Citric Acid Biotechnology

Citric Acid Biotechnology

Citric Acid Biotechnology BJØRN KRISTIANSEN Borregaard Industries Ltd, Norway MICHAEL MATTEY Department of Bioscience and Biotechnology, University of Strathclyde, UK JOAN LINDEN Gluppevelen 15, 1614 Fredikstad, Norway

UK Taylor & Francis Ltd, 1 Gunpowder Square, London EC4A 3DF USA Taylor & Francis Inc., 325 Chestnut Street, 8th Floor, Philadelphia, PA 19106

This edition published in the Taylor & Francis e-Library, 2002. Copyright © Taylor & Francis 1999 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-7484-0514-3 (cased) Library of Congress Cataloguing-in-Publication Data are available Cover design by Jim Wilkie ISBN 0-203-48339-1 Master e-book ISBN ISBN 0-203-79163-0 (Glassbook Format)

Contents

1

A Brief Introduction to Citric Acid Biotechnology 1.1 Citric acid from lemons 1.2 Synthetic citric acid 1.3 Microbial citric acid 1.4 Citric acid by the surface method 1.5 The submerged process for production of citric acid 1.6 Continuous and immobilized processes 1.7 Yeast based processes 1.8 The koji process 1.9 Uses of citric acid 1.10 Effluent disposal 1.11 Conclusions 1.12 References

page 1 1 2 2 3 4 5 6 7 7 8 8 9

2 Biochemistry of Citric Acid Accumulation by Aspergillus niger 2.1 Introduction 2.2 Glucose catabolism in A. niger and its regulation 2.3 Regulation of citric acid biosynthesis 2.4 Role of citrate breakdown in citrate accumulation 2.5 Export of citric acid from A. niger 2.6 References

11 11 12 19 21 24 25

3 Biochemistry of Citric Acid Production by Yeasts 3.1 Introduction 3.2 Synthesis of citric acid from n-alkanes 3.3 Synthesis of citric acid from glucose 3.4 Conclusions 3.5 References

33 33 35 46 50 50

4 Strain Improvement 4.1 Introduction 4.2 General aspects of strain improvement

55 55 55 v

Contents

vi

4.3 4.4 4.5 4.6 4.7

Isolation of recombinant strains using the parasexual cycle in A. niger Genetic engineering Concluding remarks Acknowledgements References

60 61 64 65 65

5 Fungal Morphology 5.1 Introduction 5.2 Factors affecting Aspergillus niger morphology in submerged culture 5.3 Effect of agitation 5.4 Effect of nutritional factors 5.5 Effect of inoculum 5.6 Conclusions and perspectives 5.7 References

69 69 69 70 74 82 82 82

6 Redox Potential in Submerged Citric Acid Fermentation Nomenclature 6.1 Introduction 6.2 Overview 6.3 Theory 6.4 Measurement of redox potential 6.5 Significance of redox potential 6.6 Redox potential in citric acid fermentation 6.7 Regulation of the redox potential 6.8 Regulation of redox potential in citric acid fermentation 6.9 Scale-up based on redox potential 6.10 Conclusions 6.11 References

85 85 85 86 87 88 89 91 95 95 101 102 103

7 Modelling the Fermentation Process 7.1 Introduction 7.2 Aspergillus based models 7.3 Yeast based models 7.4 Conclusion 7.5 References

105 105 107 113 119 119

8 Mass and Energy Balance Nomenclature 8.1 Introduction 8.2 Metabolic description of A. niger growth 8.3 Mass and energy balances 8.4 Kinetics of growth and citric acid production by A. niger 8.5 Carbon and available electron balances 8.6 Conclusion 8.7 References

121 121 122 123 125 128 130 131 132

9 Downstream Processing in Citric Acid Production 9.1 Pretreatment of fermentation broth 9.2 Precipitation

135 135 136

Contents

9.3 9.4 9.5 9.6 9.7 9.8 9.9

vii

Solvent extraction Adsorption, absorption and ion exchange Liquid membranes Electrodialysis Ultrafiltration Immobilization of micro-organisms References

139 142 143 144 145 146 146

10 Fermentation Substrates 10.1 Introduction 10.2 Molasses 10.3 Refined or raw sucrose 10.4 Syrups 10.5 Starch 10.6 Hydrol 10.7 Alkanes 10.8 Oils and fats 10.9 Cellulose 10.10 Other medium redients 10.11 Conclusion 10.12 References

