citric acid biotechnology.pdf

Citric Acid Biotechnology

Citric AcidBiotechnology

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 3DFUSA 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 retrievalsystem, or transmitted in any form or by any means, electronic, electrostatic, magnetictape, mechanical, photocopying, recording or otherwise, without the prior permission ofthe copyright owner.

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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 ISBNISBN 0-203-79163-0 (Glassbook Format)

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Contents

1 A Brief Introduction to Citric Acid Biotechnology page 11.1 Citric acid from lemons 11.2 Synthetic citric acid 21.3 Microbial citric acid 21.4 Citric acid by the surface method 31.5 The submerged process for production of citric acid 41.6 Continuous and immobilized processes 51.7 Yeast based processes 61.8 The koji process 71.9 Uses of citric acid 71.10 Effluent disposal 81.11 Conclusions 81.12 References 9

2 Biochemistry of Citric Acid Accumulation by Aspergillus niger 112.1 Introduction 112.2 Glucose catabolism in A. niger and its regulation 122.3 Regulation of citric acid biosynthesis 192.4 Role of citrate breakdown in citrate accumulation 212.5 Export of citric acid from A. niger 242.6 References 25

3 Biochemistry of Citric Acid Production by Yeasts 333.1 Introduction 333.2 Synthesis of citric acid from n-alkanes 353.3 Synthesis of citric acid from glucose 463.4 Conclusions 503.5 References 50

4 Strain Improvement 554.1 Introduction 554.2 General aspects of strain improvement 55

Contentsvi

4.3 Isolation of recombinant strains using the parasexual cycle in A. niger 604.4 Genetic engineering 614.5 Concluding remarks 644.6 Acknowledgements 654.7 References 65

5 Fungal Morphology 695.1 Introduction 695.2 Factors affecting Aspergillus niger morphology in submerged culture 695.3 Effect of agitation 705.4 Effect of nutritional factors 745.5 Effect of inoculum 825.6 Conclusions and perspectives 825.7 References 82

6 Redox Potential in Submerged Citric Acid Fermentation 85Nomenclature 85

6.1 Introduction 856.2 Overview 866.3 Theory 876.4 Measurement of redox potential 886.5 Significance of redox potential 896.6 Redox potential in citric acid fermentation 916.7 Regulation of the redox potential 956.8 Regulation of redox potential in citric acid fermentation 956.9 Scale-up based on redox potential 1016.10 Conclusions 1026.11 References 103

7 Modelling the Fermentation Process 1057.1 Introduction 1057.2 Aspergillus based models 1077.3 Yeast based models 1137.4 Conclusion 1197.5 References 119

8 Mass and Energy Balance 121Nomenclature 121

8.1 Introduction 1228.2 Metabolic description of A. niger growth 1238.3 Mass and energy balances 1258.4 Kinetics of growth and citric acid production by A. niger 1288.5 Carbon and available electron balances 1308.6 Conclusion 1318.7 References 132

9 Downstream Processing in Citric Acid Production 1359.1 Pretreatment of fermentation broth 1359.2 Precipitation 136

Contents vii

9.3 Solvent extraction 1399.4 Adsorption, absorption and ion exchange 1429.5 Liquid membranes 1439.6 Electrodialysis 1449.7 Ultrafiltration 1459.8 Immobilization of micro-organisms 1469.9 References 146

10 Fermentation Substrates 14910.1 Introduction 14910.2 Molasses 15010.3 Refined or raw sucrose 15610.4 Syrups 15610.5 Starch 15710.6 Hydrol 15710.7 Alkanes 15710.8 Oils and fats 15810.9 Cellulose 15810.10 Other medium redients 15810.11 Conclusion 15910.12 References 159

11 Design of an Industrial Plant 161Nomenclature 161

11.1 Design of an industrial plant 16311.2 Data required 16311.3 Design basis 16511.4 Scope definition 16711.5 Process package 16711.6 Raw material 16911.7 Substrate preparation 16911.8 Fermentation 17011.9 Design of a stirred tank reactor 17111.10 Airlift and bubble column reactors 17411.11 Product isolation 17611.12 Cell removal 17711.13 Purification 17811.14 Crystallization stages 18211.15 Product packaging 18311.16 Effluent and by-products 18311.17 In conclusion 18311.18 References 184

Index 187

Contributors

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

Marin BerovicDepartment of Chemistry and Biochemical Engineering, National Chemistry Laboratoryfor Biotechnology and Industrial Mycology, 1115 Slo, Ljubljana, Hajdrihova 19 POB 30,Slovenia

Pawel GluszcaDepartment of Bioprocess Engineering, Technical University of Lodz, Wolczanska 17590-924 Lodz, Poland

Bjørn KristiansenBorregaard Industries Ltd, PO Box 162, 1701 Sarpsborg, Norway

Liliana KrzystekDepartment of Bioprocess Engineering, Technical University of Lodz, Wolczanska 17590-924 Lodz, Poland

Christian KubicekInstitute for Biochemical Technology and Microbiology, University of TechnologyGetreidemarkt 9/1725, A-1060 Wien, Austria

Staniskaw LedakowiczDepartment of Bioprocess Engineering, Technical University of Lodzul, Wolczanska 17590-924 Lodz, Poland

Wladyslaw LesniakFood Biotechnology Department, Academy of Economics, Komandorska 118/120 PL 53-345 Wroclaw, Poland

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

Maria Papagianni8 Kamvounion Street, 54 621 Thessaloniki, Greece

George RuijterSection of Molecular Genetics of Industrial Microorganisms, Wageningen AgriculturalUniversity, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands

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

Jaap VisserSection of Molecular Genetics of Industrial Microorganisms, Wageningen AgriculturalUniversity, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands

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

Markus WolschekInstitute for Biochemical Technology and Microbiology, University of TechnologyGetreidemarkt 9/1725, A-1060 Wien, Austria

Contributorsx

1

1

A Brief Introduction to Citric AcidBiotechnology

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 isnot 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 famousphrase at the time that the industrial production of citric acid by fungal fermentation wasbeing 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 acidreflects the politics and economics of the era as well as the science. Indeed the productionof 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 thatbalance between practical science, fundamental understanding and economics.

Citric acid derives its name from the Latin citrus, the citron tree, the fruit of whichresembles a lemon. The acid was first isolated from lemon juice in 1784 by Carl Scheele, aSwedish chemist (1742–1786), who made a number of discoveries important to the advanceof chemistry, amongst them hydrofluoric, tartaric, benzoic, arsenious, molybdic, lactic, citric,malic, oxalic, gallic and other acids as well as chlorine, oxygen (1772, published in Englishin 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 Englandby John and Edmund Sturge, but with the increasing importance of citric acid as an item ofcommerce, production was started in Italy by the lemon growers, who established a virtualmonopoly during the rest of the nineteenth century. Lemon juice remained the commercialsource of citric acid until 1919 when the first industrial process using Aspergillus nigerbegan in Belgium.

Lemon juice itself remains an important product. World lemon production averages about3.3 million metric tonnes (US Foreign Agricultural statistics); about 75 per cent comesfrom the United States, Italy, Spain and Argentina, with the rest from some 15 other producercountries.

Citric Acid Biotechnology2

Marketing of lemons is the subject of political control both in Europe and the USA. InEurope the processing of lemons to juice carries a processing subsidy which makes it attractiveto process the lemons rather than sell them as fresh produce; additionally the EU interventionmechanism results in significant quantities of lemons being destroyed. In the USA marketingis controlled by the United States Department of Agriculture (USDA) Lemon AdministrativeCommittee which determines how many lemons will be sold into the fresh market and whatgrowing areas will be allowed to sell them.

The economic result of any monopoly tends to be to make the product expensive; withoutthe spur of competition the control of costs, the development of the process and the efficiencyof production are neglected. Citric acid in the nineteenth century was no exception; theItalian monopoly resulted in high prices that tempted the entrepreneurs of the era to seekalternative sources of the increasingly useful product. Unable to find an alternative botanicalsource of citric acid, the nineteenth century advances in chemistry and microbiology wereexamined. 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 laterfrom symmetrical dichloroacetone (i) by treating with hydrogen cyanide and hydrochloricacid 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. Allchemical methods have so far proved uncompetitive or unsuitable, mainly on economicgrounds, with the starting material worth more than the end product, although poor yieldsdue to the number of reaction steps in the synthesis and precautions necessary when handlinghazardous compounds involved have contributed to the problem.

1.3 Microbial citric acid

The concept of microbiological action yielding useful products followed from Pasteur’spioneering studies on fermentation and resulted in systematic investigations of fungi andbacteria. Amongst them Wehmer, in 1893, showed that a ‘Citromyces’ (now Penicillium)accumulated citric acid in a culture medium containing sugars and inorganic salts. Thiswork did not lead directly to a commercial process but the subsequent search for otherorganisms capable of this synthesis did. Many other organisms were found to accumulatecitric 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

Figure 1.1 Synthesis of citric acid

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 mediawith low pH values, high sugar levels and mineral salts. Prior to this A. niger was known toproduce oxalic acid; the key difference was the low pH which, as we now know, suppressedboth the production of oxalic acid, which would be toxic, and gluconic acid, which has asignificantly higher production rate from sugar than citric acid. Currie subsequently joinedChas. Pfizer & Co. Inc. and his discovery formed the basis of the citric acid plant establishedin the USA by the firm in 1923. This plant and the other similar processes established firstin Belgium then in England by J.E.Sturge, in Czechoslovakia and in Germany in the nextfew years used the ‘surface process’. The details of this process are not well documenteddespite its long history, due in part to the restriction of information by manufacturers. Inbiotechnological 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 inproductivity depend on the detail of the various processes, many of which are not easilyprotected 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 isgrown as a surface mat on a liquid medium in a large number of shallow trays with acapacity of 50 to 100 litres. Each tray has a surface area of about 5 m2 and a depth ofbetween 5 and 20 cm. The trays are manufactured from high purity aluminium or stainlesssteel and usually can be lifted by just two men. The trays are stacked in racks in a chamberto allow operation under relatively aseptic conditions. Various sucrose sources were usedinitially but cane molasses and then beet molasses soon became the norm as the sugarsource. The molasses are diluted to the required concentration, usually 15 per cent andthe pH adjusted to 5–7. After sterilization, the medium is pumped into the trays andinoculation carried out directly from spores, either by adding a liquid suspension or byblowing the spores in with the air stream. Aerating the chambers is important for twopurposes, oxygenation and heat removal. The air requirement depends on the stage ofgrowth. Initially sterile air at low rates is used to prevent contamination during thegermination stage, which takes about 12 hours. Later, when growth is maximal, rates ofup to 10 m3 per cubic metre medium per minute are needed to ensure heat dispersal. Theheat generation is considerable, around 1 kJ h-1 m-3 medium and the surface and mediumtemperatures are ideally around 28°C to 30°C. This high volume air is not necessarilysterile, as contamination is normally not a problem once the pH has fallen, after about 24hours growth. The pH falls to about 2, or slightly lower, and remains at that level until theend of the process, hence the need for high-grade materials for the construction of thetrays. The incoming air is humidified to 40–60 per cent to prevent moisture loss from thehigh surface area of the medium. Cultivation continues for 8 to 15 days, with the objectiveof 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 kgper square metre per day can be obtained and yield is up to 75 per cent of the initial sugarlevel. At the end of the process, which can be monitored by total acid production orjudged by experience, the mycelial mat is removed by filtration and washed, as it containsup to 15 per cent of the total citric acid. The washings and spent medium are treated withlime (calcium hydroxide) at about 90°C to precipitate the insoluble tri-calcium tetrahydrate


salt of citric acid. It is not possible to crystallize the acid directly from the crude molassesmedium although this can be done if pure sucrose is used as the carbon source. Theprecipitate of calcium citrate is washed and suspended in enough sulphuric acid toprecipitate the calcium as calcium sulphate. This releases the citric acid into solutionfrom where it can be treated further as required.

The surface process, though commercially profitable for many years, is labour intensiveand 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 the1940s by the development of submerged fermentation processes. When Shu and Johnsonpublished their work on the effect of medium ingredients and their concentrations oncitric acid production in submerged culture, the fundamental technology for submergedproduction 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 countriesbecause it is less labour intensive, gives a higher production rate, and uses less space. Severaldesigns of reactor have been used, particularly in pilot scale systems; the stirred tank reactoris the most common design although air-lift reactors, with a higher aspect ratio than thestirred 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 tosolubilize metal ions and the presence of manganese in stainless steels. Inferior grades ofsteel 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 processof ‘conditioning’ a reactor, whereby a few batches are processed before optimal productionlevels are achieved, may be related to this problem.

The other general requirement for reactors for citric acid production is the provision ofaeration systems that can maintain a high dissolved oxygen level. With both tank and towerreactors sterile air is sparged from the base, although extra inputs are often used with towerreactors. The reactor may be held above atmospheric pressure to increase the rate of oxygentransfer into the fermentation broth. The influence of dissolved oxygen on citric acidformation has been examined and the dissolved oxygen levels are routinely monitored. Theoxygen levels are also affected by the rheology of the broth.

A typical plant will consist of four areas: medium preparation, reactor section, brothseparation and product recovery. The medium preparation will involve dilution of themolasses, or other raw material, addition of nutrients and other pre-treatment such asferrocyanide, and sterilization, either in-line or in the reactor. Where in-line sterilization isused the reactors are steam sterilized separately. It is usual to prepare an inoculum for theproduction reactor in a smaller reactor, in which the conditions may be modified to giverapid growth rather than product formation. Primary inoculation is by spores and the initialphase of the growth is critical.

When a separate inoculum stage is used, the correct stage for transfer, characteristicallybetween 18 and 30 hours, is judged by pH level. Production temperature, like the inoculumtemperature, is about 30°C. The process is allowed to continue until the rate of citric acidproduction falls below a predetermined value, which is reached many hours before theproduction ceases altogether.


Many reports suggest that the morphology of the mycelium is crucial to the ultimateyield; not only with respect to the shape of hyphae, but also their aggregation. Severalstudies suggest that hyphae should be abnormally short, bulbous and heavily branched. It isrecognized that this condition is brought about by manganese deficiency or related to theaddition of ferrocyanide, which is probably the same thing. The mycelium should also formsmall (less than 0.5 mm) pellets with a smooth, hard surface. Such pellets are producedwhen a number of factors are controlled, such as ferrocyanide levels, manganese levels, lowiron (less than 1 ppm), low pH, control of aeration and agitation or the amount of sporeinoculum.

It is clear that this morphological appearance is not in itself necessary for a successfulyield, but is a result of the correct process parameters. Pellet formation is not necessary, butdoes give a broth with a lower energy requirement for mixing. When a change to a filamentousgrowth 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 considerationof the diffusion characteristics of pellets versus filamentous mycelium would suggest thatwhile yields may be similar, productivity should be greater without the additional diffusionalconstraint of pellets.

Aeration is a significant factor in the cost of the process, and although a constant aerationrate is used in many laboratory scale studies, the industrial practice is to use relatively lowaeration rates initially (0.1 vvm) rising to 0.5–1 vvm as growth proceeds. Such aerationrates will lead to foaming and various devices and agents are available to minimize theproblem. Although very high yields are possible, the productivity is a more importantconsideration on an industrial basis, and it is rare that the process is allowed to continue tothe maximum yield.

The processes run today owes much to the pioneering work carried out by D.S.Clarkand his co-workers at the Northern Regional Research Laboratories in Canada during the1950s and early 1960s. Here, the technology for large-scale production of citric acid withA. niger using molasses was established. After the fermentation characteristics were workedout, attention was given to the controlling mechanisms of the fermentation. Numerousreports 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 thatregulate 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 andSinclair, 1979), but no commercial application of this has been made in spite of the highproductivity values obtained (Kristiansen and Charley, 1981). The process does not use thecarbon source as the limiting substrate so that excess sugar will pass out of the reactor. Asthe carbohydrate substrate is one of the major cost factors, the continuous process will beless efficient than the batch process. This might be overcome by using several reactors inseries, but this offsets any advantage from the continuous process.

Fed-batch processes have been used industrially so that the conversion of sugarconcentrations greater than 15 per cent can be achieved, but the gain does not seem to besufficient to allow the fed-batch method to become standard.

The possibility of using the mycelium in an immobilized system has occurred toseveral workers and attempts on a small scale have been reported. Immobilization of


mycelium in alginate beads or collagen proved possible, but with very low productionrates. The difficulties of avoiding oxygen limitation when preparing beads, andpreventing further growth, which reduces oxygen transfer rates, have led to theimmobilization of conidia which are then grown under nitrogen limitation to the desiredcompact pellet. While giving a manageable system, the productivity was still too low tobe of industrial interest.

Other constructs for immobilization that have been more successful are the use of exchangefiltration, and a rotating disc with an adhering mycelial film, reminiscent of sewage treatmenttechniques. These radical methods are unlikely to gain acceptance, even were they to giveeconomic productivity gains, unless the engineering problems of scale-up can be overcomewithout 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 werebuilt to use the method. The economics have altered since then and plants that have beenbuilt to utilize both yeast technologies have apparently switched back to carbohydratefeedstocks.

The potential advantages of using yeasts rather than filamentous fungi are the higherinitial 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 molassescan be used without costly pre-treatment. Since 1968, when the patent for citric acidproduction from molasses by eight genera of yeasts was allowed, there have been manyprocess modifications reported. Candida, Hansenula, Pichia, Debaromyces, Torulopsis,Kloekera, Trichosporon, Torula, Rhodotorula, Sporobolomyces, Endomyces, Nocardia,Nematospora, Saccharomyces, and Zygosaccharomyces species are known to producecitric acid from various carbon sources. Out of these genera the Candida species, includingC. lipolytica, C. tropicalis, C. guillermondii, C. oleophila and C. intermedia have beenused.

The original process incorporated calcium carbonate into the medium to maintain a neutralpH, and generally a pH above 5.5 was used. Various additions have been proposed to reducethe isocitric acid contamination that afflicts yeasts even on carbohydrate media. Halogensubstituted alkanoic mono- or di-substituted acids, n-hexadecyl citric acid or trans-aconiticacid, and even lead acetate have been patented, despite the possibility of toxic residues inthe 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 centwithout pre-treatment of the molasses substrate, has been patented.

Tower reactors of fairly standard design are used, but with improved cooling systems asthe rate of heat production is high. A continuous process has been described where the pHis maintained at 3.5 with ammonium hydroxide.

The industrial production of citric acid from n-alkanes is not now economic, although aplant 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 withstirred, aerated tank reactors of 400 m3, operating on a 72 hour cycle. The conversion fromalkanes was reported to exceed 130 per cent (by weight). The theoretical yield is 250 percent, but part of the alkanes was converted to biomass and carbon dioxide. The yeast wasremoved by centrifugation and the purification was traditional. The medium used was based


on the process developed for the yeast strain that had a substrate concentration of 10 percent n-decane, although n-alkanes from 9 to 20 carbons could be used. The availability andcost of Libyan n-alkanes, which lead to the development of this and other plants, includingthe dual substrate plants, has changed over the last three decades. One unique feature of then-alkane process is the insolubility of the substrate. To ensure a rapid conversion the n-alkane 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 originallydeveloped in Japan where it uses the readily available rice bran and fruit wastes. It is confinedto south-east Asia and is a relatively small-scale process. The carbohydrate source, which isprincipally 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 is30°C and the process takes about four to five days. Yields are low because of the difficultyof controlling trace metals and the process parameters. The fungus produces sufficientcellulases and amylases to break down the substrate, though the low yields may reflect therate limitations of this step.

1.9 Uses of citric acid

Citric acid is used in food, confectionery and beverages, in pharmaceuticals and inindustrial 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 hasthree pKa values at pH 3.1, 4.7 and 6.4. As these three values are relatively close togetherthe second H+ is appreciably dissociated before the first is completed, and similarly withthe third. Because of this overlapping the solution is well buffered throughout the titrationcurve 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 sequesteringagent in industrial processes and as an anticoagulant blood preservative. It is also the basisof its antioxidant properties in fats and oils where it reduces metal-catalysed oxidation bychelating 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 enhanceother flavours.

A process to remove sulphur dioxide from flue gases has been developed where citricacid is used as a scrubber, forming a complex ion which then reacts with H2S to give elementalsulphur, regenerating citrate. This may become more important with increased environmentalpressures.

Citric acid esters of a range of alcohols are known; the triethyl, butyl and acetyltributylesters are used as plasticizers in plastic films and monostyryl citrate is used instead of citric acidas an antioxidant in oils and fats. A summary of the uses of citric acid is given in Table 1.1.


1.10 Effluent disposal

Regardless of the method of production the disposal of waste is an increasing problemfor manufacturers both from a cost and a regulatory viewpoint. Gypsum (calciumsulphate) is not valuable enough to purify and use in, for example, plaster. It may bedisposed 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 filtratefrom the precipitation where molasses has been used as a raw material; the waste isnon-toxic, but has a high biological oxygen demand, so that it cannot be disposed of torivers untreated. Anaerobic digestion, with fuel gas as a useful by-product, is probablythe future method of choice, although animal feedstuff formulation in the form ofcondensed molasses solubles is another possibility. It can also be used as a medium forthe 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 fermentationof carbohydrates, initially exclusively by Aspergillus niger, but in recent times by Candidayeasts as well, with the proportion derived from the Candida process increasing. The higherproductivity of the yeast-based process suggests it will be the method of choice for any newplants that may be built.

The intimate knowledge about the large-scale fermentation and subsequent recoveryprocesses are still regarded as industrial property. Nevertheless, the citric acid process is

Table 1.1 Applications of citric acid


one of the rare examples of industrial fermentation technology where academic discoverieshave 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 estimateaccurately; no international statistics are collected, but industry estimates suggest that upwardsof 400 000 tonnes per year may be produced. Citric acid is a ‘mature product’ but theupward 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, quantitiesappearing on the world market from countries such as China the situation is unlikely toimprove.

The lemon, which started it all, is doing well, with an estimated world production of 3 to4 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 ofcitric acid are still produced by lemons!

The various themes touched on in this introduction are dealt with in greater depth in thefollowing 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 PublishingCo., 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. TheCitrate 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 Aspergillusniger, Industrial and Engineering Chemistry, 40, 1202.

SHU, P and JOHNSON, M J, 1948b. The interdependence of medium constituents in citric acidproduction by submerged fermentation, Journal of Bacteriology, 54, 161.

WEHMER, C, 1893. Note sur la fermentation Citrique, Bull. Soc. Chem. Fr, 9, 728.

11

2

Biochemistry of Citric AcidAccumulation by Aspergillus niger

MARKUS F.WOLSCHEK AND CHRISTIAN P.KUBICEK

2.1 Introduction

The biochemical mechanism by which Aspergillus niger accumulates citric acid hasattracted the interest of researchers since the late 1930s when the optimization of thisaccumulation to give a commercial process began. In this sense, the various theorieswhich have been proposed to explain the accumulation of citric acid in such high yieldsalso reflect the general biochemical knowledge at the time the respective research wasdone. In view of the high input into this research through more than 50 years it is thereforerather disappointing that there is still no explanation of the biochemical basis of thisprocess which would consistently explain all the observed factors influencing thisfermentation. Reasons for this are manifold. First, citric acid is only accumulated whenseveral nutrient factors are present, either in excess (i.e. sugar concentration, H+, dissolvedoxygen), or at suboptimal levels (trace metals, nitrogen and phosphate), and thus is subjectto multifactorial influence. Hence it is unlikely that single biochemical events are solelyresponsible for citric acid overflow.

Secondly, an appreciable part of the literature consists of work which has been performedusing low or only moderately producing strains or by applying nutrient conditions not optimalfor citric acid production, and while this may be justified for special reasons in individualcases, the respective results are not comparable to those obtained by others. Moreover, theirsignificance for the understanding of the commercial citric acid fermentation is questionable.

Thirdly, the biochemical knowledge of filamentous fungi is still significantly inferior tothat of, for example, Saccharomyces cerevisiae or higher eukaryotes and, moreover, resultsfrom these sources cannot be uncritically transformed to filamentous fungi, which impedesa biochemically correct interpretation of results in several areas. Hence, although aconsiderable amount of basic biochemical research has been carried out with A. niger, thepresent state of understanding of the events relevant for citric acid accumulation (not to sayproduction) is still a poorly resolved puzzle.

This chapter attempts to draw the currently recognizable picture and to aid in the furtherfitting together of the other scattered bits and pieces.


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 Martinand Wilson (1951), that citric acid is mainly formed via the reactions of the glycolyticpathway. Like most other fungi Aspergillus spp. utilize glucose and other carbohydrates forenergy and cell synthesis by channelling glucose into the reactions of the glycolytic and thepentose phosphate pathway, respectively. The pentose phosphate pathway accounts for onlya minor fraction of metabolized carbon during citric acid fermentation, and this decreasesthroughout prolonged cultivation (Legisa and Mattey, 1986; Kubicek, unpublished data).Legisa and Mattey (1988) speculated that this may be due to inhibition of 6-phosphogluconatedehydrogenase by citrate, but evidence for this is lacking. It should be noted that botharabitol 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 isobviously not taking place.

A. niger possesses a further pathway of glucose catabolism which is catalyzed by glucoseoxidase (Hayashi and Nakamura, 1981). This enzyme is induced by high concentrations ofglucose and strong aeration in the presence of low concentrations of other nutrients (Mischaket al., 1985; Rogalski et al., 1988; Dronawat et al., 1995), conditions which are also typicalfor citric acid fermentation; glucose oxidase will hence inevitably be formed during thestarting phase of citric acid fermentation and convert a significant amount of glucose intogluconic acid. However, due to the extracellular location of the enzyme, it is directlyinfluenced by the external pH and will be inactivated at pH <3.5 (Mischak et al., 1985;Roukas and Harvey, 1988). Because of the pKa values for citric acid, its accumulationdecreases the pH of the culture filtrate to pH 1.8 thereby inactivating glucose oxidase(Mischak et al., 1985). It is not known if, and by which mechanism gluconic acid can becatabolized to citric acid during further fermentation.

The catabolism of glucose via glycolytic catabolism leads to 2 moles of pyruvate, andtheir subsequent conversion to the precursors of citrate (i.e. oxaloacetate and pyruvate).Cleland and Johnson (1954) were the first to show that A. niger uses 1 mole of the carbondioxide which is released during the formation of acetyl-CoA and 1 mole of pyruvate toform 1 mole of oxaloacetate (Figure 2.1a). This reaction is of utmost importance to highcitric acid yields, because oxaloacetate could otherwise only be formed by one turn of thetricarboxylic acid cycle, which would be accompanied by the loss of two moles of CO2 andonly two thirds of the carbon of glucose could therefore accumulate as citric acid (Figure2.1b). The enzyme catalyzing this reaction was shown to be pyruvate carboxylase (Woronickand Johnson, 1960; Bloom and Johnson, 1962), which was characterized by Feir andSuzuki (1969) and Wongchai and Jefferson (1974). Unlike the enzyme from several othereukaryotes, the pyruvate carboxylase of A. niger is localized in the cytosol (Bercovitz etal., 1990; Jaklitsch et al., 1991). Glycolytic pyruvate will therefore be converted tooxaloacetate, and further to malate by the cytosolic malate dehydrogenase isoenzyme(Ma et al., 1981), thereby also regenerating 50 per cent of the glycolytically producedNADH (cf. Figure 2.2). It has been postulated (Kubicek, 1988) that, analogous to highereukaryotes, the cytosolic malate may serve as the co-substrate of the mitochondrialtricarboxylic acid carrier, and that such an enhanced malate concentration may stimulateexport of citrate from the mitochondrion. It should be noted that the fixation of carbondioxide, while convincing and experimentally verified, does not seem to occur during theearly phases of fermentation: Kubicek et al. (1979b), by continuously quantifying carbon

Biochemistry of citric acid accumulation by A. niger 13

dioxide and oxygen in the exit air of a pilot plant citric acid fermentation, observed thatduring the first 70 hours of fermentation the respiratory coefficient (i.e. CO2 released/O2

taken up) is close to 1; it starts to decrease thereafter and reaches the level predicted fromthe operation of the pyruvate carboxylase reaction (0.66) only at stages where citrateaccumulation is already taking place at a constant rate (e.g. <120 hours). The Cleland andJohnson reaction may therefore only be important at later stages of fermentation, whereasthe initial phase of citric acid accumulation takes place without anaplerotic carbon dioxidesupply.

Although not directly within the topic of this chapter, it should be noted that A. niger isalso capable of accumulating another organic acid—oxalic acid—as a (toxic) byproduct ofcitric acid fermentation. The biosynthesis of this compound is controversial (Müller, 1975;Müller and Frosch, 1975; Kubicek et al., 1988), and appears to depend on whether glucoseor citric acid is used as the carbon source. In the latter case, the glyoxalate cycle has beenimplicated in its biosynthesis (Müller, 1975). Its biosynthesis on glucose as a carbon sourceoccurs by the hydrolysis of oxaloacetate catalyzed by oxaloacetate hydrolase, which iscytosolically located and appears to act as a valve by which the carbon overflow can bechanelled into an energetically neutral pathway (Figure 2.3) and so compete with citrateoverproduction (Kubicek, 1988). Although production of oxalate is, because of its toxicity,of considerable interest to citric acid fermentation, the regulation of its biosynthesis iscontroversial (Kubicek et al., 1988; Strasser et al., 1994).

Figure 2.1 Metabolic pathways from glucose to citric acid by (a) involvement of ananaplerotic carbon dioxide fixation (Cleland and Johnson, 1954), and (b) sole involvement ofthe citric acid cycle. Only relevant intermediates are given, and arrows may indicate morethan a single enzymatic step. Note that in (b), each of the two acetyl-CoA molecules issubject to one turn of the tricarboxylic acid cycle.

Figure 2.2 Metabolic and regulatory network of citric acid biosynthesis from sucrose in A.niger. For convenience, sucrose is assumed to be split into glucose and fructose by invertaseextracellularly (Boddy et al., 1993; Rubio and Maldonado, 1995) and only themonosaccharides are taken up. The double line indicates the plasma membrane, the hatcheddouble line the mitochondrial membrane. Circles inserted into the membranes indicateknown or assumed transport steps (hatched: characterized in A. niger; full: assumed, but notyet characterized; empty: countertransport, to be verified). Thick lines and arrows indicatemetabolic reactions; thin lines and arrows indicate regulatory interactions (*activation: //inhibition). Intermediates of regulatory importance are boxed.


2.2.2 Transcriptional regulation of the citric acid synthesizing pathway

It is uncertain to what extent the apparently high flux through the glycolytic pathway, whichis obviously necessary for citric acid accumulation, requires an activation of transcriptionof the genes encoding glycolytic and other enzymes (e.g. citrate synthase). The quantificationof enzyme activities in cell-free extracts of A. niger mutants, which were selected accordingto a reduced lag in growth on high sucrose concentrations and correspondingly increasedrates of citric acid accumulation, revealed enhanced hexokinase and phosphofructokinaseactivities (Schreferl-Kunar et al., 1989). Also, a class of A. niger mutants, resistant to 2-desoxyglucose and displaying reduced hexokinase activity, exhibited decreased rates ofcitric acid production (Fiedurek et al., 1988; Kirimura et al., 1992; Steinböck et al., 1994).Torres et al. (1996a) showed that high glucose concentrations (>50 g/l) are a prerequisitefor the formation of a low-affinity glucose transporter. However, knowledge of thetranscriptional regulation of the respective genes is still lacking.

Only preliminary data are as yet available to understand whether an enhancement oftranscription of selected glycolytic genes would increase the rate of citric acid accumulation.Ruijter et al. (1996b) have—selectively and in combination—amplified the genes encodingphosphofructokinase 1 (pfkA) and pyruvate kinase (pkiA), but the rates of citrate accumulationby the moderately citric acid producing strain used (N400) were not increased. Torres (1994a,1994b), using the biochemical system theory and a constrained linear optimization method,calculated that the activities (Vmax) of at least seven glycolytic enzymes must besimultaneously increased to obtain an effect. Clearly, such an increase can only be achievedby appropriate manipulation of the transcription factors regulating the genes encoding theenzymes for citric acid biosynthesis.

Unfortunately, transcriptional regulation of glycolytic genes has not yet been studied insufficient detail in A. niger nor in any related fungus. In Saccharomyces cerevisiae, mutationsin the GCR1 gene, which encodes a DNA-binding protein (Baker, 1986, 1991), were foundto exhibit strongly reduced levels of most glycolytic enzymes. Another protein, GCR2, wasshown to interact physically with GCR1 (Uemura and Jigami, 1992). The authors proposedthat both factors co-operate together in a transcriptional activation complex. Further factorsinvolved in the regulation of the glycolytic genes have been described in yeast (RAP1,REB1, ABF1; Brindle et al., 1990; Chambers et al., 1990; McNeil et al., 1990; Huie et al.,1992). GCR1-binding sites are generally located near RAP1-binding sites (Huie et al., 1992).Furthermore, several glycolytic genes contain consensus binding sites for binding of ABF1and REB1 in the vicinity of RAP1- and GCR1-binding sites (Brindle et al., 1990; Chamberset al., 1990; Chasman et al., 1990; Scott and Baker, 1993).

Figure 2.3 Pathway of oxalate biosynthesis by Aspergillus niger. Note that concentrations ofacetate corresponding to those of oxalate have not been detected in culture filtrates of A.niger, and the metabolism of acetate therefore requires further study


It is intriguing that the above named binding sites have so far not been detected in the 5'-noncoding sequences of the few glycolytic genes studied in A. niger or the close relativeAspergillus nidulans (Table 2.1, Figure 2.4). Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase (Punt et al., 1988, 1990, 1992) and of 3-phosphoglycerate kinase(Clements and Roberts, 1986; Streatfield et al., 1992) has been studied in some detail in theclosely related fungus A. nidulans. Its transcription depends on positive control by severalco-operating DNA-binding proteins since a truncated core promoter of the pgkA gene onlycontaining the CAAT, TATA and CT-rich elements could not trigger transcription. Punt etal. (1988) identified a ‘glycolytic box’ as responsible for transcription. No differences inexpression of gpdA were observed on 1% glucose or 0.1% fructose (Punt et al., 1990).

A 24-bp region, which shares 60 per cent similarity with the ‘glycolytic box’, is alsopresent at -638 and -488 of the pgkA promoter (Figure 2.4). However, another sequence,located between -161 and -120, in the pgkA promoter was shown to be essential for expressionof the respective gene. It consists of two non-overlapping octameric sequences that matchin seven out of eight nucleotides to the higher eukaryotic consensus ATGCAAAT (Falkneret al., 1986).

A 17-base pair sequence was found in the 5'-regions of the A. nidulans and A. niger pkiAgenes that may act as an upstream regulating sequence (de Graaff et al., 1992). This sequencewas shown to be distinct from the proposed cis-acting element mediating increasedtranscription of pyruvate kinase on glycolytic carbon sources (de Graaff et al., 1988).

Table 2.1 Genes encoding enzymes involved in the biosynthesis of citric acid by A. niger,which have already been cloned from A. niger or other Aspergillus spp.

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2.2.3 Glucose metabolism and its regulation

The disappointing result that amplification of selected genes did not lead to an increase inthe rate of citric acid accumulation by A. niger indicates the operation of very tight finecontrol of at least some of the enzymes involved. In fact, this fine control was already amajor target of investigation throughout the early 1980s, and as a consequence several ofthe enzymes involved in it have been comparably well characterized (Table 2.2). Mostrecently, the method to study the concentration of intracellular metabolites in A. niger hasalso been critically reassessed (Ruijter and Visser, 1996). Based on these data, Figure 2.2shows those regulatory interactions between metabolites and enzymes which are believedto be of major importance to the regulation of citric acid biosynthesis in A. niger. Similar tothe situation in yeast and higher eukaryotes, citrate and phosphoenolpyruvate (PEP) seemto be the major factors negatively affecting the glycolytic flux, whereas Fructose-2,6-diphosphate (Fru-2,6-P2) and Fru-1,6-P2 appear to be the major activators.

2.3 Regulation of citric acid biosynthesis

Citrate is one of the best known inhibitors of glycolysis, and the ability of A. niger tooverproduce citrate by an active glycolytic pathway has therefore attracted biochemicalinterest for a long time; it is considered to be of major consequence for the fermentation rate(cf. Habison et al., 1979). However, under appropriate nutrient conditions (see below), thisinhibition is more than counteracted by the accumulation of various positive effectors ofPFK1 (NH4

+, inorganic phosphate, AMP, Fru-2,6-P2), and hence this feedback does notoccur (Habison et al., 1983; Arts et al., 1987).

