was added to the fermentation medium with respect to the amount of urea added, was thought to be the reason for its inclusion in the medium. Results show, however, that omission of CSL from the medium had no detrimental effects on the overall outcome of the fermentation. Marginally increased pyruvate yields, attributed to increased glycolytic flux, were recorded (6.7 g/l), as was a reduction in ADH activity (benzyl alcohol yields of 0.5 g/l). These changes did not, however, translate to increased L-PAC yields, which remained essentially unchanged (12.8 g/l). The addition of whey to the medium was originally undertaken to provide a source of lactose. However, because C. utilis is unable to utilize this carbohydrate (the medium was not originally developed for the growth of C. utilis), the inclusion of whey in the medium was considered superfluous. Whey also contributes lactic acid (which C. utilis can utilize) and thiamine, but at the concentrations present combined with the amount of whey added to the medium, the contribution of both was considered to be insignificant. The omission of whey from the medium proved to be apparently beneficial, resulting in a 15% increase in the acid rollover pyruvate concentration. However, there was no effect observed on L-PAC yield, or on any of the other measured variables, including benzyl alcohol concentration. Preliminary experiments were undertaken by Oliver et al. ( 1997) to determine the effect of lowering urea and potassium dihydrogen phosphate concentrations. The results indicated that both of these materials could be reduced by up to 15% without any detrimental effect on either the L-PAC or the benzyl alcohol yields. As an extension to the work on the fermentation medium composition, the effect of benzaldehyde feed rate and acetaldehyde or ethanol addition were examined. While work by both Long and Ward (1989b) and Mahmoud et al. (1990b) demonstrated the benefits of benzaldehyde dosing over extended periods, Agarwal and co-workers (1987) showed that ADH activity remained higher than that of PDC when benzaldehyde concentrations were less than 4 m~ (Section 3.7.1). The rationale behind increasing the benzaldehyde flow rate was thus to raise the benzaldehyde concentration to a level such that ADH activity, and subsequently benzyl alcohol yield, would be significantly reduced.


39

Increasing the benzaldehyde flow rate from 1.2 ml/l.h to 2.5 mVl.h successfully brought about a reduction in benzyl alcohol concentration of 50%, although the resulting increase in L-PACyield was less than 10% (to approximately 13 gA). Increasing benzaldehyde flow rates also resulted in marginally reduced (slightly more than 10%)pyruvate concentrations. The lower benzyl alcohol concentrations were thought to be due to premature inactivation of ADH, as proposed by Agarwal et al. (1987). As noted in Section 3.6, it has been proposed that acetaldehyde can compete with benzaldehyde for sites on ADH. In studies by Oliver (1996), significant gains were made in the yield and efficiency of L-PACproduction in the presence of added acetaldehyde, although reductions in the benzyl alcohol yield were minimal. The addition of ethanol (up to a total of 9.8 g/l) was also tested as a method for increasing benzaldehyde solubility. The result was a very slight reduction in benzyl alcohol production at low benzaldehyde flow rates but minimal effect at the maximum benzaldehyde flow rate (2.5 ml/l.h) tested. The effect of added ethanol on L-PAC yield was negligible. The limited effects of ethanol dosing observed were considered to result from the addition of ethanol at inappropriately low levels in the study since the amount of ethanol added (0.22 M) was substantially less than the 2-3 M concentration shown by Rogers et al. (1997) to be required before an effect is observed on PDC activity in C. utilis. Acetaldehyde dosing was also undertaken. However, there was no effect on L-PACor benzyl alcohol yields when concentrations of 2 g/l or less were added.

5. CONCLUSION

In theory, the L-PACproduction process appears to be a straightforward condensation of added benzaldehyde with acetaldehyde generated metabolically through the decarboxylation of pyruvate. In practice, the process is far from simple because of toxicity effects from the substrate (benzaldehyde),the principal by-product (benzyl alcohol) and the product itself (L-PAC).A number of strategies have been employed to ameliorate the toxic effects, and to maximize the L-PACyield, whilst minimizing the generation of by-products. Such strategies rely heavily on increased understanding of the predominant enzyme systems involved (PDC and ADH), together with a more detailed understanding of the biochemical and physiological basis for variations in L-PAC yield between the yeast species examined and the different fermentation systems employed. The effects of nutrients on L-PAC production are less well understood and appear to be worthy of further investigation. An overall increase in the understanding of the biochemistry and physiology of yeast systems capable

40


of L-PACsynthesis will aid in the optimization of full-scale L-PACproduction through productivity increases and reduced medium-related expenses.

Agarwal, S.C. and Basu, S.K. (1989) Biotransformation of benzaldehyde to L-acetyl phenyl carbinol by fed batch culture system. J. Microb. Eiorechnol. 4, 84-86. Agarwal, S.C., Basu, S.K., Vora, V.C., Mason, J.R. and Pirt, S.J. (1 987) Studies on the production of L-acetyl phenyl carbinol by yeast employing benzaldehyde as precursor. Biotech. Eioeng. 29,783-785. Aiba, S., Shoda, M. and Nagatani, M. (1968) Kinetics of product inhibition in alcohol fermentation. Biotech. Eioeng. 10, 845-864. Antonini, E., Carrea, G. and Cremonesi, P. (1981) Enzyme catalysed reactions in water organic solvent two-phase systems. Enzyme Microb. Technol. 3,291-296. Becvarova, H. and Hanc, 0. (1963) Production of phenylacetylcarbinol by various yeast species. Folia Microbiol. 8, 4 2 4 7 . Becvarova, H., Hanc, 0. and Macek, K. (1963) Course of transformation of benzaldehyde by Saccharomyces cerevisiae. Folia Microbiol. 8, 165-169. Berry, D.R. and Brown, C. (1987) Physiology of yeast growth. In: Yeast Eiotechnology (D. R. Berry, I. Russell, and G. G Stewart, eds), pp. 159-199. Allen and Unwin, London. Boiteux, A. and Hess, B. (1970) Allosteric properties of yeast pyruvate decarboxylase. FEES Lett. 9,293-296. Bowen, W.R., Pugh, S.Y.R. and Schomburgk, N.J.D. (1986) Inhibition of horse liver and yeast alcohol dehydrogenase by aromatic and aliphatic aldehydes. J. Chem. Technol. Eiotechnol. 36, 191-196. Bringer-Meyer, S. and Sahm, H. (1988) Acetoin and phenylacetylcarbinol formation by the pyruvate decarboxylases of Zymomonas mobilis and Saccharomyces carlsbergensis. Eiocatalysis 1,321-33 1. Brown, S.W., Oliver, S.G., Harrison, D.E.F. and Righelato, R.C. (1981) Ethanol inhibition of yeast growth and fermentation: differences in the magnitude and complexity of the effect. Eul: J. Appl. Microbiol. Eiotechnol. 11, 151-155. Cardillo, R., Servi, S. and Tinti, C. (1991) Biotransformation of unsaturated aldehydes by microorganisms with pyruvate decarboxylase activity. Appl. Microbiol. Eiotechnol. 36, 300-303. Carlsen, H.N.. Degn, H and Lloyd, D. (1991) Effects of alcohols on the respiration and fermentation of aerated suspensions of baker’s yeast. J. Gen. Micmbiol. 137,2879-2883. Casey, G . P. and Ingledew, W.M. (1986) Ethanol tolerance in yeasts. CRC Crit. Rev. Microbiol. 13, 219-280. Cejka, A. (1985) Preparation of media. In: Eiotechnology: A Comprehensive Treatise, Vol. 2 (H.-J. Rehm and G. Reed, eds), pp. 630-698. Verlag-Chemie, Weinheim. Chow, Y.S., Shin, H.S., Adesina, A.A. and Rogers, P.L. (1995) A kinetic model for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde. EiotechnoL Letr. 17, 1201-1 206. Conn, E.E., Stumpf, P.K., Bruening, G. and Doi, R.H. (1987) Outlines ofEiochemistry, 5th edn. John Wiley and Sons, Singapore. Crout, D.H.G.. Dalton, H., Hutchinson, D.W. and Miyagoshi, M. (1991) Studies on pyruvate decarboxylase: acyloin formation from aliphatic, aromatic and heterocyclic aldehydes. J. Chem. Soc. Perkin Trans. 1329-1334.