149 149 150 156 156 157 157 157 158 158 158 159 159

11 Design of an Industrial Plant Nomenclature 11.1 Design of an industrial plant 11.2 Data required 11.3 Design basis 11.4 Scope definition 11.5 Process package 11.6 Raw material 11.7 Substrate preparation 11.8 Fermentation 11.9 Design of a stirred tank reactor 11.10 Airlift and bubble column reactors 11.11 Product isolation 11.12 Cell removal 11.13 Purification 11.14 Crystallization stages 11.15 Product packaging 11.16 Effluent and by-products 11.17 In conclusion 11.18 References

161 161 163 163 165 167 167 169 169 170 171 174 176 177 178 182 183 183 183 184

Index

187

Contributors

Ho Ai Meng Amy Blk 135 Pasir Ris Street 11, # 06-239, Singapore 510135

Marin Berovic Department of Chemistry and Biochemical Engineering, National Chemistry Laboratory for Biotechnology and Industrial Mycology, 1115 Slo, Ljubljana, Hajdrihova 19 POB 30, Slovenia

Pawel Gluszca Department of Bioprocess Engineering, Technical University of Lodz, Wolczanska 175 90-924 Lodz, Poland

Bjørn Kristiansen Borregaard Industries Ltd, PO Box 162, 1701 Sarpsborg, Norway

Liliana Krzystek Department of Bioprocess Engineering, Technical University of Lodz, Wolczanska 175 90-924 Lodz, Poland

Christian Kubicek Institute for Biochemical Technology and Microbiology, University of Technology Getreidemarkt 9/1725, A-1060 Wien, Austria

x

Contributors

Staniskaw Ledakowicz Department of Bioprocess Engineering, Technical University of Lodzul, Wolczanska 175 90-924 Lodz, Poland

Wladyslaw Lesniak Food Biotechnology Department, Academy of Economics, Komandorska 118/120 PL 53345 Wroclaw, Poland

Michael Mattey Department of Bioscience and Biotechnology, University of Strathclyde, Todd Centre 33 Taylor Street, Glasgow G4 0NR

Maria Papagianni 8 Kamvounion Street, 54 621 Thessaloniki, Greece

George Ruijter Section of Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands

Jacobus van der Merwe NCP, Project Engineering Division, PO Box 494, Germiston 1400, South Africa

Jaap Visser Section of Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands

Frank Wayman Department of Bioscience and Biotechnology, University of Strathclyde, Todd Centre 33 Taylor Street, Glasgow G4 0NR

Markus Wolschek Institute for Biochemical Technology and Microbiology, University of Technology Getreidemarkt 9/1725, A-1060 Wien, Austria

1

A Brief Introduction to Citric Acid Biotechnology MICHAEL MATTEY AND BJØRN KRISTIANSEN

1.1 Citric acid from lemons They are going to be squeezed, as a lemon is squeezed—until the pips squeak. My only doubt is not whether we can squeeze hard enough, but whether there is enough juice. (Sir Eric Geddes, 1918)

It is probably no more than a coincidence that Sir Eric Geddes uttered his now famous phrase at the time that the industrial production of citric acid by fungal fermentation was being developed to circumvent the high price and lack of availability of lemon juice. However, the association of taxation and squeezing lemons is appropriate, as the history of citric acid reflects the politics and economics of the era as well as the science. Indeed the production of citric acid is a ‘classical’ biotechnology phenomenon, where the science, though important, is secondary to the economics and politics of production. This book seeks to reflect that balance between practical science, fundamental understanding and economics. Citric acid derives its name from the Latin citrus, the citron tree, the fruit of which resembles a lemon. The acid was first isolated from lemon juice in 1784 by Carl Scheele, a Swedish chemist (1742–1786), who made a number of discoveries important to the advance of chemistry, amongst them hydrofluoric, tartaric, benzoic, arsenious, molybdic, lactic, citric, malic, oxalic, gallic and other acids as well as chlorine, oxygen (1772, published in English in 1780, predating the discovery by Priestly in 1774), glycerine and hydrogen sulphide. Citric acid was thus one amongst many natural organic acids. Citric acid was produced commercially from Italian lemons from about 1826 in England by John and Edmund Sturge, but with the increasing importance of citric acid as an item of commerce, production was started in Italy by the lemon growers, who established a virtual monopoly during the rest of the nineteenth century. Lemon juice remained the commercial source of citric acid until 1919 when the first industrial process using Aspergillus niger began in Belgium. Lemon juice itself remains an important product. World lemon production averages about 3.3 million metric tonnes (US Foreign Agricultural statistics); about 75 per cent comes from the United States, Italy, Spain and Argentina, with the rest from some 15 other producer countries. 1