A series of investigations by Kubicek and co-workers favour the assumption that Fru2,6-P2 may play a major role in the counteraction of citrate inhibition: Kubicek-Pranz et al.(1990) found that the triggering of citric acid accumulation by replacing A. niger in highconcentrations (14% w/v) of sucrose or glucose (Shu and Johnson, 1948b; Xu et al.,1989a) is paralleled by a rise in the intracellular concentration of Fru-2,6-P2. Also, myceliacultivated on carbon sources which allow higher yields of citric acid (i.e. those which aretaken up rapidly; Hossain et al., 1984; Kubicek and Roehr, 1986; Honecker et al., 1989;Xu et al., 1989a) showed higher concentrations of Fru-2,6-P2. The concentration of Fru-2,6-P2 correlates therefore positively with the rate of citrate production, and this fact maybe responsible for the lack of citrate inhibition of PFK1. The reason for the increased F-2,6-P2 level is not completely clear, but it appears to be due to an increased Fru-6-Psupply for PFK2, since this enzyme is only poorly regulated in A. niger (Harmsen et al.,1992).

The biosynthesis and regulation of Fru-2,6-P2 links regulation of PFK1 to that of earliersteps in glycolysis. Torres (1994a, 1994b) has recently concluded from theoreticalcalculations that a major part of the actual control of citric acid production must occur athexose uptake and/or phosphorylation, which is in accordance with such an assumption.The biochemistry of these early steps in A. niger glycolysis is not completely clear, howeverSteinböck et al. (1994) found a single hexo/glucokinase only in the citric acid producingstrain ATCC 11414, which was inhibited by citrate and weakly sensitive to trehalose-6-phosphate (Arisan-Atac et al., 1996). The inhibition by citric acid was due to chelation ofMg2+ which is required to chelate the co-substrate ATP, and is most probably irrelevantunder physiological conditions where Mg2+ is present in excess. However, the inhibition bytrehalose-6-phosphate appears to be relevant to the flux towards citric acid, since a


recombinant strain of A. niger, which carries a disrupted copy of the constitutively expressedtrehalose-6-phosphate synthase gene tpsA (Wolschek and Kubicek, 1997), produces citricacid at increased rates (Arisan-Atac et al., 1996). Similarly, a strain bearing multiple copiesof tpsA and hence overproducing trehalose-6-phosphate synthase exhibited a reduced rateof citrate production. These data indicate that the cellular level of trehalose-6-phosphateregulates the flux from glucose to citric acid and are thus in accordance with the conclusionsof Torres et al. (1996a) that hexokinase most likely accounts for the major part of regulationat the early steps of glycolysis, thereby supplying an increased concentration of substratefor PFK2.

However, most recently Panneman et al. (1996) reported on the isolation andcharacterization of a glucokinase from A. niger N400, a strain producing only low levels ofcitric acid, which has properties different from the hexo/glucokinase purified by Steinböcket al. (1994). They also concluded that by analogy with A. nidulans (Ruijter et al., 1996a)there may be also at least one separate hexokinase as well. The difference between theresults of Steinböck et al. (1994) and Panneman et al. (1996) are currently unresolved.Hybridization of an A. niger ATCC 11414 DNA with a Kluyveromyces lactis hexokinase-encoding gene as a probe showed hybridization to a single fragment only (F.Narendja andC.P.Kubicek, unpublished data). The gene from strain ATCC 11414 has recently been clonedin our laboratory, and its characterization has to be awaited for clarification of this situation.Whatever the results of this investigation, the results by Arisan-Atac et al. (1996) clearlyshow that a relief from trehalose-6-phosphate inhibition positively influences the glycolyticflux at high sugar concentrations, and the hexose-phosphorylating step is therefore a majorregulatory point in this fermentation.

Glucose uptake by A. niger was investigated by Torres et al. (1996a). A. niger ATCC11414 contains two transporters with different Km and Vmax. However, the high-affinitypermease can only be detected during growth on low glucose concentration (1% w/v),whereas the low-affinity permease is detectable in the presence of high glucoseconcentrations. The latter may therefore contribute to the increased glycolytic flux duringgrowth on high glucose concentrations.

Several lines of evidence suggest that the regulation of PFK1 by Fru-2,6-P2 may not bethe only parameter regulating citrate accumulation. Citrate inhibition of PFK1 also seemsin vivo to be antagonized by ammonium ions (Habison et al., 1979). This antagonism isfunctionally linked to the well known effect of trace metal ions (particularly manganeseions) on citric acid accumulation (Shu and Johnson, 1948b; Tomlinson et al., 1950; Trumpyand Millis, 1963), as one of the effects caused by manganese deficiency is an impairment ofmacromolecular synthesis in A. niger (Kubicek et al., 1979a; Hockertz et al., 1987), whichcauses increased protein degradation (Kubicek et al., 1979a; Ma et al., 1985). As aconsequence, mycelia accumulate elevated concentrations of NH4

+ (Kubicek et al., 1979a).Proof for the role of manganese ions in this process has been obtained by the isolation ofmutants of A. niger whose PFK1 was partially citrate-insensitive and whose citric acidaccumulation was simultaneously more tolerant to the presence of Mn2+ (Schreferl et al.,1986). Furthermore, several authors have reported that the exogenous addition of NH4

+

during citric acid fermentation even stimulates the rate of citrate production (Shepard, 1963;Choe and Yoo, 1991; Yigitoglu and McNeil, 1992), which is consistent with this effect ofNH4

+ on PFK1. The latter authors documented that both the time of addition as well as theconcentration of NH4

+ were important, and its addition during inappropriate fermentationphases even decreased acid accumulation.

The reason for the impairment of macromolecular synthesis under manganese deficientcultivation conditions had originally been assumed to be at the translational level (Ma et


al., 1985). However, Hockertz et al. (1987) have demonstrated that the absence ofmanganese ions from the nutrient medium of A. niger causes a reversible inhibition ofDNA, but not RNA biosynthesis. This is supported by the findings that the effect ofmanganese deficiency can be mimicked by addition of hydroxyurea, an inhibitor ofribonucleotide reductase (Hockertz et al., 1987). They proposed that manganese deficiencymay primarily impair DNA synthesis by causing a shortage of desoxyribonucleotidesrequired for DNA replication.

A further mechanism of regulation of PFK1 was proposed by Legisa and co-workers,who postulated that PFK1 is regulated by phosphorylation by cyclic-AMP dependent proteinkinase A (Legisa and Bencina, 1994). They speculate that a high concentration of sucrosecauses an increase in mycelial cyclic-AMP levels which trigger the phosphorylation ofPFK1, thereby converting an inactive (non-phosphorylated) form into an active(phosphorylated) form (Legisa and Gradisnik-Grapulin, 1995). The support for their modelis their observation that PFK1 was inactivated by treatment with alkaline phosphatase (Legisaand Bencina, 1994). However, this model, while intriguing, has to be treated cautiouslyuntil solid evidence for it has been obtained, as the molecular weight of the PFK1 purifiedby Legisa and Bencina (1994) and used for their studies was 48 kDa which is not that ofnative PFK1 (84 kDa). Moreover, the method section of their paper does not indicate whether(and how) the alkaline phosphatase had been removed or inactivated prior to the PFK1assay. If this was not done, the ‘inactivation of PFK1’ may have been due to a removal ofFru-6-P from the assay and thus be an artefact. Proof for a regulation of PFK1 byphosphorylation is therefore still needed.

A stimulation of citric acid accumulation by increased cyclic-AMP levels had also beenpostulated earlier (Wold and Suzuki, 1973, 1976a, 1976b). They showed that the stimulatoryeffect was dependent on the zinc concentration of the medium. Adenylate cyclase from A.niger has been described as Zn2+ dependent (Wold and Suzuki, 1974). A bottleneck of theirinvestigations, however, is that they were using 1% (w/v) sucrose throughout, and hence therelevance of their findings to the effect of zinc under citric acid fermentation conditions isunclear. Xu et al. (1989b) studied the intracellular concentration of cyclic-AMP in A. nigerduring citric acid biosynthesis on media with and without Mn2+ ions added, and with high(14%) and low (1%) sucrose concentrations. They reported that the cyclic-AMP levels weregrowth rate dependent, and comparable if phases of similar growth rates were compared.Whether or not cyclic-AMP is in fact involved in the regulation of citrate overproductionremains to be assessed.

2.4 Role of citrate breakdown in citrate accumulation

2.4.1 Role of the citric acid cycle

The reason why A. niger accumulates such massive amounts of citric acid has, since theearly studies by Ramakrishnan et al. (1955), attracted numerous investigations. Althoughcitrate has been considered an ‘overflow’ product (Foster, 1949), which implies that itaccumulates as a result of an excessive substrate supply rather than a limited catabolism,an excessive amount of work has been concerned with the attempt to identify a bottleneckin the tricarboxylic acid cycle as the reason for its accumulation. Numerous workersclaimed that inactivation of an enzyme degrading citrate (e.g. aconitase or the isocitratedehydrogenases) would be essential for the accumulation of citric acid (for review seeSmith et al., 1974; Berry et al., 1977; Roehr et al., 1983, 1996; Kubicek and Roehr,


1986). While this view has an extraordinary long half-life in the review literature, solidevidence for the presence of an intact citric acid cycle during citric acid fermentation waspresented 25 years ago (Ahmed et al., 1972), and explanations based on this view aretherefore simply incorrect.

The requirement of citric acid accumulation of a deficiency in some metal ions (e.g.Mn2+, Fe3+) has frequently been used to explain an inhibition of some enzymes of the TCAcycle (for review see Kubicek and Roehr, 1986). Thus, iron deficiency has been claimed toinhibit aconitase (Szczodrak and Ilczuk, 1985). However, the activity of this enzyme duringcitric acid accumulation has been demonstrated clearly by others both in vitro (La Nauze,1966; Ahmed et al., 1972; Mattey, 1977) as well as in vivo (Kubicek and Roehr, 1985). Itshould be kept in mind that the enzymes of the respiratory chain, which also require iron,are highly active during citric acid accumulation (Ahmed et al., 1972; Hussain et al., 1978).By a similar rationale, the necessity for Mn2+ deficiency has been used to claim an inhibitionof either of the two isocitrate dehydrogenases which require divalent metal ions for activity(cf. Gupta and Sharma, 1995). However, this requirement is for chelation of the substrate(i.e. isocitrate; cf. Bowes and Mattey, 1979; Meixner-Monori et al., 1986). In view of thefact that Mg2+ (which is present in excess) can take over the chelating role of Mn2+ efficiently,this interpretation is unlikely to explain the effect of Mn2+.

Several other explanations for citric acid accumulation are based on the postulation of ametabolic inhibition of the NADP-specific isocitrate dehydrogenase by citrate (Mattey, 1977)or glycerol (Legisa and Mattey, 1986), which would create a bottleneck in the tricarboxylicacid cycle and—because of the Keq of aconitase—lead to a spilling over of citrate.Unfortunately, none of the explanations which are based on an inhibition of NAD-or NADP-specific isocitrate dehydrogenases have ever been supported by evidence from in vivoexperiments. The ‘glycerol theory’ (Legisa and Mattey, 1986; Gradisnik-Grapulin and Legisa,1996), has recently been reassessed by studying the effect of increased mycelial glycerolconcentrations on the oxidation of 1,5–14C-citrate by mycelia and isolated mitochondria ofA. niger (Arisan-Atac and Kubicek, 1996). The appearance of 14C-labelled CO2—whichbecause of the labelling position applied can only be released during the metabolic conversionof citrate to a-ketoglutarate—was virtually unaffected by the glycerol concentration, therebyclearly disproving an effect of glycerol on the activity of isocitrate dehydrogenases andconsequently this theory. Also, in contrast to the enzyme from crude cell-free extracts (Legisaand Mattey, 1986), the purified NADP-specific isocitrate dehydrogenase was not inhibitedby citrate (Arisan-Atac and Kubicek, 1996).

It is surprising that the question of whether the isocitrate dehydrogenase step of the TCAcycle is active during citric acid fermentation or not has never been viewed from a theoreticalpoint of view: using the cellular concentration of free and protein-bound glutamic acid asan indicator of metabolic flux from glucose to a-ketoglutarate, there is no indication for asignificant change in this flux unless at late stages of fermentation where the fungal growth(and also the need for glutamic acid) has stopped, and this flux is only 17 per cent lowerthan that occurring in a culture accumulating 78 per cent less citric acid, and hence may notbe of high relevance to the mechanism of citric acid accumulation (O.Zehentgruber andC.P.Kubicek, unpublished data).

With regard to the mechanisms which trigger the initial accumulation of citrate from themitochondria, a fact completely overlooked so far is the activity of the tricarboxylatetransporter. This carrier competes directly with aconitase for citrate, and if its affinity forcitrate were much higher than that of aconitase, would pump citrate out of the mitochondriawithout any necessity for inhibition of one of the TCA cycle enzymes. As the tricarboxylatecarrier of mammalian tissues and yeast occurs by countertransport with malate (Evans et


al., 1983), such a situation is conceivable when its counter-ion malate accumulates in thecytosol. Malate accumulation has in fact been shown to precede citrate accumulation (Roehrand Kubicek, 1981). However, the mitochondrial citrate carrier of A. niger has not yet beeninvestigated, and this hypothesis clearly needs thorough investigation before it can be usedto explain citrate accumulation. It is also not known to what extent changes in the fluxthrough the NAD-dependent-, NADP-dependent isocitrate dehydrogenases, a-ketoglutaratedehydrogenase and succinate dehydrogenase, contribute to a rise in the intramitochondrialcitrate concentration. As these enzymes are known to be regulated by the mitochondrialNADH/NAD and NADPH/NADP ratios, as well as by AMP, cis-aconitate and oxaloacetate(Chan et al., 1965; Meixner-Monori et al., 1985, 1986), fluctuations in the level ofmitochondrial TCA metabolites are likely.

2.4.2 Respiratory activity and the role of NAD regeneration

Formation of citric acid is dependent on strong aeration, and dissolved oxygen tensionshigher than those required for vegetative growth of A. niger stimulate citric acid fermentation(Clark and Lentz, 1961; Kubicek et al., 1980; Dawson et al., 1988a). On the other hand,sudden interruptions in the air supply cause an irreversible impairment of citric acidproduction without any harmful effect on mycelial growth (Kubicek et al., 1980; Dawson etal., 1988b). The biochemical basis of this observation appears to be related to the presenceof an alternative respiratory pathway, which is obviously required for re-oxidation of theglycolytically produced NADH, by a continuously maintained, high oxygen tension (Kubiceket al., 1980; Zehentgruber et al., 1980; Kirimura et al., 1987, 1996), whose activity is impairedby short interruptions in the air supply (Kubicek et al., 1980). Weiss and colleagues (Wallrathet al., 1991; Schmidt et al., 1992; Prömper et al., 1993) studied the role of the standard andalternative respiratory pathways in citric acid accumulation in detail. They detected that theassembly of the proton pumping NADH:ubiquinone oxidoreductase is impaired during citricacid accumulation (Schmidt et al., 1992), which could be the reason for the importance ofthe activity of the alternative pathway. Interestingly, disruption of the gene encoding theNADH-binding subunit of complex I in a low producing strain of A. niger increased itscatabolic overflow, yet this strain excreted much less citrate than its parent (Prömper et al.,1993). These findings stress the fact that citric acid accumulation is not a mono-causalprocess, and citrate accumulation in high amounts depends on a delicate balance of severalfactors, whose interrelationship is not yet fully understood.

The requirement of a high oxygen supply is also related to another effect of Mn2+ ions onA. niger, i.e. on the morphology of the fungus: whereas A. niger grows in long and smoothfilaments when supplied with optimal concentrations of Mn2+ ions, Mn2+ deficient grownmycelia are strongly vacuolated, highly branched, contain strongly enthickened cell wallsand exhibit a bulbous appearance (Kisser et al., 1980; Papagianni et al., 1994). This type ofmorphology has been shown to provide a much better rheology (Olsvik et al., 1993) andenables a higher oxygen transfer (Fujita et al., 1994; Iwahori et al., 1995); it may thus berequired for optimal citric acid yields.

NAD regeneration may also be related to the effect of pH on citric acid fermentation:the almost quantitative conversion of glucose to citric acid, as occurs during the idiophaseof fermentation, yields 1 ATP and 3 NADH. While part of the NADH pool can be reoxidizedby the alternative, salicylhydroxamic acid (SHAM) sensitive respiratory pathway describedabove, this yield of ATP probably still exceeds that of the cell’s maintenance demands.Roehr et al. (1992) speculated that the ATP will be consumed by the plasma membrane


bound ATPase during maintenance of the pH gradient between the cytosol and theextracellular medium. The involvement of this enzyme in the maintainance of the pHgradient in citric acid producing A. niger has been shown by Mattey et al. (1988). Hencethe requirement of a low pH for citric acid accumulation may be, at least in part, relatedto a high turnover of the ATP formed, which otherwise would lead to a metabolic imbalanceand so stop acidogenesis. However, this explanation still requires experimental verification.Most recently, single-point mutagenesis of a plant ATPase and its expression in yeastresulted in increased H+-pumping and increased growth rates at low pH (Morsomme etal., 1996).

2.5 Export of citric acid from A. niger

Torres et al. (1994a) proposed that the two citric acid transport steps, i.e. that from themitochondria to the cytosol, and that from mycelia into the culture filtrate, are among themost important regulatory points for the obtention of high yields.

The mechanism of transport of mitochondrial citrate into the cytosol is still completelyunknown, except for the hypothesis that it occurs by countertransport with theglycolytically overproduced malate (see above). ATP-citrate lyase, an enzyme which inother cells uses the cytosolic citrate for lipid biosynthesis, appears to be unable to managethis high efflux but its precise regulation under citric acid producing conditions is notunderstood (Pfitzner et al., 1987; Jernejc et al., 1991). The latter authors have also purifieda cytosolic and a mitochondrial carnitine acetyltransferase from A. niger, which exhibitedsimilar kinetic and physicochemical properties (Jernejc and Legisa, 1996). As the activityof this enzyme was in considerable excess of that of ATP-citrate lyase they concluded thattransfer as a carnitine ester may be the major physiological source of acetyl-CoA for lipidbiosynthesis. If this is indeed the case it would explain why the cytosolic citrate pool israther stable. Because of their findings of a cytosolic isoenzyme of carnitineacetyltransferase, Jernejc and Legisa (1996) also speculated that this enzyme transfersacetyl-CoA to the mitochondria and thus for citrate biosynthesis. This is an intriguingspeculation, but requires the identification of a cytosolic pathway from pyruvate to acetyl-CoA which is not yet known.

Mattey and co-workers (Mattey, 1992; Kontopidis et al., 1995) explained the export ofcitrate through the plasma membrane in terms of the large pH gradient between the cytosoland the extracellular medium, and postulated that citrate efflux from the cells may occur bydiffusion of the 2(-) citrate anion, driven by a gradient. If this assumption is correct, the lowpH would be responsible for the citrate gradient necessary for transport and consequentlyless citrate would be secreted at higher pH values. However, recent studies in our laboratoryclearly showed that citrate export requires ATP, and its Vmax is not strongly affected by theexternal pH (Netik et al., 1997); this renders the diffusion hypothesis rather unlikely. Netiket al. (1997) also reported that citrate export is strongly increased in mycelia grown undermanganese deficiency, which is consistent with previous observations that the intracellularconcentration of citrate in manganese sufficient and deficient grown mycelia is not greatlydifferent (Kubicek and Roehr, 1985; Legisa and Kidric, 1989; Prömper et al., 1993), despitethe five- to seven-fold higher extracellular levels under the latter conditions. The reason forthe requirement of manganese deficiency for citrate export is not clearly understood, butmay be related to an absolute requirement of citrate uptake for manganese ions, probablybecause of a requirement for chelated citrate as a substrate for the permease (Netik et al.,1997).


The reason for the reciprocal effect of Mn2+ ions on export and import of citric acid mayalso be related to yet another effect of manganese deficiency, i.e. inhibition of triglycerideand phospholipid synthesis as well as a shift in the ratio of saturated to unsaturated fattyacids of whole mycelial lipids (Orthofer et al., 1979; Jernejc et al., 1989) and of isolatedplasma membranes (Meixner et al., 1985). The different behaviour of the citrate export andimport system of A. niger may also be seen in the light of earlier studies on the antagonismof several membrane affecting compounds on the detrimental action of manganese ions,e.g. lower alcohols (Moyer, 1953), lipids (Millis et al., 1963; Gold and Kieber, 1967), ortertiary amines (Batti, 1969). Also the technically important ability of Cu2+ ions to antagonizethe deleterious effect of Mn2+ may be related to citrate excretion, as Cu2+ strongly inhibitedthe uptake of citric acid from the medium (Netik et al., 1997). However, the effect of Cu2+

(Schweiger, 1959) may also reside in its inhibition of the uptake of Mn2+ by A. niger (Hockertzet al., 1987), which occurs by a specific, high affinity transport system (Seehaus et al.,1990). The properties of the uptake and the export system are otherwise similar (?pH drivenproton symport) and it may be speculated that they are catalyzed by the same enzymesystem.

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33

3

Biochemistry of Citric AcidProduction by Yeasts

MICHAEL MATTEY

3.1 Introduction

In terms of bulk production citric acid is widely regarded as one of the most important ofthe organic acids produced by microbiological methods, although reliable estimates of worldproduction are not easily obtained. A widespread perception has been that most of theproduction is achieved with Aspergillus niger, in what has come to be regarded as the‘traditional’ fermentation process, although the first indications of a microbiological processfor the production of citric acid were from Wehmer (1893), who noted that ‘Citromyces’(now Penicillium) could accumulate citric acid.

Indeed until around 1970 A. niger was almost exclusively the organism used for theproduction of citric acid; the ability of other filamentous fungi to excrete citric acid wasknown and has been reviewed (Röhr and Kubicek, 1992), but they are of limited importance.A more important class of production organisms found within the Candida yeasts, and anincreasing proportion of the total production of citric acid is now manufactured using strainsof Yarrowia lipolytica (the asexual form is Candida lipolytica, syn. Saccaromycopsis).

The Candida genus (family Cryptococcaceae, subfamily Cryptococcoideae) contains30 species, and six varieties, many of which are pathogenic to animals, including humans.With increasing numbers of immunodeficient people, either through retroviral disease orthe anti-rejection drugs used in organ transplantation, the pathogenic species such as C.albicans have assumed a new importance. Many Candida species have been isolated fromfruit, seeds, soil, and similar sources.

Vegetative growth consists of budding cells and pseudomycelium, or true myceliumwith blastospores. C. lipolytica was isolated from margarine (hence its name, Gr. Lipos, fat;lysis, breaking). The cells are variable, long oval to almost cylindrical and short oval. A welldeveloped pseudomycelium is frequently formed with some true mycelium. The organism,as well as hydrolysing fats, will liquify gelatine, but does not ferment sugars.

As well as the industrial importance of the organism, it is being developed as a cloningvehicle for the expression of heterologous proteins. Some understanding of the pathwaysinvolved in citric acid production has resulted from the cloning of particular genes as aresult of this development; in particular our knowledge of the peroxisomal pathways has


been advanced through developments in the understanding of protein targeting. It is rarelyknown whether the perfect or imperfect form is being used in a particular process. Indeed,C. lipolytica is a dimorphic yeast and the morphology may vary, particularly during thecourse of a batch process.

The impetus for the study of citric acid production from Candida yeasts appears to havehad two aspects: first the availability in the 1960s of relatively cheap hydrocarbons asfeedstock; and secondly the use of hydrocarbons for the production of glutamic acid byCorynebacterium (Yamada et al., 1963). Particularly in Japan the further use of this feedstockin a number of fermentation processes was explored. This phase came to a halt in 1973when the price of crude oil was increased dramatically. This process is reflected in thenumber of patents granted: from one in 1967 and two in 1968 up to 15 in 1972, then droppingto three by 1975 (source: Chemical Abstracts).

Possibly as a result of the extent of industrial commitment to yeast-based processes,alternative carbon sources were explored; the finding that alkane-utilizing yeasts canalso use sugars to produce citric acid formed the basis of the present expansion of theindustrial importance of C. lipolytica. The potential advantages of using yeasts ratherthan A. niger are the higher initial sugar concentrations that can be tolerated and thefaster conversion rates possible. The sensitivity of A. niger to metal ions, particularlymanganese, is well known, and a source of increased costs associated with the pre-treatment of molasses. This is avoided with Candida yeast that is far less sensitive tometal ions.

The fermentation with Candida yeasts appears to be biphasic (Marchal et al., 1977) withcitric acid accumulating after the growth phase, when nitrogen is exhausted. Althoughnitrogen limitation, with nitrogen usually supplied in the form of ammonium salts, is thetrigger for acid accumulation, other parameters influence the yield and productivity of theprocess. The pH is maintained above pH 5.0, unlike the situation in A. niger where a mediumpH below 2 is required for a good yield. Lowering the pH with the C. lipolytica fermentationresults in the production of polyols (Tabuchi and Hara, 1970), mainly erythritol and arabitol.

The addition of iron salts to the medium lowers the yield of citric acid, although someiron is required for normal growth. The addition of iron increases the activity of aconitatehydratase, and this is thought to result in the conversion of citric to isocitric acid (Tabuchi etal., 1973). The influence of iron on the growth and synthesis of citric and isocitric acid inethanol-containing media showed similar effects (Kamzolova et al., 1996). Changes in theconcentration of iron caused abrupt switching between the predominant formation of eithercitric or isocitric acids.

One noteworthy feature of the process is the requirement for thiamine. Unless this isadded, oxoacids, mainly oxoglutarate, accumulate and the yield of citric acid is reduced.The reason for this requirement is not known but is likely to be related to the level ofoxidative decarboxylation required, where thiamine pyrophosphate (TPP) is a co-factor.When ß-oxidation is the main assimilatory pathway the requirement for pyruvatedehydrogenase would not be significant, but the flux through oxoglutarate dehydrogenasemight be elevated. The other obvious requirement for TPP is for the transketolase reaction;although the role of the pentose phosphate cycle in the metabolism of C. lipolytica duringcitric acid accumulation is not known, the production of erythritol and arabitol underconditions of low pH might be indicative of its activity. The activities of enzymes of theTCA cycle have been measured after thiamine-limited growth with ethanol as a substrate(Morgunov et al., 1995). This will use essentially the same pathway as growth on alkanes,and thiamine limitation is similarly accompanied by oxogluarate production. The activityof the oxoglutarate dehydrogenase complex is greatly reduced and oxidative

Biochemistry of citric acid production by yeasts 35

decarboxylation of oxoglutarate becomes the limiting reaction in the TCA cycle. Thisleads to oxoglutarate accumulation within the cells and secretion into the culture medium.The glyoxylate cycle is used as an alternative pathway when the TCA cycle is impaired inthis way.

Although growth is usually limited by nitrogen exhaustion, limitation of growth bysulphur, magnesium or phosphorus gives a similar effect (McKay et al., 1994). Citric acidlevels between 50 and 220 mM were measured after 168 hours, with nitrogen and sulphurlimitation giving the highest specific production rates. Potassium limitation was ineffective(6 mM), and the glucose uptake rate was only 50 per cent of that achieved when nitrogen orsulphur was limiting.

3.2 Synthesis of citric acid from n-alkanes 3.2.1 Growth on alkanes

An important feature of the growth of yeast on alkanes is that the flow of carbon from thesubstrate to the cellular materials is significantly different to that found during conventionalgrowth on a carbohydrate, in that during growth on carbohydrates fatty acids are synthesizedwhile carbohydrates are degraded, whilst the opposite is true for growth on alkanes. Themetabolic sequence when growth on alkanes occurs is therefore:

1 Uptake of alkanes into the cell.

2 Oxidation of alkanes into the corresponding fatty acids.

3 Conversion of the fatty acids to acyl CoA esters.

4 Metabolism of fatty acyl CoA esters to acetyl CoA, or incorporation into cellular lipid.

5 Synthesis of TCA cycle intermediates.

6 Gluconeogenesis, synthesis of amino acids, nucleic acids, etc.

Since the utilization of alkanes overlaps considerably with the metabolism of fatty acids theoleaginous yeasts such as C. lipolytica were obvious targets for fermentation processeswith this type of feedstock.

3.2.2 Uptake of alkanes

Alkanes are of limited solubility in water so that the uptake of alkanes by cells could beof three types: by direct contact between the alkane droplets and the microbial cells;through the soluble phase; or by ‘solubilization’ by micelle formation in an emulsionwith subsequent uptake. All three mechanisms are believed to occur. Once contactbetween a hydrophobic alkane droplet and the hydrophobic cell membrane has beenmade the alkane will dissolve in the lipid phase and be transported across the membrane.Despite this, particular areas of the membrane may become specialized for the rapiduptake of alkanes.

Meissel et al. (1973, 1976) have observed distinctive channels in the cell wall of yeastgrown on alkanes when studied by electron microscopy. Similar channels have beenobserved by Osumi et al. (1975), together with protrusions on the cell surface whichreach the cell membrane through electron-dense channels. The hypothesis has been putforward that alkanes attach to these channels and migrate through them to the membraneand into the endoplasmic reticulum which appears to be particularly associated with the


cytoplasmic end of these channels. The endoplasmic reticulum is the site of the initialoxidation of the alkanes.

Alkane emulsions adhere to the cell wall of Candida yeast by a non-enzymatic mechanism(Einsele et al., 1975; Käppeli and Fiechter, 1976, 1977). The binding is due to alipopolysaccharide in the cell wall that is induced by alkanes. The lipopolysaccharide, whichis mannan with about 4 per cent covalently linked fatty acid, has been isolated andcharacterized (Käppeli et al., 1978).

3.2.3 Initial oxidation of n-alkanes

Three oxidation mechanisms are known but the one most likely to be operating in Candidayeast is the mixed function oxidase (mono-oxygenase). A cytochrome P-450 hydroxylasesystem, dependent on NADPH+ and H+, has been described in several species of Candida (Liuand Johnson, 1971; Lebeault, 1971; Duppel et al., 1973), see Figure 3.1. The formation of P-450 has been shown to be inducible by long-chain alkanes, alkenes, secondary alcohols andketones (Gallo et al., 1973) with hexadecane increasing the specific activity by 150-fold relativeto cells grown on glucose. As well as the P-450, a microsomal NADPH-cytochrome c-reductasewas increased (Gallo et al., 1971). The influence of carbon and nitrogen sources on a numberof NAD+- and NADP+-linked dehydrogenases was examined (Hirai et al., 1976a); no significanteffects other than on the NADP+-cytochrome c-reductase were seen.

The cytochrome P-450 concentration was linearly related to hexadecane uptake rateswhen cells were cultivated under conditions of oxygen limitation in a chemostat (Gmunder,1979), leading to the suggestion that cytochrome P-450 is the rate-limiting step in alkaneuptake and oxidation. It is unlikely however that flux control is in fact dependent on a singlestep in a steady state system such as this.

Alkane molecules are susceptible to such oxidations at one or both of the terminal methylgroups. A monoterminal oxidation pathway appears to be operating in C. lipolytica. Thefatty acids in cell lipids of active, alkane degrading cells of C. lipolytica grown on variousalkanes showed a pattern corresponding to the n-alkane chain length. The alkanes are oxidizedto the corresponding fatty acids that are incorporated into lipids, either directly, after chainelongation or by ß-oxidation.

Figure 3.1 Hydroxylation of an alkane by cytochrome P-450 dependent mono-oxygenase


Table 3.1 shows the relationship for C. lipolytica grown on a variety of substrates.The correlation between odd chain length alkane substrate and odd chain length fattyacids in the cells is clear. The high activity of a mono-oxygenase system, with its oxygenradical mechanism, suggests that protection against damage by free radicals might beimportant when yeast grows on alkanes. Indeed a considerable increase in copper andzinc superoxide dismutase (SOD) is seen during growth on n-alkanes as compared toglucose (Kujumdzievasavova et al., 1991). A correlation between SOD and catalasewas noted and resistance to oxygen free radicals observed as a result of the high levelsof copper/zinc SOD, which also protected against deleterious effects of Cu2+ and Zn2+

in the medium.

3.2.4 Oxidation of higher alcohols

The product of the microsomal oxidase system is a higher alcohol corresponding to thechain length of the alkane. These alcohols are oxidized to the corresponding fatty acidthrough the aldehyde. NAD+-linked alcohol dehydrogenase and NAD+-linked aldehydedehydrogenase, specific to long-chain substrates, carry out these reactions (Lebault et al.,1970a, 1970b). Both enzymes are inducible by alkanes as well as long-chain alcohols oraldehydes. A soluble alcohol oxidase may also be present in some strains of Y. lipolytica(Ilchenko et al., 1994). The enzyme was purified from strain H-222 grown on n-alkanes,and showed maximum activity with carbon chain lengths ranging from 10 to 18 (see Table3.2). It appeared that several other specific alcohol oxidases might have been present. The

Table 3.1 Ratio of odd chain fatty acids and C17 acids to total cellular fatty acids in Candidalipolytica cells grown on n-alkanes and glucose (Tanaka et al., 1976)


localization of these enzymes within the cells appears generalized. Early reports areconfusing, possibly because until 1974 the occurrence of peroxisomes was not known.Osumi et al. (1974) have detected both dehydrogenases in peroxisomes, mitochondria andmicrosomes.

3.2.5 Peroxisomes in yeast metabolizing n-alkanes

When grown on alkanes, specific organelles, less than 1 µm in diameter, infrequently seenin cells grown on carbohydrate substrates, become numerous (Osumi et al., 1974; Teranishiet al., 1974; Mishina et al., 1978). These organelles have been identified as peroxisomes bycytochemical staining for catalase. Indeed their appearance is directly related to the increasedcatalase activity seen during the metabolism of alkanes. These organelles contain several ofthe enzyme systems involved in the initial oxidation of alkanes and similar substrates, andtransport between the various compartments of substrates and intermediates is a complexarea. Two peroxisomal targeting signals are known (PTS1 and PTS2) and it is suggestedthat PTS receptors, which have been found in several subcellular locations, shuttle betweenthe cytosol and the peroxisomal membrane. The PTS1 protein is highly conserved and thehuman homologue (PTS1R) has been cloned as a result. Interestingly this is mutated in agroup of patients afflicted with a fatal peroxisomal disorder (Subramani, 1996). Proteinunfolding is not required for the import of peroxisomal matrix proteins, which is markedlydifferent from other mechanisms for the translocation of proteins. The gene pay5 encodes aperoxisomal integral membrane protein in Y. lipolytica, pay5p, of 380 amino acids (41.7kDa) (Eitzen et al., 1996) homologous to the mammalian PAF-1 protein which is essentialfor peroxisome assembly. Pay5p is targeted to mammalian peroxisomes in an interestingexample of the evolutionary conservation of targeting mechanisms. In humans, mutation ofPAF-1 results in the Zellweger syndrome. Mutants of Y. lipolytica (pay5-1) also show defectiveperoxisome synthesis.

3.2.6 Activation of fatty acids to CoA esters

Two acyl CoA synthetases have been isolated from C. lipolytica (Mishina et al., 1978) withdifferent locations, specificity functions and regulation. Their properties are summarized inTable 3.3. Synthetase I is constitutive while synthetase II is inducible by fatty acids. Theenzymes could be distinguished immunochemically (Hosaka et al., 1979). The synthetase Iis widely distributed including mitochondria where glycerophophate acyltransferase is alsolocated, while synthetase II is located in the peroxisomal compartment where ß-oxidationoccurs. Evidence for ß-oxidation has been obtained from the study of peroxisomes from C.tropicalis (Kawamoto et al., 1978). The stoichiometry of the process demonstrated that theß-oxidation system was similar to that described for castor bean (Cooper and Beevers,1969) and liver (Lazarow and de Duve, 1976).

Acyl CoA esters are oxidized by acyl CoA oxidase, a FAD-containing enzyme, to enoylCoA, forming hydrogen peroxide from molecular oxygen. The catalase present in theperoxisome breaks down the hydrogen peroxide. The enoyl CoA is then metabolized togive acetyl CoA with CoA and NAD+ as hydrogen acceptor. Acyl CoA oxidase has beenpurified from C. lipolytica and C. tropicalis (Shimizu et al., 1979) from which organism ithas been crystallized. Its substrate specificity is summarized in Table 3.4.