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Nikolova, P. and Ward, O.P. (1992b) Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems. Biotechnol. Bioeng. 39, 870-876. Nikolova, P. and Ward, O.P. ( 1 992c) Whole cell yeast biotransformations in two-phase systems: effect of solvent on product formation and cell structure. J. Industrial Micmbiol. 10, 169-177. Nikolova, P. and Ward, O.P. (1993) Effect of organic solvent on biotransformation of benzaldehyde to benzyl alcohol by free and silicone-alginate entrapped cells. Biotechnol. Technol. 7 , 897-902. Nikolova, P. and Ward, O.P. (1994a) Effect of support matrix on ratio of product to by-product formation in L-phenylacetyl carbinol synthesis. Biotechnol. Lett. 16,7-10. Nikolova, P. and Ward, O.P. (1994b) Reductive biotransformation of benzaldehyde derivatives by baker’s yeast in non-conventional media: effect of substrate hydrophobicity on the biocatalytic reaction. Biocutulysis 9,329-341. Nikolova, P., Long, A. and Ward, O.P. (1991) Colorimetric determination of L-phenylacetyl carbinol produced by biotransformation of benzaldehyde and pyruvate using Saccharomyces cerevisiae. Biotechnol. Technol. 5 , 3 1-34. Noronha, S . and Moreira, A.R., (1993). Bioconversion of benzaldehyde by yeast. Ahstr: Pup. Am. Chem. SOC.205, Biot, 173. Novak, M., Strehaiano, P., Moreno, M. and Goma, G. (1981) Alcoholic fermentation: on the inhibitory effect of ethanol. Biotechnol. Bioeng. 23, 201-21 1. Oliver, A. L. (1996) Influence of medium components on the production of phenylacetylcarbinol (PAC) by yeast. M. App. Sci. Thesis. Royal Melbourne Institute of Technology, Melbourne, Australia. Oliver, A.L., Roddick, F.A. and Anderson, B.N. (1997). Cleaner production of phenylacetylcarbinol through productivity improvements and waste minimization. Pure Appl. Chem. 69,2371-2385. Ose, S . and Hironaka, J. (1957) Studies on production of phenyl acetyl carbinol by fermentation. Proceedings of the International Symposium on Enzyme Chemistry 2, 457460. Oura, E. (1983) Biomass. In: Biotechnology: A Comprehensive Treatise, Vol. 3 (H.-J. Rehm and G. Reed, eds), pp. 18-19. Verlag-Chemie, Weinheim. Pascual, C., Alonso, A., Garcia, I., Romay, C. and Kotyk, A. (1988) Effect of ethanol on glucose transport, key glycolytic enzymes, and proton extrusion in Saccharomyces cerevisiae. Biotechnol. Bioeng. 32, 374-378. Pohl, M. (1997) Protein design on pyruvate decarboxylase (PDC) by site-directed mutagenesis Adv. Biochem. Eng. Biotechnol. 58, 15-43. Reed, G. and Peppler, H.J. (1973) Yeast Technology. AVI, Connecticut. Rogers, P.L. (1990) ICI Internal Report. Orica Ltd, Ascot Vale, Victoria, Australia. Rogers, P.L., Shin, H.S. and Wang, B. (1995) Review of biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC), an intermediate in L-ephedrine production. Proceedings of the 4th Pac$c Rim Biotechnology Conference, pp. 2 10-2 11. Rogers, P.L. Shin, H.S. and Wang, B (1997) Biotransformation for ephedrine production. Adv. Biochem. Eng. Biotechnol. 56,33-59. Romano, P. and Suzzi, G. (1996) Origin and production of acetoin during wine yeast fermentation. Appl. Environ. Microhiol. 62, 309-3 15. Sambamurthy, K., Ellaiah, P. and Krishna, K.T. (1984) Studies on the production of phenyl acetyl carbinol from benzaldehyde by Saccharomyces cerevisiae. Indian J. Pharm. Sci. Jan-Feb, 62. Schmitt, H.D. and Zimmermann, F.K. (1982) Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis. J. Bacteriol. 151, 1146-1 152.

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Schure, E.G. ter, Flikweert, M.T., Dijken, J.P. van, Pronk, J.T. and Venips, C.T. (1998) Pyruvate decarboxylase catalyses decarboxylation of branched-chain 2-oxoacids but is not essential for fuse1 alcohol production by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 64, 1303-1 307. Seely, R.J., Heefner, D.L., Hageman, R.V., Yarus, M.J. and Sullivan, S.A. (1989a) US patent 89/04421. Seely, R.J., Hageman, R.V., Yarus, M.J. and Sullivan, S.A. (1989b) US patent 89/04423. Shacar-Nishri, Y. and Freeman, A. (1993) Continuous production of acetaldehyde by immobilked yeast with in situ product trapping. Appl. Biochem. Biotechnol. 39140,387-399. Shin, H.S. and Rogers, P.L. (1995a) Kinetics of biotransformation of benzaldebyde to Lphenylacetylcarbinol (L-PAC) by immobilised pyruvate decarboxylase from Candidu utilis. Proceedings ofthe 4th Pacgc Rim Biotechnology Conference, pp. 328-329. Shin, H.S. and Rogers, P.L. ( I 995b) Biotransformation of benzaldehyde to L-phenylacetylcarbinol, in intermediate in L-ephedrine production, by immobilised Candidu utilis. Appl. Microbiol. Biotechnol. 4 7 - 1 4 . Shin, H.S. and Rogers, P.L. (1996a) Production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde using partially purified pyruvate decarboxylase (PDC). BiotechnoL Bioeng. 49, 52-62. Shin, H.S. and Rogers, P.L. (1996b) Kinetic evaluation of biotransformation of benzaldehyde to L-phenylacetylcarbinol by immobilised pyruvate decarboxylase from Candidu utilis. Biotechnol. Bioeng. 49,429-436. Sims, A.P. and Barnett. J.A. (1991) Levels of activity of enzymes involved in anaerobic utilization of sugars by six yeast species: observations towards understanding the Kluyver effect. FEMS Microbiol. Lett. 77, 295-298. Sims, A.P., Stalbrand, H. and Bamett. J.A. (1991) The role of pyruvate decarboxylase in the Kluyver effect in the food yeast, Candidu utilis. Yeast 7,479-487. Smidsrod, 0 . and Skjak-Braek, G. (1990) Alginate as immobilization matrix for cells. Trends Biotechnol. 8, 71-78. Smith, P.F. and Hendlin, D. (1953) Mechanism of phenylacetylcarbinol synthesis by yeast. J. Bacteriol. 65,440-445. Smith, P.F. and Hendlin, D. (1954) Further studies on phenylacetylcarbinol synthesis by yeast. Appl. Microbiof. 2, 294. Stanbury, P.F. and Whitaker, A. ( 1 984) Principles of Fermentation Technology, pp. 74-90. Pergamon Press, Oxford. Stanley, G.A. (1993) Acetaldehyde effects in Saccharomyces cerevisiae. Ph.D. thesis, University of Melbourne, Australia. Stanley, G.A., Douglas, N.G., Every, E. J., Tzanatos, T. and Pamment, N. B. (1993) Inhibition and stimulation of yeast growth by acetaldehyde. Biotechnol. Lett. 15, 1199-1204. Tripathi, C.K.M., Basu, S.K., Vora, V.C., Mason, J.R. and Pirt, S.J. (1988) Continuous cultivation of a yeast strain from biotransformation of L-acetyl phenyl carbinol (L-PAC) from benzaldehyde. Biotechnol. Lett. 10,635-636. Tripathi, C.K.M., Basu, S.K., Vora, V.C., Mason, J.R. and Pirt, S.J. (1991) Biotransformation of benzaldehyde to L-acetyl phenyl carbinol (L-PAC)by immobilised yeast cells. Res. Industry 36, 159-160. Tripathi, C.K.M., Agarwal, S.C., Bihari, V., Joshi, A.H. and Basu, S.K. (1997) Production of L-phenylacetylcarbinol by free and immobilised yeast cells. Indian J. Exp. Biol. 35, 886-889. Urk, H. van, Mak, P.R., Scheffers, W.A. and Dijken, J.P. van ( I 988) Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess. Yeast 4, 283-291.