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Citric Acid Biotechnology

Figure 1.1 Synthesis of citric acid

Marketing of lemons is the subject of political control both in Europe and the USA. In Europe the processing of lemons to juice carries a processing subsidy which makes it attractive to process the lemons rather than sell them as fresh produce; additionally the EU intervention mechanism results in significant quantities of lemons being destroyed. In the USA marketing is controlled by the United States Department of Agriculture (USDA) Lemon Administrative Committee which determines how many lemons will be sold into the fresh market and what growing areas will be allowed to sell them. The economic result of any monopoly tends to be to make the product expensive; without the spur of competition the control of costs, the development of the process and the efficiency of production are neglected. Citric acid in the nineteenth century was no exception; the Italian monopoly resulted in high prices that tempted the entrepreneurs of the era to seek alternative sources of the increasingly useful product. Unable to find an alternative botanical source of citric acid, the nineteenth century advances in chemistry and microbiology were examined. By the turn of the century both possibilities existed.

1.2 Synthetic citric acid Citric acid had been synthesized from glycerol by Grimoux and Adams (1880) and later from symmetrical dichloroacetone (i) by treating with hydrogen cyanide and hydrochloric acid to give dichloroacetonic acid (ii), and converting this into dicyano-acetonic acid (iii) with potassium cyanide, which on hydrolysis yields citric acid (iv), as shown in Figure 1.1. Several other routes using different starting materials have since been published. All chemical methods have so far proved uncompetitive or unsuitable, mainly on economic grounds, with the starting material worth more than the end product, although poor yields due to the number of reaction steps in the synthesis and precautions necessary when handling hazardous compounds involved have contributed to the problem.

1.3 Microbial citric acid The concept of microbiological action yielding useful products followed from Pasteur’s pioneering studies on fermentation and resulted in systematic investigations of fungi and bacteria. Amongst them Wehmer, in 1893, showed that a ‘Citromyces’ (now Penicillium) accumulated citric acid in a culture medium containing sugars and inorganic salts. This work did not lead directly to a commercial process but the subsequent search for other organisms capable of this synthesis did. Many other organisms were found to accumulate citric acid including strains of Aspergillus niger, A. awamori, A. fonsecaeus, A. luchensis, A. phoenicus, A. wentii, A. saitoi, A. lanosius, A. flavus, Absidia sp., Acremonium sp., Aschochyta

A brief introduction to citric acid biotechnology

3

sp., Botrytis sp., Eupenicillium sp., Mucor piriformis, Penicillium janthinellum, P. restrictum, Talaromyces sp., Trichoderma viride and Ustulina vulgaris. Currie (1917) found strains of A. niger that produced citric acid when cultured in media with low pH values, high sugar levels and mineral salts. Prior to this A. niger was known to produce oxalic acid; the key difference was the low pH which, as we now know, suppressed both the production of oxalic acid, which would be toxic, and gluconic acid, which has a significantly higher production rate from sugar than citric acid. Currie subsequently joined Chas. Pfizer & Co. Inc. and his discovery formed the basis of the citric acid plant established in the USA by the firm in 1923. This plant and the other similar processes established first in Belgium then in England by J.E.Sturge, in Czechoslovakia and in Germany in the next few years used the ‘surface process’. The details of this process are not well documented despite its long history, due in part to the restriction of information by manufacturers. In biotechnological terms, citric acid is known as a bulk, or low value, product. The market is, and always has been, very competitive, so the profit margins are small. Improvements in productivity depend on the detail of the various processes, many of which are not easily protected by patents, so that secrecy is important and understandable.