The peroxisome contains an NAD+-dependent glycerol-3-phosphate dehydrogenase(Kawamoto et al., 1979) which is thought to act as a shuttle hydrogen carrier with the FAD-dependent glycerol-3-phosphate dehydrogenase present in the mitochondria, regeneratingNAD+ and generating energy.

3.2.7 Synthesis of intermediates of the tricarboxylic acid cycle

While growing on alkanes it is clear that the substrate is degraded to the level of acetylCoA, or propionyl CoA in the case of odd chain length acids, and while lipids may beincorporated from the fatty acids all other intermediates must be synthesized from thetwo-carbon precursor. In general, yeast growing under gluconeogenic conditions utilizesthe glyoxylate cycle as an anaplerotic mechanism. The role of this cycle has beendemonstrated in Candida yeast grown on alkanes (Hildebrandt and Weide, 1974). Thetwo characteristic enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase,are induced by growth on n-alkanes (Nabeshima et al., 1977). However, while the level ofisocitrate lyase is considerably elevated compared to the levels in glucose grown cells, thelevel of NAD dependent isocitrate dehydrogenase is lower (Hirai et al., 1976a; Tanaka etal., 1977). The distribution of the flux of intermediates between the TCA cycle and theglyoxylate cycle is determined by the relative activities of these two enzymes which thereforesuggests that a high level of glyoxylate cycle activity occurs during growth on n-alkanes.Much of the isocitrate lyase is present in the particulate fraction of the cells, and theenzymes of the glyoxylate cycle have been localized to the peroxisomal compartment(Hirai et al., 1976b). However, citrate synthase, aconitase and malate dehydrogenase,

Table 3.3 Comparison of the properties of acyl CoA synthetases for C. lipolytica


the characteristic enzymes of the TCA cycle, are present in the mitochondrial compartmentas might be anticipated (Kawamoto et al., 1977). Fatty acid ß-oxidation is not present in themitochondria of C. lipolytica (Mishina et al., 1978) or C. tropicalis (Kawamoto et al., 1978)but appears confined to the peroxisome. This implies that acetyl CoA required for citratesynthesis must be transported to the mitochondria from the peroxisome, probably by thecarnitine acyltransferase system (Kawamoto et al., 1978). Methyl isocitrate cycle

The propionyl CoA, derived from odd chain length n-alkanes, is metabolized by a cyclicpathway analogous to the first steps in the TCA cycle (Tabuchi, 1975a, b). This pathway isbased on the accumulation of pyruvate and seven carbon tricarboxylic acids in C. lipolyticagrown on odd chain length alkanes. Methyl citrate, methylaconitate and methylisocitratewere detected as were the key enzymes described in the methyl citrate cycle. In this pathwaypropionyl CoA from the odd chain fatty acid ß-oxidation sequence reacts with oxaloacetate

Figure 3.2 Methyl isocitrate cycle in C. lipolytica


via a methylcitrate-condensing enzyme, analogous to citrate synthase (Uchiyama andTabuchi, 1976). The resulting methylcitrate is isomerized to methylisocitric acid, possiblyby aconitase and the methylisocitrate is cleaved in a manner analogous to citrate lyase togive succinate and pyruvate (Tabuchi and Satoh, 1976, 1977).

The two key enzymes, methylcitrate synthase and methylisocitrate lyase, clearly differfrom citrate synthase and isocitrate lyase. Both the methyl tricarboxylic acid convertingenzymes seem to be constitutive, like the TCA cycle enzymes, while the key enzymes of theglyoxylate cycle are inducible. The overall effect of this path is to convert propionate topyruvate. The main factor in the level of citric acid production from alkanes is the amountof isocitrate lyase (Behrens et al., 1977; Tanaka et al., 1977; Aiba and Matsuoka, 1979;Matsuoka et al., 1980, 1984).

The role and control of isocitrate lyase has been examined in C. lipolytica and C.tropicalis, and it has been suggested to be a rate limiting enzyme for the process. Thecharacteristics of the enzyme were determined in crude extracts by Marchal et al. (1977);the main features were a Km value of 0.6 mM (at pH 7.0) with inhibition byphosphoenolpyruvate and succinate. Significantly, citrate at 5 mM was not inhibitory.When grown on glucose the level of isocitrate lyase was only 2 per cent of that foundwhen grown on alkanes, where the level was four times that found when grown on acetate,the classic two-carbon substrate. Induction of this enzyme is clearly greater in the presenceof alkanes.

The role of isocitrate lyase in the production of citrate from carbohydrate substrates isunlikely to be significant and it might well be that the critical factor in the overproduction isnot the details of the regulation but that maximal fluxes are obtained, that is, the absence ofeffective regulation! A similar situation may occur in A. niger. The control of flux throughthe TCA cycle versus the glyoxylate cycle is usually thought of in terms of competitionbetween NAD+-dependent isocitrate dehydrogenase and isocitrate lyase, with adeninenucleotide regulation of isocitrate dehydrogenase and repression/de-repression of isocitratelyase but with no metabolite level control of isocitrate lyase, which is the situation in E. coli(Nimmo, 1984). This situation cannot occur in Y. lipolytica as isocitrate lyase is apparentlyin the peroxisome and the NAD+-dependent isocitrate dehydrogenase is mitochondrial, withno common pool of isocitrate.

The isocitrate lyase from Yarrowia lipolytica has been purified and characterized (Honeset al., 1991). The active form was obtained as a single peak from an ion exchange column,with a specific activity of 7.4 U/mg. The molecular mass was estimated to be between200 and 210 kDa, and appears to have four subunits of about 50 kDa. The pH optimumwas pH 6.0 and a Km of 0.3 mM was estimated. The enzyme was non-competitivelyinhibited by succinate and oxalacetate. The gene for isocitrate lyase has been cloned bycomplementation of a mutation (acuA3) in the structural gene of isocitrate lyase of E. coli(Barth and Scheuber, 1993). The open reading frame was 1668 bp long and had no intronsin contrast to the genes sequenced from other filamentous fungi. The deduced proteinwas 555 amino acids with a molecular mass of 62 kDa, which is similar to that observedfor the purified monomer. The enzyme has a putative glyoxosomal targeting sequence S–L–K at the carboxy-terminus and contained a partial repeat which is typical for eukaryoticisocitrate lyases, but is absent from the E. coli sequence. Deletion mutants, as expected,were unable to utilize acetate, ethanol, fatty acids or alkanes, but surprisingly the growthon glucose was also reduced.

Citrate synthase from several strains of Y. lipolytica which are citrate producers havebeen isolated and purified to homogeneity (Morgunov et al., 1994). The enzyme was adimer with a subunit molecular mass of 40 kDa, and exhibited a Km value of 10 and 5 µM


Figure 3.3 Compartmentation in C. lipolytica during growth on n-alkanes


with acetyl CoA and oxalacetate respectively. The enzyme activity observed in extracts isgreater than that of isocitrate lyase, aconitase or isocitrate dehydrogenase (Behrens et al.,1977; Omar and Rehm, 1980). Candida lipolytica has both an NAD+ and an NADP+-dependent isocitrate dehydrogenase. The NADP+-dependent enzyme had Michaelis–Menten type kinetics with respect to isocitrate, and a Km of 80 µM for isocitrate at pH 7.0.There was inhibition by oxalacetate at 5 mM of about 40 per cent. The energy metabolismof C. lipolytica has been examined during growth on n-alkanes; in particular, theconcentration of adenine nucleotides during a batch fermentation was measured (Marchalet al., 1977).

After 25 hours there was a sharp drop in the concentration of ADP and AMP while theATP level rose. The total adenine nucleotide levels fell slightly, then recovered. These changescoincided with the exhaustion of nitrogen in the medium and the effective cessation ofgrowth. At this point citric acid excretion began, together with isocitric acid. The proportionof isocitric to citric acid was high, about 40 per cent, although the intracellular citric toisocitric ratio was close to that expected from the aconitase equilibrium at about 90 per centcitric:10 per cent isocitric acid. The adenylate energy charge (Atkinson, 1970) reflected thechanges in adenine nucleotides, rising to approach 1. However, the dramatic changes areseen in the ATP:AMP ratio, and the most common allosteric effectors amongst the adeninenucleotides are AMP and ATP, so that the ATP:AMP ratio is a better indicator of regulatorychanges. The enzyme that is regarded as a significant target for allosteric regulation duringgrowth on alkanes is mitochondrial NAD+-specific isocitrate dehydrogenase (Marchal etal., 1977). The activity of this enzyme with respect to isocitrate and AMP is sigmoidal,consistent with its structure which has four co-operative binding sites. The enzyme is totallydependent on AMP for activity, with maximal activity shown at 0.1 mM AMP and 50 percent activity at 0.05 mM. At values below 0.01 mM the enzyme is virtually inactive.

Magnesium also behaved as an allosteric activator of the enzyme, apparently with twoco-operative sites. Since the substrate for the enzyme is magnesium isocitrate this is perhapssurprising. There was no correlation between the rate of n-alkane uptake and nitrogen

Table 3.5 Adenine nucleotide levels (mM) during a batch fermentation


exhaustion or changes in adenine nucleotide levels. This is unexpected since the cessationof growth should greatly reduce the energy demand and hence the substrate uptake. Theimplication is that the coupling between electron transport and ATP synthesis becomes‘loose’, or that energy is used in, for example, a transport process.

The mitochondrial ATP synthase genes have been studied in Y. lipolytica (Matsuoka etal., 1994) and a 6.6 kilobase region sequenced. This closely resembled the humanmitochondrial genome with ATP synthase subunits 8 and 6 being followed by the genes forcytochrome c oxidase subunit 3, NADH-ubiquinone oxidoreductase subunit 4 and ATPsynthase subunit 9. All the genes were transcribed from the same strand of DNA intomultigenic RNAs starting from a nonanucleotide sequence, 5'-ATA-TAAATA-3', similar toother yeast mitochondrial promoters. In addition to these apparently normal mitochondrialgenes there is a cyanide-resistant oxidase (Medentsev and Akimenko, 1994) located in theinner membrane. Its activity is typically blocked by benzohydroxamic acid. This resemblesthe situation found in A. niger and the circumstances of uncoupled electron transport arealso similar.

The activity of the NAD+ isocitrate dehydrogenase, which is already low compared tothe level found in cells grown on glucose, is almost totally inhibited by the drop in AMP,and the evolution of carbon dioxide mirrors this, being sharply reduced to very low levelswhen growth ceases. The metabolic production of carbon dioxide from acetate via theTCA cycle during growth is stopped by the inhibition at the level of isocitratedehydrogenase.

Isocitrate lyase was high compared to cells grown on glucose so that the entire carbonflux through the mitochondrial compartment is via the glyoxylate cycle. The activity ofcitrate synthase in C. lipolytica has not been extensively studied, but it is reported to showlimited inhibition by ATP (40 per cent at 5 mM ATP). Since the concentration of ATPreported in the whole cell during the citric acid accumulation phase varied from 0.6 mM atthe start to 0.8 mM at the end, it is unlikely to rise much above 5 mM in the vicinity ofcitrate synthase, even if most of the ATP is in the mitochondrial compartment. Further, thelevel of acetyl CoA, which will be high during growth on n-alkanes, will reduce the ATPinhibition of citrate synthase.

The 3-phosphoglycerate kinase gene has been isolated from a genomic library, by probingwith a PCR fragment amplified with primers deduced from two highly conserved regionsof various pyruvate kinases (Ledall et al., 1996). It encodes a polypeptide of 417 residueswith extensive homology to other kinases. The expression of the gene is higher ongluconeogenic substrates, such as alkanes, than on glycolytic ones. Pyruvate kinase hasalso been cloned as part of the development of expression/secretion systems for heterologousproteins (Buckholz and Gleeson, 1991; Strick et al., 1992). Genomic clones were selectedby their specific hybridization to synthetic oligodeoxyribonucleotide probes based onconserved sequences. The gene predicts a protein that is highly homologous to thecorresponding Saccharomyces cerevisiae enzyme and the gene further transforms wild typeY. lipolytica with a twofold increase in pyruvate kinase activity. The gene sequence containedan intron, the first reported in a Y. lipolytica gene.

3.2.8 Transport of citric acid

Although the outline of the biochemical pathways for the over-production of citric acidfrom n-alkanes is clear, two problems remain: the secretion mechanism and the reason forthe simultaneous production of isocitric acid. The transport mechanism(s) could involve


direct citrate excretion across the plasma membrane by some form of facilitated diffusion,active transport, or vacuolar transport, possibly by accumulation in vacuoles and exocytosis.Studies of the activities of enzymes in acetate mutants of Y. lipolytica (Rymowicz et al.,1993) indicated that the excretion of isocitric and citric acids depended more on the transportsystem than metabolite levels within the cell.

The vacuolar transport idea appeared promising when vacuoles from Y. lipolytica isolatedduring exponential growth showed the ability to concentrate citric acid through a citrateuniporter. The vacuoles showed high ATPase activity (1000 mU/mg protein at six hoursgrowth, falling to 270 mU/mg after 48 hours), which was not sensitive to orthovanadate,nor was it inhibited by azide or oligomycin. The citrate transport rate was up to 12 nmol/mgprotein/min after 12 hours growth, and calcium was also transported (140 nmol/mg/min).The vacuoles generated both a proton gradient and a membrane potential. However duringthe stationary phase, after nitrogen exhaustion, the transport ability fell to zero for bothcalcium and citrate. This observation was found to be true regardless of the growth limitingsubstrate, the carbon source, or whether citrate was released from the cells or not. Theconclusion was that the citrate transporting system of the vacuolar membrane was not involvedin the citrate release into the medium, and that that process was associated with transportsystems in the plasma membrane.

The ratio of excreted isocitric to citric acid is higher than would be expected from thethermodynamic equilibrium of aconitase, being as high as 40 per cent in wild-type yeasts,rather than the 7 per cent expected on an equilibrium basis. Nonetheless it is apparent thatthe strategies used to mitigate this unwanted production of isocitric acid all have a commontheme in that they inhibit or delete aconitase. By limiting the formation of isocitrate in thefirst place the problem is resolved. The strategies reported include: the use of iron-freemedium, which resulted in impairment of aconitase activity (Kimura and Nakanishi, 1973);the addition of sodium tetraborate, which may complex with iron to give a similar outcome(Furakawa et al., 1982); and the selection of mutants with low aconitase activities(Benckiser, 1974). These have been selected by their ability to grow on n-alkanes but notcitric acid and the low aconitase results in improved citrate to isocitrate ratios and decreasedbiomass, but the complete absence of aconitase would presumably be lethal. Otherstrategies are the use of inhibitors such as monofluoroacetate (Benckiser, 1974) which ismetabolized to monofluorocitrate and acts as a competitive inhibitor of aconitase (Akiyamaet al., 1972, 1973a, 1973b), and 2,4-dinitrophenol, an uncoupler of oxidativephosphorylation; and the addition of alcohols, up to oleyl alcohol (Kimura and Nakanishi,1973). The first two strategies are of use in selecting mutants but would be undesirable ina commercial fermentation. The relative levels of isocitrate lyase and aconitase indetermining the ratio of isocitrate to citrate was underlined by Finogenova et al. (1986)with a study of a series of C. lipolytica mutants. Mutants with a high isocitrate lyaseactivity and a low aconitase level synthesized citric acid almost exclusively regardless ofwhether the carbon source was glucose, alkane, ethanol, acetate or glycerol. The mutantlow in isocitrate lyase but with a high level of aconitase produced primarily isocitric acidon alkanes, where the ratio of citrate to isocitrate was 1:3.6, while on glucose the ratiowas 1.8:1. Wild-type strains with high levels of both enzymes gave intermediate results.In the wild-type strains the ratio could be shifted towards isocitrate synthesis by inhibitingisocitrate lyase with aconitate, the reverse of the industrial strategy.

The explanation advanced by Marchal et al. (1980) is still valid. They suggested thatthe high isocitrate ratio was a result of compartmentation within the cell. Whereas citrateis mainly mitochondrial, isocitrate is in the mitochondrial, the cytoplasmic and theperoxisomal compartments. Isocitrate will be exported from the mitochondrial


compartment to the cytosol and then to the peroxisome where it will be converted toglyoxylate and succinate. The absence of aconitase from the cytoplasmic compartmentwill result in higher isocitrate levels with lower citrate levels, and it is presumably fromthe cytoplasm that the acids are exported. It is further possible that citrate and isocitratehave differential transport, but no mutants have been reported to suggest that there is aseparate export mechanism for each acid.

3.3 Synthesis of citric acid from glucose

3.3.1 Introduction

The production of citric acid from glucose by C. lipolytica was established before theindustrial production from alkanes was initiated (Tabuchi et al., 1969; Abe et al., 1970),although an inhibitor of aconitase was required to minimize isocitric acid production (Pfizer,1972). The productivity on glucose was found to be similar to that on n-alkanes, so that theindustrial process, which in some cases had started out to make citric acid from alkanes,was adapted to synthesis from glucose without major problems. Such industrial plants weredesigned for yeasts, but the feedstock could be altered. To over-produce citrate from glucose,the same obstacles must be overcome as with synthesis from n-alkanes: an undiminishedsupply of precursors for citrate synthesis in the form of oxaloacetate and acetyl CoA, areduction in the catabolism of the citrate, an unregulated citrate synthase and a transportmechanism.

3.3.2 Pathway for citrate synthesis from glucose

The pathways involved in the synthesis of citrate in Candida yeasts are similar to those ofother organisms in basic properties; the over-accumulation is a result of differences inregulation rather than differences in mechanisms. The outline of the biochemical pathwaysis shown in Figure 3.4. The basic difference between the pathways on n-alkanes andglucose lies in the source of acetyl CoA: in the case of n-alkanes, ß-oxidation from fattyacids; in the case of glucose, by glycolysis. In both cases oxalacetate is synthesized by ananaplerotic route, either the glyoxylate cycle or in the case of glucose, pyruvatecarboxylase; the immediate reaction leading to citrate is citrate synthase with bothsubstrates. The differences lie in the direction of the pathways: with n-alkanes as a substrate,gluconeogenesis is required for the synthesis of metabolites derived from the glycolyticsequence; when glucose is the substrate, fatty acids must be synthesized from acetylCoA. Pyruvate carboxylase was shown to be the source of oxalacetate by Aiba andMatsuoka (1978). Its relative activity in glucose-grown cells is almost ten times that incells grown on n-alkanes (Finogenova et al., 1986), but it was only 10 per cent of theactivity of citrate synthase.

The incorporation of carbon dioxide into pyruvate and thence into citrate appears toinvolve both carbon dioxide from metabolism within the cell and from the medium. Intheory, however, the amount of carbon dioxide released in the pyruvate dehydrogenasereaction to yield acetyl CoA should be enough to form the oxaloacetate needed for citratesynthesis. When grown at a medium pH of 4.5, C. lipolytica showed 20 per cent incorporationof exogenous carbon dioxide into one of the carboxyl groups of citrate, but this fell to 8 percent at pH 6.0 (Zyakun et al., 1992).


Figure 3.4 The metabolic relationships of citrate metabolism in yeasts with n-alkanes orglucose as substrate. 1, Alkane monooxygenase; alkane, reduced rubridoxin:oxygen 1-oxidoreductase, 1.14.15.3. 2, Alcohol dehydrogenase; alcohol:NAD+ oxidoreductase,1.1.1.1. 3, Aldehyde dehydrogenase; aldehyde:NAD+ oxidoreductase, 1.2.1.3. 4, ß-oxidation. 5, Hexokinase; ATP:D-hexose 6-phosphotransferase, 2.7.1.1. 6, Glycolysis. 7,Pyruvate carboxylase; pyruvate:carbon dioxide ligase (ADP), 6.4.1.1. 8, Pyruvatedehydrogenase; pyruvate:lipoate oxidoreductase (acceptor acylating), 1.2.4.1. 9, Citratesynthase; citrate:oxaloacetate lyase (CoA acylating), 4.1.3.7. 10, Aconitase; citrate (isocitrate)hydrolyase, 4.2.1.3. 11, Isocitrate dehydrogenase; threo-DS-isocitrate:NAD oxidoreductase(decarboxylating), 1.1.1.41(42). 12. Isocitrate lyase; threo-DS-isocitrate:glyoxylate-lyase,4.1.3.1. 13, Malate synthase; 1-malate glyoxylate-lyase (CoA-acetylating), 4.1.3.2., 14,Gluconeogenesis


The subcellular location of the various enzymes has been determined (Sokolov et al.,1995a). Pyruvate carboxylase was found in the cytoplasmic compartment in C. lipolytica,and many other yeasts. The NADP+-dependent isocitrate dehydrogenase was found to bedistributed in both cytoplasmic and mitochondrial compartments, while ATP-citrate lyasewas found in the cytoplasmic compartment. The presence of this latter enzyme would berequired for lipid synthesis, but is presumably regulated so that it does not degrade asignificant amount of the cytoplasmic citrate. The number of peroxisomes in yeasts grownon glucose is very small.

The properties of the NAD+-dependent isocitrate dehydrogenase are thought to be centralto citrate accumulation when glucose is used as a substrate as well as n-alkanes. The AMPrequirement for activity means that the very low AMP levels found during the stationaryphase induced by nitrogen depletion will result in very low activity of the isocitratedehydrogenase (Bartels and Jensen, 1979). The enzyme was shown to be allostericallyregulated by AMP (Sokolov et al., 1995b) although with excess isocitrate the rate becameAMP independent. It is also inhibited by ATP which is high during citric acid accumulation.

The export of isocitrate from the mitochondial compartment is presumably reduced whenglucose is a substrate, as the glyoxylate cycle is non-functional because of the low level ofisocitrate lyase and the absence of malate synthase (Finogenova et al., 1986). This may bethe reason for the improved ratio of citrate to isocitrate produced when glucose is a substrate.The presence of the NADP+-dependent isocitrate dehydrogenase in both cytoplasm andmitochondria has been noted but its role, if any, is not known.

An important factor in the over-production of citric acid is the maintenance of the fluxthrough glycolysis when metabolite levels rise. In particular the inhibition ofphosphofructokinase by citrate might be expected to regulate the precursors for citrateproduction when citrate levels are high. In citrate producing strains of Y. lipolytica the citrateinhibition of phosphofructokinase appears to be weak (Sokolov et al., 1996), while AMPhas no effect. Ammonium suppressed the inhibitory effect of citrate and activated the enzyme,a similar mechanism to that suggested for A. niger (Habison et al., 1983). However in Y.lipolytica under conditions of nitrogen exhaustion, when growth has ceased it is less likelythat there is a significant pool of intracellular ammonium.

The entry of glucose into the cell is normally regulated, and under conditions of citrateaccumulation there is indeed a reduction in the glucose uptake rate (Aiba and Matsuoka,1978), suggesting that the regulation is present to some extent. The regulation of hexokinasehas been shown to be sensitive to trehalose-6-phosphate, which occurs in yeasts at about0.2 mM (Blazques et al., 1993). This is well above the apparent Ki for Y. lipolytica hexokinaseand it was concluded that this compound was physiologically significant. There was, however,

Table 3.7 Activities of enzymes of a Candida lipolytica strain grown on glucose


no activity against glucokinase up to 5 mM so that high levels of glucose might avoid theregulatory step at hexokinase.

A related substrate, glycerol, has attracted some attention, and the activities of glycerolkinase and the NAD+ and FAD-dependent glycerol-3-phosphate dehydrogenases, involvedin the glycerol phosphate shuttle between cytoplasm and mitochondria, were determined(Morgunov et al., 1991). Glycerol kinase was localized in the cytoplasm but both glycerolphosphate dehydrogenases were associated with the membrane fraction of the cells. Theglycerol kinase was purified and found to be inhibited by AMP, but insensitive to fructose-1,6-bisphosphate.

3.3.3 Nitrogen metabolism during growth on glucose

Yeasts and fungi contain both NAD+ and NADP+ dependent forms of glutamatedehydrogenase as well as glutamine synthetase. Glutamate dehydrogenase functions bothas an anabolic and catabolic enzyme:

The NADPH form acts primarily in the direction of glutamate synthesis although it isreversible; the NADH form acts as a catabolic enzyme providing a-oxoglutarate for thecitric acid cycle. The activity of the NADPH enzyme is increased under the nitrogen depletionwhich precedes citric acid excretion in Y. lipolytica (Peskova et al., 1996), while that ofglutamine synthetase is decreased as might be expected. Both the NADPH and the NADHglutamate dehydrogenases were located in the cytosolic compartment in Y. lipolytica whichis consistent with a role in synthesis of glutamate rather than energy metabolism. Glutaminesynthetase was also cytoplasmic. Interestingly the enzymes in the closely related C. maltosaare mitochondrial, and the organism does not produce citric acid. Aspartate aminotransferasewas located in the mitochondria in Y. lipolytica. Glutamate dehydrogenases are normallyallosterically regulated by inhibition by ATP or GTP and activation by ADP or GDP. It isnot known whether this situation occurs in Y. lipolytica, but it would be consistent with thehigh level of ATP and low ADP seen during the period of nitrogen starvation.

The importance of nitrogen levels to citric acid production was demonstrated by Moresi(1994) who determined kinetic constants for a Y. lipolytica strain at different initial glucoseconcentrations in the medium. Although increasing the glucose concentration from 40 to108 g/l gave a negative effect on the growth rate, the yield coefficients for glucose andnitrogen were approximately constant. By using a production medium without nitrogen, acitrate lag phase was observed during which the intracellular nitrogen fraction decreasedfrom about 8 per cent to a new low equilibrium value of less than 3 per cent. The idiophasewas found to be a non-growth associated process, and the citric acid formation rate wasdependent only on the cellular nitrogen concentration. The strain used in this study wascapable of equalling the productivity of the best A. niger mutants (about 1.05 g/l/h), but notthe selectivity as citric acid was only 85.5 per cent of the acid excreted; the majority of therest was isocitric acid.

3.3.4 Transport of citric acid during growth on glucose

The effect of various inhibitors on the excretion of citric acid has indicated that the exportof citric acid is energy requiring. The addition of protein synthesis inhibitors to cultures of


C. lipolytica at the time of nitrogen exhaustion inhibited the production of citric acid (Trutkoet al., 1993). At the same time, dinitrophenol (an uncoupler of oxidative phosphorylation),reumycin (respiratory chain shunting agent), or arsenate (which forms ADP-arsenyl insteadof ATP) all decreased the yield of citric acid in proportion to the concentration of the agent.There was no significant effect on biomass yield. Since the over-production of citric acidappears to involve some ‘uncoupling’ of the electron transport chain from ATP synthesis,with maximal levels of ATP resulting, the requirement for ATP shown here may be connectedwith export rather than synthesis, as may be the requirement for protein synthesis.

On the other hand, Kulakovskaya et al. (1994) showed that the activity of the plasmamembrane ATPase of Y. lipolytica decreased by a factor of ten during the course of nitrogenlimited growth with glucose as a carbon source. Citric acid excretion was independent ofglucose concentration and resistant to diethylstilboestrol, an inhibitor of the plasma membraneATPase, for the first 30 minutes of the excretion process. They concluded that the process isindependent of energy provision.

3.4 Conclusions

The over-production of citric acid by yeasts from both alkane and carbohydrate sources isnow well established, both commercially and scientifically. The basic pathways and someof the enzymology are understood, although many details remain to be resolved. With bothsubstrates the overproduction appears to represent a mechanism for recycling reducingequivalents and energy produced by unbalanced growth conditions in the form of the absenceof, and subsequent intracellular restriction on, a primary substrate from the growth medium.

Further developments in enzymology may arise coincidentally from the use of theorganism as a cloning vehicle, but one of the main unresolved problems is the mechanismof excretion, which is central to the problem of high productivity.

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55

4

Strain Improvement

GEORGE J.G.RUIJTER AND JAAP VISSER

4.1 Introduction

Many factors need to be considered by citric acid producers to obtain the economicallymost favourable process. Strain breeding is one of these factors. In this chapter we willsummarize ways to improve citric acid production genetically. Commercial production ofcitric acid is performed mainly with Aspergillus niger and to some extent with Candida (orYarrowia) lipolytica. As the existing fermentation processes usually give high yields, themain objective of strain breeding nowadays is shortening of fermentation time. However,other factors may also be relevant for strain improvement. For example, accumulation of ahigh concentration of citric acid by A. niger results from quite extreme culture conditionsand strain breeding may decrease the sensitivity of the process to these conditions.

The number of reports considering strain improvement that have appeared in literatureis limited. Röhr et al. (1983), Kubicek and Röhr (1986) and Mattey (1992) have reviewedmuch of the older work. However, some research and screening activities are ‘hidden’,i.e. performed by industry and not published for obvious reasons. We have structured thischapter more or less on the basis of the methodology used for strain improvement of A.niger:

1 mutagenesis and selection;

2 the use of the parasexual cycle; and

3 genetic engineering.

As strain breeding involves fungal genetics, some aspects of the genetic methodology usedhave been included.

4.2 General aspects of strain improvement

The initial A. niger production strains were isolated from their natural habitat, soil. Betterstrains have been derived from these isolates by various procedures. Basically, two methodscan be distinguished for strain selection. In the first method, acid production is tested for


individual colonies obtained from single spores (single spore method). Such a methodrequires automated screening procedures that enable testing of thousands of colonies andis therefore usually done by plate tests. A pH indicator is commonly included in themedium to estimate acid production, but since a pH indicator does not distinguish betweencitric acid and other acids, improved methods have been developed, e.g. using p-di-methylaminobenzaldehyde, which specifically measures citric acid (Röhr et al., 1979).Yields are evaluated by determining the ratio between acid zone and colony diameter.Obviously, statistical analysis of screening results is quite important to evaluate thesignificance of a difference in acid production between the parental strain and strainsderived from it. Liquid cultures, such as shake flasks, are not suitable in the initial stageof screening, but can be used in later steps for a limited number of selected strains. Analternative to screening by plates would be the use of ‘high throughput’ screeningprocedures, making use of microtitre plate technology. This has an even higher capacitythan plates as it can be automated to a large extent. Nowadays, microtitre plate technologyis commonly used by industry in all kinds of screening processes, but it is not clear whethercitric acid producers also employ it.

The second method comprises selection of mutants with a specific trait from a largepopulation using a suitable discriminative growth condition (passage method). Selectionmay be on the basis of resistance against an antimetabolite (Kirimura et al., 1992) or failureto grow on a particular carbon source (Akiyama et al., 1973a). Mutants can arisespontaneously or be produced by mutagenic treatment. A variety of methods are used formutagenesis including exposure to chemicals, UV light, g- and X-ray radiation (see e.g.Begum et al., 1990; Hamissa et al., 1991; Avchieva and Vinarov, 1993; Gupta and Sharma,1995). A serious drawback of mutagenic treatment is that high doses increase the chancesof obtaining more than one mutation per genome at a time. Thus, in addition to a mutationthat results in improved citric acid production, an isolate may have other mutations thatmight, for example, result in (slightly) reduced viability. To minimize the chances to introducesuch unwanted mutations, mutagenic treatment should be performed in such a way that ahigh percentage of survival is obtained.

When a better producing mutant is isolated it should be maintained in a proper way toprevent decay, i.e. lose its particular characteristics favourable for citric acid production.Decay is most pronounced during the vegetative stage and therefore storage of spores is thebest way to preserve a strain. The optimal storage method depends on the organism. A.niger conidiospores are usually stored on silica beads at 4°C or suspended in a 20 to 30 percent glycerol solution and frozen. Apart from natural variation certain mutations may beparticularly unstable, i.e. losing such mutation may be advantageous for the fungus. Forexample a certain mutation may result in improved citric acid production, but concomitantlycause reduced vitality. This necessitates careful preservation of original strains and possiblyfrequent re-isolation.

The biochemistry of citric acid biosynthesis has been reviewed before (Kubicek andRöhr, 1986; Mattey, 1992) and will not be treated at length here. Some aspects will howeverbe discussed in order to understand the rationale behind some strategies. Biosynthesis ofcitric acid from hexoses is depicted in Figure 4.1. Following uptake, hexoses are degradedmainly via glycolysis yielding pyruvate. Part of the pyruvate is converted to acetylCoA, part to oxaloacetate. Finally, these two compounds are condensed to citric acid,which is secreted and accumulated in the medium. Only in a few cases is the geneticbasis or biochemical mechanism of the improved performance by a mutant known.Schreferl-Kunar et al. (1989) isolated several mutants that grew better than the parent

Strain improvement 57

Figure 4.1 Schematic representation of biosynthesis of organic acids and polyols with A.niger. The following steps are depicted: 1, glucose oxidase; 2, lactonase; 3, glucose transport;4, hexokinase or glucokinase; 5, phosphoglucose isomerase; 6, fructose transport; 7,hexokinase; 8, mannitol–1-phosphate dehydrogenase; 9, mannitol-1phosphate phosphatase;10, mannitol transport; 11, phosphofructokinase; 12, aldolase; 13, triosephosphateisomerase; 14, glyceraldehyde–3-phosphate dehydrogenase; 15, phosphoglycerate kinase;16, phosphoglycerate mutase; 17, enolase; 18, pyruvate kinase; 19, pyruvate dehydrogenase;20, pyruvate carboxylase; 21, oxaloacetate hydrolase; 22, oxalate transport; 23, malatedehydrogenase; 24, citrate synthase; 25, tricarboxylate carrier; 26, citrate transport. Dashedarrows are used for multiple steps in biosynthesis of erythritol and glycerol. PPP, pentosephosphate pathway


on 14 per cent sucrose. The rationale of this selection procedure is the notion that a high rateof citric acid production requires the ability for fast sugar metabolism. Four mutants consumedsucrose faster and gave higher citric acid yields than the parental strain. Unfortunately, it isnot clear whether the productivity or just the final yield is improved in these mutants, althoughfaster sucrose consumption suggests increased productivity. Interestingly, all four mutantshad about twofold higher activity of the glycolytic enzymes hexokinase andphosphofructokinase, suggesting that the activity of these two enzymes is important incontrolling the rate of sugar consumption. In the following sections a few specific objectivesfor strain improvement will be discussed.

4.2.1 Improved yield on alternative substrates

In most liquid fermentation processes glucose, obtained from hydrolysed starch, or sucrose,in beet or cane molasses, are used as substrates. The semi-solid ‘Koji’ process uses agriculturalraw materials containing polysaccharides, such as starch and cellulose. Polysaccharidesgive low productivities in submerged fermentation processes (Begum et al., 1990),supposedly because their rate of hydrolysis to sugars is too slow. However, sincepolysaccharides are less expensive than glucose syrups or molasses, there have been someattempts to improve strains aiming at direct use, i.e. without prior hydrolysis, ofpolysaccharides (e.g. starch) in liquid fermentation (Rugsaseel et al., 1993; Suzuki et al.,1996). A mutant originally isolated as being 2-deoxyglucose resistant during growth oncellobiose (Sarangbin et al., 1993) showed enhanced citric acid production from solublestarch (Suzuki et al., 1996). The mutant had increased glucoamylase activity during citricacid fermentation on starch and the most probable explanation for these observations isdecreased repression by glucose of both ß-glucosidase, the enzyme catalysing hydrolysisof cellobiose to glucose, and glucoamylase, one of the enzymes catalysing hydrolysis ofstarch. Although some mutants give improved yields on starch, these yields are still lowcompared to those obtained on glucose and sucrose.

4.2.2 Decreased formation of by-products

During citric acid fermentation, conversion of substrate into compounds other than citricacid is undesirable for two major reasons. Firstly, by-products reduce the final yield andsecondly they complicate recovery of citric acid from the broth. In addition to citric acid, A.niger readily accumulates other acids, mainly gluconic acid and oxalic acid, and polyolcompounds, e.g. mannitol, erythritol and glycerol. Polyol compounds are formed from sugars,but will be reconsumed once the sugar substrate is exhausted. Therefore, polyol compoundsare probably not a major problem for the final yield of citric acid as long as the sugarsubstrate is completely consumed. The subsequent production and reconsumption of polyolsmay, however, reduce the rate of citric acid production. To our knowledge, no data areavailable on strains with reduced polyol production, but this may be related to the functionsof polyols in fungal physiology. It has been shown that conidiospores have a high mannitolcontent, probably functioning as carbon storage, whereas glycerol is the major solute inosmotic adjustment of the mycelium (Witteveen and Visser, 1995). Thus, polyol productionis probably vital and not liable to alterations.