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Urk, H. van, Schipper, D., Breedveld, G.J., Mak, P.R., Scheffers, W.A. and Dijken, J.P. van ( 1989) Localization and kinetics of pyruvate-metabolising enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim. Biophys. Acta 992.78-86. Urk, H. van, Voll, W.S.L., Scheffers, W.A. and Dijken, J.P. van (1990) Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl. Environ. Microbiol. 56, 281-287. Verduyn, C., Breedveld, G.J., Scheffers, W.A. and Dijken, J.P. van (1988) Substrate specificity of alcohol dehydrogenase from the yeasts Hansenula polymorpha CBS 4732 and Candida utilis CBS 621. Yeast 4, 143-148. Voets, J.P., Vandamme, E.J. and Vlerick, C. (1973) Some aspects of the phenylacetyl carbinol biosynthesis by Saccharomyces cerevisiae. Z. Allg. Mikrobiol. 13,355. Vojtisek, V. and Netrval, J. (1982) Effect of pyruvate decarboxylase activity and of pyruvate concentration on the production of 1 -hydroxy- 1-phenylpropanone in Saccharornyces carlshergensis. Folia Microbiol. 27, 173-177. Wang, B. (1993) Kinetic study of fed-batch and continuous bioconversion processes for Lphenylacetylcarbinol (L-PAC) production by the yeast Candida utilis. Ph.D. thesis, University of New South Wales, Sydney, Australia. Wang, B., Shin, H.S. and Rogers, P.L. (1994) Microbial and enzymatic biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC),an intermediate in L-ephedrine production. In: Better Living Through Innovative Biochemical Engineering (W.K. Teo, M. G.S. Yap and S.W.K. Oh, eds), p. 249. Continental Press, Singapore. Ward, O.P. and Young, C. S. (1990) Reductive biotransforrnations of organic compounds by cells or enzymes of yeast. Enzyme Microb. Technol. 12,482-493. Zabriskie, D.W., Arrniger, W.B., Phillips, D.G. and Albano, P.A. (1980) Traders’ Guide to Fermentation Media Formulation.Traders’ Protein Division, Traders’ Oil Mill Company, Memphis, TN.


Fungal Production of Citric and Oxalic Acid: Importance in Metal Speciation, Physiology and Biogeochemical Processes Geoffrey M. Gadd Department of Biological Sciences, University of Dundee, Dundee, DDI 4HN9 UK

ABSTRACT

The production of organic acids by fungi has profound implications for metal speciation, physiology and biogeochemical cycles. Biosynthesis of oxalic acid from glucose occurs by hydrolysis of oxaloacetate to oxalate and acetate catalysed by cytosolic oxaloacetase, whereas on citric acid, oxalate production occurs by means of glyoxylate oxidation. Citric acid is an intermediate in the tricarboxylic acid cycle, with metals greatly influencing biosynthesis: growth limiting concentrations of Mn, Fe and Zn are important for high yields. The metal-complexing properties of these organic acids assist both essential metal and anionic (e.g. phosphate) nutrition of fungi, other microbes and plants, and determine metal speciation and mobility in the environment, including transfer between terrestrial and aquatic habitats, biocorrosion and weathering. Metal solubilization processes are also of potential for metal recovery and reclamation from contaminated solid wastes, soils and low-grade ores. Such ‘heterotrophicleaching’can occur by several mechanisms but organic acids occupy a central position in the overall process, supplying both protons and a metal-complexing organic acid anion. Most simple metal oxalates [except those of alkali metals, Fe(II1) and All are sparingly soluble and precipitate as crystalline or amorphous solids. Calcium oxalate is the most important manifestation of this in the environment and, in a variety of crystalline structures, is ubiquitously associated with free-living, plant symbiotic and pathogenic fungi. The main forms are the monohydrate (whewellite) and the dihydrate (weddelite) and their formation is of significance in biomineralization, since they affect nutritional heterogeneity in soil, especially Ca, P, K and A1 cycling. The formation of insoluble toxic metal oxalates, e.g. of Cu, may confer ADVANCES IN MICROBIAL PHYSIOLOGY VOL 41 ISBN 0-12-027741-7

Copyright 0 1999 Academic Press All rights of reproduction in any form reserved

4a

GEOFFREY M. GADD

tolerance and ensure survival in contaminated environments. In semiarid environments, calcium oxalate formation is important in the formation and alteration of terrestrial subsurface limestones. Oxalate also plays an important role in lignocellulose degradation and plant pathogenesis, affecting activities of key enzymes and metal oxidoreduction reactions, therefore underpinning one of the most fundamental roles of fungi in carbon cycling in the natural environment. This review discusses the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal speciation, physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation, plant pathogenesis and metal biogeochemistry. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Metal chemistry of oxalic and citric acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Fungal biosynthesis of oxalic acid and calcium oxalate formation . . . . . . . . . . . 3.1. Oxalic acid biosynthesis ......................................... 3.2. Calcium oxalate ................................................ 4. Role of metals and oxalate in lignocellulose degradation and plant pathogenesis ...................................................... 4.1. Lignocellulose degradation ...................................... 4.2. Plant pathogenesis ............................................. 5. Catabolism of oxalic acid ............................................ 6. Fungal biosynthesis of citric acid ...................................... 6.1. Role of metals in citric acid production ............................. 7. Fungal organic acid production and metal biogeochemistry . . . . . . . . . . . . . . . 7.1. Metal solubilization and anion mobility ............................. 7.2. Role of organic acids in corrosion of stone and building materials . . . . . . 7.3. Role of fungal oxalate in limestone biomineralization . . . . . . . . . . . . . . . . . 8. Fungal organic acid production and metal biotechnology ................. 8.1. Metal solubilization for recovery and bioremediation . . . . . . . . . . . . . . . . . Acknowledgements ................................................. References ........................................................