1.4 Citric acid by the surface method The general details of the original process are straightforward. The fungal mycelium is grown as a surface mat on a liquid medium in a large number of shallow trays with a capacity of 50 to 100 litres. Each tray has a surface area of about 5 m2 and a depth of between 5 and 20 cm. The trays are manufactured from high purity aluminium or stainless steel and usually can be lifted by just two men. The trays are stacked in racks in a chamber to allow operation under relatively aseptic conditions. Various sucrose sources were used initially but cane molasses and then beet molasses soon became the norm as the sugar source. The molasses are diluted to the required concentration, usually 15 per cent and the pH adjusted to 5–7. After sterilization, the medium is pumped into the trays and inoculation carried out directly from spores, either by adding a liquid suspension or by blowing the spores in with the air stream. Aerating the chambers is important for two purposes, oxygenation and heat removal. The air requirement depends on the stage of growth. Initially sterile air at low rates is used to prevent contamination during the germination stage, which takes about 12 hours. Later, when growth is maximal, rates of up to 10 m3 per cubic metre medium per minute are needed to ensure heat dispersal. The heat generation is considerable, around 1 kJ h-1 m-3 medium and the surface and medium temperatures are ideally around 28°C to 30°C. This high volume air is not necessarily sterile, as contamination is normally not a problem once the pH has fallen, after about 24 hours growth. The pH falls to about 2, or slightly lower, and remains at that level until the end of the process, hence the need for high-grade materials for the construction of the trays. The incoming air is humidified to 40–60 per cent to prevent moisture loss from the high surface area of the medium. Cultivation continues for 8 to 15 days, with the objective of minimizing the residence time to maximize the plant productivity. The details of time, productivity and yield are closely guarded secrets, but productivity of the order of 1 kg per square metre per day can be obtained and yield is up to 75 per cent of the initial sugar level. At the end of the process, which can be monitored by total acid production or judged by experience, the mycelial mat is removed by filtration and washed, as it contains up to 15 per cent of the total citric acid. The washings and spent medium are treated with lime (calcium hydroxide) at about 90°C to precipitate the insoluble tri-calcium tetrahydrate

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Citric Acid Biotechnology

salt of citric acid. It is not possible to crystallize the acid directly from the crude molasses medium although this can be done if pure sucrose is used as the carbon source. The precipitate of calcium citrate is washed and suspended in enough sulphuric acid to precipitate the calcium as calcium sulphate. This releases the citric acid into solution from where it can be treated further as required. The surface process, though commercially profitable for many years, is labour intensive and inefficient in its use of space; there is a limit as to how high a large tray can be lifted! The production of citric acid by surface culture was challenged at the beginning of the 1940s by the development of submerged fermentation processes. When Shu and Johnson published their work on the effect of medium ingredients and their concentrations on citric acid production in submerged culture, the fundamental technology for submerged production was ready to be exploited on an industrial scale (Shu and Johnson, 1948a, 1948b).

1.5 The submerged process for production of citric acid The submerged process has become the method of choice in the industrialized countries because it is less labour intensive, gives a higher production rate, and uses less space. Several designs of reactor have been used, particularly in pilot scale systems; the stirred tank reactor is the most common design although air-lift reactors, with a higher aspect ratio than the stirred tank reactor are also used. The reactors are constructed of high-grade stainless steel, an important requirement in view of the low pH levels developed, the ability of citric acid to solubilize metal ions and the presence of manganese in stainless steels. Inferior grades of steel have caused problems in the past, both of leaching and pitting or general corrosion. Industrial rumours suggest it may still happen though not by design! The empirical process of ‘conditioning’ a reactor, whereby a few batches are processed before optimal production levels are achieved, may be related to this problem. The other general requirement for reactors for citric acid production is the provision of aeration systems that can maintain a high dissolved oxygen level. With both tank and tower reactors sterile air is sparged from the base, although extra inputs are often used with tower reactors. The reactor may be held above atmospheric pressure to increase the rate of oxygen transfer into the fermentation broth. The influence of dissolved oxygen on citric acid formation has been examined and the dissolved oxygen levels are routinely monitored. The oxygen levels are also affected by the rheology of the broth. A typical plant will consist of four areas: medium preparation, reactor section, broth separation and product recovery. The medium preparation will involve dilution of the molasses, or other raw material, addition of nutrients and other pre-treatment such as ferrocyanide, and sterilization, either in-line or in the reactor. Where in-line sterilization is used the reactors are steam sterilized separately. It is usual to prepare an inoculum for the production reactor in a smaller reactor, in which the conditions may be modified to give rapid growth rather than product formation. Primary inoculation is by spores and the initial phase of the growth is critical. When a separate inoculum stage is used, the correct stage for transfer, characteristically between 18 and 30 hours, is judged by pH level. Production temperature, like the inoculum temperature, is about 30°C. The process is allowed to continue until the rate of citric acid production falls below a predetermined value, which is reached many hours before the production ceases altogether.