Gluconic acid is formed from glucose with concomitant reduction of oxygen to hydrogenperoxide by the enzyme glucose oxidase. Glucose oxidase is an extracellular enzyme,


localized mainly in the cell wall (Witteveen et al., 1992) and induced by hydrogen peroxideand high glucose concentration (Witteveen et al., 1993). The enzyme is not stable at pHvalues below 2 to 3 and hence not induced since no H2O2 is formed. Oxalic acid is producedby oxaloacetate hydrolase, which is a cytoplasmic enzyme (Kubicek et al., 1988).Biosynthesis of oxaloacetate hydrolase is also regulated by external pH, but in this case themechanism is unclear. Induction of the enzyme is optimal at pH 5 to 6, whereas a very lowoxaloacetate hydrolase activity is observed at pH 2 (Kubicek et al., 1988). In pure sugarfermentations, production of gluconic and oxalic acid can thus be kept to a minimum bystarting the fermentation at a relatively low pH. In fermentations using molasses as a substrate,an initial pH of 5 to 6 is commonly employed, because conidiospores will not germinate atlower pH values. Therefore, in processes using molasses, production of gluconic and oxalicacid may be a problem favouring production strains lacking glucose oxidase and oxaloacetatehydrolase. In our laboratory a number of gox mutants have been isolated. One of themutations, goxC, results in the absence of glucose oxidase activity and strains carryinggoxC do not produce gluconic acid from glucose (Witteveen et al., 1990). Interestingly, agoxC mutant produces more oxalic acid from glucose than wild-type A. niger (Van de Merbelet al., 1994).

The major problem with production of citric acid by C. lipolytica is the simultaneousproduction of considerable amounts of isocitric acid. Wild-type C lipolytica strains produceapproximately equimolar amounts of citric acid and isocitric acid from n-alkanes, whereasless isocitric acid is produced from sugar substrates (Finogenova et al., 1986). Akiyama etal. (1973a) reasoned that a low activity of aconitase, the enzyme catalyzing the conversionof citric acid to isocitric acid, was essential to reduce production of isocitric acid. Theyselected a mutant that was more sensitive to fluoroacetate than the wild-type strain. Thismutant had approximately 1 per cent of the wild-type aconitase activity and produced virtuallyno isocitric acid (Akiyama et al., 1973a, 1973b).

4.2.3 A. niger mutants with a decreased sensitivity towards manganese

It is commonly known that the manganese concentration should be extremely low duringcitric acid fermentation with A. niger. Any addition of manganese results in a loweryield (Kubicek and Röhr, 1986; Mattey, 1992). Manganese deficiency has multipleeffects on physiology, e.g. increased protein turnover and altered cell wall composition,which probably means that the manganese effect is not clearly related to a particularcellular function. In pure sugar fermentations, manganese is usually removed by cationexchangers, whereas in molasses, manganese is precipitated with ferrocyanide.Obviously, mutants with a higher manganese tolerance would be advantageous, as thiswould make removal of manganese less critical. Gupta and Sharma (1995) reported anA. niger mutant which was more tolerant to manganese; it seems however that theirparental strain is already quite tolerant as addition of 0.5 ppm manganese does notdecrease the yield, while usually a level below 1 ppb is recommended (Mattey, 1992).Nevertheless, in the presence of 1.5 ppm manganese, citric acid production by the mutantwas threefold higher than obtained with the parental strain and similar to production inthe absence of manganese.

One of the effects of manganese deficiency is a relatively high intracellular NH level,which presumably is due to increased protein turnover (Kubicek et al., 1979). This high NHconcentration partially counteracts inhibition of the glycolytic enzyme, phosphofructokinase,by citrate (Arts et al., 1987). A mutant isolated by Schreferl et al. (1986) contained a


phosphofructokinase that was less sensitive to citrate than the one in the parental strain; thismutant accumulated approximately threefold more citric acid compared to the parent on amedium containing 20 mM manganese. However, the citric acid yield of the mutant in thepresence of manganese was only half that obtained with the parental strain on manganesedeficient medium, indicating that the effects of manganese cannot be attributed tophosphofructokinase alone.

4.2.4 Morphology of A. niger

Characteristic for citric acid fermentation with A. niger is a rather abnormal morphology,which has been attributed to manganese deficiency, although other process conditions, suchas pH, impeller speed and seeding level also affect morphology. Hyphae are abnormallyshort and stubby and the mycelium shows excessive branching. The aggregation of myceliuminto compact pellets is also reported to be important, but this may vary between strains andwith process conditions. An important benefit of such compact pellets is better rheology ofthe broth. A lower viscosity of the broth makes it easier to mix, requiring a lower powerinput for mixing and resulting in a higher dissolved oxygen tension. Efficient aeration isquite important as productivity decreases at lower dissolved oxygen tension and interruptionof the oxygen supply even results in cessation of citric acid formation. For processes operatingwith a filamentous mycelium, mutants with altered morphology, i.e. more branching, resultingin more compact aggregates, might be beneficial. Such mutants were easily obtained in thecase of Fusarium graminearum (Wiebe et al., 1989), but we are not aware of such mutantsin A. niger.

4.3 Isolation of recombinant strains using the parasexual cycle inA. niger

Crossing these strains might combine beneficial characteristics of different strains. A. nigerdoes not have a sexual cycle and crossings therefore involve the so called ‘parasexual cycle’,which is not a life cycle, but a series of independent steps, i.e. fusion of hyphae resulting inheterokaryon formation, fusion of the nuclei of the different parents to form a diploid,mitotic recombination and finally haploidization of the diploid strain to yield haploid strainsagain (Pontecorvo et al., 1953). If crossing of strains is impossible due to heterokaryonincompatibility, fusion of protoplasts can be used to obtain heterokaryons. Protoplasts canbe prepared by treatment of mycelium with cell wall lysing enzymes in an osmoticallystabilized medium.

Usami and coworkers (Kirimura et al., 1988a, 1988b; Sarangbin et al., 1994) haveinvestigated the application of A. niger diploids and haploid recombinants in citric acidfermentation. They have fused protoplasts of a strain optimized for submerged fermentationand a strain optimal for semi-solid fermentation. Some of the resulting diploid strains andhaploid recombinants were better producers than both parents (Kirimura et al., 1988a, 1988b),but most were without significant improvement. The reason for higher production by diploidsor haploid recombinants may be combination of beneficial mutations or complementationof adverse mutations introduced in the parents during previous mutagenic treatment. In thecase of diploids the presence of two copies of the genome might result in overproduction ofcertain enzymes.


4.4 Genetic engineering

Strain improvement by the techniques described in the previous sections is largely a trialand error process involving laborious screening procedures. Moreover, in many cases theimproved performance is ‘magic’, as the underlying mechanism is not identified. Geneticengineering, on the contrary, is a rational approach as particular metabolic steps aremanipulated. The use of recombinant DNA technology to improve citric acid productionhas been employed only recently, although transformation of A. niger was reported in1985 (Buxton et al., 1985; Kelly and Hynes, 1985). C. lipolytica transformation is alsopossible (Davidow et al., 1985), but we are not aware of any reports of genetic engineeringof C. lipolytica to improve citric acid production. Different protocols for transformationof A. niger exist, but the most commonly used method involves polyethyleneglycolmediated uptake of DNA by protoplasts, followed by regeneration on a suitable selectivemedium. Introduction of DNA fragments (either circular or linearized) into A. niger resultsin integration of the DNA into the genome of the recipient strain. Integration can occureither at the homologous locus or at other loci. Multiple copies (tandemly integrated orscattered over the genome) of the gene introduced can be obtained. Expression of thegene introduced depends on the copy number and on the site of integration. To date thereare three cases of genetic engineering concerning citric acid production by A. niger, whichwill be addressed in Sections 4.4.2 and 4.4.3, but first we will discuss some aspects ofmetabolic modelling.

4.4.1 Quantitative analysis of metabolism

For genetic engineering it is necessary to have at least some idea of which enzymatic stepshould be altered to increase the metabolic flux through the pathway of citric acidbiosynthesis. However, to find the optimal strategy for metabolic engineering, it isnecessary to analyze the metabolism involved quantitatively. For example, the simplefinding that an enzyme has a low activity in vitro does not mean that it is ‘rate-limiting’ invivo, since the activity of an enzyme in the cell also depends on the concentrations of itssubstrates, products and possible effectors. To understand the control properties of ametabolic pathway, two major theoretical frameworks have been developed. Metaboliccontrol analysis (MCA) was established independently by Kacser and Burns (1973) andHeinrich and Rapoport (1974), whereas biochemical systems theory (BST) was developedby Savageau (1976). The majority of the literature concerns MCA and the formalism ofMCA and its applicability in biotechnology have been reviewed extensively (Kell andWesterhoff, 1986; Fell, 1992, 1997). Only a few of the basic concepts of MCA and BSTwill be discussed here. Both theories use the characteristics of the metabolic pathwayunder study, i.e. the kinetic properties of the enzymes, to describe it quantitatively. Withthis description it is possible to perform a sensitivity analysis. The effect of a small variationin, for example, the activity of an enzyme on the steady-state flux through the pathway(which is the rate of conversion of the primary substrate to the final product) can becalculated. In MCA the ‘flux control coefficient’ (C) was introduced, which is defined asthe fractional change in flux (J) divided by the fractional variation in enzyme activity (e):(dJ/J)/(de/e). In most cases flux control coefficients have values between 0 (the flux doesnot change upon an increase in enzyme activity, i.e. no flux control) and 1 (the change influx is proportional to the change in enzyme activity, i.e. the enzyme is completely rate-limiting).


An important feature of MCA is the summation theorem, which states that the sum ofthe flux control coefficients of the enzymes in a pathway is equal to 1. As a consequence,the flux control coefficient of any enzyme in a very long pathway is probably very small asthere are many enzymes contributing to control. Moreover, when an enzyme with someflux control is overproduced, control readily shifts to another step in the pathway. In practicethis means that genetic engineering is not easy in complex pathways. The benefit of controlanalysis in designing strategies to optimize biotechnological processes depends heavily onthe availability of enzyme kinetic data and on the reliability of these data. In the case ofcitric acid biosynthesis by A. niger quite a few enzymes have been studied now but a few,such as the transport steps, are less well or not at all investigated, hampering a preciseanalysis.

Recently, a few attempts have been performed to analyse flux control in citric acidbiosynthesis by A. niger (Torres, 1994a, 1994b; Ruijter et al., 1996; Torres et al., 1996a).Torres performed modelling of the first part of the pathway, i.e. up to pyruvate (see Figure4.1) using BST formalism and suggested that sugar transport and phosphorylation, whichare lumped into one step in the model, form the most important step in controlling the fluxthrough the pathway. Thus, according to this model, the cellular amount of sugar transporterand/or hexokinase should be increased to obtain a higher metabolic flux. To a certain extentthese findings correlate with experimental data. As described in Section 4.2 certain mutantswith improved citric acid production had increased activity of hexokinase andphosphofructokinase (Schreferl-Kunar et al., 1989) and Steinböck et al. (1994) found thatsome 2-deoxyglucose resistant mutants had lower hexokinase activity and produced lesscitric acid than the parent. From an investigation of glucose transport in A. niger, Torres etal. (1996b) concluded that hexokinase contributed more to flux control in glycolysis thanglucose transport.

In a subsequent study (Torres et al., 1996a) it was concluded from flux optimizationcalculations that simultaneous overproduction of seven enzymes was required for a significantincrease in flux. For practical reasons this is not achievable at the moment. Firstly, most ofthe A. niger genes required for this approach are not available and secondly, simultaneousoverexpression of seven enzymes in a controlled way is experimentally difficult toaccomplish. Notably, this model has not incorporated the metabolism from pyruvate toextracellular citric acid and hexokinase might have flux control in the conversion of glucoseto pyruvate, but the control in the complete pathway (hexose to citric acid) might be in latersteps i.e. between pyruvate and citric acid. Nevertheless, a modelling approach is worthwhile.It may not produce an exact solution to improve the process, but it provides a guideline forgenetic engineering of A. niger.

4.4.2 Manipulation of the respiratory chain in A. niger

In addition to the normal respiratory chain, A. niger possesses alternative respiratoryenzymes, including an NADH oxidase and an ubiquinol oxidase (Zehentgruber et al.,1980; Kirimura et al., 1987; see also Figure 4.2). In the course of a citric acid fermentationthe activities of the normal respiratory enzymes decrease whereas the activities of thealternative oxidases increase (Kirimura et al., 1987; Wallrath et al., 1991). The alternativeoxidases do not pump protons concomitantly with electron transport and their physiologicalfunction is thought to be removal of excess reducing equivalents. Such a function is inagreement with the presence of the alternative oxidases during citric acid production.Conversion of hexoses to citric acid results in net production of ATP and NADH. Since


there is no growth in the stage of citric acid accumulation, the cells probably do not requiremuch ATP, and a switch from normal respiration to alternative oxidases would enable thefungus to reoxidize its NADH without concomitant ATP production.

A very attractive hypothesis has been put forward by the group of Weiss. They foundthat the proton-pumping NADH:ubiquinone oxidoreductase (complex I) is very fragile inA. niger B-60, which is a good citric acid producer, compared to a wild-type A. niger strain(Schmidt et al., 1992; Wallrath et al., 1992). The selective loss of complex I might result inan increased NADH/NAD+ ratio in the cell, because the affinity of the alternative NADHoxidase for NADH is approximately one order of magnitude lower than that of complex I.Excretion of citric acid is a possibility in order to get rid of the excess reducing equivalents.As such, the switch to alternative oxidases is not a reaction of the fungus to citric acidproduction, but the loss of complex I results in initiation of citric acid accumulation. To testthis hypothesis one of the subunits of complex I was inactivated in a ‘wild-type’ (badproducing) A. niger strain by disruption of the corresponding gene, nuo51, by moleculargenetic techniques (Prömper et al., 1993). The mutant was unable to form a functionalcomplex I and should accordingly accumulate citric acid as B-60 does. Unexpectedly, themutant excreted virtually no citric acid, whereas the wild-type A. niger strain producedapproximately 30 per cent of the yield obtained with B-60. However, the mutant accumulatedhigh intracellular levels of TCA cycle intermediates, including citrate. Apparently, the mutantis indeed unable to reoxidize NADH under these conditions, resulting in accumulation ofTCA cycle intermediates. Prömper et al. propose that, in contrast to wild-type A. niger andstrain B-60, the mutant is unable to excrete citric acid (or other TCA cycle intermediates).This postulate, i.e. the presence of a citrate carrier, may explain the differences in citric acidproduction between wild-type A. niger and B-60, but does not resolve the discrepancybetween wild-type A. niger and the mutant lacking complex I. It would be interesting to testthe effect of disruption of nuo51 in strain B-60. In addition to the effect it might have oninitiation of citric acid accumulation, it might also bring about an increase in the rate of acidproduction. Assuming an excess of ATP during citric acid production, inactivation of complexI would be a way to decrease such an excess, since less ATP is produced per NADH.

4.4.3 Engineering of glycolysis in A. niger

Obviously a high metabolic flux is necessary for fast citric acid accumulation. To date, tworeports have been published in which attempts to increase metabolic flux and henceproductivity are described. Arisan-Atac et al. (1996) describe an increase in the rate of

Figure 4.2 Schematic representation of the normal and alternative respiratory chains. Thenormal respiratory chain (lower part) contains three complexes: NADH:ubiquinoneoxidoreductase (complex I), ubiquinol:cytochrome c oxidoreductase (complex III) andcytochrome c oxidase (complex IV). In the alternative respiratory chain (top part) electronsare tranferred directly from ubiquinol to oxygen


citric acid accumulation by a mutant of A. niger strain B-60 in which the gene encoding asubunit of trehalose-6-phosphate synthase, ggsA, was disrupted. This mutant lacks trehalose-6-phosphate synthase activity and the rationale for construction of this strain was thefollowing. Trehalose-6-phosphate and the enzyme catalyzing its biosynthesis have recentlybeen shown to play a role in regulation of glycolytic flux in the yeast Saccharomycescerevisiae (Thevelein and Hohmann, 1995). Trehalose-6-phosphate inhibits hexokinaseactivity in S. cerevisiae in vitro (Blázquez et al., 1993) and this was found to be also the casein A. niger (Arisan-Atac et al., 1996; Panneman et al., 1996). Inactivation of trehalose-6-phosphate synthase would result in the inability to synthesize trehalose-6-phosphate and if,under citric acid producing conditions, trehalose-6-phosphate inhibits glycolysis in A. niger,the absence of trehalose-6-phosphate synthase might result in an increased glycolytic fluxand increased citric acid production. This was indeed found to be the case. The ggsAdisruption strain produced the same final yield of citric acid as the wild-type strain, butreached this yield in a shorter fermentation time. This is the only case where geneticengineering of A. niger results in improved citric acid production.

Recently we have studied in our laboratory, the effects of overproduction of two glycolyticenzymes, phosphofructokinase and pyruvate kinase (Figure 4.1) on citric acid productionby A. niger (Ruijter et al., 1997). A few experimental studies have suggested thatphosphofructokinase might be an important step in control of the glycolytic flux. Firstly,cultivation on a high concentration of sucrose, glucose or fructose stimulated citric acidaccumulation by A. niger and these conditions also led to increased intracellular levels offructose-2,6-bisphosphate, a potent activator of phosphofructokinase (Kubicek-Pranz et al.,1990). Secondly, as already addressed in Section 4.2, mutants selected for the ability togrow fast on high concentrations of sucrose exhibited increased citric acid production andin these strains the activities of hexokinase and phosphofructokinase were twofold higherthan in the parental strain (Schreferl-Kunar et al., 1989). We have overexpressedphosphofructokinase and pyruvate kinase, both individually and simultaneously, in A. nigerN400 (Ruijter et al., 1997). Unfortunately, moderate overexpression of these enzymes (threeto five times the wild-type level) did not enhance citric acid production by the fungussignificantly (Figure 4.3). Overexpression of pyruvate kinase even appeared to have a negativeeffect on citric acid production. Thus, phosphofructokinase and pyruvate kinase do notseem to contribute in a major way to flux control of the metabolism involved in the conversionof glucose to citric acid. However, it must be noted that in cells overproducingphosphofructokinase, the concentration of fructose-2,6-bisphosphate was decreasedapproximately twofold compared to the wild-type. Hence, the fungus appears to adapt tooverexpression of phosphofructokinase by decreasing the specific activity of the enzymethrough a reduction in the level of fructose-2,6-bisphosphate. From his modelling studiesTorres (1994b) also concluded that phosphofructokinase and pyruvate kinase did not haveflux control. In the model of Torres, however, regulation of phosphofructokinase by fructose-2,6-bisphosphate was not included. Our data suggest that overproduction ofphosphofructokinase, while maintaining or increasing fructose-2,6-bisphosphate levels, maystill increase glycolytic flux in A. niger.

4.5 Concluding remarks

Although the strains utilized for commercial production of citric acid are undoubtedly high-yielding, further strain improvement will most certainly be attempted. At the moment theprimary strategy for strain breeding is probably still mutagenesis and selection. Quantitative


analysis of metabolism and metabolic pathway engineering are only just being implemented,but in our view this is a promising approach, not so much as an alternative to the traditionalstrain breeding methods, but complementary to it.

4.6 Acknowledgements

GR is financially supported by the Dutch Ministry of Economic Affairs, the Ministry ofEducation, Culture and Science, The Ministry of Agriculture, Nature Management andFishery in the framework of an industrial relevant research programme of The NetherlandsAssociation of Biotechnology Centres (ABON).

4.7 Reference

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5

Fungal Morphology

MARIA PAPAGIANNI

5.1 Introduction

In submerged culture the morphology of filamentous micro-organisms varies between pelletsand free filaments depending on culture conditions and the genotype of the strain. All thegrowth forms have their own characteristics concerning growth kinetics, nutrient consumptionand broth rheology. Of the two extremes, pellet suspensions exhibit Newtonian rheologicalbehaviour, while the filamentous form produces more viscous media with consequent effectsof poor mixing and mass transfer. This is unfortunate, as it is very often the case that thedisperse filamentous form is the productive form. Another drawback with the pelletedsuspension is that cell growth occurs only at the surface of the pellets where contact withoxygen and other nutrients is adequate, and the cells growing within a pellet respond to a verydifferent environment. Further into the pellet, mass transfer limitation will gradually occurand cells could autolyse.

This chapter deals with the factors that affect Aspergillus niger morphology in submergedculture and the influence of morphology on productivity in citric acid fermentation.

5.2 Factors affecting Aspergillus niger morphology in submerged culture

According to many reports, the morphology of the mycelium is crucial to the process offermentation, not only in relation to the shape of the hyphae themselves and the aggregationinto microscopic clumps (micro-morphology), but also in the pelleted form of growth(macro-morphology). In all cases reported, the mycelium of acidogenic Aspergillus nigerwas found to conform to the morphological type described by Snell and Schweiger (1951):short, swollen branches which may have swollen tips. The mycelial pellets should besmall with a hard, smooth surface (Clark, 1962). It is known that this is brought about byadjustment of aeration and agitation (Svenska Sockerfabrik, 1964), adjustment of pH(Fried and Sandza, 1959), concentration of manganese and other trace metals (Shu andJohnson, 1948; Clark et al., 1966; Kisser et al., 1980), and inoculum level (Berry et al.,1977). However, it is not known whether the pelleted or filamentous form is more desirablefor citric acid production.


Since the morphological form can strongly influence the overall process productivity,research on various aspects of morphological development has attracted the interest ofacademia and industry and attempts have been made to induce a particular form of growthand to relate morphology to product synthesis.

Initial investigations of mycelial morphology relied on manual measurements fromphotographs and little quantitative work was presented until the 1970s. Detailedmorphological characterization of the free filamentous form was first presented by Metz etal. (1981). Their method, which made use of an electronic digitizer to make measurementsfrom microphotographs, was time consuming, inaccurate and also difficult to automate. In1988, Adams and Thomas presented the first image analysis method for morphologicalmeasurements of a filamentous fungus, using images taken directly from the microscope toan image analyzer. Since then, highly automated methods have been developed which havemany applications and allow detailed characterization of growth and simple differentiationof filamentous micro-organisms (Thomas, 1992). With the large variety of products producedby filamentous organisms and their complex physiology, a method that provides accurateand reproducible quantitative morphological characterization is invaluable in studies ofprocess optimization and modelling.

In this chapter, the effects of agitation, nutritional factors (type and concentration ofcarbon source, nitrogen and phosphate limitation, pH, dissolved oxygen tension, trace metalslevels) and inoculum size will be discussed with respect to the micro-morphology of A.niger.

5.3 Effect of agitation

In submerged fermentation, agitation is important for adequate mixing, mass transfer andheat transfer. For aerobic fermentation, mixing is required to ensure sufficient oxygen transferthroughout the reactor vessel and aeration has been shown to have a critical effect on thesubmerged process of citric acid fermentation. Agitation creates shear forces that affectmicro-organisms in several ways, causing morphological changes, variation in their growthand product formation and also damage in the cell structure.

For the dispersed form, in filamentous fermentation the effects of agitation superimposedon the fermentation process are difficult to quantify. Changes in morphology of filamentousfungi as a result of intensive agitation conditions have been observed in many cases (Dionet al., 1954; Belmar-Beiny and Thomas, 1991; Papagianni et al., 1994). Under theseconditions hyphae were thick, short and densely branched and this morphological type isusually associated with increased product yields. However, high impeller speeds were foundto promote mycelial growth and possibly to stimulate the occurrence of metabolic pathwayswhich resulted in low productivity of citric acid. The effect of stirrer speed on growth andproductivity of three Aspergillus niger strains was reported by Ujcova et al. (1980). Higherspeeds resulted in thicker and highly branched filaments. There was a drop in productivityat higher speeds although growth remained rapid. A similar effect for penicillin fermentationwas reported by König et al. (1981). At higher speeds only a short period of penicillinproduction was maintained and a large fraction of the substrate was converted into carbondioxide.

It has been reported that increased agitation can lead to breakage of hyphae for anumber of micro-organisms (Märkl and Bronnenmeier, 1985; Belmar-Beiny and Thomas,1991). Although A. niger cultures are normally resistant to shear damage, mycelialfragmentation due to mechanical forces has been reported. The damage of hyphae and the

Fungal morphology 71

consecutive release of intracellular material may account for the decreased productivitiesreported in many cases under intensive agitation conditions. This has been proven forPenicillium chrysogenum. Smith et al. (1990) and Makagiansar et al. (1993) observed thatthe lower rates of penicillin synthesis at high agitation speeds were due to increased damageof hyphae, since it involved a greater frequency of circulation of mycelia through the highenergy dissipation zone around the impeller.

Case study 1

The effect of agitation on A. niger morphology and citric acid production has been studiedin a tubular loop (TLR) and a stirred tank reactor (STR), through a series of batch experimentscarried out at different circulation times (4 to 18 s) in the case of the TLR and at stirrerspeeds from 100 to 600 rpm for the STR (Papagianni, 1995). Both reactors were inoculatedwith a vegetative inoculum. The inoculum filaments started to clump within 24 hours offermentation in all experiments.

Morphological measurements using image analysis showed that by increasing the intensityof agitation, the size of clumps (P1) decreased, as did the length of the filaments (L) thatarose from the cores of clumps, while the diameter of filaments (d) increased. The perimeterof the clumps was measured by joining the tips of the filaments that arose from the core ofthe clumps. For the estimation of the core of clumps (P2), lines were drawn around the coreand their combined length was measured. For the estimation of L, the length of the filamentsand branches that arose from the core of the clump was measured.

In both fermenters, specific rates of citric acid formation, sp.rp, increased with agitation;the amount of citric acid produced at the end of the runs (at 168 h) was dependent on thecirculation time and the stirrer speed. In the STR, as the stirrer speed increased citric acidproduction increased up to a point (300 rpm) beyond which it remained constant. In Figures5.1 and 5.2 the effect of agitation on citric acid production and the relation between productionand morphology is shown. In both reactors low dissolved oxygen levels were observedunder conditions of low agitation intensities.

If the intensity of agitation was changed, different patterns of morphologicaldevelopment were observed in both fermenters. The effect of intensive agitation wasmore pronounced in the stirred tank reactor, since the length of filaments was reduced bya factor of three, while in the loop rector the reduction was much smaller. Figure 5.3 shows the

Figure 5.1 Effect of agitation on citric acid production and the relation between productionand morphology parameters in the tubular loop reactor


time course of P1 in the STR fermentations whilst the time course for the hyphal length inthe TLR is shown in Figure 5.4.

The mean diameters of filaments also changed during fermentation; filaments becamethinner with time in all experiments performed in both fermenters. The reduction of hyphaldiameter was found to be dependent on agitation: the faster the broth circulation and thehigher the stirrer speed, the more rapid was the reduction.

These differences in morphological development during fermentation could not beexplained by the assumption of increased branching alone (newly formed branches would

Figure 5.2 Effect of agitation on citric acid production and the relation between productionand morphology in the stirred tank reactor

Figure 5.3 Time course for the clump perimeter, P1, at different stirrer speeds in the stirredtank fermentations


lower the mean values of L and P1) under conditions of intensive agitation. It is known thatincreased branching frequency is associated with increased specific growth rates (Katz etal., 1971; Morrison and Righelato, 1974). This is not the case for these experiments. For theruns in which values for the specific growth were comparable rate (e.g. at circulation times4s and 10s in the TLR), very different time courses of L as well as of P1 and P2 wereobserved. This also applied to stirred tank fermentations; at 500 rpm, L decreased rapidlyfor the first 100 hours and for the rest of the run decrease was slower, while m values for theperiod 30 to 72 hours were lower than those noted at 300 rpm. Hyphal fragmentation as aresult of increased agitation intensity could explain the different patterns of time coursesobserved in these studies. Thus, under intensive agitation conditions a cycle of fragmentationand regrowth takes place while at low agitation intensities a gradual ageing processpredominates and the filaments grow long, with few branches remaining; mean values of Land P1 are higher.

Mitard and Riba (1988), studying the effect of stirrer speed on A. niger growth andmorphology, observed that there was a relationship between the specific growth rate of theorganism and the rupture of mycelial aggregates. As the aggregates were broken, the specificgrowth rate reduced; it increased again as the liberated filaments went on growing. Thiscould explain the lower specific growth rates observed at 500 rpm during the period between30 and 72 hours and the rapid reduction of the length of filaments. At this stirrer speed P1and P2 also decreased for 100 hours from inoculation; after this period their mean valuesincreased again towards the end of the run. This indicates that fragmentation and regrowthtook place.

From Figures 5.1 and 5.2, it can be observed that beyond a stirrer speed of 300 rpm,changes in morphology were not followed by changes in citric acid production. However,the specific rates of citric acid formation were stirrer speed dependent as was themorphology. As the specific production rate increased with the stirrer speed, the parameters

Figure 5.4 Time course for the specific growth rate and hyphal length, L, as a function ofcirculation time in the tubular loop reactor


P1 and L reduced. The core perimeter, P2, appeared to be not affected by agitation and itcannot be directly linked to citric acid production.

As Figures 5.1 and 5.2 show, the morphological characteristics of the broth are similar inthe two reactor systems and there appear to be no fundamental differences between the resultsobtained from the loop and the stirred tank reactor. To compare the two different systems thecirculation times in the STR were estimated and they were found to be 1.1, 1.8, 2.2, 3, 3.8 and6 seconds, while the mixing times were 8, 12, 14, 15, 16 and 22 seconds for the stirrer speedrange of 600 to 100 rpm. Compared to those of the loop reactor, both circulation and mixingtimes in the STR were smaller. As a means of comparing the mixing characteristics of the twoferments, the dimensionless parameter relative mixing time, tm, can be used. The relativemixing time is equal to the mixing time divided by the circulation time (tm = tm/tc). It wascalculated and found to be within the range of 4 to 8 in both reactors.

In Figure 5.5, the final values for citric acid concentration and P1 have been plottedagainst tm for both reactors. As shown in this figure, by increasing tm, citric acid productionincreased, while the perimeter of clumps decreased, with good agreement between the tworeactor systems. It appears that the amount of product and morphology is a function of tmand for this fermentation, production and morphology are strongly linked.

5.4 Effect of nutritional factors

Citric acid accumulation is strongly influenced by the composition of the nutrient medium.The medium constituents which have been found to exert an effect on citric acid fermentationare: type and concentration of the carbon source, supply of nitrogen and phosphate, pH,dissolved oxygen levels and concentration of certain trace metals. The influence on

Figure 5.5 Plots of the final values for citric acid concentration and the morphologyparameter P1 against the relative mixing time in the loop and stirred reactors


morphology of A. niger or other filamentous micro-organisms in submerged culture formost of these factors has been studied.

5.4.1 Type and concentration of carbon source

The carbon source for citric acid fermentation has been the focus of much study, frequentlywith a view to the utilization of polysaccharide sources (Gupta et al., 1976; Hossain et al.,1984; Xu et al., 1989). The nature of the source has been shown in many cases to affectcitric acid production, since it exerts a strong effect on levels of enzyme activity within theTCA cycle. In general, only sugars, which are rapidly taken up by the fungus, allow a highfinal yield of citric acid. Polysaccharides, unless hydrolyzed, are not included in this category.Information concerning the role of the nature of sugar source on A. niger morphology incitric acid fermentation is limited. However, it has been observed that factors favouringincreased growth rates, such as media rich in easily assimilated nutrients, affect morphologyby reducing pellet formation in filamentous organisms (Hemmersdorfer et al., 1987).

Not only the type, but also the concentration of the carbon source is critical to thisfermentation, influencing the rate of production and the final yield, in addition to growth ofthe fungus. In the following two case studies, the influence of glucose concentration onmorphology and productivity of A. niger in batch and fed-batch culture will be discussed.

Case study 2

Studies in conventional batch culture confirmed that the initial glucose concentration in thefermentation medium affects the rates of citric acid fermentation and morphology of A.niger (Papagianni, 1995). In batch experiments performed in an STR at initial glucoseconcentrations of 150 g l-1, 100 g l-1 and 60 g l-1 it was found that the specific production ratedecreased with the initial glucose concentration. For the first 48 hours, the specific growthrate increased as initial glucose concentrations decreased.

Initial glucose concentration clearly affected the length of filaments, L, as Figure 5.6shows. In early fermentation stages, L decreased with decreasing glucose levels. Towardsthe end of the fermentation, when the differences in the sugar level diminish, it seems thatthe length of the filaments converges. The parameter L could be regarded as an indication ofbranching, since the high degree of clumping made impossible the counting of branchingpoints. As these fermentations were performed at the same stirrer speed and pH and thespecific growth rate was found to increase for the first 48 hours of fermentation, thereshould be a link between specific growth rate and branching frequency for these experiments;a large value for L would indicate few branches. Increased branching frequency and reductionin the hyphal growth unit with increasing specific growth rates have been reported in theliterature (Katz et al., 1972; Morrison and Righelato, 1974).

Case study 3

To eliminate the effect of a constantly changing glucose concentration in the batchexperiments, a series of fed-batch runs, where glucose levels were maintained constantduring the citric acid production phase, were performed under otherwise identicalfermentation conditions. The range of glucose levels included the following concentrations:130 g l-1, 88 g l-1, 70 g l-1, 44 g l-1, 17 g l-1 and 5 g l-1. The specific rate of citric acidformation was found to increase with glucose levels (see Figure 5.7). In contrast, for


Figure 5.6 Effect of initial glucose concentration on length of filaments in batch fermentations

Figure 5.7 Effect of glucose levels on the specific rate of citric acid production in fed-batchfermentations


the early stages of fermentation (48 hours), the highest specific growth rate values wereobserved when glucose was maintained at the lowest glucose concentration of 5 g l-1. Laterin the fermentation, glucose level had little influence on the specific growth rate. The meanlength of filaments at 24 hours in fed-batch runs with glucose levels in the range from 130g l-1 to 17 g l-1, decreased with decreasing glucose levels (Figure 5.8). Mean values of P1(Figure 5.9) and L were found to be smaller in fed-batch runs with glucose levels between130 g l-1 and 17 g l-1 after 24 hours of fermentation, than those observed in the batch runwith initial glucose at 150 g l-1. The values of the specific growth rate were significantlyhigher in fed-batch experiments for the first two days of fermentation than those obtained inthe batch run at 150 g l-1 glucose. Since it was observed in both cultures that the specificgrowth rate values for the first 48 hours were increased with decreasing glucose levels, thereduction in L could be a result of increased branching.

Fermentation rates and morphology developed in a different way when glucose waskept at 5 g l-1 throughout fermentation. The very low glucose levels in this run affectedmetabolism, since citric acid formation was reduced in favour of cell growth and thechanges in metabolism were accompanied by changes in morphology, as shown in Figures5.8 and 5.9. Morphological changes also included the appearance of pellets after threedays of fermentation, although the bulk of the mycelium was in the form of clumps. Ithas been suggested that factors favouring increased growth rates may reduce pelletformation in fungi (Hemmersdorfer et al., 1987). This is in contrast to our observations

Figure 5.8 Time course for the mean length of filaments under different glucose levels in fed-batch fermentations


of the values for the specific growth rate which were the highest noted and for the first timepellets appeared in the broth.

It seems clear that the carbon source concentration affects citric acid production ratesand A. niger growth and morphology. For this system morphology and product formationwere closely related, since the reduction of the length of filaments and size of clumps wasassociated with increased specific production rates.

5.4.2 Nitrogen and phosphate limitation

In order to accumulate citric acid, growth must be restricted, but it is not clear whetherphosphate or nitrogen is the necessary limiting factor. According to Shu and Johnson (1948),phosphate does not have to be limiting, but when trace metal levels are not limiting, additionalphosphate results in side reactions and increased growth. Kubicek and Röhr (1977) showedthat citric acid accumulated whenever phosphate was limited even when nitrogen was not.In contrast, Kristiansen and Sinclair (1979), using continuous culture, concluded that nitrogenlimitation was essential for citric acid production.

Pellet formation in filamentous fungi has been discussed in many cases and among thefactors considered to induce it, is the limitation of particular nutrients, including nitrogen(Braun and Vecht-Lifshitz, 1991). On the other hand, factors favouring increased growthrates, including excess phosphate concentrations, have been shown to reduce pellet formation.The following case study examines the effect of increased phosphate concentration onmorphology of the free filamentous form.