48 50 53 53 55 61 61 64 65 65 67 68 68 72 74 76 76 78 79

1. INTRODUCTION

In the terrestrial environment,fungi are of fundamental importance as decomposers, plant pathogens and symbionts (mycorrhizas),playing important roles in carbon, nitrogen and other biogeochemical cycles (Wainwright, 1988).They are often dominant in acidic conditions and, in soil, can comprise the largest pool of biomass (including other microorganisms and invertebrates)(Metting, 1992). This, combined with their branching filamentous explorative growth habit and high surface area to mass ratio ensures that fungal-metal interactions

FUNGAL PRODUCTION OF CITRIC AND OXALIC ACID

49

are an integral component of major environmental cycling processes. Metals, both essential and inessential, and their derivatives can interact with fungi in various ways depending on the metal species, organism and environment, while fungal metabolic activities can also influence speciation and mobility (Gadd, 1993;Wainwright and Gadd, 1997). Certain mechanisms may mobilize metals into forms available for cellular uptake and leaching from the system, e.g. complexation with organic acids, other metabolites and siderophores (Francis, 1994), while immobilization may result from sorption on to cell components and exopolymers, transport and intracellular and extracellular sequestration or precipitation (Morley and Gadd, 1995; Gadd, 1996; Sayer and Gadd, 1997; White et al., 1997). Such apparently opposing processes of solubilization and immobilization are important for biogeochemical cycles for indigenous or introduced metals, and fundamental determinants of fungal growth, morphogenesis and physiology (Morley et al., 1996; Ramsay et al., 1999). Furthermore, several processes are relevant to environmental bioremediation (White et al., 1997; Sayer et al., 1998). Organic acids have important roles in fungal nutrition and physiology, apart from their possible utilization as carbon and energy sources, which include contributions to intracellular osmotic potential, charge balance and pH homeostasis (see Jennings, 1995). In addition, the production of metal-complexing organic acids assists both essential metal and anionic nutrition of fungi and plants via the solubilization of phosphate and sulphate, from insoluble metalcontaining substances, including salts and minerals. Production of such acids is also important in biodeterioration and weathering. Although much information on fungal organic acid production has been obtained as a result of the commercial importance of citric acid (Mattey, 1992; Kubicek, 1998),the wider significanceof citric and oxalic acids in affecting metal speciation and mobility should not be overlooked. As well as the relevance to metal nutrition and toxicity, metal solubilization by the formation of organic acid complexes is important in environmental metal mobility and transfer between terrestrial and aquatic habitats, in plant nutrition and productivity, metal recovery from wastes and low-grade ores (‘heterotrophic leaching’) (Burgstaller and Schinner, 1993), and bioremediation (Francis et al., 1992; Dodge and Francis, 1994; Francis and Dodge, 1994). Metal immobilization by insoluble metal oxalate formation is again a process of marked environmental significance regarding fungal survival, biodeterioration, pathogenesis, soil weathering, mineral formation and metal detoxification. Oxalate-containing or oxalate-derived minerals, including humboldtine (ferrous oxalate dihydrate), whewellite (calcium oxalate monohydrate) and weddelite (calcium oxalate dihydrate), occur in the geosphere, with significant microbiological involvement in their production. Many plants contain calcium oxalate, which can render them poisonous to herbivores and humans, and this can also constitute a significant fraction of the dry weight of lichen thalli (Purvis and Halls, 1996). In a fungal

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GEOFFREY M. GADD

context, oxalic acidkalcium oxalate has long been known to be of ubiquitous occurrence and associated with fungi from different groups (Hamlet and Plowright, 1877; De Bary, 1887). The scope of this review is the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation and plant pathogenesis, and metal biogeochemistry.

2. METAL CHEMISTRY OF OXALIC AND CITRIC ACIDS

Organic acids can form coordination compounds or complexes with metals. These may be non-ionic, anionic or cationic depending on the sum of the charges of the central metal atom or ion and surrounding ions and molecules (Basolo and Johnson, 1964; Munier-Lamy and Berthelin, 1983). If the organic acid, e.g. citric or oxalic acid, contains two or more electron donor groups so that one or more rings are formed, then the organic acid can be termed a chelating agent and the resulting complexes termed metal chelates (Martell and Calvin, 1952). Such complexation is dependent on the relative concentrations of the anions and metals in solution, the pH and the stability constants of the various complexes (DenCvre et al., 1996). Oxalic acid is a relatively strong acid and crystallizes from water as monoclinic prisms of oxalic acid dihydrate. In the biosphere, oxalic acid occurs in rocks, microorganisms, including fungi, plants and animals as the free acid but more commonly as the K or Ca salt (Hodgkinson, 1977). Indeed, in a microbiological context, raphides of calcium oxalate were, with microbes, among the first objects to be observed under the optical microscope (Leeuwenhoek, 1675). Like monocarboxylic acids, oxalic acid can be converted into salts and, as with other dicarboxylic acids, it is possible to obtain compounds where only one of the carboxyl groups has been derivatized, or where both carboxyl groups have been converted into the same or different derivatives (Hodgkinson, 1977). The oxalate ion, C 0 2-, is a bidentate 2. 4 ligand, forming a five-membered chelate ring when it binds to a metal (Fig. 1). A metal (M) which normally forms octahedral six-coordinate complexes (e.g. A13+,Cr3+,Fe3+)can bind three oxalates to form an anionic complex (Fig. 1):

This can be crystallized as a potassium salt K3[M(C204),].3H20.Metals which form square planar four-coordinate complexes (e.g. Cu2+,Zn2+) can complex two oxalates (Fig. l),

51


L

1

B

A

C

3-

D

E

Figure I Metal complex formation by oxalic acid. (A) oxalic acid; (B) oxalate; (C) bidentate metal (M) complex formation; (D) complex anion formation with metals which form square planar four-coordinatecomplexes, e.g. Cu2+,(Cu oxal)2-; (E) complex anion formation with metals which form octahedral six-coordinate complexes, e.g. A13+, Fe3+, C?+, (A1 ~ x a l ) ~(Fe - , oxal)j-, (Cr ~ x a l ) ~ - .

M2++ 2C20,2- + M(C20,)22-

(2)

with the crystallized potassium salt being K2[M(C20,),].2H20. Some metals, e.g. Al, Cr, Zn and Fe, form acid complexes, e.g. H,[Cr(C,O,)]. Most simple oxalates are sparingly soluble in water except those of the alkali metals (Li, Na, K), ammonium and Fe(III), the last probably because of the formation of the Fe[Fe(C,O,),] complex. Divalent metal oxalates are of similar solubilities with the most soluble being magnesium oxalate and the least soluble being calcium and lead oxalates (Table l). Precipitation occurs according to the following equation: M,qn+

+ n/2C20,2- + M(C2O,),,.xH20

(3)

Because of the coordinating properties of the bidentate oxalate ion, most metals of particular interest to biologists form both simple and complex oxalates; these metals include Mg, Ca, Sr, Ba, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ag, Cd, Sn, Hg, Pb, and a range of actinides and lanthanides. Many simple oxalates are crystalline or amorphous solids with solubility constants ranging from lo4 to The values for divalent metal oxalates generally lie between

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GEOFFREY M. GADD

Table I

Solubility products of some metal oxalates (Adapted from Chang, 1993)

Metal oxalate

Temperature (“C)

Solubility product

Barium (dihydrate) Cadmium (trihydrate)

18 18 25 18 25

I .20 x 1.53 x 1.42 x 1.78x 2.57 X 2.87 x 2.10 x 2.74 x 8.51 x 8.57 x 4.83 X 1.70 x I .75 x 5.40 x 5.61 x 1.35x 1.37 x

Calcium (monohydrate)

25 25

Copper (11) Ferrous Lead

18

Magnesium (dihydrate) Manganese (11) (dihydrate) Mercury (I) Silver (I) Strontium (monohydrate) Zinc (dihydrate)

25 25 25 25 25 25 18 18

25

10-7 10-9

lW9 10-7

lo-” 1O-Io 10-5 10“ 10-7

I 0-13 lo-’, 10-9 10-9

CH2COOH

I

HO-C-COOH

I

CH2COOH A

B

Figure 2 Metal complex formation by citric acid. (A) citric acid; (B) bidentate complex, e.g. (Ca cit)-, (Ni cit)-, (Fe(OH), cit)2-; (C) tridentate complex, e.g. (Cd tit)-, (Fe tit)-, (FeOH tit)*-, (Pb cit)-, (Cu tit)% (D) binuclear complex - (UO,), tit:-. Adapted from Francis et al. (1992).