A brief introduction to citric acid biotechnology

5

Many reports suggest that the morphology of the mycelium is crucial to the ultimate yield; not only with respect to the shape of hyphae, but also their aggregation. Several studies suggest that hyphae should be abnormally short, bulbous and heavily branched. It is recognized that this condition is brought about by manganese deficiency or related to the addition of ferrocyanide, which is probably the same thing. The mycelium should also form small (less than 0.5 mm) pellets with a smooth, hard surface. Such pellets are produced when a number of factors are controlled, such as ferrocyanide levels, manganese levels, low iron (less than 1 ppm), low pH, control of aeration and agitation or the amount of spore inoculum. It is clear that this morphological appearance is not in itself necessary for a successful yield, but is a result of the correct process parameters. Pellet formation is not necessary, but does give a broth with a lower energy requirement for mixing. When a change to a filamentous growth type occurs, the dissolved oxygen level may fall by 50 per cent for a fixed input. That filamentous growth can give satisfactory yields has been demonstrated and consideration of the diffusion characteristics of pellets versus filamentous mycelium would suggest that while yields may be similar, productivity should be greater without the additional diffusional constraint of pellets. Aeration is a significant factor in the cost of the process, and although a constant aeration rate is used in many laboratory scale studies, the industrial practice is to use relatively low aeration rates initially (0.1 vvm) rising to 0.5–1 vvm as growth proceeds. Such aeration rates will lead to foaming and various devices and agents are available to minimize the problem. Although very high yields are possible, the productivity is a more important consideration on an industrial basis, and it is rare that the process is allowed to continue to the maximum yield. The processes run today owes much to the pioneering work carried out by D.S.Clark and his co-workers at the Northern Regional Research Laboratories in Canada during the 1950s and early 1960s. Here, the technology for large-scale production of citric acid with A. niger using molasses was established. After the fermentation characteristics were worked out, attention was given to the controlling mechanisms of the fermentation. Numerous reports have been published on the role of metal ions on the citric acid cycle, in particular. After decades of academic discussion, there is general agreement about the factors that regulate the fermentation and give rise to the high yields obtained in industry (Mattey, 1992).

1.6 Continuous and immobilized processes A process for continuous production of citric acid has been described (Kristiansen and Sinclair, 1979), but no commercial application of this has been made in spite of the high productivity values obtained (Kristiansen and Charley, 1981). The process does not use the carbon source as the limiting substrate so that excess sugar will pass out of the reactor. As the carbohydrate substrate is one of the major cost factors, the continuous process will be less efficient than the batch process. This might be overcome by using several reactors in series, but this offsets any advantage from the continuous process. Fed-batch processes have been used industrially so that the conversion of sugar concentrations greater than 15 per cent can be achieved, but the gain does not seem to be sufficient to allow the fed-batch method to become standard. The possibility of using the mycelium in an immobilized system has occurred to several workers and attempts on a small scale have been reported. Immobilization of

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Citric Acid Biotechnology

mycelium in alginate beads or collagen proved possible, but with very low production rates. The difficulties of avoiding oxygen limitation when preparing beads, and preventing further growth, which reduces oxygen transfer rates, have led to the immobilization of conidia which are then grown under nitrogen limitation to the desired compact pellet. While giving a manageable system, the productivity was still too low to be of industrial interest. Other constructs for immobilization that have been more successful are the use of exchange filtration, and a rotating disc with an adhering mycelial film, reminiscent of sewage treatment techniques. These radical methods are unlikely to gain acceptance, even were they to give economic productivity gains, unless the engineering problems of scale-up can be overcome without making the capital costs too large.