Figure 5.9 Time course for the perimeter of the clumps under different glucose levels in fed-batch fermentations


Case study 4

Figure 5.6 shows the time courses of the morphological parameters P1, P2 and L, of A. nigerclumps, for two experiments with KH2PO4 concentration in the fermentation medium of 0.1 gl-1 and 0.5 g l-1. Both experiments were carried out in the loop fermenter of case study 1, at thecirculation time of 18 s. As Figure 5.10 shows, a small increase in phosphate level led todrastic changes in morphology. The clump perimeter became almost three times larger, whilethe perimeter of the core remained small. The clumps lost their compact structure and theform of an extended growth around a small core predominated. The length of filaments increasedduring fermentation, while at the lower phosphate level, after an early growth phase, L remainedat the same levels until the end of the run. The fermentation itself was also drastically affected,since the yield of citric acid on glucose consumed fell from 70 per cent to 39 per cent onincreasing the phosphate level, while biomass concentration increased from 6.1 g l-1 at thelower to 11.5 g l-1 at the higher phosphate level. The differences in specific growth rates andbiomass concentrations could explain the different time course for the hyphal length L in thetwo runs. In contrast to the very high increase of P1, L remained comparatively small. Thiscould be an indication of increased branching with increasing phosphate levels.

Although the strain used in this work did not form macroscopic pellets but microscopicclumps, factors such as increased phosphate concentrations seem to exert similar controlson fundamentally different morphological types.

5.4.3 pH

Culture pH can have a profound effect on citric acid production by A. niger, since certainenzymes within the TCA cycle are pH sensitive. The maintenance of a low pH duringfermentation is vital for a good yield and it is generally considered necessary for the pH

Figure 5.10 Effect of phosphate level on A. niger morphology in the loop reactor at acirculation time of 18 s


to fall to around pH 2 within a few hours of the initiation of the process, otherwise the yieldsare reduced (Mattey, 1992). Information concerning the effect of pH upon the morphologyof citric acid producing A. niger is very limited. Reports on the effect of pH on morphologyfor other fungi are contradictory; either it influences morphology greatly or it has no effectat all (Pirt and Callow, 1959; Van Suijdam and Metz, 1981; Miles and Trinci, 1983). Thefollowing case study examines the effect of culture pH on citric acid production and A.niger morphology in the stirred tank reactor.

Case study 5

Fermentations were carried out at 500 rpm and uncontrolled pH (resulting in a final pH of1.6) and controlled pH (by addition of titrants) at 2.1 and 3 (Papagianni, 1995). Citric acidproduction was highest when pH was maintained at 2.1, with 122 g l-1 at the end of the run(168 hours of fermentation), compared to 75 g l-1 at pH 3 and 65 g l-1 at pH 1.6, as shown inFigure 5.11. Biomass levels were slightly increased with increasing pH: 5.65 g l-1 at pH 1.6,6.21 g l-1 at 2.1 and 7.40 g l-1 at pH 3. The highest values of specific rates of citric acidformation were obtained at pH 2.1, with a maximum value 0.35 h-1 while it reached 0.18 l-

1 in the other two runs.Mean values of the morphological parameters P1, P2, L and d at the end of the runs

performed at 500 rpm and pH 2.1, 3 and uncontrolled are shown in Table 5.1. P1, P2 andL increased with pH whilst there was no unidirectional response for the diameter offilaments. In addition to the small size of clumps and small length of filaments at final pH1.6, there was an unusually high number of swollen cells and tips in the mycelium in thisrun, as shown in Figure 5.11. A number of swollen cells were always present at stirrerspeeds above 400 rpm in the STR. The low pH in this experiment seemed to aid thedevelopment of this morphological form which also gave low citric acid concentrations.

Figure 5.11 Effect of culture pH on citric acid production in the stirred tank reactor


These experiments also indicated that productivity and morphology are linked. As withagitation, the conditions which promote a certain morphological type, i.e. that of smallclumps and short filaments, favour citric acid production, much as observed with therelationship between macro-morphology and citric acid production.

5.4.4 Dissolved oxygen tension

It has been shown that oxygen acts as a direct regulator of citric acid accumulation as it isfavoured by increasing the dissolved oxygen tension of the fermentation medium (Kubiceket al., 1980). As mentioned in the agitation case study, lower dissolved oxygen levels occurredwith morphologies not associated with high yields on citric acid. However, reports on theeffect of dissolved oxygen tension on the macro-morphology of A. niger suggest that no directrelationship exists between the two. Gomez et al. (1988), in their work on citric acid productionfrom A. niger, found that no difference in morphology for pellets and filaments could beascribed to dissolved oxygen levels, although production on citric acid was enhanced,particularly from pellets, by increasing the dissolved oxygen at different fermentation stages.Similarly, Van Suijdam and Metz (1981) showed that oxygen tension in the range of 12 to 300mg Hg had no influence on the morphology of P. chrysogenum. These reports contradict thelimitation hypothesis made by Hemmersdorfer et al. (1987), which suggests that lack of anyparticular nutrient, including oxygen, induces pellet formation.

5.4.5 Trace metal level

A number of divalent metals have been suggested as being required in limiting amounts fora successful citric acid process. These include Fe2+, Cu2+, Zn2+, Mn2+ and Mg2+ (Shu andJohnson, 1948; Mattey, 1992). Only the effect of manganese concentration has been shownto influence A. niger morphology. Manganese ions are known to be specifically involved inmany cellular processes, such as cell wall synthesis, sporulation and production of secondarymetabolites (Kubicek and Röhr, 1977). Cellular anabolism of A. niger is impaired underMn deficiency and/or nitrogen and phosphate limitation. The protein breakdown under Mndeficiency results in a high intracellular concentration. This causes inhibition of the enzymephosphofructokinase (essential enzyme in the conversion of glucose and fructose to pyruvate),leading to a flux through glycolysis and the formation of citric acid (Habison et al., 1979;Röhr and Kubicek, 1981).

Kisser et al. (1980) studied morphology and cell wall composition of A. niger underconditions of Mn sufficient and deficient cultivation in an otherwise citric acid producing

Table 5.1 The influence of pH on selected morphological parameters (measurements taken atthe end of fermentation in a lab-scale fermenter operating at constant stirred speed of 500rpm)


medium. Omission of Mn ions (less than 10-7) from the nutrient medium resulted in abnormalmorphological development that was characterized by increased spore swelling and squat,bulbous hyphae. The inhibition of glucoprotein turnover caused by the presence of Mn ionsled to a possible loss of hyphal polarity and increased branching and chitin synthesis. Clarket al. (1966) also discussed changes in A. niger morphology following the addition of Mn.The authors noticed an undesirable change in morphology from the pellet like form tofilamentous form with the addition of 2 ppb Mn to ferrocyanide-treated molasses.Morphological changes, which included prevention of clumping, absence of swollen cellsand reduced diameters of filaments, accompanied by a 20 per cent reduction in citric acidyield, following the addition of 30 mg l-1 Mn to a Mn-free medium, were also reported byPapagianni (1995).

5.5 Effect of inoculum

Among the factors that determine morphology and the general course of fungal fermentations,the amount and type of inoculum is of prime importance. Early attempts have been made tostandardize inocula for citric acid production in submerged culture (Martin and Waters,1952; Steel et al., 1955). Van Suijdam et al. (1980) reported that A. niger pellets would onlyform at inoculum sizes below 1011 spores per m3. However, the effect of inoculum on mycelialmorphology in submerged culture has been assessed mainly by the presence or absence ofpellets and their characteristics (Smith and Calam, 1980; Vecht-Lifshitz et al., 1990). Thereason for this was the lack of an adequate method to monitor mycelial morphology duringfermentations. Morphology was quantified by an image analysis method (Tucker et al.,1992) in the work of Tucker and Thomas (1992); a sharp transition from pelleted to dispersedforms of growth for Penicillium chrysogenum was reported, as inoculum levels rose towards5 × 105 spores per m3. This suggests that research on the inoculum in citric acid fermentationcould now be more systematic, making use of the technological advances in characterizationand monitoring of morphology in fungal fermentations.

5.6 Conclusions and perspectives

Discussion of the factors influencing A. niger morphology in submerged culture shoulddistinguish between macro- and micro-morphology although a number of similaritiesexist in relation to citric acid production and responses to the environment. This chapterhas concentrated on micro-morphology. The case studies presented suggest a strongrelationship between morphology and productivity in citric acid fermentation. Theobservations indicate that it might be possible to manipulate the morphology parametersin order to improve bioreactor performance and process yields. Image analysis providesthe tools for monitoring these parameters; however, further research is required to revealpossible general trends in metabolite regulation in relation to morphology of the producermicro-organism.

5.7 References

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BRAUN, S and VECHT-LIFSHITZ, S E, 1991. Mycelial morphology and metabolite production,Trends in Biotechnology, 9, 63–68.

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CLARK, D S, ITO, K and HORITSU, H, 1966. Effect of manganese and other heavy metals onsubmerged citric acid fermentation of molasses, Biotechnology and Bioengineering, 8, 465–471.

DION, W M, CARILLI, A, SERMONTI, G and CHAIN, E B, 1954. The effect of mechanical agitationon the morphology of Penicillium chrysogenum Thom in stirred fermentors. Rend. Ist. Super. deSanita, 17, 187–205.

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GUPTA, J K, HELDING, L G and JØRGENSEN, O B, 1976. Effect of sugars, hydrogen ionconcentration and ammonium nitrate on the formation of citric acid by Aspergillus niger, ActaMicrobiologie Academy of ScienceHungary, 23, 63–67.

HABISON, A, KUBICEK, C P and RÖHR, M, 1979. Phosphofructokinase as a regulatory enzyme incitric acid producing A. niger, FEMS Microbiology Letters, 5, 39–42.

HEMMERSDORFER, H, LEUCHTENBERGER, A, WARDSACK, C and RUTTLOFF, H, 1987.Journal of Basic Microbiology, 27, 309–315.

HOSSAIN, M, BROOKS, J D and MADDOX, I S, 1984. The effect of the sugar source on citric acidproduction by Aspergillus niger, Applied Microbiology and Biotechnology, 19, 393–397.

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KISSER, M, KUBICEK, C P and RÖHR, M, 1980. Influence of manganese on morphology and cellwall composition of A. niger during citric acid fermentation, Archives in Microbiology, 128, 26–33.

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KUBICEK, C P and RÖHR, M, 1977. Influence of manganese on enzyme synthesis and citric acidaccumulation by Aspergillus niger, European Journal of Applied Microbiology, 4, 167–173.

KUBICEK, C P, ZEHENTGRUBER, O, HOUSAM, E K and RÖHR, M, 1980. Regulation of citricacid production by oxygen: effect of dissolved oxygen tension on adenylate levels and respirationin Aspergillus niger, Applied Microbiology and Biotechnology, 9, 101–115.

MAKAGIANSAR, H Y, AYAZI-SHAMLOU, P, THOMAS, C R and LILLY, M D, 1993. The influenceof mechanical forces on the morphology and penicillin production of Penicillium chrysogenum,Bioprocess Engineering, 9, 83–90.

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85

6

Redox Potential in Submerged CitricAcid Fermentation

MARIN BEROVIC

Nomenclature

a,b constants (-)ae electron activity (-)aox activity of oxidized form (-)ared activity of reduced form (-)k redox reaction balance constant (-)n number of electrons in redox reaction (-)EO2/H2O standard potential—1223 mV (mv)Eh potential measured in a solution, (mV)

based on standard hydrogen electrodeEo standard redox potential of a 50 per cent reduced substance (mV)

based on standard hydrogen electrodeF Faraday constantN stirred speed (s-1)Qg volumetric gas flow rate (vvm)pO2 dissolved oxygen partial pressure (atm)pO2crit critical dissolved oxygen partial pressure (atm)P citric acid concentration (g/l)R gas constant (cal/°C mol)rH negative log of partial pressure of gaseous hydrogen (-)S sugar concentration (g/l)T temperature (°C)X biomass concentration (g/l)t residence time (s)Y yield factors (-)

6.1 Introduction

In living organisms oxidation–reduction systems play such an intimate and essential a part,that life itself might be defined as a continuous oxidation–reduction reaction. It is not


surprising, therefore, that theoretical speculations and experimental studies on oxidationand reduction processes in animals and plants have been actively pursued since the isolationof oxygen over 150 years ago (Hewitt, 1950).

Helmholtz (1883) was the fast to describe the decolorization of litmus in a mediumcontaining decaying protein. This was a reductive process since on passing air into solution,the original colour could be obtained again. Ehrlich (1885) injected redox dyes into livinganimals, killed them and investigated the redox state of the dyes in the organs. He attributedthe varying state of reduction to the oxygen uptake of the organs. These dyes could thereforebe used as indicators for particular reducing conditions. (Potter (1910) carried out the fastelectrometric measurement of reducing conditions in bacterial cultures. He detected with aplatinum electrode that the bacterial culture had a more negative potential than un-inoculatednutrient medium.)

Gillespie (1920) followed the development of bacterial cultures and showed that stronglynegative potentials become more positive when air is passed into the culture. Gillespie wasalso the first who applied the physical–chemical term ‘redox potential’ although the termsredox potential, reduction–oxidation potential, electrode potential and reduction potentialwere and are still used synonymously by various authors.

Redox potential detectors are usually not added to standard bioreactor instrumentationfor a number of reasons, most of them related to conventional thinking in bioreactorinstrumentation practices. As pH measurement represents the sum of all pH influencingcompounds, redox potential measurement represents the sum of all redox potentialinfluencing compounds in fermentation broth.

6.2 Overview

Redox potential is, however, a parameter that can give valuable information about metabolismtaking place in various aerobic and anaerobic microbial cultures (Kjærgaard, 1977). Thesignificance of redox potential levels for high yielding citric acid biosynthesis has beendemonstrated in submerged citric acid fermentation (Berovic, 1996; Berovic and Cimerman,1993).

Although only limited attention has been paid to this phenomenon in the past, someinteresting and informative research work has been presented. Some workers have advocatedthe use of redox potential measurements for monitoring and controlling dissolved oxygen(Shibai et al., 1975; Radjai et al., 1984). At constant pH the relation between redox potentialand dissolved oxygen partial pressure can be simplified by logarithmic relation (Jacob,1970; Memmert and Wandrey, 1987).

During the last few years a great deal of the attention for redox potential measurementand it uses, has been given to anaerobic bioprocesses (Beck and Schink, 1995). Theimportance of redox potential measurements was referred to in articles on waste waterbioprocessing, as in the case of propionate degrading Methanospirillum andMethanocorpusculum bacteria in a fluidized bed reactor, where degradation was inhibitedat redox potential below –300 mV (Heppner et al., 1992), and in anaerobic digestion inmethanogenic fermentation where volatile fatty acids were used as the substrate (Peck andChynoweth, 1992).

Redox potential measurements have also been found to be important in extremelythermophilic Thermotoga sp. bioprocessing, where most thermodynamic problems wereassociated with the relatively high redox potential (Janssen and Morgan, 1992). In variousaerobic processes the importance of the redox potential has been observed. In the case of

Redox potential in submerged citric acid fermentation 87

the biochemical transformation of l-sorbose to 2-keto-l-gluconic acid by a mutant strain ofPseudomonas (Tengerdy, 1961), it was found that the redox potential indicated the oxygendemand of the culture. The importance of redox potential was also very significant infermentations with Proteus vulgaris, Clostridium paraputrificium and Candida utilis (Jacob,1970; Balakireva et al., 1974), Lactobacillus sanfrancisco (Stolz et al., 1993) and Lactoccocuslactis (Vonktaveesuk et al., 1994). In Acetobacterium malicum degradation of fatty acids,there were differences in redox potentials at which electrons were released during oxidativepyruvate formation (Strochaker and Schink, 1991). In acetone–butanol fermentation byClostridium acetobutylicum, redox potential measurements were used in batch and continuousfermentation. A correlation between redox potential and switch from an acidogenic tosolventogenic metabolism was reported (Penguin et al., 1994). Although the redox conditionin a fermentation broth is reflected in the redox potential values measured, its characteristicscannot be generalized and the role of redox potential should be studied for each microbialprocess.

Publications on regulation of redox potential levels are rare. In the experiments of Lengeland Nyiri (1965) and Kjærgaard (1977), on various bioprocesses, the redox potential wasregulated by addition of reductants, while in Candida guilermondii fermentation by Huangand Wu (1974), the addition of n-paraffins was used.

6.3 Theory

Oxidation is a process in which a substance, molecule or ion loses or gives up electrons.Reduction, on the other hand, is a process in which a substance, molecule or ion, is involvedin the taking up of electrons. Whenever one substance in a system is oxidized, anothersubstance must be reduced. The relation between reduction and oxidation may be expressedas:

Reduced form � Oxidized form + electron(s)

However, since free electrons never exist in any noteworthy concentration, reductionand oxidation reactions are always coupled together, so that one reaction releases just asmany electrons as the other one consumes. Thus a pair of reactions always takes part in sucha process. These simultaneous and complementary reduction and oxidation processes aregenerally known as redox reactions (Bühler and Galster, 1980). The oxidation (or reduction)capacity of a solution is characterized by the free electron activity in it. Despite the fact thatthe lifetime of a free electron is extremely short (10-1–10-15 seconds) there is a statisticalpossibility of free electron existence at the moment of transformation from electron-donorsystems to electron-acceptor systems. (Balakireva et al., 1974).

The thermodynamic probability of electron emergence under activated reaction-capableconditions (Inczedy, 1970) is understood as electron activity in a solution:

ae = (1/k)1/n (ared/aox) (6.1)

The oxidation potential is a quantitative measure of redox capacity of a solution. It is anelectrical unit of charge of free energy in a redox interaction of the given system with astandard system. The system:

2H++ 2e- « H2

is a standard one. The oxidation potential is related to the electron activity in solution:Eh = -RT/F lnae (6.2)


Eh = -RT/F ln[(1/k)1/n (ared/aox)] (6.3)

Eh = kRT/nF + RT/nF ln(ared/aox) (6.4)

In the first part of equation (6.4), kRT is equal to Eo, the standard redox potential of a 50 percent reduced substance, based on a standard hydrogen electrode.

Eh = Eo + RT/nF ln(ared/aox) (6.5)

Equation (6.5) is the well-known Nernst equation.The redox potential of the measured substance, or substrate, depending on pH, is expressed

in the Kjærgaard equation (Kjærgaard, 1977):

Eh = EO2/H2O + RT/4F lnaO2 - RT/4F ln2.303 pH (6.6)

The potential values measured are dependent on pH, so that in each case measurements ofredox potential should be accompanied by a statement of the pH value at which they weretaken. In general a pH variation of one unit causes a potential variation of 57.7 mV (Jacob,1970).

6.4 Measurement of redox potential

In principle there are two ways of measuring a redox potential: by redox dyes and byelectrodes. Measurement of the redox potential by dyes is not exact and requires a numberof different dyes to obtain semi-quantitative measurements; furthermore, many of thesedyes may be toxic to the cells or may inhibit the enzyme activities in biological liquids(Hill, 1973). Therefore this method is not used in biochemical engineering. In bioreactors,combined sterilizable platinum as indicator and calomel or silver/silver chloride electrodeas reference electrodes are employed. As electrolyte 3M KC1 solution or sometimes KCl-gel are used.

It has been suggested that a decrease in Eh for a tenfold decrease in concentration ofdissolved oxygen amounts to 14.8 mV (Ishizaki et al., 1974). Clark and Cohen (1923)introduced the concept of rH in order to eliminate pH dependence on the potential (Clarkand Cohen, 1923):

rH = -logaH2 (6.7)

An rH of 0 corresponds to a pO2 of 0 atm and pH = 0, and rH = 42 corresponds to a solutionin which pO2 = 1 atm and pH = 0 (see Figure 6.1).

6.4.1 Calibration of redox electrodes

For calibration of the redox electrodes various redox buffers are in use. In this case twosaturated solutions of quinhydrone at two different pH values at 25 °C are recommended(Kjærgaard, 1977):

Eh qinhydrone = 699 - 59.1 pH (6.8)

A relatively easier method is to use ascorbic acid at various pHs (Hewitt, 1950):Eh ascorbic acid = 375 - 60 pH (6.9)


6.5 Significance of redox potential

Redox potential in microbial cultures is caused by the existence of reversible oxido– reductioncouples, irreversible reductors, and the action of free oxygen and free hydrogen (Rabotnova,1963). It is dependent on pH value, dissolved oxygen concentration, equilibrium constantand oxido–reduction potentials in the liquid (Ishizaki et al., 1974). Mass transfer of oxygenin aerobic cultures requires a potential difference between oxygen concentration in the celland the surrounding medium. The concentration of oxygen decreases from the solutiontowards the cells, and it is highly probable that the intracellular redox potential of micro-organisms is always slightly more negative than the extracellular redox potential (Jacob,1970).

Several investigations have revealed that the redox potential yields more informationabout the oxidative status in aerobic or partially aerobic microbial cultures than concentrationof dissolved oxygen (Wimpenny, 1969; Andreeva, 1964; Kjærgaard, 1976, 1977). Mostcommercial dissolved oxygen probes, when used in industrial conditions, are oftensusceptible to failure or erratic signal behaviour during the fermentation cycle, especiallywhen dissolved oxygen is a limiting factor. In a L-leucine fermentation, the redox signal

Figure 6.1 Electrode potential versus pH. Continuous line, theoretical curves, broken line,actual system (from Hewitt, 1950)


was useful in determining the oxygen transfer requirements when dissolved oxygen waspractically zero (Shibai et al., 1974; Akashi et al., 1978).

At constant pH the relation between redox potential and dissolved oxygen partial pressurecan be simplified by the following equation (Shibai et al., 1975), demonstrated in Figure6.2:

logpO2 = aEh + b (6.10)

A similar relationship between pO2 and Eh has been observed in amino acid production byCorynebacterium glutamicum (Radjai et al., 1984):

logpO2 = 0.0157Eh - 0.071 (6.11)

Shibai et al. (1975) carried this further for inosine production by Bacillus subtilis; pO2crit

was determined by measuring the dissolved oxygen, the redox potential and cell respirationrate in pH and temperature controlled culture. When the dissolved oxygen partial pressurewas above 1.10-2 atm, the redox potential had a linear relationship with the logarithm of thedissolved oxygen partial pressure. Therefore pO2 = 1.10-2 atm was estimated by determiningthe redox potential, on the assumption that there was a linear relationship even at the pO2

level less than 1.10-2 atm. The redox potential was markedly lowered by the physiologicalchange in the cells, when cell respiration was inhibited at Eh = -180 mV, which correspondedto pO2 = 2.10-4 atm.

pO2 in this culture was recorded as nearly zero when the cell rapidly biosynthesized theproduct. It went up above 1.10-2 atm at the end of fermentation, when the substrate wasalmost completely assimilated. The data showed that maximum production was obtainedunder limited oxygen supply, where cell respiration was inhibited. When cell respiration

Figure 6.2 Relationship between oxygen tension and redox potential (from Shibai et al.,1975)


was not inhibited, as the pO2 level rose above 1.10-2 atm, the cell did not produce the maximumamount of L-leucine.

The lowest values of pO2crit that have been reported were 4.10-3 atm for Saccharomycescerevisiae, 3 × 10-4 atm for inosine and 2 × 10-4 atm for the leucine producer (Akashi et al.,1978).

6.6 Redox potential in citric acid fermentation

Although citric acid production is the oldest industrial process, in addition to our own work(Berovic and Cimerman, 1982; Berovic and Roselj, 1997), there are only two otherpublications on redox potential measurements (Matkovicz and Kovacz, 1957; Tengerdy,1961). In our research on submerged citric acid fermentation using beet molasses as asubstrate, the relevance of redox potential levels for high product yielding biosynthesis hasbeen demonstrated. For a high citric acid yielding fermentation there is an optimal courseof the redox potential profile with two maxima of 260 and 280 mV and two minima of 180and 80 mV of essential importance. This redox potential course has been evaluated byanalysis of more than 200 fermentations (Berovic, 1996; Berovic and Cimerman, 1993).The time course for a typical batch fermentation is shown in Figure 6.3.

Beet molasses contains different organic and inorganic redox couples, substances andseveral metal ions that could significantly influence redox potential of the wholefermentation broth. Addition of K4Fe(CN)6, a well known redox substance, to the substrate

Figure 6.3 Process parameters of high citric acid yielding fermentation


causes the formation not only of metal ion complexes, but also the Fe3+/Fe2+ redox couple,which regulates the ion balance of the substrate (Clark and Cohen, 1923). The balance ofvarious redox couples and especially metal ion in fermentation broth is of essential importancefor citric acid biosynthesis. Related to this the influence of various influent factors andsubstances on redox potential levels of beet molasses substrate have been studied (Figure6.4).

A reference redox potential profile was obtained for sterile medium only, with noaddition of K4Fe(CN)6 (curve 1). After 24 hours of aeration, the redox potential reacheda stationary phase that was unchanged until the end of the experiment. In experimentswhere inoculated substrate was used in absence of any addition of K4Fe(CN)6 (curve 2),and with an initial addition of this compound (curve 3), the redox potential profile exhibiteda typical single peak. Only in the case where inoculated substrate with primary andsecondary addition of K4Fe(CN)6 was used (results shown in Figure 6.3), was the twin

Figure 6.4 Redox potential measurements and citric acid formation by Aspergillus niger.Curve 1: aerated sterile sugar beet molasses substrate including potassium ferrocyanide;curve 2: inoculated and aerated sterile sugar beet molasses substrate excluding potassiumferrocyanide; curve 3: inoculated and aerated sterile sugar beet molasses substrate includingpotassium ferrocyanide


peak redox potential course observed. The different metabolic activities in the fermentationprocess are summarized in all the redox reactions and detected in redox potentialmeasurement. From these experiments we concluded that only in the presence of Aspergillusniger were the relevant changes detected. Similar observations were made by Kwong andRao (1991, 1992) in amino acid fermentation using Corynebacterium glutamicum.

Redox potential measurements in citric acid fermentation might also give valuableindications of product biosynthesis in the fermentation. From evaluation of more than 200batches, we concluded that a high yielding fermentation is directly related to levels andtime course of redox potential.

The yield of citric acid is reflected in the time course of the redox potential. This isshown in Figures 6.5 and 6.6, where experiments with different histories are shown.Figure 6.5 presents the characteristics of an unsuccessful fermentation with respect to

Figure 6.5 Process variables in a low yielding, abnormal citric acid fermentation


citric acid production. Growth was diffused and the low citric acid production was thereforeexpected. This is also reflected in the course of the redox potential. The second peak is lowand almost negligible (190 mV).

The effect of temperature change is well reflected in the course of redox potential. Figure6.6 presents the data from an experiment started at an initial temperature of 20°C. Thetemperature was changed after 20 hours to 30°C. The effect of this change can be clearlyseen from the redox curve. In the same experiment foaming caused a loss of the substrate(89 hours), which was also indicated in a new peak of the redox potential.

In a high yielding fermentation on beet molasses substrate, the redox potential coursestarts at a level of 0 to 20 mV, as shown in Figure 6.3. After 12 hours, the culture reaches a

Figure 6.6 Effect of temperature shift on citric acid fermentation with Aspergillus niger andsuger beet molasses medium


level of oxygenation which significantly influences germination of conidia in the lag phaseand the subsequent development of bulbous cells that appears at the first peak of the redoxpotential at 260 mV. After the first peak, a period of inhibition followed by the first redoxminimum at 180 mV occurs. In this phase it seems that microbial activity stops. Oxygenpartial pressure in fermentation broth increases and the carbon dioxide and redox potentialdecreases, indicating a reduced level of activity for the micro-organism. This phase is aprogressive transition from glucose to fructose consumption. For this reorganization, a lowredox potential level is needed, resulting in the change in morphology (Smith, 1983).

After this phase, the microbial growth mode changes to spherical pellets. This wasindicated by the second redox peak at 280 mV. After a decrease in redox potential to 80 mV,the second minimum, citric acid production starts. As reported by Tengerdy (1961), at thelowest redox potential level, the peak oxygen demand and initiation of rapid excretion ofcitric acid can be observed. The low redox potential reveals the reducing state of the complexredox system of the fermentation broth, where the respiratory enzyme system signifiesstrong metabolic activity. It seems that citric acid biosynthesis (Matkovicz and Kovacz,1957), as well as some other microbiological reactions, proceeds favourably at the redoxpotential near the minimum of the redox curve for the particular culture involved (Hewitt,1950; Tengerdy, 1961). This was found to be true in riboflavin fermentation.

The redox potential time-course in a high citric acid yielding fermentation reaches afinal level of 180 mV. Interestingly if significant amounts of oxalic acid, up to 20 mg/l, areproduced, the redox potential will only reach levels of 100 to 120 mV at the end offermentation. It has also been found that oscillations in redox potential greater than ±20 mVhave a strong influence on further development of fermentation (Berovic, 1996).

6.7 Regulation of the redox potential

Although measurements and observation of redox potential have been published in severalarticles, its regulation and process control have only rarely been discussed. In a fewfermentation processes, as in Bacillus lichenoformis cultivation, a chemical method basedon addition of glucose (Kjærgaard and Jørgensen, 1976, 1979) has been used. Huang andWu (1974) added n-paraffins for regulation of redox potential in a Candida guilermondiifermentation. In a continuous process for production of xylanase by Bacillusamiloliquefaciens, a physical method based on regulation of dilution rate and agitation(Memmert and Wandrey, 1987) was used. Constant maintenance of redox potential in variousbioprocesses were reported by Lengel and Nyiri (1965). Radjai et al. (1984) found that theredox potential minimum for amino acid fermentations with Corynebacterium glutamicumwas directly influenced by the agitation rate. The minimum redox potential of the culturebecame less negative as the rate of agitation was increased. This is consistent with theincrease obtained in the oxygen transfer rate and subsequently in dissolved oxygen partialpressure as agitation speed is increased.

6.8 Regulation of redox potential in citric acid fermentation

The aim of using regulation of redox potential in citric acid fermentation was to establisha method for redox potential regulation that will conduct a fermentation process towardsthe essential redox levels needed for high citric acid production. According to this, twodifferent methods, a chemical method, similar to those used for pH control by using

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oxidants and reductants, and a physical method, based on simultaneous agitation and aerationcontrol were tested (Berovic and Cimerman, 1993).

6.8.1 Chemical methods

In the first experiment, 0.1% hydrogen peroxide was used as oxidant, and 0.1% sodiumsulphite as reductant. The results of the experiment are presented in Figure 6.7. The firstredox peak reached 260 mV. At this level no oxidant was added. The microbial growth formwas bulbous cells. After the first redox maximum, the first minimum of 180 mV was obtainedby addition of 80 ml of the sodium sulphite. After this addition microbial growth turnedfrom bulbous to filamentous hyphae growth forms. At the second redox maximum, 280mV, reached by addition of 20 ml of the hydrogen peroxide solution, filamentous hyphalaggregates were the dominant growth form. The second redox minimum was obtained byaddition of 195 ml of sodium sulphite solution. After this the microbial form did not change.Shear in the bioreactor caused formation of hyphal fragments which were present until theend of fermentation. This period also exhibited an unchanged redox potential of 80 mV.Although this method gave a redox time-course which was similar to the time-course inhigh yielding fermentations, the microbial growth after addition of sodium sulphite turnedcompletely to low citric acid producing filamentous growth forms. The addition of reductantdid not stop microbial growth, but it produced an unproductive growth form. At the end offermentation a biomass level of 5.5 g/l and 12.8 g/l citric acid were obtained.

In a further experiment, a water solution of 0.1% hydrogen peroxide as oxidant and a20% solution of glucose as reductant were used. In Figure 6.8 the results of such an experimentare presented. The first maximum of 260 mV was obtained at 25 hours. In this period,bulbous cell agglomerates appeared. Following this the regulation of redox potential levelsstarted. The first redox minimum of 180 mV was obtained at 30 hours by addition of 275 mlof the glucose solution. The second maximum of 280 mV was reached soon after by additionof 26 ml of hydrogen peroxide. This phase was characterized by formation of small sphericalpellets with short and thin peripherial hyphae. The second minimum of 200 mV appearedafter the second peak soon after the regulation was stopped. This was followed by a slow,unaided drift up to a third peak of 240 mV. In this phase mycelium growth in the pellet formwith thin and long peripherial hyphae appeared. The redox then fell slowly to a third minimumof 100 mV followed by a gentle increase to 180 mV at the end of fermentation.

Using these agents, regulation of redox potential of the fermentation broth was possible.The redox potential time-course was close to the optimal and addition of either compounddid not inhibit the development of a productive growth mode, mycelial pellets with thickand long peripherial hyphae being the typical morphology feature. At the end of thefermentation, a biomass level of 11.1 g/l and 40 g/l of citric acid were obtained (see Figure6.8).

6.8.2 Physical methods

Physical parameters such as temperature, head space pressure, agitation and aerationstrongly influence the oxygen transport coefficient in the liquid phase. Therefore bylowering the temperature and increasing head space pressure, agitation and aeration, thedissolved oxygen partial pressure pO2, will increase. Increasing pO2 influences stronglythe potential difference between oxygen level in the liquid phase and the oxygen level in


the microbial cell. According to the Nernst equation (equation 6.5), increasing aox in a cellcould strongly influence the metabolic and enzyme activities in micro-organisms (Harrison,1972). In submerged citric acid fermentation with Aspergillus niger beet molasses, in additionto the maxima and minima redox levels, timing of these events is of essential importance(Berovic and Cimerman, 1982, 1993).

The possibilities of regulating both redox maxima and the first redox minimum weretested, using agitation and aeration as a physical means of regulating redox potential. Thefirst maximum of Eh = 220 mV appeared at 23 hours (Qg = 0.4 vvm speed of agitation = 400rpm). As the level of 220 mV was too low for further process development, increasing theaeration rate Qg to 1 vvm and agitation to 600 rpm gave a redox level of 260 mV. By furtherreducing the aeration rate to 0.3 vvm and agitation to 200 rpm, at 30 hours the first redoxminimum Eh = 180 mV was obtained. After this step, aeration was increased to 1.2 vvm andagitation to 700 rpm. The second redox maximum Eh = 280 mV at 36 hours appeared. Thefermentation then proceeded at constant conditions of Qg = 1 vvm and N = 600 rpm untilthe end of the process. As the course of redox potential was not maintained by aeration andagitation during the last phase, it started to deviate from the optimal course with a thirdmaximum (265 mV) occurring at 48 hours and a third minimum (120 mV) at 75 hours. Thisgave final biomass and citric acid concentrations of 11.4 and 68.5 g/l respectively (seeFigure 6.9).

Figure 6.8 Process parameters of citric acid fermentation at chemical regulation of redoxpotential using 20% glucose as reductant and 0.1% hydrogen peroxide as oxidant

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Finally, optimized redox level profiles were followed using simultaneous regulation ofaeration and agitation during the whole course of the fermentation. This resulted in 14.7 g/l of biomass and 95 g/l of citric acid at the end of the fermentation. The results are given inFigure 6.10.

6.9 Scale-up based on redox potential

The aim of scale-up is to develop a method based on the physiological needs of the micro-organism that would give high yielding and reproducible results on various scales. Scale-upis usually based on criteria such as: geometrical similarity, power input, volumetric oxygentransfer coefficient, mixing time, etc. (Nienow, 1992; Dunn et al., 1992, Dubuis et al.,1993). However, we decided on scale-up based on redox potential, being the most relevantprocess parameter for our process. As redox potential indicates oxygen demand of the culture,the basic idea was to use a physiological criterion of our bioprocess for scale-up. If redoxpotential indicates a microbial demand for oxygen, it could also reflect information on theappropriate aeration and agitation conditions needed to meet this demand.

6.9.1 Bioreactor dimensions

The experiments were performed in 10 l Bioengineering AG, and 100 and 1000 l ChemapAG bioreactors. These were all equipped with Rusthon turbines, but were not geometricallysimilar. The reactor dimensions are given in Table 6.1.

6.9.2 Media composition

The fermentation substrate consisted of diluted beet molasses with 12.5 per cent of totalreducing sugars. It was treated by addition of potassium hexacyanoferrate K4[Fe(CN)6],which balanced the ratio of heavy metals ions by the formation of metal complexes (Clarket al., 1965). K4[Fe(CN)6] was added in two stages, before sterilization (primary addition)and after (secondary addition) (Cimerman et al., 1974; Berovic and Cimerman, 1982). Thefermentations were carried out at T = 30°C.