53

FUNGAL PRODUCTION OF CITRIC AND OXALlC ACID

(Table 1). It is generally believed that only simple oxalates can and occur in biological systems because the excess oxalate ion required for complex stability would be toxic. Citric acid can form mononuclear, binuclear or polynuclear complexes depending on the metal (Fig. 2). Ca2+,Fe3+and Ni2+form bidentate, mononuclear complexes with two carboxylic acid groups while Cu2+,Fe2+,Cd2+and Pb2+form tridentate mononuclear complexes with two carboxylic acid groups and the hydroxyl group. Uranium forms a binuclear complex involving four carboxylic acid groups and two hydroxyl groups from two citric acid molecules (Francis et al., 1992) (Fig. 2). Such complex formation affects metal mobility, toxicity and biodegradation: the recalcitrance of metal citrate complexes may play a role in the migration of hazardous metals from metal and nuclear disposal sites (Francis et al., 1992).

3. FUNGAL BIOSYNTHESIS OF OXALlC ACID AND CALCIUM OXALATE FORMATION 3.1. Oxalic Acid Biosynthesis

Oxalic acid may be considered as a toxic by-product of citric acid production, and its synthesis appears to depend on whether glucose or citric acid is used as the carbon source (Wolschek and Kubicek, 1999). Biosynthesis on glucose occurs by hydrolysis of oxaloacetate to oxalate and acetate catalysed by cytosolic oxaloacetase [oxaloacetate (acety1)hydrolasel (Fig. 3); this can be considered to be a valve whereby carbon overflow is channelled into an energetically neutral pathway and so competes with citrate production (Kubicek, 1988;Wolschek and Kubicek, 1999).Where citric acid is used, oxalic acid production occurs by means of the glyoxylate cycle (Fig. 4)(Hodgkinson, 1977; Dutton and Evans, 1996; Wolschek and Kubicek, 1999).

2 ADP

Glucoseu

2 ATP

2 ADP

2 b m v a t e y 2 woacetate

co2

3.5, with optimal activity at pH 9 (Balmforth and Thomson, 1984). Glyoxylate oxidation to oxalate by glyoxylate dehydrogenase has also been observed in other fungi including Fomes annosus and Tyromycespalustris (Hutterman et al., 1980;Akamatsu, 1993).In S. rolfsii, glyoxylate dehydrogenase and isocitrate lyase appeared to be located in microbodies (Maxwell et al., 1972; Armentrout et al., 1978), which could be analogous to plant glyoxysomes (Dutton and Evans, 1996). The ectomycorrhiza Puxillus involutus was able to use bicarbonate for oxalate biosynthesis in a nitrate-nitrogen medium, the oxalate being synthesized either directly from oxaloacetate, or via citrate, isocitrate and glyoxylate (Lapeyrie, 1988).As well as these mechanisms of oxalate production, ascorbic acid analogues may also act as precursors of oxalic acid synthesis in certain fungi, e.g. Sclerotinia sclerotiorum, although further work is needed to confirm the steps involved (Franceschi and Loewus 1995; Loewus et al., 1995). Oxalic acidoxalate production is widespread in fungi with factors affecting biosynthesis including carbon and nitrogen source, and medium or environment pH (Punja and Jenkins, 1984a; Lapeyrie etal., 1987; Bennett and Hindal, 1989; Pierson and Rhodes, 1992; Akamatsu et al., 1993c, 1994; Dutton et al., 1993; Micales, 1994, 1995a; Wang and McNeil, 1995; Dutton and Evans, 1996; Shimada et al., 1997; Gharieb and Gadd, 1999). For example, nitrate as a nitrogen source and maintenance of the culture pH above 3.0 enhanced

55


oxalate production by S. rolfsii (Maxwell and Bateman, 1968a,b). Effects of C, N and the pH have also been noted on a wide variety of wood-rotting basidiomycetes, including both white-rot and brown-rot species, especially when carbon sources are depleted in medium (Dutton et al., 1993; Gharieb and Gadd, 1999). While it is dificult to generalize, it appears that brown-rot fungi tend to produce most oxalate in low-nitrogen culture media, whereas white rots prefer high-nitrogen media (Akamatsu et al., 1994; Henriksson et al., 1995; Shimada et al., 1997). However, differences in yield may reflect differences in production through the growth phase. For example, the white-rot basidiomycetes, Coriolus versicolor, Heterobasidium annosum, Pleurotus jlorida and Phanerochaete chrysosporium, produced millimolar concentrations of oxalate in the stationary phase, but little was produced during earlier active growth and no lowering of medium pH was observed. In contrast, some brown-rot fungi, Amyloporia xantha, Coniophora marmorata, Coniophora puteana and Poria vaporaria produced oxalate throughout growth and concentrations up to 20 mM reduced the medium pH (Dutton et al., 1993). Differences in oxalate yield may also reflect differential expression of the oxalate-degrading enzyme, oxalate decarboxylase, in the different species (Micales, 1995b, 1997). Under nutrient-rich conditions, oxalate was not produced by l? chrysosporium (Kuan and Tien, 1993), while Poria placenta increased oxalate production under nitrogen limitation (Micales, 1994) or during growth on amorphous cellulose (Ritschkoff et al., 1995). Carbon and nitrogen sources influence oxalate production by plant pathogenic and mycorrhizal fungi (see Dutton and Evans, 1996), with nitrate being the preferred nitrogen source for Paxillus involutus (Lapeyrie et al., 1987). Oxalate production by P. involutus was enhanced by the presence of carbonatehicarbonate (Lapeyrie et al., 1987; Lapeyrie, 1988).

3.2.Calcium Oxalate In the environment, the main forms of calcium oxalate are the monohydrate (whewellite) and the dihydrate (weddelite), which extensively occur in fossil rocks, microorganisms, plants and human urinary calculi. The monohydrate is monoclinic while the dihydrate is tetragonal in crystallization, although both can crystallize in a variety of forms. The solubility and instability of hydrated calcium oxalate increases with increasing water of crystallization, and this is reflected in the pattern of crystallization of calcium oxalate from aqueous solution. The initial precipitation phase is the trihydrate which loses water of crystallization to form either the monohydrate or the dihydrate, depending on conditions: CaC20,.3H20,

+ CaC,0,.2H20, + H,O

(4)

56

GEOFFREY M. GADD

CaC20,.2H,O,

+ CaC,O,H,O, + H,O

(5)

CaC20,.3H,0,

+ CaC,O,.H,O, + 2H,O

(6)

or

The solubility of calcium oxalate increases with decreasing pH because of the formation of dioxalate ions and oxalic acid: Ca2++ C,O,2-

CaC,O, C,OZ-

t)

(7)

+ H+ t)H.C,O,

H.C,04-

+ H+t)H,C,O,

(9)

Solubility increases markedly below pH 5 and may also be increased by the presence of substances which will form soluble complexes with either calcium or oxalate ions, such as citric acid and magnesium. Conversely, solubility may be decreased by the presence of a common ion, e.g. by the addition of calcium chloride or ammonium oxalate. The ubiquitous association of oxalic acidcalcium oxalate with many and diverse kinds of fungi from all major classes has long been evident in natural, laboratory and industrial environments (De Bary, 1887; Foster, 1949; Arnott, 1982a,b, 1995; Malajczuk and Cromack, 1982; Dutton et al., 1993; Dutton and Evans, 1996).In fact, the absence of calcium oxalate may be a more significant systematic character in some species (Krisai and Mrazek, 1986).Calcium oxalate crystals can be associated with the hyphae of oxalic acid-producing strains, as well as fruiting bodies, with the main forms being the monohydrate (whewellite) and the dihydrate (weddelite), the latter being the most ubiquitous (Fig. 5 ) . Sometimesthe two hydration states can be distinguished by their morphology as the monohydrate belongs to the monoclinic system, while the dihydrate belongs to the hexagonal system (Arnott, 1995). It is thought that monohydrate crystals may arise from previously produced dihydrate in a recrystallization process , the monohydrate being the more stable form. However, (Verrecchia et ~ l . 1993), Horner et al. (1995) found that the youngest portions of fungal rhizomorphs (from an oak wood) possessed monohydrate crystals, whereas older parts of the hyphae were associated with the dihydrate, while only monohydrate was found in mantle hyphae of larch ectomycorrhiza (Jones et al., 1992).This could reflect changes that occur over development and maturation of the calcium oxalate, although contrasts with the proposed sequence of events for biomineralizationin Quaternary calcretes (Verrecchiaet al., 1993).The relative contribution of fungal metabolism or diagenesis in effecting changes in crystal morphology and hydration state is therefore unclear at present.