1.7 Yeast based processes From about 1965 methods using yeasts were developed, first from carbohydrate sources, then from n-alkanes. At this time hydrocarbons were relatively cheap and plants were built to use the method. The economics have altered since then and plants that have been built to utilize both yeast technologies have apparently switched back to carbohydrate feedstocks. The potential advantages of using yeasts rather than filamentous fungi are the higher initial sugar concentrations that can be tolerated and the faster conversion rates possible. Further, the insensitivity to metal ions means that crude (and hence cheaper) grade molasses can be used without costly pre-treatment. Since 1968, when the patent for citric acid production from molasses by eight genera of yeasts was allowed, there have been many process modifications reported. Candida, Hansenula, Pichia, Debaromyces, Torulopsis, Kloekera, Trichosporon, Torula, Rhodotorula, Sporobolomyces, Endomyces, Nocardia, Nematospora, Saccharomyces, and Zygosaccharomyces species are known to produce citric acid from various carbon sources. Out of these genera the Candida species, including C. lipolytica, C. tropicalis, C. guillermondii, C. oleophila and C. intermedia have been used. The original process incorporated calcium carbonate into the medium to maintain a neutral pH, and generally a pH above 5.5 was used. Various additions have been proposed to reduce the isocitric acid contamination that afflicts yeasts even on carbohydrate media. Halogen substituted alkanoic mono- or di-substituted acids, n-hexadecyl citric acid or trans-aconitic acid, and even lead acetate have been patented, despite the possibility of toxic residues in the resulting citric acid. Many mutants have been selected for reduced isocitrate production. An osmophilic strain, which would convert sugar concentrations as high as 28 per cent without pre-treatment of the molasses substrate, has been patented. Tower reactors of fairly standard design are used, but with improved cooling systems as the rate of heat production is high. A continuous process has been described where the pH is maintained at 3.5 with ammonium hydroxide. The industrial production of citric acid from n-alkanes is not now economic, although a plant was built, and operated, around 1970 at Saline, Reggio Calabria, Italy (Liquichimica). This process was based on a low aconitase mutant of C. lipolytica in a batch process with stirred, aerated tank reactors of 400 m3, operating on a 72 hour cycle. The conversion from alkanes was reported to exceed 130 per cent (by weight). The theoretical yield is 250 per cent, but part of the alkanes was converted to biomass and carbon dioxide. The yeast was removed by centrifugation and the purification was traditional. The medium used was based

A brief introduction to citric acid biotechnology

7

on the process developed for the yeast strain that had a substrate concentration of 10 per cent n-decane, although n-alkanes from 9 to 20 carbons could be used. The availability and cost of Libyan n-alkanes, which lead to the development of this and other plants, including the dual substrate plants, has changed over the last three decades. One unique feature of the n-alkane process is the insolubility of the substrate. To ensure a rapid conversion the nalkane has to be thoroughly dispersed, so additives such as polyoxypropylene glycol ether, at concentrations from 20 to 200 ppm, are used to enhance this.

1.8 The koji process A third method for the production of citric acid is the koji process, using Aspergillus species. This is the solid state equivalent of the surface process described previously. It was originally developed in Japan where it uses the readily available rice bran and fruit wastes. It is confined to south-east Asia and is a relatively small-scale process. The carbohydrate source, which is principally starch and cellulose, is sterilized by steaming and the resulting semi-solid paste (about 70 per cent water), at a pH of about 5.5, is inoculated by spraying on spores of A. niger. Additions of ferrocyanide or copper may be made. The incubation temperature is 30°C and the process takes about four to five days. Yields are low because of the difficulty of controlling trace metals and the process parameters. The fungus produces sufficient cellulases and amylases to break down the substrate, though the low yields may reflect the rate limitations of this step.

1.9 Uses of citric acid Citric acid is used in food, confectionery and beverages, in pharmaceuticals and in industrial fields. Its uses depend on three properties: acidity, flavour, and salt formation. Chemically citric acid is 2-hydroxy-1,2,3-propane tricarboxylic acid (77-92-9). It has three pKa values at pH 3.1, 4.7 and 6.4. As these three values are relatively close together the second H+ is appreciably dissociated before the first is completed, and similarly with the third. Because of this overlapping the solution is well buffered throughout the titration curve and there are no breaks from about pH 2 (the approximate pH of a 0.2M solution) to pH 7. Citric acid forms a wide range of metallic salts including complexes with copper, iron, manganese, magnesium and calcium. These salts are the reason for its use as a sequestering agent in industrial processes and as an anticoagulant blood preservative. It is also the basis of its antioxidant properties in fats and oils where it reduces metal-catalysed oxidation by chelating traces of metals such as iron. There are two components to its use as a flavouring: the first is due to its acidity, which has little aftertaste; the second to its ability to enhance other flavours. A process to remove sulphur dioxide from flue gases has been developed where citric acid is used as a scrubber, forming a complex ion which then reacts with H2S to give elemental sulphur, regenerating citrate. This may become more important with increased environmental pressures. Citric acid esters of a range of alcohols are known; the triethyl, butyl and acetyltributyl esters are used as plasticizers in plastic films and monostyryl citrate is used instead of citric acid as an antioxidant in oils and fats. A summary of the uses of citric acid is given in Table 1.1.