6.9.3 Laboratory scale experiments

Basic research for scale-up was performed in a 10 l laboratory fermentor. The best redoxprofile was determined from some 200 fermentations. The objective of the scale-up was toobtain a similar redox profile in the larger reactors by regulating the agitation and aeration.

Table 6.1 Stirred tank reactor dimensions


6.9.4 Pilot scale experiments

Fermentations were carried out in the 100 l and 1000 l reactors; aeration and agitation wereincreased and decreased stepwise as outlined above to obtain the two desired redox potentialmaxima and minima. This was achieved as indicated in Table 6.2. The results from theexperiments are summarized in Table 6.3, with similar results obtained on all scales.

6.10 Conclusions

For high citric acid yielding submerged fermentation on beet molasses the optimal redoxpotential time course and its typical redox levels, with two maxima, 260 and 280 mV, andtwo minima, 180 and 80 mV, are essentially important (Berovic, 1996; Berovic andCimerman, 1993). It is possible to influence the fermentation by changing the redox potentialprofile as well as the magnitude of the maxima and minima. Regulating the redox by usinghydrogen peroxide as oxidant and sodium sulphite or glucose as reductant, resulted in afavourable redox profile for the whole process, but the fermentation was affected to such anextent that poor growth and reduced citric acid yields were obtained.

A better method for regulating the redox potential during fermentation is through alterationin aeration and agitation. The desired redox profile is attained by respectively increasing,and decreasing the aeration and agitation to obtain the desired maximum and minimumvalues. It is a simple practical approach based on changing the gradient of oxygen transferin the fermentation broth, which influences changes in intracelluar oxygen concentrationand therefore the microbial physiology of the cell.

Table 6.2 Scaling up redox profiles


This method of regulating the redox profile was used as a scale-up criterion with theprocess successfully scaled up from 10 to 1000 l. Considering the results obtained, it isevident that this new scale-up method leads to very reproducible results even in geometricallynon-similar bioreactors.

6.11 References

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BEROVIC, M and ROSELJ, M, 1997. Possibilities of redox potential regulation in submerged citricacid fermentation on beet molasses substrate (unpublished).

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CIMERMAN, A, SKAFAR, S and JOHANIDES, V, 1974. YU Patent P2481/74.CLARK, W M and COHEN, B, 1923. Public Health Report Washington, 38, 666.CLARK, O S, ITO, K and TYMCHUK, P, 1965. Effects of potassium ferrocyanide addition on the

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DUBUIS, B, PLÜSS, R, ROMETTE, J L, KUT, O M and BORNE, J, 1993. Physical factors affectingthe design and scale-up of fluidized bed bioreactors for plant cell culture, 3rd InternationalCongress on Bioreactor & Bioprocess Fluid Dynamics. Ed. A.NIENOW (MEP Publications),pp. 89–100.

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ERLICH, P, 1965. Recommendations 1964 of the International Union of Biochemistry (Elsevier).GILLESPIE, L J, 1920. Soil Science, 9, 199.HARRISON, D E F, 1972. Physiological effects of dissolved oxygen tension and redox potential on

growing populations of micro-organisms, Journal of Applied Chemistry and Biotechnology, 22,417–440.

HELMHOLTZ, H, 1883. Archives of Anatomy and Physiology.HEWITT, L F, 1950. Oxidation–reduction potentials. In Potentials in Bacteriology and Biochemistry,

6th edition (Livingstone, Edinburgh).HEPPNER, B, ZELLNER, G and DIEKMANN, H, 1992. Start-up and operation of a propionate

degrading fluidised bed reactor, Applied Microbiology and Biotechnology, 36, 810–816.HILL, R, 1973. Bioenergetics, 4, 229.HUANG, S Y and WU, C S, 1974. Redox potential in yeast cultivation broth using n-paraffins as

carbon source, Journal of Fermentation Technology, 52, 818–827.INCZEDY, J, 1970. Period Polytechnic Chemical Engineering, 14, 2.ISHIZAKI, A, SNIBAI, H and HIROSE, Y, 1974. Basic aspects of electrode potential change in

submerged fermentation, Agricultural and Biological Chemistry, 38, 2399.JACOB, H E, 1970. Methods in Microbiology, Vol. 2. Eds J R NORRIS and D W RIBBONS (Academic

Press).JANSSEN, P H and MORGAN, H W, (1992). Heterotrophic sulphur reduction; end product inhibition,

FEMS Microbiology Letters, 2, 213–218.KJÆRGAARD, L, 1976. European Journal of Applied Microbiology, 2, 215.


KJÆRGAARD, L, 1977. Advances in Biochemical Engineering, 77, 131.KJÆRGAARD, L and JØRGENSEN, B B, 1976. Maintenance of a constant redox potential during

fermentation by automatic addition of glucose, 5th International Fermentation Symposium, Berlin.KJÆRGAARD, L and JØRGENSEN, B B, 1979. Redox potential as state variable in fermentation

systems, Biotechnology and Bioengineering Symposium, No. 9, 85–94.KWONG, S C W and RAO, G, 1991. Utility of culture redox potential for identifying metabolic state

changes in amino acid fermentation, Biotechnology and Bioengineering, 22, 1034–1040.KWONG, S C W and RAO, G, 1992. Effect of reducing agents in an aerobic amino acid fermentation,

Biotechnology and Bioengineering, 40, 851–857.LENGEL, Z L and NYIRI, L, 1965. An automatic aeration control system for biosynthetic processes,

Biotechnology and Bioengineering, 7, 91–100.MATKOVICZ, B and KOVACZ, E, (1957). Untersuchung der zitronnensäureproduction und der

potentialveränderung in oberflächen und tiefkulturen, Naturwissenschaften, 44, 447.MEMMERT, K and WANDREY, C, 1987. Proceedings 4th European Congress on Biotechnology, 3,

153.NIENOW, A E, 1992. Scale-up and scale-down of stirred tank reactors, EFB Bioreactor Engineering

Course Lecture Notes. Eds M BEROVIC and T KOLOINI, pp. 209–230.PECK, M and CHYNOWETH, D P, 1992. On-line fluorescent monitoring of the metanogenic

fermentation, Biotechnology and Bioengineering, 39, 1151–1160.PENGUIN, S, GOMA, G, DELORME, P and SOUCAILLE, P, 1994. Metabolic flexibility of

Clostridium acetobutylicum in response to methyl viologen addition, 42, 611–616.POTTER, M C, 1910. Durham University, Philosophical Society Proceedings, 3.RABOTNOVA, I L, 1963. Die Baedeutung physikalisch-chemischer Faktoren fur die Lebenstatigkeit

der Bakterien (Fischer-Verlag).RADJAI, M K, HATCH, R T and CADMAN, T W, 1984. Optimisation of amino acid production by

automatic self tuning digital control of redox potential, Biotechnology and BioengineeringSymposium, No. 14, 657.

SHIBAI, H, ISHIZAKI, A, KOBAYASH, K and HIROSE, Y, 1974. Studies on oxygen transfer insubmerged fermentations, Agricultural and Biological Chemistry, 37, 91–97.

SHIBAI, H, ISHIZAKI, A, KOBAYASH, K and HIROSE, Y, 1975. Studies on oxygen transfer insubmerged fermentations, Agricultural and Biological Chemistry, 38, 2407–2410.

SMITH, J E, 1983. University of Strathclyde, Glasgow, United Kingdom (private communication).STOLZ, P, BOCKER, V, VOGEL, R F and HAMMES, W P, 1993. Utilisation of maltose and glucose

by Lactobacilli isolated from Sourdough, FEMS Microbiology Letters, 109, 237–242.STROCHAKER, J and SCHINK, B, 1991. Energetic aspects of malate and lactate fermentation by

Acetobacterium malicum, FEMS Microbiology Letters, 90, 83–88.TENGERDY, R P, 1961. Redox potential changes in 2-keto-l-gulonic acid fermentation. Correlation

between redox potential and dissolved oxygen concentration, Biotechnology and Bioengineering,3, 255.

VONKTAVEESUK, P, TONOKAWA, M and ISHIZAKI, A, 1994. Simulation of the rate of l-lactatefermentation using Lactococcocus lactis Io-1 by periodic electrodialysis, Journal of Fermentationand Bioengineering, 7, 508–512.

WIMPENNY, J W T, 1969. Biotechnology and Bioengineering, 11, 623.

105

7

Modelling the Fermentation Process

FRANK WAYMAN, HO AI MENG AMY AND BJØRN KRISTIANSEN

7.1 Introduction

This chapter will focus on the kinetic modelling of industrial citric acid production byAspergillus niger and Candida (= Saccharomycopsis = Yarrowia) lipolytica. A good workingdefinition of kinetic modelling is the mathematically expressed correlation between therates and concentrations of reactants and products. When applied to appropriate massbalances, it is possible to predict the utilization of substrates and the yield of individualproducts. A well constructed model can be used to express the course of a whole fermentationexperiment based on a small set of initial values for the fermentation variables. Such modelscan then be used as a basis for simulations which are essential for the optimal design andoperation of a given process.

7.1.1 Different types of kinetic models

Unstructured models use a single biomass component to describe the total biomassconcentration in steady state conditions. They are deterministic in their approach (i.e., theyare primarily used to fit a restricted set of data) and perform poorly when applied tosignificantly different operating conditions.

Structured models are also deterministic, but are an improvement on unstructured modelsas they may use more than one set of equations to represent different phases of growth andproduction. It is also possible to represent the biomass as more than one component todescribe the physiological state of the micro-organisms during a fermentation. Both typesof model involve the specific growth rate (µ) as a function of the concentration of substrate(S), product (P) and biomass (X).

A mechanistic model describes the conversion from substrates into products byapplying the known concentrations of metabolites and properties of all the enzymes inthe reaction sequence. This obviously requires a great deal of preliminary research intothe physiology of the chosen micro-organism, and so far has only been possible tomodel simple reaction sequences with a small number of enzymatic steps. Such modelsare closely related to the models used for metabolic control analysis, where the activity


of individual enzymes is related to their effect upon the overall reaction rate. This secondprocess has been carried out for the production of citric acid through the glycolyticpathway by A. niger (Torres 1994a, 1994b) and was successful in showing that theactivities of glycolytic enzymes have little influence on the rate of product formation inthis system. A shortage of information currently prohibits the creation of a mechanisticmodel for biomass production.

7.1.2 Unstructured models based on the Monod equation and other equations

The 1942 Monod equation (equation 7.1) relies on the principle that even when there aremany substrates the rate of biomass production depends on the concentration of just onelimiting substrate. At low concentrations of this substrate (S), µ is proportional to S, butfor increasing values of S an upper value µmax for the specific growth rate is graduallyreached. The maximum specific growth rate (µmax) and the saturation coefficient (Ks)must be determined experimentally. This model has been shown to correlate withfermentation data for many different micro-organisms, but fits best with well mixedunicellular systems.

7.1.3 Other growth modelling equations

Trinci (1970) measured the growth of Aspergillus nidulans colonies and pellets in terms ofradius and dry weight. He showed that the growth of pellets could be described as exponentialat the start of growth (equation 7.2), changing to a cube-root phase (equation 7.3) andending in a linear phase (equation 7.4):

Log of growth linear with time lnXt = lnX0 + µt (7.2)

Cube root of growth linear with time (7.3

Growth linear with time Xt = X0 + ktt (7.4)

This model was used to explain that part of the pellets was either not growing, or wasgrowing sub-optimally, because it would otherwise be expected that the pellets wouldcontinue to grow exponentially until exhaustion of the nutrients. These equations are usefulwhen attempting to describe hyphal growth in filamentous fungi, particularly in conditionsthat lead to the formation of pellets.

The rates of formation and depletion within the fermentation are linked to the formationof biomass by yield coefficients, and are described mathematically as follows:

(7.5)

(7.6)

(7.1)

Modelling the fermentation process 107

where rX, rS and rN are the rates of biomass accumulation, carbon source consumption andnitrogen source consumption, respectively.

The Luedeking–Piret (1959) equation (equation 7.8) is used to relate the formation ofproducts to either the biomass concentration or the rate of biomass accumulation:

rP = (α · rX) + ( ß · X) (7.8)

This equation is often simplified by substituting zero for one of the product formationconstants, α or ß, giving equations for growth specific and non-growth specific productformation.

7.2 Aspergillus based models

Filamentous micro-organisms have growth kinetics that are quite distinct from those ofunicellular micro-organisms. All cells within the mycelium may contribute towards themaintenance of internal conditions, but it has long been established that growth only occursat the hyphal tips. In A. niger, citrate excretion is also restricted to the apical cells (Kristiansenand Sinclair, 1979).

The modelling of filamentous fungi has been advanced to a stage where structured modelsthat describe the growth, differentiation and secondary metabolite production have beendeveloped. One example of this is the structured model developed for penicillin fermentationby Thomas and Paul (1994). However, the modelling of citric acid fermentation has yet toreach such an advanced stage.

7.2.1 A simple struct ured model for growth and citrate productionin A. niger

A typical growth curve for an A. niger under citric acid producing conditions hasbeen presented by Kubicek and Röhr (1989) as shown in Figure 7.1. This shows afast-growth phase followed by a slow-growth phase. This change in growth rate isdue to a change in the physiological state of the mycelium from the normal growthform to the citrate excreting form. An examination of the kinetics of citric acidproduction by Aspergillus niger growing on sucrose was carried out in a pilot plant(Röhr et al., 1981). Cell growth and product formation were subdivided into severalphases, each described by a simple deterministic model. The growth phases identifiedwere the hyphal growth phase (Bx), pellet growth phase (Cx), restricted growth phase(Dx), transition period between trophophase and idiophase (Ex) and idiophase growth(Fx). The growth in each phase was described by logarithmic, cube root and linearequations and the best fitting equation was identified by evaluating the degree oflinearity within a limit of 5 per cent maximum deviation. The three phases areillustrated in Figure 7.2, where the cell growth during citric acid fermentation byAspergillus niger B60/B3 has been plotted.

Product formation was related to the growth rate by a modified Luedeking–Piretequation. However, although the same descriptions were applicable for both growth and

(7.7)


acid formation (Figure 7.3), the acid formation kinetics usually differentiated from the growthkinetics by a term that represented the lag time. This lag time is also known as the maturationtime, when the culture has taken up all the ammonium ions but does not yet produce citricacid. The respective rate of product formation was then said to be proportional to the rate ofcells entering this physiological state and was expressed as follows:

rPt = krX(t–tm) (7.9)

where rP, t, X have their usual meanings and k and tm are the product formation rate constantand maturation time respectively.

One problem with this equation is that k was found not to be constant but increases invalue during the fermentation. It was assumed that there were at least two different types ofcells within the mycelium with different productivities, and a production term for each typecould be described by the Luedeking–Piret equation. Therefore, equation 7.9 was modifiedto become:

rPt = k1rX(t–tm) + k2(X)t-tm (7.10)

where k1 is a growth-associated constant and k2 is a non-growth associated constant.

Figure 7.1 Typical example of growth and citric acid accumulation by A. niger in submergedculture (from Kubicek and Röhr, 1989)

Figu

re 7

.2 G

row

th d

urin

g ci

tric

aci

d fe

rmen

tatio

n by

A. n

iger

B60

/B3

(from

Röh

r et

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198

1).

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re 7

.3 C

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aci

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trat

ion

duri

ng c

itric

aci

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B60

/B3

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Röh

r et

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198

1).


The constants k1 and k2 were determined by a computer-based optimization procedurefollowing the determination of tm, and are shown in Table 7.1. Figure 7.4 shows a comparisonof the experimental values for citric acid production and the line calculated by the modifiedequation above. It can be clearly seen that the model closely resembles the data from citricacid production (Röhr et al., 1981).

7.2.2 Expressions for mixed biomass types

In the above model, the state of cells present in the fermentation was not differentiated, i.e.,the description for rate of cell growth was considered applicable to describe all states ofcells. Since it is known that at least two types of active cells are present, a separate expressionfor the rate of cell growth of different states of active cells should be more practical in themodelling of such a bioprocess.

An example of a set of different rate expressions for the different types of cells present inthe fermentation was established by Kristiansen and Sinclair (1979) for a single-stage ideallymixed CSTR:

rXb = µbXb - ktXb - DXb (7.11)

rXc = µcXc + ktXb - kdXc + DXc (7.12)

rXd = kdXc - DXb (7.13)

where D is the dilution rate and subscripts b, c and d refer to basic, citric acid-producing(carbon storage) and deactivated cells respectively. The dilution rate (D) in these equationscan be substituted by the overall growth rate (µ) if they are to be applied to a batch typefermentation (Sinclair et al., 1987).

It was assumed that the rate constants of the above equations took the following forms:

(7.16)

where the first rate constant, µb, was the Monod expression for growth on a limiting substrate,the second constant, µc, accounted for the increase in mass of bulbous citrate producingcells and kt was the rate of transformation of basic to storage cells.

Table 7.1 Calculated values for tm and k (from Röhr et al., 1981)

(7.15)

(7.14)

Figu

re 7

.4 C

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aci

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ncen

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duri

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aci

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n, a

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and

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1 (fr

om R

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.


7.2.3 A model of the A. niger process using initial conditions

The only published A. niger citric acid model to date which calculates the outputs from theinitial conditions has been that written by Ho et al. (1994). This model used four differentMonod type growth rate equations with different values for µmax and ks. The influence ofeach on the overall growth rate was altered by the calculated concentration changes of eachsubstrate and product. A value representing available intracellular nitrogen is also calculated.The volumetric rates of all components in the system were then calculated from the differentgrowth rates with a combination of different yield coefficients that were derived from batchdata. The equations were then linked together, producing the model results that are plottedalongside the original experimental data in Figure 7.5.

7.3 Yeast based models

7.3.1 A model of the S. lipolytica process using initial conditions

Klasson et al. (1991) described the citric acid process by Saccharomycopsis lipolytica NRRLY-7576 growing on glucose using the logistic growth curve equation:

rX = KX(1 - X/Xmax) (7.17)

The equation has no expression for the limiting substrate, ammonia, which is not present inthe medium during most of cell growth, as shown in Figure 7.6. However, the model doeslimit the maximum cell concentration to the initial level of ammonia with a yield coefficient.

Xmax = X0 + YX/N · N0 (7.18)

This simplifies the model as there is no need to calculate a value for intracellular availablenitrogen. The concentration of glucose never becomes limiting because citric acid productionwill continue as long as it is present in the medium (Klasson et al., 1991).

The formation of product was related to the physiological state of the cells. TheLuedeking–Piret equation was used to model the rate of product formation of this batchfermentation and was written as follows:

rP = (K · rX) + (qm · X) (7.19)

where qm represents the non-growth associated constant and K denotes the growth associatedconstant. K was found to be negative which confirms the proposed model where productionwas initiated at an intermediate point in the bioprocess and that citric acid is producedmainly by resting yeast cells.

The glucose substrate consumption rate was modelled generally by the followingexpression:

-rS = (1/YX/S · rX) + (1/YP/S · rP) + mX (7.20)

where the first term on the right-hand side of the equation describes the rate of substrateconsumed to synthesize new cell material, the second term describes the rate of substrateconsumed to synthesize excreted product and the last term describes the maintenance energyrequired by the cell. The model accurately predicted the levels of the broth componentsthroughout the batch (Figure 7.7), but with a few exceptions. It did not predict the disruptionof cells caused by the high shear forces present in the bioreactor. This leads to a fall inbiomass concentration after growth ceases and this effect could be added to the model as adeath rate term (kd). This first inaccuracy then leads to further inaccuracies in productformation and substrate removal which accumulate as the model progresses.

Figu

re 7

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iger

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el w

ith e

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ata.

X, B

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ass;

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rom

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994)

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. lip

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odel

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, (su

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; P, (

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(fro

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199

1)


7.3.2 A phase related yeast model

The production of citric acid by Yarrowia lipolytica ATCC 20346 growing on glucose andthe fermentation was schematically subdivided into three different phases which could becharacterized by unstructured kinetic models (Moresi, 1994). The three phases weretrophophase (when ammonia is removed from the medium and growth occurs), citric acidlag phase (where rapid growth from stored ammonia occurs) and idiophase (when citricacid production occurs) (Figure 7.8).

Figure 7.8 Citric acid production in batch culture by Yarrowia lipolytica ATCC 20346.Concentrations of biomass (X) and citric acid (P) and weight fraction of intracellular nitrogen(ZN) as a function of the fermentation time (t). Continuous lines were calculated using theequations and yield coefficients reported (from Moresi, 1994)


A linear type growth equation for trophophase was found to give an acceptablecorrelation to the experimental profiles of X, S and N. After the nitrogen source is depleted,the exhaustion does not prevent further growth but growth is accompanied by asimultaneous decrease in the intracellular nitrogen content (ZN) before the start of citricacid excretion (Figure 7.8).

The cell growth rate of this citric acid lag phase was modelled by the followingexpression:

rX = -X (d ln zN/dt) (7.21)

Figure 7.9 Repeated batch citric acid production by Yarrowia lipolytica ATCC 20346.Concentrations of biomass (X) and citric acid (P) and weight fraction of intracellular nitrogen(ZN) as a function of the fermentation time (t) (from Moresi, 1994)


The citric acid production phase or idiophase was modelled using Luedeking–Piret kinetics:

rp = (YP/S/YX/S) (rX) + mPX (7.22)

However, as citric acid is formed by resting yeast cells, rX approximates to 0, so the aboveLuedeking–Piret equation is reduced to:

rP » mPX (7.23)

Confirmation of this can be seen in Figure 7.9, where the citric acid concentration increaseslinearly with time while the biomass concentration remains constant in a repeated-batchexperiment.

7.4 Conclusion

Kinetic modelling is a powerful tool in the design and optimization of all biotechnologicalprocesses, and the citric acid process is no different. The Candida lipolytica process is quitewell understood, and so it is not surprising that the published models are accurate andapplicable to a wide range of fermentation conditions.

On the other hand, the physiological change that occurs in Aspergillus niger myceliumduring the citric acid process is far from being well defined, and models which concentratepurely on the production phase are therefore less susceptible to error. The single publishedmodel which covers this transition phase is only capable of predicting the course of thefermentation within a very narrow range of starting parameters. Outside this range, errorsstart to accumulate after the transition phase.

If Aspergillus models are to reach the same degree of predictive accuracy as the Candidamodels, a greater knowledge is needed of the internal metabolic changes that occur duringthe transition phase, when the organism appears to become stressed.

7.5 References

HO, S F, KRISTIANSEN, B and MATTEY, M, 1994. Phase-related mathematical model of theproduction of citric acid by Aspergillus niger, European Federation of Biotechnology InternationalConference on Modelling of Filamentous Fungi, Otocec, Slovenia, p. 57.

KLASSON, T K, CLAUSEN, E C and GADDY, J L, 1989. Continuous fermentation for the productionof citric acid from glucose, Applied Biochemistry and Biotechnology, 20, 491–509.

KLASSON, T K, CLAUSEN, E C, GADDY, J L and ACKERSON, M D, 1991. Modelling lysine andcitric acid production in terms of initial limiting nutrient concentrations, Journal of Biotechnology,21, 271–282.

KRISTIANSEN, B and SINCLAIR, C G, 1979. Production of citric acid in continuous culture,Biotechnology and Bioengineering, 21, 297–315.

KUBICEK, C P and RÖHR, M, 1989. Citric acid fermentation, CRC Critical Reviews in Biotechnology,4, 331–373.

LUEDEKING, R and PIRET, E L, 1959. A kinetic study of the lactic acid fermentation, Journal ofBiochemistry, Microbiology, Technology and Engineering, 1, 393–412.

MONOD, J, 1942. Recherches sur la croissance des cultures bacteriannes (Hermann and Cie, Paris).MORESI, M, 1994. Effect of glucose concentration on citric acid production by Yarrowia lipolytica,

Journal of Chemical Technology and Biotechnology, 60, 387–395.RÖHR, M, ZEHENTGRUBER, O and KUBICEK, C P, 1981. Kinetics of biomass formation and

citric acid production by Aspergillus niger on pilot plant scale, Biotechnology and Bioengineering,23, 2433–2445.


SINCLAIR, C G, KRISTIANSEN, B and BU’LOCK, J D, 1987. Kinetics and Modelling (Taylor andFrancis, Open University Press), p. 56.

THOMAS, C R and PAUL, G C, 1994. Modelling of the penicillin fermentation, European Federationof Biotechnology International Conference on Modelling of Filamentous Fungi Abstract, Otocec,Slovenia, p. 19.

TORRES, N V, 1994a. Modelling approach to control of carbohydrate metabolism during citric acidproduction by Aspergillus niger: I. Model definition and stability of the steady state, Biotechnologyand Bioengineering, 44, 104–111.

TORRES, N V, 1994b. Modelling approach to control of carbohydrate metabolism during citric acidproduction by Aspergillus niger: II. Sensitivity analysis, Biotechnology and Bioengineering, 44,112–118.

TRINCI, A P J, 1970. Kinetics of the growth of mycelial pellets of Aspergillus nidulans, Archiv fürMikrobiologie, 73, 353–367.

121

8

Mass and Energy Balance

LILIANA KRZYSTEK AND STANISKAW LEDAKOWICZ

Nomenclature

C concentration (C-mol m-3)K ATP consumption for polymerization of

biomass precursors (mol ATP (C-mol DM)-1)mATP specific maintenance requirements of ATP (mol ATP (C-mol DM)-1 h-1)mS specific maintenance requirements of

substrate (C-mol (C-mol DM)-1 h-1)mO specific maintenance requirements of oxygen (mol O2 (C-mol DM)-1 h-1)N moles ATP generated per 1 C-mol of

substrate by substrate level phosphorylation (mol ATP (C-mol)-1)NP moles ATP generated per 1 C-mol of

substrate before the biomass or productformation diverges from the catabolicpathway (mol ATP (C-mol)-1)rate of ATP consumption (mol ATP m-3 h-1)rate of ATP consumption in maintenanceprocesses (mol ATP m-3 h-1)rate of ATP conversion of compound j (C-mol m-3 h-1)maximum true yield of biomass on ATP (C-mol DM (mol ATP)-1)maximum true yield of product on ATP (C-mol DM (mol ATP)-1)yield on available electrons (C-mol DM (mol e-)-1

true yield on available electrons (C-mol DM (mol e-)-1

yield of ATP on substrate (moles ATPgenerated per 1 C-mol of substrate from thecatabolic breakdown reaction) (mol ATP (C-mol)-1)yield of ATP on substrate (moles ATPrequired for maintenance) (mol ATP (C-mol)-1)yield of oxygen on substrate (mol O2 (C-mol)-1)yield factor for compound j on compound i (C-mol (C-mol)-1)


Y¢i,j true yield factor for compound j oncompound i (C-mol (C-mol)-1)

YC upper limit of Yi,j based on carbonavailability (-)

YE upper limit of Yi,j based on energyavailability (-)

YH upper limit of Yi,j based on reducing potential (-)d P/O ratio (mol ATP (0.5 mol O2)-1)g¢j generalized degree of reduction of

compound j (-)sj weight fraction of C in compound j (-)h fraction of available electrons transferred to

the biomass (-)e fraction of available electrons transferred

to oxygen (-)xP fraction of available electrons transferred

to products (-) Subscripts

O oxygenP productX biomassS substratemax maximumpre biomass precursorsr real

8.1 Introduction

For any bioprocess, rapid formation of an appropriate amount of biomass is of greatimportance, both when it is the only expected product and when the purpose is to synthesizedefinite chemical compounds. The simplest of the generally applied criteria of bioprocessefficiency are mass yield coefficients. Precise determination of the amount of biomassproduced and substrate used is a starting point for the evaluation of all other process yieldsand for mass and energy balances as well as elementary balances of carbon, oxygen, nitrogen,etc. What makes the elementary balances much easier is the assumption of a constant averageweight fraction of carbon in the biomass (Erickson et al., 1979). Thus, it is specified howmuch substrate an organism has used in the synthesis of its own mass and how much in theproduction of energy. Furthermore, it is also important to understand the possibilities toincrease or decrease yields. This understanding is only possible by an extensive knowledgeof the biochemical intracellular reactions and processes that lead to biomass or products.Such knowledge is best presented in the form of a metabolic model. The model is based onthe specification of a set of intracellular chemical reactions, which are derived from theavailable biochemical information.

This chapter deals with mass and energy balances for Aspergillus niger growth kinetics.The relations obeyed by observed and true yield coefficients resulting from balance equations for carbon, reduction potential and energy during intensive cell growth and citric

Mass and energy balance 123

acid overproduction are derived and discussed. Quantitative balances based on one macro-chemical equation for checking the consistency of experimental data and evaluation of theefficiency of conversion of organic substrates by A. niger are also presented.

8.2 Metabolic description of A. niger growth

The process of submerged citric acid production carried out by A. niger in sugar mineralmedium is characterized by the following features (Figure 8.1): in the phase of fast mycelialgrowth (trophophase), formation of citric acid is observed; during the stationary phase(idiophase), product formation is maximized, but hardly any growth occurs. The substrate,being the source of carbon and energy, is converted either to biomass, CO2 and H2O or tocitric acid. Citric acid accumulation results from the disruption of the tricarboxylic acidcycle, in which destruction of citric acid is blocked. NADH formed during intensive growthof A. niger is a substrate for transformations of a respiratory chain in the presence ofcytochromes, with which the synthesis of ATP is coupled in the process of oxidativephosphorylation. The main pathway of electron transport during citric acid accumulation isan alternative respiratory system (Kubicek and Röhr, 1986). The functioning of this‘alternative oxidase’ stimulates glycolysis since it permits oxidation and NADH recirculationwithout ATP synthesis and contributes to citric acid overproduction.

A list of the most important metabolic processes accompanying citric acid accumulationwith sucrose as a carbon source is shown in Table 8.1. The complex machinery of cellularprocesses has been arranged into fundamental reaction patterns: catabolic pathways(breakdown of substrates into energy and small molecules), anabolic pathways (synthesisof precursors for biomass), polymerization of the precursors to biomass, and the maintenancemetabolism keeping the cellular machinery operative (Roels, 1983). The reactions

Figure 8.1 Typical example of growth and citric acid accumulation by A. niger in submergedculture (air-life bioreactor) (reprinted from Krzystek et al. (1996) with kind permission ofElsevier Science)


listed in Table 8.1 such as synthesis of biomass precursors (I), production of citric acid (II),catabolism of sucrose (VII), oxidative phosphorylation (VIII), polymerization of biomassprecursors (IX) and maintenance processes (X) are considered predominant. The formationof any other by-products (i.e. polyhydric alcohols (III–VI)) is assumed to be negligible,since their total concentration is at a low level in comparison to citric acid (Röhr et al.,1987). The stoichiometry determines the mass and energy balances of energy carriers (ATP,GTP) and reducing equivalents (NADH2, NADPH2, FADH2):

Table 8.1 Stoichiometry of main metabolic pathways appearing in the citric acid productionby A. niger on sucrose (reprinted from Krzystek et al. (1996), with kind permission of ElsevierScience)


The rate equations for substrate consumption and product formation could be derived afterapplying a quasi-steady state approximation (QSSA) to biomass precursors, energy carriersand reducing equivalents (Krzystek et al., 1996):

The equations obtained have the structure in which separate terms occur for substrate andoxygen consumption associated with growth, product formation and maintenance. Formally,they are identical to the most common assumption made on an a priori base, postulated byPirt (1975):

but now the yields are related to stoichiometric coefficients resulting from metabolic reactions(Roels, 1983):

The above values were calculated taking the molecular mass MX of biomass 28.3 g DM(C-mole DM)-1 assuming an ash content of 8 per cent DM. The P:O ratio was estimated asd = 2.17 mol ATP (0.5 mol O2)-1 (Roels, 1983; Garret and Grisham, 1995), while a meanvalue of the true biomass yield on ATP, Ym

Aa

TxP,X, of 0.371 C-mol DM (mol ATP)-1, and the true

citric acid yield YmA

aT

xP,X = -6 C-mol citric acid (mol ATP)-1 (Andrews, 1989; Roels, 1983).

The citric acid formation pathway generates 1/6 mol ATP from 1 C-mol of sucrose (Table8.1), and, for simple products whose synthesis does not diverge from the catabolic pathwayYATP,P = ¥.

8.3 Mass and energy balances

The yield coefficients are essentially thermodynamic quantities. They result from a balancebetween the energy generated by the catabolic reactions and that consumed by the anabolicreactions for the production of new cell mass. The following equations can be shown tohold the mass and energy balances of ATP and reducing equivalents (Andrews, 1989):


Formation of mycelium of A. niger is an energy consuming process (Table 8.1, pathways (I)and (IX)) and it implies that cell growth is energy limited. In turn, energy is generated in thereaction of citric acid formation (Table 8.1, pathway (II)), so this is clearly a carbon limitedproduct. The yields YC, YH and YE represent their upper limits, referred to as carbon-limited,reduction-limited and energy-limited, respectively. However, the mass and energy balanceson the reactions (equations (8.1)–(8.3)) give the relations (8.9)– (8.11) which are obeyed,not only by the observed yields, but also the true yields.

In the case of theoretical yield all the substrate was used for production of a singleproduct and the cell maintenance is ignored. For the biomass the theoretical yield equals thesmallest of the following:

The value of YEX corresponds to the value of Yav,e = 0.11 C-mol DM (mol)-1. Similarly, thecalculations of theoretical citric acid yield give:

YCP = 1

YHP = 4/3 = 1.33

YEP = 1.33

and:

(YSP)max = YCP = 1 C-mol citric acid (C-mol sucrose)-1.

Simultaneous consideration of the mass and energy balances allows the calculation of thehighest biomass and product yields to be expected in practice (real values):


The graphical interpretation is given in Figure 8.2, where the citric acid yield is shown asa function of dimensionless biomass. The highest theoretical biomass and citric acid yieldscorrespond to the point where the energy balance line for the value of YEX/YCX = 0.5,crosses the diagonal line representing the mass balance (taking equality in relation (8.12)and ignoring cell maintenance). The maximum real yield for citric acid is 0.8 C-mol citricacid (C-mol sucrose)-1 and the corresponding maximum real yield of biomass is 0.18 C-mol DM (C-mol sucrose)-1 (i.e. 0.9 g citric acid (g sucrose)-1 and 0.18 g DM (g sucrose)-

1, respectively.The yield coefficient YE (equation (8.10)) can also be determined taking into account

the ATP requirement for maintenance. Calculated values for cells and product are asfollows:

The formation of biomass is also an energy consuming process as well as the production ofcitric acid is a carbon-limited process. The calculated value of Y¢av,e now equals 0.16 C-molDM (mol)-1.

Figure 8.2 Possible biomass and product yields during citric acid production (reprinted fromKrzystek et al. (1996) with kind permission of Elsevier Science)

¢


The mass and energy equations (8.8)–(8.11) written in terms of rates are as follows:

8.4 Kinetics of growth and citric acid production by A. niger

The form of equations (8.15) and (8.16) is identical to that of (8.4, 8.5) and (8.6, 8.7) if theenergy yield coefficients YE have the values of Y¢E (i.e. taking into account the ATPrequirement for maintenance). This makes the linear growth equations represent in fact theoverall energy balance where:

The energy and reduction limitation are the same in the case of formation of citric acidsince YATP,P = ¥ C-mol citric acid (mol ATP)-1 and the maintenance requirement for ATP ismet approximately by substrate-level phosphorylation. In addition, for carbon limitedproducts (YSP)max = YCP = YHP, thus from (8.14) and (8.15) the following equation can beshown to hold:


The verification has been performed using the data from submerged citric acid processes insucrose mineral medium at the initial pH value about 2.5 carried out in a pilot plant air-liftbioreactor (Gluszcz and Michalski, 1994). An agreement between the theory andexperimental results was observed, confirming the linear growth equation to be an energy

Citric acid is formed by A. niger during the growth phase as well as in the stationary phase,although mainly in the stationary phase when citric acid formation is maximized and hardlyany growth occurs (Kubicek and Röhr, 1986). It implies that in the intensive growth phasethe substrate consumption rates (sucrose and oxygen) can be described as:

On the basis of batch experimental data the yield coefficients in equations (8.37) and (8.38)were verified (Krzystek et al., 1996). The equations are as follows:


balance with the true yields coefficients. Experimentally obtained yield coefficients of citricacid and biomass on sucrose and oxygen are presented in Table 8.2 and compared withtheoretical and true yield coefficients. The yield of citric acid on sucrose reached 83 percent of real maximum theoretical values. Yields of biomass on sucrose and oxygen were 96per cent and 109 per cent, respectively.