57

Figure 5 Scanning electron micrographs of calcium oxalate crystals produced in solid malt extract agar medium supplemented with 0.5% (w/v) gypsum by (a) Serpulu himantioides, (b) Aspergillus niger and (c,d) in leaf litter microcosms. In (c), note the small needle-like crystals characteristic of calcium oxalate monohydrate, as well as prismatic crystals of calcium oxalate dihydrate, and, in (d), the prism-like crystal of calcium oxalate dihydrate within the hyphal network. Scale bar markers: 10 pm (Gharieb, Sayer, Tait and Gadd, unpublished; see Gharieb et al., 1998).

In the environment, calcium oxalate formation by free-living and symbiotic mycorrhizal fungi is frequently observed both in soil, decomposing wood and in leaf litter (Graustein et al., 1977; Cromack e f al., 1979; Lapeyrie et al., 1984; Snetselaar and Whitney, 1990; Cairney and Clipson, 1991; Homer et al., 1995; Shinners and Tewari, 1997; Tewari et al., 1997), and also associated with certain plant pathogenic fungi (Punja and Jenkins, 1984b; Punja et al., 1985; Yang et al., 1993; Arnott, 1995). A variety of calcium oxalate crystal formations have been described with many fitting into the four groups: tetragonal bipyramids, prisms, tablets and needles (Keller, 1985; Amott, 1995; Whitney

58

GEOFFREY M. GADD

and Amott, 1986a,b, 1988; Shinners and Tewari, 1997). However, morphology is extremely variable and crystals can be separate or arranged in arrays, and may also arise from twinning (Arnott, 1995). The most common types in plants are large single needles (styloids),bundles of needles (raphides), stellate conglomerates (druses), rhombohedra1 (prismatic) and packets of angular microcrystals (crystal sand); analogous morphologies also arise in fungi (Franceschi and Loewus, 1995). Some crystal shapes appear to be transient, although in some cases their shape and location appears to be species specific (Horner et al., 1995). While most reports demonstrate calcium oxalate forrnation external to the biomass, an obvious consequence of oxalic acid excretion (Sayer and Gadd, 1997; Gharieb et al., 1998), other reports have described intracellular formation of calcium oxalate, with some crystals apparently covered by a wall or membrane (De Bary, 1887; Arnott, 1982a,b, 1995; Powell and Arnott, 1985; Whitney and Arnott, 1987) or deduced to arise in specific wall chambers (Amott, 1995), or in vacuolar vesicles (Lapeyrie et al., 1990). For fungi growing in the soil, the regularity of calcium oxalate deposits over the surface of hyphae has suggested a more complex mode of formation than precipitation of oxalate with exogenous calcium, hence the proposal that deposition of calcium oxalate arises within the hyphae and is not a simple surface precipitation (Arnott, 1982a,b, 1995;Arnott and Webb, 1983;Arnott and Fryar, 1984). However, it seems doubtful whether truly intracellular calcium oxalate deposition occurs in fungi (Franceschi and Loewus, 1995). In several fungi, semi-mature crystals are encased in an organic sheath covering the hyphae. Such sheaths are carbohydrate/proteinbased (Nicole et al., 1993) but are subject to distortions and shrinkage when treated with conventional electron microscopy reagents (Daniel, 1994; Connolly et al., 1995). It is likely that shrinkage during dehydration could make the extracellular matrix of fungi appear like the cell wall and lead to possible misinterpretation of electron micrographs (Connolly and Jellison, 1995; Connolly et al., 1995). In the white rot Resinium bicolor, the hydrated hyphal sheath is considerably thicker than the dehydrated sheath and contains numerous calcium oxalate crystals. Calcium oxalate druses nucleated and grew only in the more mature hyphal sections, suggesting that only the hyphal sheath possessed the extensibility necessary to accommodate them (Connolly and Jellison, 1995). The pattern of crystal formation along hyphae may reflect the nature and location of oxalate andor Ca2+secretion, although exogenous calcium is generally abundant (Connolly and Jellison, 1995). It is pertinent that, in plants, oxalates may accumulate in vacuoles, but there is no significant information relating to the role of the fungal vacuole in oxalate storage or deposition (Franceschi and Loewus, 1995). In basidiomycete rhizomorphs, some of the interwoven hyphae are encrusted with crystals and ‘vessel hyphae’ within may also possess crystalline deposits (Cairney et al., 1989; Cairney, 1990; Cairney and Clipson, 1991). Because of this, it is thought that the ability of litter degraders to


59

translocate and precipitate calcium as calcium oxalate could contribute to nutritional heterogeneity in soil, with concomitant influences on P, A1 and K (see later) (Connolly and Jellison, 1995, 1997).While the abundance of oxalate and importance in P and Ca cycling is clear for forest ecosystems, in arid ecosystems, oxalate production by mycorrhizal fungi appears limited to matforming fungi, such as Hysterangium separabile, and arbuscular hyphae do not have associated crystal structures (Allen et al., 1996). In addition to calcium oxalate, fungi are also able to precipitate other metal oxalates (Figs 6 and 7). The production of oxalic acid by fungi provides a means of immobilizing soluble metal ions, or complexes, as insoluble oxalates, decreasing bioavailability and conferring tolerance (Sayer and Gadd, 1997).As mentioned earlier, most metal oxalates are insoluble, some exceptions being Na, K, Li and Fe (Strasser et al., 1994). Copper oxalate (moolooite) has been observed around hyphae growing on wood treated with copper as a preservative (Murphy and Levy, 1983; Sutter et al., 1983, 1984).The copper appeared on the surface of the wood and around hyphae as copper oxalate, which was reported to be non-toxic because of its insolubility. A. niger can form metal oxalate crystals after 1-2 days when grown on medium amended with a wide range of metal compounds, including insoluble metal phosphates (Fig. 6)

Figure 6 Solubilization of insoluble Co,(PO,), by organic acid production and subsequent reprecipitationas insoluble cobalt oxalate. Aspergillus niger was grown at 25 "C for 6 days on malt extract agar containing 0.5% (wlv) Co,(PO,),. The photograph shows the clear zone of solubilization around the colony and the precipitation of cobalt oxalate crystals within this zone (see Gadd, 1996; Sayer and Gadd, 1997).

60

GEOFFREY M. GADD

Figure 7 Scanning electron micrographs of purified insoluble metal oxalate crystals produced by Aspergillus niger. (a) Cobalt oxalate, (b) copper oxalate, (c) zinc oxalate, (d) manganese oxalate, (e) strontium oxalate dihydrate and (f) strontium oxalate dihydrate with needle-like crystals of strontium oxalate monohydrate; (a-d) were obtained after growth on solid malt extract agar (MJZA) supplemented with 0.5% (w/v) of the corresponding metal phosphates; (e) and (9 were obtained after growth on MEA containing 15 m~ %(NO,),. Scale bar markers: (a,d) 100 pm; (b,c,e,f) 10 pm (Sayer, Whatley and Gadd, unpublished; see Sayer and Gadd. 1997).