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Citric Acid Biotechnology

Table 1.1 Applications of citric acid

1.10 Effluent disposal Regardless of the method of production the disposal of waste is an increasing problem for manufacturers both from a cost and a regulatory viewpoint. Gypsum (calcium sulphate) is not valuable enough to purify and use in, for example, plaster. It may be disposed of to landfill sites, at a cost, and in some cases may be pumped out to sea, where tidal conditions permit. A more serious problem is the disposal of the filtrate from the precipitation where molasses has been used as a raw material; the waste is non-toxic, but has a high biological oxygen demand, so that it cannot be disposed of to rivers untreated. Anaerobic digestion, with fuel gas as a useful by-product, is probably the future method of choice, although animal feedstuff formulation in the form of condensed molasses solubles is another possibility. It can also be used as a medium for the growth of yeasts for animal feeds.

1.11 Conclusions Books must follow science, not science books. (Francis Bacon, Propositions touching Amendment of Laws)

For the last 80 years citric acid has been produced on an industrial scale by the fermentation of carbohydrates, initially exclusively by Aspergillus niger, but in recent times by Candida yeasts as well, with the proportion derived from the Candida process increasing. The higher productivity of the yeast-based process suggests it will be the method of choice for any new plants that may be built. The intimate knowledge about the large-scale fermentation and subsequent recovery processes are still regarded as industrial property. Nevertheless, the citric acid process is

A brief introduction to citric acid biotechnology

9

one of the rare examples of industrial fermentation technology where academic discoveries have worked in tandem with industrial know-how, in spite of an apparent lack of collaboration, to give rise to a very efficient fermentation process. The current world market for citric acid and its derivatives is difficult to estimate accurately; no international statistics are collected, but industry estimates suggest that upwards of 400 000 tonnes per year may be produced. Citric acid is a ‘mature product’ but the upward trend in its use seen over many years is an annual 2–3 per cent increase. The price is such that profit margins are low, and with significant, but erratic, quantities appearing on the world market from countries such as China the situation is unlikely to improve. The lemon, which started it all, is doing well, with an estimated world production of 3 to 4 million tonnes per year. Commercial varieties such as ‘Eureka’ are all high acid lemons, with the acid content exceeding 4.5 per cent by weight, so that some 140 000 tonnes of citric acid are still produced by lemons! The various themes touched on in this introduction are dealt with in greater depth in the following chapters.

1.12 References COOPER, W C and CHAPOT, H, 1977. Fruit Production—with special emphasis on fruit for processing. In Citrus Science and Technology, Vol. 2. Eds S Nagy, P E Shaw and M K Veldhuis (AVI Publishing Co., Westport, CT, USA). CURRIE, J N, 1917. The citric acid fermentation of A. niger, Journal of Biological Chemistry, 31, 5. GRIMOUX, E and ADAMS, P, 1880. Synthese de l’acide citrique, C.R.Hebd. Seances Acad. Sci., 90, 1252. KRISTIANSEN, B and CHARLEY, R C, 1981. The effect of medium composition on citric acid production in continuous culture, Presented at 2nd European Congress of Biotechnology, UK. KRISTIANSEN, B and SINCLAIR, C G, 1979. Production of citric acid in continuous culture, Biotechnology and Bioengineering, 21, 297. MATTEY, M, 1992. The production of organic acids, CRC Critical Reviews in Biotechnology, 12, 81. PASTEUR, L., 1875. Nouvelle observations sur la nature de la fermentation alcoolique, C.R. Acad. Sci., 80, 452. REUTHER, W, CALAVAN, E C and CARMAN, G E, 1967. The Citrus Industry, Vol. 1. History, World Distribution, Botany and Varieties. Univ. Calif. Div. Agric. Nat. Res., San Pablo, California. ROSENBAUM, J B, MCKINNEY, W A, BEARD, H L, CROCKER, L and NISSEN, W I, 1973. Sulphur Dioxide Emission Control by Hydrogen Sulphide Reaction in Aqueous Solution. The Citrate System. US Bureau of Mines, Report 1774. SCHEELE, C, 1793. Crells Ann. 2, 1 1784, from Sämmtliche Physische und Chemische Werke. Hermbstädt (Berlin). SHU, P and JOHNSON, M J, 1948a. Citric acid production submerged fermentation with Aspergillus niger, Industrial and Engineering Chemistry, 40, 1202. SHU, P and JOHNSON, M J, 1948b. The interdependence of medium constituents in citric acid production by submerged fermentation, Journal of Bacteriology, 54, 161. WEHMER, C, 1893. Note sur la fermentation Citrique, Bull. Soc. Chem. Fr, 9, 728.