On the basis of experimental data the maintenance coefficients were also estimated:

mS = mO = 0.026 C-mol sucrose (mol O2) (C-mol DM)-1(h)-1.

The resulting specific maintenance requirement of ATP was:

mATP = 0.015 mol ATP (C-mol DM)-1(h)-1,

which is of the order of common value for mATP (Solomon and Erickson, 1981).

8.5 Carbon and available electron balances

Material and energy balances and their regularities based on the concept of one macroscopicequation introduced by Minkevich and Eroshin (1973) were used to evaluate sugar conversionto citric acid, mycelium, CO2 and to check the accuracy of experimental data by Röhr et al.(1983, 1987) and Nowakowska-Waszczuk and Sokolowski (1987). In submerged citric acidproduction by A. niger in sugar mineral medium the carbon balances may be based on themeasurements of sugar consumed and biomass, citric acid and CO2 produced, according tothe equations:

Table 8.2 Yield coefficients in citric acid production by A. niger. (reprinted from Krzystek etal. (1996), with kind permission of Elsevier Science)

The energy in the organic substrate is incorporated into biomass, evolved as heat (released as aresult of combustion or as electron equivalents that can be transferred to oxygen) or incorporatedinto extracellular products. Available electron balance may be written in the form:


A balance analysis for sugar and oxygen uptake made by Röhr et al. (1983, 1987) showedthat during the first phase of citric acid accumulation (up to 130 hours) more sugar is takenup than the production of biomass, CO2 and citric acid can account for. In contrast, duringlater phases of fermentation, more citric acid, CO2 and mycelium are formed than sugaruptake would theoretically allow.

A similar pattern was also reflected in a balance for oxygen uptake, where less uptakeoccurs during the early phase of fermentation than needed for complete balance; thereverse was observed during the late stage of fermentation. This was caused by theintermediate accumulation and partial re-consumption of a number of polyhydric alcoholssuch as glycerol, arabitol, erythritol and mannitol, up to almost 9 g l-1. This findingexplained earlier observations (Shu and Johnson, 1948) of accumulation of more citricacid (9.9 g l-1) during the late stages of fermentation than sugar uptake can account for(6.7 g l–1), since the polyols become degraded during the late stages of fermentation. Thepolyols as by-products of citric acid production account for 70 per cent of ‘lacking material’(Röhr et al., 1983, 1987).

Nowakowska-Waszczuk and Sokolowski (1987), calculating the amounts of glucosecarbon utilized during the fermentation and its distribution to mycelium, citric acid andCO2, also observed that the carbon content of consumed sugar and products did not balance.During the first 24 hours of the process carried out in an air-lift bioreactor of 0.8 m3 workingvolume (height about 11 m) only about 76 per cent sugar carbon was found in the products(biomass, citric acid and CO2). From the second day a surplus of carbon was found in theproducts. When the sugar content was very low or had been completely consumed, thesurplus carbon in the products was reduced to 0.9–5.3 per cent. In two different experimentsthe conversion of glucose carbon to citric acid, mycelium and CO2 was 76.4 and 81 percent, 13.8 and 12.1 per cent and 13.54 and 11.8 per cent respectively. The pool of electronstransferred to oxygen in the two runs was 4.89 and 4.55, corresponding to 1.22 and 1.14mmol O2 dm-3. The carbon content in mycelium and the degree of reduction of its carbonwas accepted as 0.46 and 4.29, respectively.

8.6 Conclusion

Substrate and oxygen requirements as well as biomass and product yields, which are someof the basic parameters that need to be considered in determining the feasibility of thefermentation process, may only be estimated properly if material and energy balances canbe applied to the bioprocess. Available electron, ATP and carbon balances as well as thecomparison of estimated values of yields and maintenance parameters can be used to testthe consistency of the data in fermentations and to gain insight into the possibility of by-


product formation. Material balance for carbon is in particular widely examined during thecourse of the process since it can be readily confirmed by actual proof on the basis ofexperimental data.

The assumption that the micro-organisms are alike in their chemical and biochemicalproperties leads to formulation of a macro-chemical equation. Obviously, this is a grosssimplification that only holds as a first rough approximation. However, for many bio-technological processes it is important to know accurately the yield values. This can berealized by formulating the metabolic model.

A useful metabolic model should represent, in a concise way, the main biochemicalproperties of a specific micro-organism specified as a limited number of reactions.Such simple metabolic models give the proper formulation of the Pirt type relationlinking as well as more accurate yield values and the biochemical insight how these canbe manipulated. In linear relations following from restrictions out of the metabolicnetwork the yield values are a function of the biochemical ATP and decarboxylationstoichiometry.

For citric acid production by A. niger the assumption of typical mechanisticquantities such as and P:O enables the calculation of theoretical and true yields forgrowth and citric acid production appearing in kinetic equations based on knownmechanism of the process. The analysis of the distribution of carbon and energy sourcefor biomass growth, product synthesis and maintenance processes stresses theimportance of maintenance requirements in the process. Thus, it is a useful way forprocess design and optimization.

8.7 References

ANDREWS, G, 1989. Estimating cell and product yields, Biotechnology and Bioengineering, 33,256–265.

ERICKSON, L E, MINKEVICH, I G and EROSHIN, V K, 1979. Utilization of mass-energy balanceregularities in the analysis of continuous culture data, Biotechnology and Bioengineering, 21,575–591.

GARRET, R H and GRISHAM, Ch M, 1995. Biochemistry (Saunders).GLUSZCZ, P and MICHALSKI, H, 1994. Cultivation of A. niger in a pilot plant external loop airlift

bioreactor, FEMS Microbiological Reviews, 14, 83–88.KRZYSTEK L, GLUSZCZ P and LEDAKOWICZ S, 1996. Determination of yield and maintenance

coefficients by A. niger, The Chemical Engineering Journal, 62, 215–222.KUBICEK, C P and RÖHR, M, 1986. Citric acid fermentation. CRC Critical Reviews of Biotechnology,

3, 331–373.MINKEVICH, I G and EROSHIN, V K, 1973. Productivity and heat generation of fermentation under

oxygen limitation, Folia Microbiologica, 18, 376–385.NOWAKOWSKA-WASZCZUK, A and SOKOLOWSKI, A, 1987. Application of carbon balance to

submerged citric acid production by A. niger, Applied Microbiology and Biotechnology, 26, 363–364.

PIRT, S J, 1975. Principles of Microbe and Cell Cultivation (Blackwell).ROELS, J A, 1983. Energetics and Kinetics in Biotechnology (Elsevier).RÖHR, M, KUBICEK, C P, ZEHENTGRUBER, O and ORTHOFER, R, 1983. A balance of carbon

and oxygen conversion rates during pilot plant citric acid fermentation by A. niger: identificationof polyols as major by-products, International Journal of Microbiology, 1, 19–25.

RÖHR, M, KUBICEK, C P, ZEHENTGRUBER, O and ORTHOFER, R, 1987. Accumulation andpartial re-consumption of polyols during citric acid fermentation by A. niger, Applied Microbiologyand Biotechnology, 27, 235–239.

SHU, P and JOHNSON, M J, 1948. Citric acid: production by submerged fermentation by A. niger,Industrial Engineering Chemistry, 40, 1202–1204.


SOBOTKA, M, MACHON, V, SEICHERT L, UJCOVA, E and MARSCHALKOVA, Z, 1985. Chemicalengineering aspects of submerged production of citric acid, Folia Microbiologica, 30, 381–392.

SOLOMON, B O and ERICKSON, L E, 1981. Biomass yields and maintenance requirements forgrowth on carbohydrates, Process Biochemistry, February–March, 44–49.

135

9

Downstream Processing in Citric AcidProduction

PAWEL GLUSZCZ AND STANISLAW LEDAKOWICZ

9.1 Pretreatment of fermentation broth

On completion of the citric acid fermentation the obtained solution contains, besides thedesirable product, the mycelium and varying amounts of other impurities, e.g. mineral salts,other organic acids, proteins, etc. The method of citric acid recovery from the fermentationbroth may vary depending on the technology and raw materials used for the production(Grewal and Kalra, 1995).

In the surface process the fermentation fluid is drained off the trays and hot water isintroduced to wash out the remaining amount of citric acid from the mycelial mats. Thoroughwashing at this stage is necessary, because the mycelium retains about 15 per cent of theproduct formed in the fermentation. After 1–1.5 hours the wash water is drained off andthen added to the fermentation liquor and mycelial mats are removed from trays, disintegratedand flushed into the washing vessel using limited amounts of water. In this vessel themycelium is heated to about 100°C by steam. The hot pulp is subsequently dewatered bypressure filtration. The solution containing 2–4 per cent of citric acid is added to thefermentation fluid, whereas the filtration cake, containing not more than 0.2 per cent ofcitric acid, is dried to yield a protein-rich feed-stuff.

In the submerged fermentation the mycelium is far more difficult to separate from thefermentation broth. After the fermentation process is completed the mycelium containingbroth is heated to a temperature of 70°C for about 15 minutes, to obtain partial coagulationof proteins, and then filtered, usually by means of the continuous filters (e.g. a rotatingvacuum drum filter or a belt discharge filter). Because of the slimy consistency of myceliumforming in the submerged process, filter aids may be required. If the mycelium is to be usedas a feedstuff, the filter aid must also be digestible, e.g. from cellulosic materials.

If during the fermentation process oxalic acid is formed as a side product due to suboptimalcontrol of the fermentation process, it has to be removed from the broth. This is usuallyachieved by increasing the pH of the fermentation fluid with the calcium hydroxide to pH =2.7–2.9 at a temperature of 70–75°C. Calcium oxalate thus precipitated may be removedfrom the solution by filtration or centrifugation, and the citric acid remains in solution asthe mono-calcium citrate. Oxalate removal increases the rate of filtration of the calcium


citrate and gypsum in the subsequent steps of downstream processing and reduces the yellowhue of the citric acid solution.

Recovery of citric acid from pretreated fermentation broth may be accomplished byseveral procedures: classical method of precipitation, solvent extraction, adsorption/absorption on ion-exchange resins, and recently developed, more sophisticated methodssuch as electrodialysis, ultra- and nanofiltration or application of liquid membranes.

9.2 Precipitation

The standard method of citric acid recovery has involved precipitating the insoluble tri-calcium citrate by the addition of an equivalent amount of lime to the citric acid solution.Successful operation of the precipitation depends on citric acid concentration, temperature,pH and rate of lime addition. To obtain large crystals of high purity, milk of lime containingcalcium oxide (180–250 kg/m3) is added gradually at a temperature of 90°C or above andpH below, but close to, 7. The concentration of citric acid in the solution should be above 15per cent. The process of neutralization usually lasts about 120–150 minutes. The minimumloss of citric acid due to solubility of calcium citrate is 4–5 per cent.

If precipitation is properly done, most impurities remain in the solution and may beremoved by washing the filtered calcium citrate. Washing is performed with the smallestamount possible of hot water (approx. 10 m3 of water per tonne of acid at the temperature90°C) until no saccharides, chlorides or coloured substances can be detected in the effluent.The calcium citrate is then filtered off and subsequently treated with concentrated sulphuricacid (60–70 per cent) to obtain citric acid and the precipitate of calcium sulphate (gypsum).After filtering off the gypsum a solution of 25–30 per cent of citric acid is obtained. Thefiltrate is treated with activated carbon to remove residual impurities or may be purified inion-exchange columns. The purified solution is then concentrated in vacuum evaporators attemperature below 40°C (to avoid caramelization), crystallized, centrifuged and dried toobtain citric acid crystals. If crystallization is performed at temperatures below 36.5°C, thecitric acid mono-hydrate is formed and above this transition temperature citric acid an-hydrate may be obtained. The schematic flow-chart of the standard precipitation method isshown in Figure 9.1.

The disadvantage of this technology is the large amount of lime required for citric acidneutralization and of sulphuric acid for calcium citrate decomposition. Moreover, it resultsin the formation of large amounts of liquid and solid wastes (solution after calcium citratefiltration and gypsum). For one tonne of citric acid, 579 kg of calcium hydroxide, 765 kg ofsulphuric acid and 18m3 of water are consumed and approximately one tonne of wastegypsum is produced.

With the aim of decreasing the amount of lime and sulphuric acid by about one third,Ayers (1957) has proposed recovery of citric acid by precipitation of di-calcium acid citrate.An additional advantage of this method is that di-calcium acid citrate has a definite crystallinestructure and washes cleaner than the amorphous tri-calcium citrate. Moreover, fewerimpurities are precipitated from a fermentation fluid with the di-calcium salt than with thenormal salt, when the reaction mixture is completely neutralized.

Di-calcium acid citrate precipitates from a citric acid solution that has been partiallyneutralized by the addition of calcium hydroxide, calcium oxide or calcium carbonate atan elevated temperature. It is believed that an equilibrium exists between tri-calciumcitrate and citric acid on the one side, and di-calcium acid citrate on the other. At roomtemperature the rate of calcium hydrogen citrate formation is negligible, but if the

Downstream processing in citric acid production 137

Figure 9.1 Flowsheet of the standard precipitation method of citric acid recovery fromfermentation broth


temperature is elevated above 40°C the complete conversion of tri-calcium citrate mixedwith aqueous solution of citric acid occurs within a reasonable length of time (about 24hours). According to this principle a new method of citric acid recovery has beendeveloped.

The citric acid solution, obtained from the fermentation broth, is divided in two parts.The first part, about two thirds of the total volume, is completely neutralized with milk oflime, and the tri-calcium citrate is filtered off and added to the remaining part of theoriginal citric acid solution. If the obtained mixture is heated above 40°C, a precipitate ofdi-calcium acid citrate will result. As an alternative method, an amount of calciumhydroxide no greater than two thirds of that required for complete neutralization may beadded directly to a citric acid solution. This mixture of tri-calcium citrate and citric acidmay then be converted to di-calcium acid citrate by heating above 40°C, preferably to80–95°C (depending on the boiling point of the solution). It has been found that theresults of the process may be improved, both by shortening the time and by increasing theyield, if the mixture is seeded with di-calcium acid citrate crystals (practically about 10 to25 per cent of the expected yield).

As an alternative to the classical methods of precipitation, separation and purification ofcitric acid from fermentation solutions, Schultz (1963) has suggested isolating the citricacid from the fermentation solution in the form of its alkali metal salts and recovery of theacid from such salts directly in one single operation. This process is based on the fact thatcertain alkali metal salts of citric acid crystallize from a fermentation solution afterneutralization of the acid by the addition of alkaline alkali metal compounds (hydroxides,bicarbonates or carbonates) in such a manner that the mono-, di- or tri-alkali metal citratesare obtained.

The impurities contained in fermentation broth influence or even inhibit crystallizationof salts, so not all the theoretically possible alkali metal salts of citric acid can be producedin crystalline form according to the process. Of the sodium salts, however, all three possiblesalts can be recovered in the form of crystals.

Before neutralization the fermentation solution may be concentrated by vacuumevaporation to a concentration of at least 40 per cent, calculated for free citric acid. Afterneutralizing the alkali metal salts crystallize on standing or on slowly stirring the solution;seed crystals may be added to enhance the rate of the process. Crystallization is ordinarilycompleted within 24 hours. Separation of the crystals from the solution is performed by theusual methods (filtration, centrifugation). After washing the crystals with a small amount ofwater, an almost white or slightly yellowish-brown precipitate is obtained, depending uponthe type of alkali metal citrate recovered. Subsequent purification of citric acid may beperformed by ion exchange on cation exchange resins or by electrodialysis.

The yield of citric acid on recovering it in the form of its alkali metal salts is between 50per cent and about 80 per cent depending on the salt used. Citric acid remaining in thefermentation broth may be recovered by the ‘classical’ method of precipitation in the formof a calcium citrate and following treatment with the sulphuric acid. According to this processconsiderable savings in chemicals are achieved and the amount of the spent gypsum producedis reduced. Moreover, the obtained gypsum filters more rapidly, due to the presence ofalkali metal ions, than gypsum from the ‘classical’ technology, produced in the absence ofthe alkali metal ions.

The use of purer raw materials than molasses (e.g. sucrose or glucose) in citric acidproduction leads to simplified methods for its recovery and purification. Crystalline or rawsugar are the best raw materials in view of the high acid yield and relatively short fermentationtimes attained. Crystalline sugar is also favoured by the reduced risk of infection with foreign


micro-organisms due to the low initial pH value of the nutrient medium (2.5 to 3), and bythe considerable reduction of the total amount of wastes and effluents.

Crystalline sugar based fermentation makes it possible to use a modified, citrate-freemethod of citric acid recovery (Lésniak, 1989), applied in industrial practice in severalcitric acid manufacturing plants in Poland and the Slovak Republic. This technology consistsof direct removal of impurities from the post-fermentation liquor, i.e. colloids (proteins),mycelium derived substances, coloured substances formed on heating the fermentationsolution, and mineral salts introduced with the nutrient medium, substrate and water. Theseimpurities must be removed as they interfere with the subsequent crystallization process.The first step of purification of the solution is achieved using suitably selected coagulatingagents and activated carbon and then filtering off the precipitates (Adamczyk et al., 1985).Further treatment involves the removal of the remaining impurities by ultrafiltration andretention of the mineral salts using ion-exchange resins. The purified citric acid solution isconcentrated, crystallized, centrifuged and dried according to the classical production processflowsheet.

After the separation of citric acid crystals the supernatant liquid from the centrifuge isrecycled back to the concentration section where the so-called second crop and then a thirdcrop of crystals is obtained. The supernatant liquid obtained after removing the third cropcrystals by centrifugation contains a large amount of impurities and must be purified by theclassical method involving the precipitation of calcium citrate. Thus the citrate-free methodcan be used for purifying only up to 80 per cent of the whole amount of citric acid. Thisnecessitates the construction of a separate process line in order to avoid plants using theabove technology to manufacture merely a 50 per cent solution of citric acid, making itnecessary to purify a part of the citric acid by the calcium citrate method.

It is also possible to produce half of the acid amount in crystalline form and the rest inliquid form. In this case, citric acid solution purified by the citrate free method is thickened,crystallized and centrifuged to obtain the first crop. The supernatant liquid from the centrifuge(citric acid concentration of about 50 per cent) is purified by the described method so as tomeet the quality standard requirements in liquid form. The advantage of this technologylies in the fact that about half of the product is obtained in crystalline form and the use oflime and sulphuric acid is eliminated as well as the formation of large amounts of effluentsand solid wastes. The flowsheet of the simplified, non-citrate method of citric acid recoveryis shown in Figure 9.2.

9.3 Solvent extraction

An alternative method of citrate-free recovery of citric acid from a fermentation broth isextraction by means of a selective solvent which is insoluble or only sparingly soluble inthe aqueous medium (Kertes and King, 1986; Hartl and Marr, 1993; Schügerl, 1994). Thesolvent should be chosen so as to extract the maximum amount of citric acid and the minimumamount of impurities. The citric acid can then be recovered from the extract either by distillingoff the solvent or by washing the extract with the water. From the aqueous solution purifiedcitric acid is subsequently crystallized by concentration.

In the first patent concerning citric acid solvent extraction (Chemische Fabrik J A Benkiser,1932) it has been proposed to apply n-butanol and then to wash the solution of citric acid inn-butanol with water. Since the first report a number of solvent combinations have beensuggested and a great amount of information and patents have been published. In general,extraction methods may be divided into three basic groups:


Figure 9.2 Flowsheet of the simplified non-citrate method of citric acid separation andpurification


• Extraction with organic solvents which are partly or wholly immiscible with water,such as certain aliphatic alcohols, ketones, ethers or esters (Kasprzycka Guttman etal., 1989).

• Extraction with organophosphorus compounds, such as tri-n-butylphosphate (TBP) (Pageland Schwab, 1950) and alkylsulphoxides, e.g. trioctylphosphine oxide (TOPO) (Nikitinand Egutkin, 1974; Grinstead, 1976).

• Extraction with water-insoluble amines or a mixture of two or more of such amines, as arule dissolved in a substantially water-immiscible organic solvent, and extraction withamine salts (Baniel, 1981; Bauer et al., 1988; Bizek et al., 1992; King, 1992; Prochazkaet al., 1994; Juang and Huang, 1995).

Each solvent used for extraction is characterized by its equilibrium distribution coefficientwhich is defined as the ratio of the acid concentration of the extract to the acid concentrationof the aqueous phase. For low concentrations of citric acid in the raw fluid the distributioncoefficient depends strongly on the type of solvent; at higher acid concentrations differencesbetween solvents are much reduced.

Extraction with organic solvents (in practice ketones and alcohols are used) may beuseful in cases where the acid has a relatively high concentration in the aqueous systemfrom which it is to be extracted. These solvents have rather low distribution coefficients(0.02–0.36), thus the extract is always more diluted than the raw liquor and multistageextraction is necessary as a rule. Moreover, solvents with relatively higher distributioncoefficients (such as butanols) are too water-miscible, so they require energy-consumingsteps of subsequent solvent recovery. Thus, these extraction systems are relativelyinefficient for acid recovery from the dilute aqueous solutions found in most fermentationstreams.

Organophosphorus extractants have a significantly higher distribution ratio than carbon-bonded solvents under comparable conditions, e.g. using undiluted TPB for citric acidextraction a distribution ratio of about 2 may be obtained at a 0.1 mol initial acidconcentration at 25°C (Pagel and Schwab, 1950). Alkylosulphoxides have been shown toextract carboxylic acids with a distribution ratio even higher than that of TBP (Nikitinand Egutkin, 1974). The value of the distribution coefficient is influenced not only byacid concentration but also by temperature. In TBP the distribution ratio for citric aciddecreases by a factor of 4 in the 0–80°C range. This property allows perfect control of theprocess: extraction at low temperature (10–30°C) and re-extraction with water at highertemperature (70–95°C).

For the extraction by means of amines, aliphatic, araliphatic or aromatic amines, or theirmixture, preferably with the average aggregate number of carbon atoms at least 20 for eachamino group, may be used. These reagents have the advantage of providing a favourablecoefficient of distribution of the citric acid between the aqueous and amine phases so theacid may be extracted even from highly dilute solutions. On the other hand there is a problemof decomposing the amine salt and recovering the acid and the amine separately, since theamines are too expensive to be thrown out. Usually the amine is liberated by treatment ofthe salt with an inorganic base (e.g. calcium hydroxide) or inorganic acid, and the salt isthus obtained instead of free citric acid. In addition to the expenditure of chemicals, thisprocess has the disadvantage of requiring a number of processing steps.

The extraction by the amine salts may be considered as a variant of the extraction withamines. In some cases the amount of acid that can be extracted with the water-immiscibleamine is stoichiometrically considerably in excess of the amine present in the amine solution.


The possible excess amount of extracted acid depends on several parameters, e.g.concentration of the acid in the raw liquor, the nature of the amine and its solvent. In somecases this phenomenon may be applied for extracting the acid from its concentrated aqueoussolution by means of salts of amines with the same acid. From the extract the excess acidcan be recovered by washing with water.

The organophosphorus and aliphatic amine extractants were developed initially for theneeds of inorganic extractive separation technologies. When these solvents are used for therecovery of citric acid intended for the food industry, the question concerning their toxicityshould be settled. It is known that some of these compounds show teratogenic effects. Onthe other hand the amine extractant patented by Baniel et al. (1981) and Baniel (1982) hasreceived approval by the US Food and Drug Administration for the use in food and drugtechnology (Melsom, 1987; US Food and Drug Administration, 1975). Of the great amountof patents concerning recovery of citric acid from the fermentation broth by extraction onlythis one has been applied in large scale production.

9.4 Adsorption, absorption and ion exchange

As crystalline sugar or other pure raw materials are used more often in citric acid production,methods of its recovery and purification by adsorption and ion exchange on polymericresins are gaining interest. One of the methods, sometimes used as a step in other non-citrate recovery technologies mentioned above, involves adsorption of contaminants onto anon-ionic resin based on polystyrene or polyacrylates and collection of the citric acid in therejected phase. The patent literature suggests more efficient adsorption/absorption methodsthat make it possible to separate the citric acid from fermentation broth in a single step(Fauconnier et al., 1996). Kulprathipanja (1988, 1989), Kulprathipanja et al. (1989),Kulprathipanja and Strong (1990) and Kulprathipanja and Oroskar (1991) have proposedseveral methods based on a similar principle, involving polymeric adsorbents of differenttypes. One group of such adsorbents may be neutral, non-ionogenic, macro-reticular, water-insoluble styrene-based polymers cross-linked with di-vinylbenzene. Better selectivity andhigher capacity of the adsorbent may be achieved using weakly basic anionic exchangeresins, impregnated with tertiary amine or pyridine (Kulprathipanja et al., 1989; Juang andHuang, 1995; Juang and Chou, 1996), or strongly basic anionic exchange resins containingquaternary ammonium functional groups.

In the simplest case the adsorbent may be applied in the form of a dense compact fixedbed which is alternatively contacted with the feed mixture and desorbent. Any of theconventional equipment employed in static bed fluid–solid contacting may be used for sucha semi-continuous process. The citric acid is recovered from the adsorbent by desorptionwith water or dilute inorganic acid (preferably sulphuric acid of a concentration of 0.1–0.2N). According to the patents mentioned, the complete separation of citric acid from saltsand carbohydrates is achieved by adjusting the pH of the feed solution below the firstionization constant of citric acid. The pH value required to maintain adequate selectivity isinversely proportional to the concentration of citric acid in the feed mixture.

Polymeric resins proposed for use in citric acid recovery are manufactured by severalchemical companies and sold under different trade names, so they are commerciallyavailable. They may differ slightly in physical properties such as porosity, skeletaldensity, specific surface area and dipole moment. The preferred adsorbents should havea surface area of 100–1000 m2/g. The various types of polymeric adsorbents wereoriginally designed for different chemical technologies, e.g. for decolorizing dye wastes,


decolorizing pulp mill bleaching effluent or removing pesticides from waste effluent.Their effectiveness in the separation of citric acid from A. niger fermentation broth israther unexpected.

The efficiency of the ion-exchange separation process may be greatly enhanced byapplying a so called simulated moving bed counter-current flow system. In this case theapparatus consists of at least two static beds, connected with appropriate valving so that thefeed mixture is passed through one adsorbent bed while the desorbent material can be passedthrough the other. Progressive changes in the function of each ion-exchange bed simulatethe counter-current movement of the adsorbent in relation to liquid flow. In such a system,the adsorption and desorption operations are continuously taking place, which allows bothcontinuous production of an extract and a raffinate stream and the continual use of feed anddesorbent streams. The simulated moving bed system applied for citric acid recovery in apilot scale is proposed by Edlauer et al. (1990).

The disadvantage of the ion-exchange method may be seen in the fact that elution ofcitric acid from the adsorption bed may require a large amount of desorbent, due to thetailing effect known in chromatography, causing considerable dilution of the resulting citricacid solution. The periodical regeneration of the ion-exchange resins by inorganic basesmay also be a source of unwanted effluent wastes.

9.5 Liquid membranes

Recently more sophisticated methods of citric acid separation with the application of liquidmembranes are being developed (Basu and Sirkar, 1991; Friesen et al., 1991; Juang, 1995;Albulescu and Guzun-Stoica, 1996). Liquid membranes containing mobile carriers consistof an inert, micro-porous support impregnated with a water-immiscible, mobile ion-exchangeagent. The mobile carrier, which is held in the pores of the support membrane by capillarity,acts as a shuttle, picking up ions from an aqueous solution on one side of the membrane,carrying them across the membrane and releasing them to the solution on the opposite sideof the membrane (Baker et al., 1977). The flow of the complexed ion is coupled to the flowof the second ion (e.g. the hydrogen ion). This process is categorized as ‘coupled transport’,and the membranes in which it takes place are called coupled transport membranes. Thecoupling of the flows of the two ions permits one of the ions to be pumped ‘up-hill’ from asolution in which it is dilute to a solution in which it is more concentrated (Fyles et al.,1982).

For citric acid separation by liquid membranes, the tertiary amines which give the bestresults in solvent extraction can also be used. In the extraction step, the basic amine reactswith hydrogen ions in the feed solution to form a tertiary alkylammonium cation. Thiscation then associates as an ion pair with the citrate anion to form an alkylammonium salt,which is transported across the membrane and stripped from the organic carrier solutioninto the aqueous product phase. This reaction regenerates the tertiary amine, which thendiffuses back to the feed side of the membrane, where it recomplexes with hydrogen andcitrate ions.

Supported liquid membranes have not been adopted for industrial scale, primarily due toa lack of long-term stability resulting from loss of membrane by solubility, osmotic flow ofwater across the membrane, progressive wetting of the support pores, and pressure differentialacross the membrane (Danesi et al., 1987). To eliminate these problems microporous hollowfibres have been employed by Basu and Sirkar (1991). In this case the permeator consists oftwo sets of identical hydrophobic microporous hollow fibres. One set carries the feed solution


of citric acid and the other the strip solution flowing in the lumen. The organic liquidmembrane is contained in the shell side between these two sets of hollow fibres. Thistechnique has been shown to be promising for citric acid separation even in the large scale,as the extent of citric acid recovery of up to 99 per cent was linear with the membrane area,suggesting easy scale-up.

The use of liquid membranes for the recovery of citric acid from fermentation brothsoffers unique advantages over conventional techniques: lower energy consumption, higherseparation factors in a single stage, the ability to concentrate citric acid during separationand smaller size of the complete separation apparatus. These advantages may result in areduction in overall recovery costs and in amount of wastes.

9.6 Electrodialysis

Another environmentally friendly alternative to the conventional methods of citric acidrecovery may be electrodialysis. This process enables separation of salts from a solutionand their simultaneous conversion into the corresponding acids and bases using electricalpotential and mono- or bipolar membranes. Bipolar membranes are special ion exchangemembranes which, in an electrical field, enable the splitting of water into H+ and OH- ions(Strathmann et al., 1993). By integrating bipolar membranes with anionic and cationicexchange membranes a three- or four-compartment cell may be arranged, in whichelectrodialytic separation of salt ions and their conversion into base and acid takes place(Voss, 1986; Sappino et al., 1996). According to Karklins et al. (1996), completetransformation of sodium tri-citrate into citric acid in a four-compartment cell may beachieved a little faster, but voltage on electrodes is higher than in a three-chamber cell.Specific electroenergy consumption of the four-compartment cell was about 40 per centhigher than that of a three-chamber apparatus.

When converting organic salts, high final acid concentrations may be achieved, as opposedto mineral salts. It makes the process especially advantageous for citric acid recovery, as theevaporation step normally required can be omitted. On the other hand organic salts such assodium citrate have a relatively large molecular weight and the solution also shows relativelylow conductivity. These properties make the separation more difficult and lead to higherenergy consumption, as in the case of inorganic compounds. The energy consumption(excluding pumping) for the separation of 1 kg of citric acid using bipolar membranes is inthe range of 6.1 × 103 to 7.2 × 103 kWs (Novalic et al., 1995). Due to low mass transfer atlow pH values it is advantageous to adjust the pH of the feed acid stream to 7.5 (Moresi andSappino, 1996; Novalic et al., 1996).

Before the fermentation solution comes to the electrodialysis some pretreatment stepsare normally necessary: filtration of the broth, removal of ionogenic substances (especiallyCa++ and Mg++ ions) and neutralization by means of sodium hydroxide. In the subsequentelectrodialytic step the sodium citrate solution is converted into base and citric acid, whichis simultaneously concentrated and for the most part purified. The produced NaOH may bereused for the neutralization (Novalic and Kulbe, 1996).

Although there have been several patents published concerning recovery andpurification of organic acids by electrodialysis (Gomez et al., 1991), this method is stillapplied only in laboratory scale and requires optimization. The economics are mainlyinfluenced by the relatively high energy consumption, the membrane costs and the membranelife time. However, due to the wider commercial availability of bipolar membranes in thepast few years and various advantages of the electrodialysis technique it is expected that


this technology will soon be competitive with other processes (Novalic et al., 1996). Besidesthe elimination of environmental problems, the use of electrodialysis enables continuousseparation of the citric acid from the broth during fermentation, leading to the decrease ofan inhibiting influence of the product. It is also possible to apply this technique for recoveryof the citric acid in continuous fermentation processes. The scheme of the proposed method(Novalic and Kulbe, 1996) for citric acid separation by means of electrodialysis with bipolarmembranes is shown in Figure 9.3.

9.7 Ultrafiltration

Continuous separation and concentration of citric acid may be also achieved by ultra and/or nanofiltration. Visacky (1996) verified in a laboratory scale a two-stage membrane processfor citric acid recovery from the broth obtained in A. niger cultivation on sucrose.Polysulphone membrane with cut-off 10 000 used in the first stage allowed the product topass through to the permeate stream, while the retentate stream contained most of peptidesand proteins from the broth. The rejection coefficient for the product in this step was 3 percent, for the reducing sugars 14 per cent and for the proteins 100 per cent. Tighternanofiltration membrane with cut-off 200 in the second stage rejected approximately 90per cent of citric acid and 60 per cent of reducing sugars (mono-saccharides). Concentrationof the product in the retentate stream was increased three times in comparison to the feed. Asimilar two-stage membrane technique was adapted by Bohdziewicz and Bodzek (1994)for simultaneous separation and concentration of pectinolytic enzymes and citric acid from

Figure 9.3 Scheme of citric acid separation by means of electrodialysis with bipolarmembranes (from Novalic and Kulbe, 1996)


a fermentation broth. The dilute citric acid solution obtained as a permeate in the first stepof the post-fermentation fluid ultrafiltration was then concentrated up to 20 per cent usingreverse osmosis. Such membrane processes may give important benefits in industrialtechnologies of citric acid recovery: low energy consumption, no wastes in comparison tothe conventional chemical methods, possibility of use in continuous processes. However,they require practical verification and optimization in a pilot and industrial scale.

9.8 Immobilization of micro-organisms

It is worth noting that some of the problems arising in the downstream processing of citricacid produced by submerged cultivation, especially in a continuous process, might beminimized by immobilization of micro-organisms in the bioreactor. In the past few years,immobilization of microbial cells has received increasing interest. The successful applicationof immobilized micro-organisms as living biocatalysts, involving more careful handlingand often having higher production rates than free micro-organisms, has prompted a rapiddevelopment of this technique. Citric acid production by immobilized A. niger has beenperformed on a laboratory scale with the use of calcium alginate gel (Eikmeier and Rehm,1984; Tsay and To, 1987), polyacrylamide gel (Gary and Sharma, 1992; Mittal et al., 1993),polyurethane foam (Lee et al., 1989; Sanroman et al., 1994; Pallares et al., 1996) andcryopolymerized acrylamide (Wang and Liu, 1996). The profitable effect of theimmobilization of A. niger mycelium in view of the citric acid recovery from the fermentationbroth depends on the type of the support material and process conditions. Further researchis required to take full advantage of this technology, but it seems to be promising, especiallyin combination with other recently developing recovery techniques, such as ultrafiltrationor ion-exchange.

9.9 References

ADAMCZYK, E, LESNIAK, W, PIETKIEWICZ, J, PODGÓRSKI, W, ZIOBROWSKI, J andKUTERMANKIEWICZ, M, 1985. Polish Patent 128,527.

ALBULESCU, C and GUZUN-STOICA, A, 1996. Emulsion liquid membrane extraction of citricacid, Proceedings of the International Conference Advances in Citric Acid Technology, Bratislava,October, p. 32.