61


(Sayer and Gadd, 1997) and powdered metal-bearing minerals (Fig. 5) (Sayer et al., 1997; Gharieb et al., 1998). A. niger has been shown to produce metal oxalates with many different metals, e.g. Ca, Cd, Co, Cu, Mn, Sr and Zn (Fig. 7) (Sayer and Gadd, 1997). Morphological examination of fungal-produced metal oxalate crystals and comparison, where possible, with chemically synthesized oxalates, has often shown some clear differences in crystallographic form (Vivier et al., 1994; Sayer and Gadd, 1997). Thus, the formation of oxalates containing potentially toxic metals may provide a mechanism whereby oxalate-producing fungi can tolerate environments containing high concentrations of toxic metals. Copper oxalate has also been observed in lichens growing on copper-rich rocks where it is also thought that the precipitation of copper oxalate could be a detoxification mechanism (see later) (Purvis, 1984, Purvis and Halls, 1996).

4. ROLE OF METALS AND OXALATE IN LIGNOCELLULOSE DEGRADATION AND PLANT PATHOGENESIS 4.1. Lignocellulose Degradation

Brown-rot and white-rot fungi are the main wood-rotting basidiomycetes, with brown rots, e.g. Coniophoraputeana and Serpula lacrymans, unable to metabolize lignin (leaving an amorphous brown residue) and white rots, e.g. Phanerochaete chrysosporium and Coriolus versicolor, able to degrade all plant cell wall components (Dutton and Evans, 1996). Oxalate plays an important role in affecting the activity of key enzymes and processes in both these groups of fungi (Goodwin et al., 1994; Khindaria et al., 1994; Shimada et al., 1994; Tanaka et al., 1994). In brown rots, oxalic acid production lowers the external pH, which aids cellulose degradation but also gives rise to oxygen radicals. The pH of wood can fall to around 2.5 after the growth of brown rots (Green et al., 1991; Espejo and Agosin, 1991; Dutton et al., 1993; Micales, 1994); such low pH values arise because of the strength of oxalic acid as an organic acid (pK, = 1.1) (Hyde and Wood, 1997). The oxalic acid is believed to act as an electron donor in the reduction of Fe(II1) to Fe(II), with the resultant Fe(I1) being oxidized in the Fenton reaction, yielding hydroxyl radicals for the oxidative degradation of cellulose and hemicellulose (Hirano et al., 1995; Dutton and Evans, 1996; Hyde and Wood, 1997): Fe2++ H,02

+ Fe3++ HO'+ HO-

(10)

However, direct reduction of Fe(II1) by oxalate has been dismissed because of the slow speed of reaction and requirement for light (Horne, 1960; Wood,

62

GEOFFREY M. GADD

2.5 3.5 4.5 pH 3.0 4.0

Figure 8 Model for hydroxyl radical production by brown-rot fungi without damage to the hyphae (adapted from Hyde and Wood, 1997). Secretion of cellobiose dehydrogenase (CDH) provides a mechanism for Fe(II1) reduction in the presence of oxalate: diffusion of Fe(I1) away from the hyphae promotes autooxidation. Generation of Fe(II)/H,O, away from the hyphae means resultant hydroxyl radicals will not be deleterious to the fungus.

1994; Zuo and Hoigne, 1994; Hyde and Wood, 1997). Another hypothesis has been proposed where Fe(II1) is reduced to Fe(I1) by extracellular cellobiose dehydrogenase, meaning that cellobiose or cellulose act as an electron source for both reduction of Fe(II1) and 0, to generate H,O, for the Fenton reaction (Fig. 8) (Hyde and Wood, 1997). It should be noted that oxalate may promote the Fenton reaction in a bifunctional manner, since increasing concentrationsof oxalate (< 5 mM or an oxalate : Fe ratio of 50) can inhibit oxidative breakdown of cellulose catalysed by Fenton oxidation, and also H,O, and Fe(1II) (Shimada et al., 1997).Thus, while both Fe(II1) and H,O, are important for Fenton-type oxidation, if oxalate exceeds 5 mM in wood, then non-enzymatic cellulose hydrolysis may be more important in brown-rot wood decay (Akamatsu et al., 1991; Shimada et al., 1997). It is possible that lignin scavenges the hydroxyl radicals first, and then phenoxy and other indirectly formed lignin-derived radicals may attack cellulose and hemicellulose because there is no selectivity for hydroxyl radicals to oxidize cellulose (Magara et al., 1994; Shimada et al., 1997). In brown-rot fungi, polygalacturonase and oxalic acid formation are also induced by pectin. The enzyme and oxalic acid may act synergistically to hydrolyse pectin in pit membranes and middle lamellae (Green et al., 1995). Here, a possible involvement of calcium oxalate should be alluded to since calcium is mainly located as calcium pectate in middle lamellae. Thus, in pectin degradation, the calcium is removed by calcium oxalate formation, which may perturb cell wall structure, perhaps favouring entry of lignocellulosic enzymes

63


(Dutton ef al., 1993).However, there are relatively few reports on pectinase production by wood-rotting fungi (Dutton and Evans, 1996). In white-rot fungi, the low production of oxalic acid observed has been explained as a result of the presence of oxalate decarboxylase, which degrades oxalate to formate and CO, (Micales, 1995b; Green and Highley, 1997). However, decomposition of oxalate is also a result of interactions with whiterot lignin and manganese peroxidases. In the absence of oxalate, veratryl alcohol and Mn(I1) are oxidized by lignin peroxidase (Lip) and manganese peroxidase (MnP), respectively, producing veratrylaldehyde (VA) and Mn(II1). Such a system is metal regulated because high [Mn] induces the MnP system, while repressing the Lip system. At low [Mn], MnP is repressed and Lip is induced (Perez and Jeffries, 1993). However, if oxalate is present, VA cation radicals and Mn(II1) are reduced back to the substrate level by oxalate which, at the same time, yields CO, and formate radicals; these are further oxidized to superoxide anion radicals under aerobic conditions (Fig. 9) (Shimada et al., CH20H

LiP or MnP

[Mn3+] or

c02 O * - x +

02

co2:

700-

coo-

[VAtl

6

or

[Mn3+]

OCH3 OCH3

Figure 9 Reaction mechanisms for oxidative decomposition of oxalate by lignindegrading systems with lignin peroxidase (Lip) and manganese-dependent peroxidase (MnP) (adapted from Shimada ef a[., 1997). In the absence of oxalate, veratryl alcohol (VA) and Mn(I1) are oxidized by LIP or MnP, respectively, to veratraldehyde via VA cation radicals and Mn(II1). In the presence of oxalate, VA cation radicals and Mn(II1) are reduced back to the substrate level with production of CO, and formate radical, which further results in superoxide anion radicals. Thus, lignin degradation can be inhibited by the presence of oxalate.