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Biochemistry of Citric Acid Accumulation by Aspergillus niger MARKUS F.WOLSCHEK AND CHRISTIAN P.KUBICEK

2.1 Introduction The biochemical mechanism by which Aspergillus niger accumulates citric acid has attracted the interest of researchers since the late 1930s when the optimization of this accumulation to give a commercial process began. In this sense, the various theories which have been proposed to explain the accumulation of citric acid in such high yields also reflect the general biochemical knowledge at the time the respective research was done. In view of the high input into this research through more than 50 years it is therefore rather disappointing that there is still no explanation of the biochemical basis of this process which would consistently explain all the observed factors influencing this fermentation. Reasons for this are manifold. First, citric acid is only accumulated when several nutrient factors are present, either in excess (i.e. sugar concentration, H+, dissolved oxygen), or at suboptimal levels (trace metals, nitrogen and phosphate), and thus is subject to multifactorial influence. Hence it is unlikely that single biochemical events are solely responsible for citric acid overflow. Secondly, an appreciable part of the literature consists of work which has been performed using low or only moderately producing strains or by applying nutrient conditions not optimal for citric acid production, and while this may be justified for special reasons in individual cases, the respective results are not comparable to those obtained by others. Moreover, their significance for the understanding of the commercial citric acid fermentation is questionable. Thirdly, the biochemical knowledge of filamentous fungi is still significantly inferior to that of, for example, Saccharomyces cerevisiae or higher eukaryotes and, moreover, results from these sources cannot be uncritically transformed to filamentous fungi, which impedes a biochemically correct interpretation of results in several areas. Hence, although a considerable amount of basic biochemical research has been carried out with A. niger, the present state of understanding of the events relevant for citric acid accumulation (not to say production) is still a poorly resolved puzzle. This chapter attempts to draw the currently recognizable picture and to aid in the further fitting together of the other scattered bits and pieces. 11

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Citric Acid Biotechnology

2.2 Glucose catabolism in A. niger and its regulation 2.2.1 The citric acid biosynthetic pathway It is well known, since the famous tracer studies by Cleland and Johnson (1954), and Martin and Wilson (1951), that citric acid is mainly formed via the reactions of the glycolytic pathway. Like most other fungi Aspergillus spp. utilize glucose and other carbohydrates for energy and cell synthesis by channelling glucose into the reactions of the glycolytic and the pentose phosphate pathway, respectively. The pentose phosphate pathway accounts for only a minor fraction of metabolized carbon during citric acid fermentation, and this decreases throughout prolonged cultivation (Legisa and Mattey, 1986; Kubicek, unpublished data). Legisa and Mattey (1988) speculated that this may be due to inhibition of 6-phosphogluconate dehydrogenase by citrate, but evidence for this is lacking. It should be noted that both arabitol and erythritol are accumulated as by-products until late stages of the fermentation (Roehr et al., 1987); hence a complete blockage of the pentose phosphate pathway is obviously not taking place. A. niger possesses a further pathway of glucose catabolism which is catalyzed by glucose oxidase (Hayashi and Nakamura, 1981). This enzyme is induced by high concentrations of glucose and strong aeration in the presence of low concentrations of other nutrients (Mischak et al., 1985; Rogalski et al., 1988; Dronawat et al., 1995), conditions which are also typical for citric acid fermentation; glucose oxidase will hence inevitably be formed during the starting phase of citric acid fermentation and convert a significant amount of glucose into gluconic acid. However, due to the extracellular location of the enzyme, it is directly influenced by the external pH and will be inactivated at pH