64

GEOFFREY M. GADD

1997).The oxalate is important in Lip repression at high [Mn] because, as long as Mn(I1) and Mn(II1) are kept in solution by chelation, repression continues. However, Mn(IV)02 is eventually precipitated, which relieves repression and allows lignin degradation to proceed (Perez and Jeffries, 1993). Thus, in the presence of oxalate, lignin degradation is inhibited and this has been shown to be non-competitive in nature, both for VA and Mn(I1) oxidation (Akamatsu et al., 1990; Popp et al., 1990; Ma et al., 1992; Shah et al., 1992; Shimada et al., 1997). Despite this, the formation of superoxide anion radicals will lead to H202production (see above). Oxidation of phenolic moieties of lignin is not inhibited by oxalate and if these accumulate, lignin degradation may be inhibited (Akamatsu et al., 1990). The presence of oxalate decarboxylase and phenol oxidase (laccase) in white-rot fungi may provide a mechanism for removal of oxalate and phenolics, so that the Lip system may function adequately (Shimada et al., 1997).Apart from the roles described, oxalate also acts as a Mn chelator and stabilizes Mn(II1) in the MnP system (Perez and Jeffries, 1993; Kishi et al., 1994). This interaction facilitates Mn(II1) dissolution from the Mn(III)-enzyme complex to be followed by Mn(I1I) catalysed oxidation of phenolic components (Popp et al., 1990; Dutton and Evans, 1996; Shimada et al., 1997). 4.2. Plant Pathogenesis

Oxalic acid has been implicated in the phytopathogenesis of several fungi with its effects being due to several mechanisms which may act singly or in concert (Rowe, 1993). The decrease in pH of infected tissues on oxalic acid production may enhance the activity of extracellular lytic enzymes, many of which have pH optima below 5 , as well as being of general detriment to plant tissues (Dutton and Evans, 1996). As mentioned above, some fungi secrete oxalic acid with cell wall degrading enzymes, and this may assist the solubilization of pectin in membranes in the middle lamellae (Green et al., 1995). The occurrence of calcium oxalate in necrotic plant tissue infected by numerous fungal plant pathogens (Punja and Jenkins, 1984a) provides further evidence for the important role of oxalic acid in calcium removal, which in turn allows polygalacturonase to hydrolyse pectates more easily (Green et al., 1995; Dutton and Evans, 1996). Interestingly, mutants of Sclerotinia sclerotiorum that are unable to synthesize oxalic acid were, in contrast to wild-type strains, non-pathogenic in bioassays (Godoy et al., 1990),while plant cultivars resistant to S. sclerotiorum were more oxalic acid-tolerant than sensitive cultivars (Noyes and Hancock, 198l). Several other similar examples occur in the literature (Kritzman et al., 1977; Havir and Anagnostakis, 1983; Marciano et al., 1983; Wang and Tewari, 1990; Callahan and Rowe, 1991). Numerous other debilitating or toxic effects of oxalic acid have also been

65

FUNGAL PRODUCTION OF CITRIC AND OXALlC ACID

documented. These include calcium removal from plasma membranes, copper removal causing inhibition of enzymes, such as polyphenol oxidase, and magnesium oxalate formation arising from Mg removal from ribosomal subunits, enzymes, ATP and chlorophyll (Rao and Tewari, 1989; Ferrar and Walker, 1993).

5. CATABOLISM OF OXALlC ACID

Oxalic acid is decarboxylated by oxalate decarboxylase to CO, and formate: (COOH),

+ CO, + HCOOH

(1 1)

The formate may subsequently be dehydrogenated by formate dehydrogenase, yielding NADH and CO, (Shimada et al., 1997). Oxygen-requiring oxalate decarboxylase is found in a variety of fungi. In some wood-rotting basidiomycetes, oxalate decarboxylase is induced by the presence of oxalic acid (Magro et al., 1988; Mehta and Datta, 1991; Dutton et al., 1994; Micales, 1997), although in Aspergillus niger, the enzyme was non-inducible and only synthesized when the culture pH fell below 2.5 (Emiliani and Bekes, 1964). The optimum pH for oxalate decarboxylase is in the range pH 1.75-2.20 (Micales, 1995b). In Coriolus versicolor, oxalate decarboxylase was found intracellularly and extracellularly: secretion occurred at the end of exponential growth, which accounts for the lack of detectable oxalate in the medium at this growth stage. However, oxalate levels rose after this because the pH of the medium became too high for oxalate decarboxylase activity (Dutton et al., 1994). Addition of calcium carbonate to medium, which raises the pH, also inhibits oxalate decarboxylase activity (Takao, 1965). In Postia placenta, oxalate decarboxylase was induced by growth inhibitory concentrations of oxalic acid in low- and highdecay isolates, and was associated with the hyphal surface and hyphal sheath (Micales, 1997). The oxalate decarboxylase-mediated prevention of oxalate overproduction may maintain a non-toxic, low pH microenvironment which facilitates decay (Micales, 1997). Such a process may be enhanced by the formate produced during oxalic acid breakdown combining with the remaining oxalic acid to form a low pH buffer (Agosin et al., 1989).

0. FUNGAL BIOSYNTHESIS OF CITRIC ACID

Citric acid is an intermediate in the TCA cycle and therefore is ubiquitous in living organisms. It is extensively used in the food and beverage industry and

66

GEOFFREY M.GADD Acetyl-CoA ,

Figure 10 Citrate formation from gluocose via anaplerotic CO, fixation (see Wolschek and Kubicek, 1999).

is produced predominantly by A. niger. World annual production is estimated around 40 000 tons (Roehr et al., 1992). It forms complexes with metals (Fe, Cu) and is therefore used for stabilization of oils and fats, and to prevent metal-ion catalysed oxidation of ascorbic acid (Kubicek, 1998).A. niger forms citric acid by the conversion of pyruvate, which arises from glycolytic catabolism of glucose to the precursor of citrate, oxaloacetate (Roehr et al., 1996; Kubicek, 1998).A key step in this process is the use of 1 mol pyruvate (of the 2 mol arising from glycolytic conversion of 1 mol glucose) and the CO, released during acetyl CoA formation to form oxaloacetate (Fig. 10). If oxaloacetate was only formed by one turn of the TCA cycle, 2 mol CO, would be lost and only two-thirds of the glucose carbon would give rise to citric acid. Such anaplerotic CO, fixation is catalysed by pyruvate carboxylase, itself induced by high carbohydrate concentrations, and this explains high commercial yields of citric acid (Kubicek, 1998). In A. niger, pyruvate carboxylase is cytosolic in location (Bercovitz et al., 1990) and pyruvate is directly converted to oxaloacetate and then to malate by malate dehydrogenase (Ma et al., 1981). It is thought that cytosolic malate is the co-substrate of the mitochondrial tricarboxylic acid carrier and enhanced intracellular malate may therefore stimulate citrate export from mitochondria (Kubicek, 1988, 1998). A. niger possesses a further glucose catabolism pathway catalysed by glucose oxidase, which converts glucose to gluconic acid, and this enzyme is induced by high glucose concentrations and aeration (Dronawat et al., 1995; Wolschek and Kubicek, 1999). Therefore, gluconic acid may also be present at least at the initial phase of a citric acid fermentation, although glucose oxidase (extracellular) is eventually inactivated once the pH falls below 3.5 (Mischak er al., 1985).A. niger may also produce oxalic acid as a by-product of citric acid fermentation, depending on whether glucose or citric acid is used as the carbon source (see elsewhere) (Kubicek, 1988; Kubicek et al., 1988; Wolschek and Kubicek, 1999). The concentration and kind of carbon source are important parameters for citric acid production, and only easily assimilated sugars allow both high yields and rates of citric acid production (Kubicek and Roehr, 1986). Other important factors include dissolved oxygen tension and aeration (Kubicek et


67

al., 1998), pH (Kubicek and Roehr, 1986), nitrogen source (Dawson et al., 1989; Choe and Yoo, 1991; Yigitoglu and McNeil, 1992), phosphate and the presence of certain metals (see below) (Kubicek, 1998). Citric acid is reported to appear in large amounts when the pH falls below 2.5. Such a low pH may influence the growth phase and pH

Advances in Microbial Physiology Volume 41

Advances in Microbial Physiology Volume 41

Advances in Microbial Physiology Volume 01