ALTER, J E, BLUMBERG, R, 1981. US Patent 4,251,671.AYERS, R, JR, 1957. US Patent 2,810,755.BAKER, R W, TUTTLE, M E, KELLY, D J and LONSDALE, H K, 1977. Coupled-transport

membranes, Journal of Membrane Science, 2, 213–221.BANIEL, A M, 1981. Eur. Patent 0049,429.BANIEL, A, 1982. US Patent 4,334,095.BANIEL, A M and GONEN, D, 1991. US Patent 4,994,609.BANIEL, A M, BLUMBERG, R and HAJDU, K, 1981. US Patent 4,275,234.BASU, R and SIRKAR, K K, 1991. Hollow fibre contained liquid membrane separation of citric acid,

AIChE Journal, 37, 383–393.BASU, R and SIRKAR, K K, 1992. Citric acid extraction with microporous hollow fibres, Solvent

Extraction and Ion Exchange, 10, 119–144.BAUER, U, MARR, R, RUECKL, W and SIEBENHOFER, M, 1988. Extraction of citric acid from

aqueous solutions, Chemical and Biochemical Engineering, 2, 230–232.BIZEK, V, HORACEK, J, RERICHA, R and KOUSOVA, M, 1992. Amine extraction of

hydroxycarboxylic acids. 1. Extraction of citric acid with l-ocyanol/n-heptane solutions oftrialkalyamine, Industrial Engineering Chemistry Research, 31, 1554–1562.


BOHDZIEWICZ, J and BODZEK, M, 1994. Ultrafiltration preparation of pectinolytic enzymes fromcitric acid fermentation broth, Process Biochemistry, 29, 99–107.

CHEMISCHE FABRIK J A BENCKISER, 1932. German Patent 555,810.DANESI, P R, REICHLEY-YINGER, L and RICKERT, P G, 1987. Lifetime of supported liquid

membranes: the influence of interfacial properties, chemical composition and water transport onthe long-term stability of the membranes, Journal of Membrane Science, 31, 117–124.

EDLAUER, R, KIRKOVITS, A E, WESTERMAYER, R and STOJAN, O, 1990. Eur. Patent 377,430.EIKMEIER, H and REHM, H J, 1984. Production of citric acid with immobilized Aspergillus niger,

Applied Microbiology and Biotechnology, 20, 363–370.FAUCONNIER, N, BEE, A, ROGER, J and PONS, J N, 1996. Adsorption of gluconic and citric acids

on maghemite particles in aqueous medium, Progress in Colloid & Polymer Science, 100, 212–220.

FRIESEN, D T, BABCOCK, W C, BROSE, D J and CHAMBERS, A R, 1991. Recovery of citric acidfrom fermentation beer using supported-liquid membranes, Journal of Membrane Science, 56,127–141.

FYLES, T M, MALIK-DIEMER, V A, MCGAVIN, CA and WHITFIELD, D M, 1982. Membranetransport systems. III. A mechanistic study of cation-proton coupled countertransport, CanadianJournal of Chemistry, 60, 2259–2266.

GARY, K and SHARMA, C B, 1992. Continuous production of citric acid by immobilized whole cellsof Aspergillus niger, Journal of General and Applied Microbiology, 38, 605–615.

GOMEZ, O, RAMON, J, RAMON, M, LUIS, J and ZORI, D, 1991. Eur. Patent 438,369.GREWAL, H S and KALRA, K L, 1995. Fungal production of citric acid, Biotechnology Advances,

13, 209–234.GRINSTEAD, R R, 1976. US Patents 3,980,701–4.HARTL, J and MARR, J, 1993. Extraction processes for bioproduct separation, Separation Science

and Technology, 28, 805–819.JUANG, R S, 1995. Recovery of citric acid from aqueous streams by supported liquid membranes

containing various salts of tri-n-octylamine, presented at the AIChE Annual Meeting, Miami,paper 28f.

JUANG, R S and CHANG, H L, 1995. Distribution equilibrium of citric acid between aqueous solutionsand tri-n-ocytlamine-impregnated macroporous resins, Industrial Engineering ChemistryResearch, 34, 1294–1301.

JUANG, R S and CHOU, T C, 1996. Sorption kinetics of citric acid from aqueous solution bymacroporous resins containing a tertiary amine, Journal of Chemical Engineering Japan, 29,146–151.

JUANG R S and HUANG W T, 1995, Kinetics studies of extraction of citric acid from aqueoussolution with tri n-octylamine, Journal of Chemical Engineering Japan, 28, 274–281.

KARKLINS, R, SKRASTINA, I and LEMBA, J, 1996. Electrodialysis method in citric acid and itssalts recovery process, Proceedings of the International Conference Advances in Citric AcidTechnology, Bratislava, October, p. 30.

KASPRZYCKA GUTTMAN, T, JAROSZ, K, SEMENIUK, B, MYSLINSKI, A, WILCZURA, Hand KURCINSKA, H, 1989. Polish Patent 160,397.

KERTES, A S and KING, C J, 1986. Extraction chemistry of fermentation product carboxylic acids,Biotechnology and Bioengineering, 28, 269–282.

KING, C J, 1992. Amine based system for carboxylic acids recovery, CHEMTECH, 22, 285–291.KULPRATHIPANJA, S, 1988. US Patent 4,720,579.KULPRATHIPANJA, S, 1989. US Patent 4,851,574.KULPRATHIPANJA, S and OROSKAR, A R, 1991. US Patent 5,068,419.KULPRATHIPANJA, S and STRONG, S A, 1990. US Patent 4,924,027.KULPRATHIPANJA, S, OrOSKAR, A R and PRIEGNITZ, J W, 1989. US Patent 4,851,573.LEE, Y, LEE, C W and CHANG, H N, 1989. Citric acid production by A. niger immobilised on

polyurethane foam, Applied Microbiology and Biotechnology, 30, 141–143.LESNIAK, W, 1989. A modified method of citric acid production, Polish Technical Review, 5, 17–19.MILSOM, P E, 1987. Organic acids by fermentation, especially citric acid. In: R D KING and P S J

CHEETHAM, eds, Food Biotechnology Vol. 1 (Elsevier), pp. 273–307.MITTAL, Y, MISHRA, I M and VARSHNEY, B S, 1993. Characterisation of metabolically active

developmental stage of Aspergillus niger cells immobilized in polyacrylamide gel,BiotechnologyLetters, 15, 41–46.


MORESI, M and SAPPINO, F, 1996. Effect of temperature and pH on sodium citrate recovery fromaqueous solutions by electrodialysis, Proceedings of International Conference Advances in CitricAcid Technology, Bratislava, October, p. 29.

NIKITIN, YU E and EGUTKIN, N L, 1974. Neftekhimiya, 14, 780–785.NOVALIC, S and KULBE, K D, 1996. Separation and concentration of citric acid by means of

electrodialytic bipolar membrane technology, Proceedings of International Conference Advancesin Citric Acid Technology, Bratislava, October, pp. 41–44.

NOVALIC, S, JAGSCHITS, F, OKWOR, J and KULBE, K D, 1995. Behaviour of citric acid duringelectrodialysis, Journal of Membrane Science, 108, 201–205.

NOVALIC, S, OKWOR, J, and KULBE, K D, 1996. The characteristics of citric acid separation usingelectrodialysis with bipolar membranes, Desalination, 105, 277–282.

PAGEL, H A and SCHWAB, K D, 1950. Analytical Chemistry, 22, 1207.PALLARES, J, RODRIGUEZ, S and SANROMAN, A, 1996. Citric acid production by immobilised

Aspergillus niger in a fluidised bed reactor, Biotechnology Techniques, 10, 53–57.PROCHAZKA, J, HEYBERGER, A, BIZEK, V, KOUSOVA, M and VOLAUFOVA, E, 1994. Amine

extraction of hydroxy-carboxylic acids. 2. Comparison of equilibria for lactic, malic and citricacids, Industrial Engineering Chemistry Research, 33, 1565–1573.

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TMB 2022, Biotechnology and Bioengineering, 29, 297–304.US FOOD and DRUG ADMINISTRATION, 1975. Federal Register, 40, 49080–49082.VISACKY, V, 1996. Membrane nanofiltration for citric acid isolation, Proceedings of the International

Conference Advances in Citric Acid Technology, Bratislava, October, p. 31.VOSS, H, 1986. Deacidification of citric acid solutions by electrodialysis, Journal of Membrane Science,

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in gels and cryogels of polyacrylamide, Journal of Industrial Microbiology, 16, 351–353.

149

10

Fermentation Substrates

WLADYSLAW LESNIAK

10.1 Introduction

Fermentation industries have an advantage over some other manufacturing industries inthat their raw materials can be altered, within limits, allowing some buffering againstincreasing world prices. However, the past 20 years have seen global changes in the pricesof all raw materials and consequently all fermentation substrates have suffered increases tovarying extents.

For processes where different substrates can be used, or both synthetic and biologicalproduction routes exist, process economics is of paramount importance for survival. Forprocesses where the product is only obtainable through fermentation, profit margins can besustained by passing the price increases resulting from substrate cost increases on to theconsumer. Production of bulk products such as citric acid and antibiotics are obviousexamples.

These products therefore may have had less pressure on them than the others to searchfor the cheapest possible substrate, but even here there is competition between rival companiesand ways to lower costs and increase profits are thus continually being sought. The choiceof substrate is therefore always under review (Ratledge, 1977).

There is always pressure to find a cheaper or better substrate, but the new substrate maypresent storage problems, may be difficult to sterilize or have an unwanted variability incomposition. Increased productivity is not the only yardstick to be used. The substrate mayhave a residue which poses product recovery and purification problems. The cheapestsubstrate is therefore not often the best. In addition to these problems, any change in substrateor amendment to the formulation of the medium will influence the characteristic of thefermentation process, and has to be carefully evaluated.

A substrate must be readily available throughout most of the year. Seasonably producedcrops from which process wastes are used as fermentation feedstock are not suitable if theharvest period is short and the material to be used is subject to contamination and spoilage.Thus the industry must have substrates that are relatively stable and can be stored reasonablyeasily for more than half a year.

A process, for example, citric acid production, can be changed to accommodate a newsubstrate. The advent of cheap hydrocarbons in the 1960s led to many companies switching


over to this substrate. Aspergillus niger, the traditional producer, cannot grow on alkanes,but a variety of yeast can and some will accumulate citric acid sufficiently enough forindustrial processes to be established (Shennan and Levi, 1974).

The price of the substrate is crucial. However, it is important to take into considerationthe amount of available carbon. This differs according to the type of substrate being used(see Table 10.1). This suggests that if the choice of substrate is not limited, a carbohydratecould be replaced by alkanes with no loss in process productivity (an important optimizationparameter for the citric acid process). However, others factors have to be taken intoconsideration before this is accepted—increased aeration or agitation rates may be necessarywith alkanes (being a more reduced substrate) and this factor must be met by the savingsfrom the change of substrate.

Transport costs for substrates from the collection or production point to the fermentationplant have to be considered. These costs may become significant if too much water is presentand will mitigate against the use of some waste materials at sites removed from their pointof production. One substrate may be more attractive to use than another simply because itposes fewer problems in the processes both before and after the fermentation.

Fermentation media for citric acid biosynthesis should consist of substrates necessaryfor growth of the producer micro-organism and its citric acid biosynthesis, primarily thecarbon, nitrogen, phosphorus and microelements sources. Moreover process water and aircan be included as fermentation substrates.

The basic substrate for citric acid fermentation in plants using the surface method offermentation is beet or cane molasses. Plants using submerged fermentation can use notonly beet or cane molasses, but a substrate of higher purity such as hydrolysed starch,technical and pure glucose, refined or raw sugar, purified and condensed beet or cane juice.This is because use of a pure substrate may result in increases in yield, or reduction infermentation time.

10.2 Molasses

Molasses is a widely used substrate, coming in a variety of qualities. High quality molassesis usually demanded for citric acid production while poorer quality molasses is used mainlyin the production of low value products such as alcohol, where the producer micro-organismhas a much greater tolerance to impurities in the medium.

Table 10.1 Relative carbon contents of fermentation substrates (from Ratledge, 1977)

Fermentation substrates 151

The composition of cane and beet molasses has recently been compared and the uses ofmolasses as a fermentation feedstock have been discussed elsewhere (Hastings, 1971). Caneand beet molasses are not identical in composition; often one type will be preferred to theother. They are sometimes mixed to take advantage of the additional nutrients arising fromthe differences in composition.

Besides substrate type (sugar beet, sugar cane), the chemical composition of molassesdepends on many factors such as soil and climate conditions, fertilization type, cropmethod, time and conditions of storage, production technology, technical equipment ofplant, etc.

10.2.1 Beet molasses

Beet molasses consists of about 65–80 per cent dry substance and 20–25 per cent water.The main ingredient of molasses is sucrose, 44–54 per cent by weight. Other sugars(carbohydrates) which can be found in higher amounts are inverted sugar 0.4–1.5 per cent,raffinose 0.5–2.0 per cent and kestose and neokestose 0.6–1.6 per cent. Raffinose is a naturalpart of sugar beet, while kestose is the result of microbial action during sugar beet treatment.Other sugars in molasses are arabinose, xylose and mannose in amounts of 0.5–1.5 percent. All sugars (except sucrose) are included in the non-nitrogen organic substances ofmolasses. Products of chemical and thermal sugar decomposition (melanoidines, caramel)and organic acids also belong to this group. Caramel consists of sugar anhydride and colouringmatters; melanoidines are made in hot solution as the result of a reaction between reducingsugars and amino acids. In addition to the non-volatile dark coloured compounds, there areabout 40 volatile compounds as aliphatic aldehyde, methylglyoxal, diacetyl, acetoin, acetone,oxymethylfurfurol and others.

The non-volatile organic acids present in molasses are glutaric, malonic, succinic, aconitic,malic and lactic acid; the remainder are oxalic, citric and tartaric acid. These can all reactwith calcium to form insoluble salts that can influence the precipitation and recovery of thecitric acid crystals. Molasses contain such volatile acids as formic, acetic, propionic, butyricand valeric acid. Almost all organic acids, volatile and non-volatile, are potassium or calciumsalts.

The colour of molasses ranges between 1.2 and 4.6 cm3 of 0.1 N iodine solution (towhich should be added 94 cm3 of water to get the colour identical to that of 2 per centmolasses solution). Molasses containing higher amounts (over 1 per cent) of volatile acidsare normally too dark to be used as feedstock for the citric acid fermentation, though theexact relationship between content of these substances and fermentation yield has not beenestablished.

Other ingredients of molasses that have a negative influence on colour and thusfermentation yield are colloidal substances. Beet molasses contains about 4–6 per cent ofcolloids, whose chemical constitution has only recently been documented. Mostly, theyare high-molecular coloured complexes. Some of these colloids (of negative potential)can be removed from solution by acid coagulation (pH 3.2, molasses dilution 20–30 percent, temperature 80°C) and colloids of positive potential by alkaline coagulation (pHover 8.0).

Nitrogen compounds contained in molasses are mostly betaine (about 60–70 per centof total nitrogen), amino acids (20–30 per cent of nitrogen), protein (3–4 per cent ofnitrogen) and traces of nitrogen in ammonium nitrate and amide. Betaine comes frombeet and is not used by micro-organisms as a nitrogen source. It is not known to influence


the fermentation. The amino acids content in molasses depends on the soil and climateconditions and beet cultivation. Amino acid content of beet molasses is shown in Table10.2.

The content of mineral substances in beet molasses amounts to 8.5–14.0 per cent. Themain ingredient of the mineral ash is K2O (60–70 per cent of the total), CaO (4.5–7.0 percent) and MgO (about 1 per cent). The level of P2O5 in ash is normally very low (0.2–0.6per cent), because over 90 per cent of phosphorus contained in beet is removed in thesugar extraction process. If the method of juice alkalization by Na3PO4 (pH 8.3–8.5) isused in the sugar production, the contents of P2O5 in molasses ash can reach 1.2–2.0 percent.

There are also many other elements, so-called microelements, which have a greateffect on the citric acid fermentation process. The amount of particular microelementsin different molasses can range widely as indicated in Table 10.3. Another important

Table 10.2 Amino acid content of beet molasses (from Smirnow, 1983)

Table 10.3 Content of microelements in beet molasses (from Smirnow, 1983)


ingredient of molasses is vitamins, especially those that are known to stimulate microbialactivity. The content of vitamins (mg/100 g) in beet and cane molasses is shown in Tables10.4 and 10.5 respectively.

The pH of molasses depends on the sugar extraction technology. It was considered thata neutral, or slightly alkaline molasses gave the best citric acid yields. However, a fermentationtechnology to tolerate the slightly acidic molasses produced in modern refineries has beendeveloped. Today, it is considered that for citric acid fermentation the buffering capacity ofthe medium is more important than the pH value of the molasses. It is defined as the amountof 1N solution of sulphuric acid (in cm3) used to reduce pH from 5.0 to 3.0 in 100 g molassessolution diluted in 1:1 ratio with water and acidified to pH 5.0. The buffer capacity of beetmolasses usually ranges from 60 to 95 cm3. Citric acid production needs molasses with lowbuffer ability, to make possible the required rapid fall of medium pH during fermentation.

10.2.2 Cane molasses

Cane molasses differs from beet molasses in its chemical composition. It contains less sucroseand more inverted sugar, has lower content of nitrogen and raffinose, more intensive colourand lower buffer capacity. Cane molasses of raw sugar conversion also differs from beetmolasses and even from blackstrap cane molasses. The composition of cane molasses isshown in Table 10.6.

Table 10.4 Content of vitamins in beet molasses (from Smirnow, 1983)


Beet and cane molasses can also contain other substances which appear in small amounts,but are often crucial in deciding whether the molasses are suitable for use in citric acidbiosynthesis. These are pesticides, fungicides and herbicides used in beet and cane cultivationand also substances used for defoaming in sugar production process. All have mostly toxicproperties and negatively affect molasses usability. It is considered that the best molassesfor citric acid fermentation can be, as a rule of thumb, characterized as shown in Table 10.7.According to all cited requirements, beet molasses is more suitable for citric acid fermentationthan cane molasses. It is especially relevant in submerged fermentation where the quality ofthe substrate is more important for productivity and fermentation yield.

The microflora of molasses can be an agent of negative influence on yield andproductivity of fermentation. Molasses will always contain a certain number and type ofmicro-organisms, sometimes the count can be higher than 10 000 per g of molasses. Themost common micro-organism in molasses is sporulating rods of Bacillus species (over90 per cent of total molasses microflora), bacteria producing acids and gases (E. coli,Pseudomonas and others), heterofermentative lactic acid bacteria (Leuconostocmesenteroides), sometimes yeasts of Candida species, and very rarely, moulds ofPenicillium, Aspergillus and other species.

Table 10.6 Composition of cane molasses (from Smirnow, 1983)

Table 10.7 Alternative analysis of cane molasses sample


Bacteria of Bacillus species appear in molasses because their spores are present inbeet and are unaffected by high temperatures, even 125°C (Bacillus subtilis). They aredestructive because some of them (B. megaterium, B. mesentericus) are able to reducenitrates to nitrites. Strains of Aspergillus niger can be very sensitive to nitrites (a NO2

concentration in medium of 0.05 per cent will retard growth and cut the citric acidproduction by 50 per cent).

The greatest antagonists of Aspergillus niger among non-sporulating bacteria are E. coliand Pseudomonas. They grow very quickly in many media over a wide temperature range,decomposing sugar in solution to unwanted acids, alcohol and gases, and are able to reducenitrates to nitrites. Bacteria of Leuconostoc species convert sucrose to dextran. They alsoproduce unwanted volatile acids such as formic, acetic and propionic acid. Yeasts of Candidaspecies can propagate over a wide range of temperature (5–55°C) and pH value of medium(2–8). They can be very undesirable to Aspergillus niger strains, especially in submergedfermentation, where they can stop citric acid biosynthesis.

10.2.3 Treatment of molasses for citric acid production

Due to the varying chemical composition of molasses it is always required to evaluateany new delivery in a scaled down version of the citric acid production vessels. Evenvery good molasses is no guarantee for high yields of citric acid biosynthesis withoutspecial pretreatment. The basic operation in molasses preparation is a treatment forheavy metal ions removal. Potassium ferrocyanide or other complex compounds arecommonly used.

Potassium ferrocyanide reacts with many heavy metals, mostly causing theirprecipitation. It was noted that for 21 microelements found in molasses, potassiumferrocyanide reacts with 18 of them (Leopold and Valtr, 1964). Potassium ferrocyanideremoves not only metals of negative influence but also some of the microelements necessaryfor mycelium growth. Therefore its addition to molasses has to be strictly regulated. Theoptimum amount of ferrocyanide depends on molasses type and ranges from 200 to 1000mg/dm3 of medium (about 300 g of molasses); of the metals 80–85 per cent of the total iscomplexed as precipitate, 7–14 per cent is complexed in solution and 7–10 per cent is inelemental free state. At the optimum dose level of ferrocyanide, the part in elemental stateis usually constant and ranges between 50 and 100 mg/dm3, depending on strain andfermentation type. This has been used to develop a quick method of optimal ferrocyanidedosage in molasses media. (Lesniak, 1976). Ferrocyanide is normally added beforesterilization. However it can also be partially added before and after sterilization or thetotal amount can be added after sterilization.

Another compound complexing with heavy metals is the sodium salt of ethylene-diamineacetic acid (EDTA). This compound reacts with metals of I and II valency at pH7.0, with metals of III valency at pH 3–5 and with multivalency metals at pH 1. Ca and Mgions give Trilon B soluble salts and they are not removed from solution. Other heavy metalcomplexing compounds can also be used, e.g. sodium polyphosphates, potassium rhodanate,2,4-dinitrophenols and 8-oxyquinoline. Molasses media are sometimes purified by ionites,especially on cation exchanger. Not all microelements should be removed during this process,as some of them are necessary for growth of the Aspergillus niger mycelium.

To protect the fermentation process from unwanted micro-organisms, the molasses mustbe sterilized. The most economical method is steam sterilization. For sporulating bacteria atemperature of 130°C or above for 30 minutes is recommended. However, steam sterilization


of the medium may not be sufficient to ensure total sterility because some micro-organismscan enter the fermentation broth via addition ports or from the air. Because of this, othersterilizing agents such as formaline (at 0.006–0.01 per cent) (in particular for the surfacefermentation) and furan derivatives are used.

Sulphamide preparations do not totally destroy the bacteria, but antibiotics, though theydo not have any negative influences, are too expensive (Karklinsh and Probok, 1972).Applying chemical sterilizing agents enables softening of sharp thermal sterilizationconditions that have a negative effect on molasses quality. Other methods of sterilizationtested are UV and gamma radiation, ultrasound, and ultrafiltration. They are not used inpractice as they are cost-prohibitive compared with steam sterilization.

In tropical countries where date production is considerable, date syrup is a major product.The chemical composition of this material differs from that of sugar beet molasses, butwhen mixed with an equal volume of beet molasses it gives the same yield of citric acid asfor beet molasses based on the amount of sugar converted (Shadafza et al., 1976). Molassesfrom the starch industry (hydrol molasses) is also widely used in citric acid fermentation.

10.3 Refined or raw sucrose

Refined sugar of beet or cane is almost pure sucrose which Aspergillus niger strains fermentvery well (Lesniak, 1989). This sugar is a very good substrate for the submerged fermentationbecause in surface fermentation, the rate of diffusion of acid in sugar solutions is too low.Preparation of a refined sugar solution as a fermentation medium is based on its dilutingwith water to a concentration of 15–22 per cent, adding necessary nutrients (NH4NO3,KH2PO4, MgSO4) and acidifying with hydrochloric or sulphuric acid to pH 2.6–3.0 (Lesniak,1972). Normally the batch medium is sterilized in the fermentation vessel. In this case, allthe ingredients of the fermentation medium are added straight into the bioreactor or areprepared separately by diluting in hot water (85–95°C) and then pumped into the bioreactor.In this case, sugar is diluted to 50–60 per cent concentration and pumped into the fermenterthat has had an exact amount of sterile water added, resulting in a total sugar concentrationof 15–22 per cent.

Sterilization in the fermenter lasts about 0.5–1 hour at 110–120°C. The solution isthen cooled to 32–35°C with continuous stirring and aeration before the inoculum ofAspergillus niger spores or mycelium is added. The use of continuous sterilizers, wherethe sugar solution is sterilized separately from the other ingredients, is becoming morecommon.

10.4 Syrups

Syrups of beet or cane sugar can also be used as basic substrate for the submerged citricacid fermentation. The great advantage with this substrate is its purity; however, the qualityof the syrups deteriorates rapidly during storage. Because of this they can only be usedduring the sugar campaign season and only if the citric acid plant is not too far from thesugar factory because of the large transport costs.

Preparation of the syrups for fermentation entails dilution with water to a sugarconcentration of 15–20 per cent, addition of necessary nutrients (NH4NO3, KH2PO4, MgSO4,(NH4)2C2O4), acidification with hydrochloric or sulphuric acid to pH 4–5 and sterilizationat 121°C for 0.5–1 hours (Kutermankiewicz et al., 1980).


10.5 Starch

Starch can be an attractive feed stock for many fermentation processes. It can be useddirectly by many micro-organisms and is frequently incorporated into fermentationmedia as a partial ingredient. Starch is widely used as the principal substrate for theproduction of amylases and amyloses in the food and brewing industries. The productionof citric acid from sources of starch such as corn, wheat, tapioca and potato is widelyused.

The suitability of these substrates for citric acid fermentation depends on their purityand method of hydrolysis. Acid hydrolysis, enzymatic hydrolysis, or a combination of thetwo, are used. Preparation of starch substrates for fermentation is based on their enzymaticliquefaction and saccharification to a defined hydrolysis level. Additional nutrients are added,depending on which starch is used. The pH is adjusted to 3–4 using hydrochloric or sulphuricacid and the medium is sterilized at 121°C for 0.5–1 hour.

Good citric acid yields have been obtained using pure starch (potatoes, wheat or maize),hydrolysed only to 10–15 DE with a-amylase (Bolach et al., 1985). This was possible, asthe applied Aspergillus niger strain had the ability to produce its own amylolytic enzymeswhich helped in the saccharification of the starch to available sugars. Dextrose syrup, obtainedby enzymatic hydrolysis of starch, is now employed as a basic substrate for citric acidbiosynthesis in laboratory and industrial scale. In this case it is especially important torestrict the amount of heavy metals below critical levels; heavy metals should therefore beremoved by ion exchange.

When using an Aspergillus niger strain resistant to higher concentrations of heavy metals,practically the same yield may be obtained on decationized and non-decationized dextrosesyrup (Pietkiewicz et al., 1996).

10.6 Hydrol

This is a paramolasses obtained as a by-product during crystalline glucose production fromstarch. Because of the high glucose content (40–45 per cent) and high purity coefficient it isa very good substrate for citric acid production (Lesniak et al., 1986). Preparation of hydrolfor fermentation involves dilution to a sugar concentration of 15– 18 per cent, addition ofnecessary nutrients and adjustment of pH with hydrochloric or sulphuric acid to 3.0–4.0.The solution is sterilized at 121°C for 0.5 hour and cooled to 32–35°C.

10.7 Alkanes

The low price of alkanes, coupled with the ability of many organisms to utilize them, producedmajor changes in the fermentation industry during the 1960s and 1970s. Citric acidproduction, using Candida lipolytica, is a typical example and has been the subject of manypatents (Maldonado and Charpentier, 1975; Kimura and Nakanishi, 1985). However, thereare few industrial citric acid processes that are based on alkanes. There are two main reasonsfor this. Firstly, in these processes isocitric acid would also be produced at concentrationsthat would cause product recovery problems, as well as reduced citric acid yields(Wojtatowicz and Sobieszczanski, 1981). Secondly, a fourfold increase in price since 1973no longer makes alkanes a cheap substrate.


10.8 Oils and fats

Oils and fats are also being increasingly used as substrates in many fermentations. Theoils should be liquid at the temperature of fermentation; the concentration of the oils maybe up to 10 per cent but there is no reason to believe that concentrations up to 30 per centmay not be used. The prices of oils and fats vary according to their fatty acid composition,and often are very cheap. The price of the cheapest oils is such that, because of their highcarbon content, they are not much more expensive that raw sugar. For citric acid production,oils are now being used as principal carbon source in a manner analogous to the previoususe of alkanes. With palm oil as carbon source, a yield of citric acid of 145 per cent usinga mutant of Candida lipolytica has been reported (Ikeno et al., 1975).

There are examples of oil being added in small concentrations to Aspergillus nigerfermentation (Gutcho, 1973) and even being used as a sole carbon source for Aspergillusniger fermentation. It was found that citric acid could be produced on these substrateswith good yield. In particular with an initial 8 per cent concentration of vegetable oil, ayield of 104 per cent was obtained (Elimer, 1994). These oils and fats may replace alkanesin several fermentations, but it is unlikely that they will remain at their current low prices.

10.9 Cellulose

Cellulose is the major renewable form of carbohydrate in the world: about 1011 tonnes aresynthesized annually and much of this is waste. To use it as fermentation feedstock, it mustbe first hydrolyzed to starch and then to sugar, either chemically or by cellulases. Thetechnology and economics of these processes are constantly being improved, but it is stillnot apparent when the production of sugar syrups by this route is going to become profitable.In the long term, cellulose could become a major resource of the fermentation industry ingeneral, including citric acid fermentation.

10.10 Other medium ingredients

10.10.1 Other nutrients

Other substances are used as sources of nitrogen, phosphorus and micro and macro-elements. Organic compounds (ammonia, amino acids) or non-organic compounds(ammonia salt, nitrates) can be used as nitrogen source. The most commonly usephosphorus source is phosphoric acid or its salts. Whenever high purity carbon substrates(refined sugar and starch) are used, ammonium nitrate or ammonium sulphate will beused as nitrogen source and monopotassium phosphate as phosphorus source (Lesniakand Kutermankiewicz, 1990).

When using molasses, additional nitrogen is rarely required, as it will contain sufficientamounts of organic and inorganic nitrogen compounds to support the metabolic growthprocess. If the nitrogen level becomes too high, some of the sugar is converted into productionof excess biomass and not citric acid.

The most important microelements are magnesium, sulphur, zinc, iron, copper andmanganese. They are very seldom added to the medium. In complex media the level oftrace metals will normally be too high, and the main concern is simply to remove them. This


is very different from academic research into citric acid fermentation. Here, a refined sugaris invariably used as the carbon source and much work has been done on the level of nutrients,in particular trace metals required for optimal acid yields and the role of individual metalions.

10.10.2 Water

Water used for diluting basic substrates should be at least of drinking water quality. Thereshould not be organic compounds and products of their decomposition (NH3, and H2S) andthe level of trace metals must be controlled. All the water must be sterilized to removecontaminating micro-organisms.

10.11 Conclusion

Citric acid is a bulk product, with the substrate cost being a major part of the plant operatingcost. In terms of bulk, the carbon source is the most important substrate. The efficiency ofits conversion to citric acid will determine the profitability of the fermentation process.For this reason, the carbon source is also the most important substrate for processeconomics. This chapter has, therefore, concentrated on the various forms of carbon sourcesused. Most processes are based on molasses, although the use of cleaner sources is gainingground. Whatever the source, its cost and preparation in order to permit optimalfermentation conditions are two important aspects of the technology in citric acidproduction.

10.12 References

BOLACH, E, LESNIAK, W and ZIOBROWSKI, J, 1985. Acta Aliment. Polonica, 11, 1.ELIMER, E, 1994. Studies on Use of Plant Fats for Citric Acid Production by Aspergillus niger, PhD

thesis, University of Wroclaw, Poland.GUTCHO, S J, 1973. Chemicals by Fermentation (Noyes Data Corporation, Park Ridge, NY, USA).HASTING, J J H, 1971. Advances in Applied Microbiology, 14, 1.IKENO, Y, MASUDA, M, TANNO, K, OOMORI, I and TAKAHASHI, N, 1975. Journal of

Fermentation Technology, 53, 752.KARKLINSH, R J and PROBOK, A K, 1972. Organic acid biosynthesis (in Russian), Zinatne, Riga.KIMURA, K and NAKANISHI, T, 1985. German Patent 2 065 206.KUTERMANKIEWICZ, M, LESNIAK, W and BOLACH, E, 1980. Przem. Ferm. i Owoc.-Warzyw,

6, 27.LEOPOLD, H and VALTR, Z, 1964. Die Nahrung 1, 37.LESNIAK, W, 1972. Studies on Submerged Citric Acid Fermentation, PhD Thesis, University of

Wroclaw, Poland.LESNIAK, W, 1976. Przem.Ferm. i Rolny, 6, 22.LESNIAK, W, 1989. Polish Technical Review, 5, 185.LESNIAK, W and KUTERMANKIEWICZ, M, 1990. Citric Acid Production—Basic Review (in

Polish), STC, Warsaw.LESNIAK, W, PODGORSKI, W and PIETKIEWICZ, J, 1986. Przem. Ferm. i Owoc.-Warzyw, 6, 22.MALDONADO, P and CHARPENTIER, M, 1975. German Patent 2 551 469.PIETKIEWICZ, J, PODGORSKI, W and LESNIAK, W, 1996. Proceedings of the International

Conference on Advances in Citric Acid Technology (Bratislava, Slovak Republic), p. 9.RATLEDGE, C, 1977. Fermentation substrates, Annual Reports on Fermentation Processes, Vol. 1,

Chapter 3.


SHADAFZA, D, OGAWA, T and FAZELI, A, 1976. Journal of Fermentation Technology, 54, 67.SHENNAN, L and LEVI, J D, 1974. Progress in Industrial Microbiology, 13, 3.SMIRNOW, W A 1983. Food Acids (in Russian), Moscow, 105.WOJTATOWICZ, M and SOBIESZCZANSKI, J, 1981. Acta Microbiologica Polonica, 30, 69.

161

11

Design of an Industrial Plant

JACOBUS D VAN DER MERWE

Nomenclature

a specific area (m2 m-3)A area (m2)B permeability coefficient (m2)Cn (n = 1 – 5) constantsCp specific heat capacity (J kg-1 °C-1)dhole pore diameter (m)D diameter (m)D liquid diffusivity (m2 s-1)e fractional voidage (-)g acceleration due to gravity (m s-2)h bed height (m)hf film heat transfer coefficient (W m-2 °C-1)HD dispersion height (m)J flux (m3 m-2 s-1)k thermal conductivity (W m-1 °C)kL liquid side mass transfer coefficient

at the gas–liquid interface (m s-1)K fluid consistency index (Pa s)K² Kozeny constant (-)lp pore length (m)L liquid height in the reactor (m)L characteristic length in dimensionless numbers (m)N impeller speed (s-1)Np power number (-)P power input (kW)DP pressure drop (N m-2)Q volumetric gas flow rate at NTP (m3 s-1)r filtration resistance of the filter medium (m-1)R universal gas constant


T temperature (°C)u superficial gas velocity (m s-1)V volume (m3)

Greek letters

a ratio of maximum hydrostatic head to the (-)pressure at liquid surfaceor; average specific cake resistance (m-1)

em membrane porositys interfacial tension (N m-1)s dynamic viscosity (Pa s)f gas hold-up (-)y density (kg m-3)mf fraction of filter area immersed (-)µ viscosity (N s m-2)w mass of dry solids in a filtrate volume V (kg m-3)

Abbreviation and dimensionless numbers

BOD biological oxygen demand (mg 1-1)Bo Bond number gDrr/sDe Deborah number ug(1+f)l/fds

Flg aeration number Q/ND3

Fr Froude number N2D/gGa Galilei number gDR

3/heff

HMF hydroxy-methyl-furfuralNF nanofilterPr Prandtl number Cpµ/kPFD process flow diagramRCS readily carbonizable substrateSc Schmidt number µ/rDSVC standard variable cost of productionUF ultrafiltrationVVM volumetric flow of air per unit reactor volume per minute (min)

Subscripts

a apparentd downcomerD total areaeff effectiveg gasi impellerL liquido unaerated

Design of an industrial plant 163

r riserR reactorT top

11.1 Design of an industrial plant

11.1.1 The customer requirement

The focus of this chapter is to outline the many facets that are integral to the design of anindustrial plant. It is assumed that the request to design the plant originated with a customerrequirement: either internal or external to the process engineering team. Although the exactterminology used might differ from company to company, the project cycle will more orless follow the scheme presented in Figure 11.1.

All the necessary planning, project documentation, detail design and project executionaspects cannot be covered in detail: such a treatise would warrant a separate volume. Ratheran approach is presented which will provide a person with a reasonable technical backgroundwith a framework from which to proceed. The methodology is generic to the design of any(fermentation) process plant. As such the designer is advised to consult the standard chemicalengineering texts on the subject. For the purpose of this chapter, the process engineer ispresumed to be working as a member of a multidisciplinary team, within an environmentthat has formal project procedures in place. Thus aspects such as tender enquiries,procurement and project documentation will not be discussed. Where applicable, the use ofspecialists in the field will strongly be recommended. For certain unit operations, it is costeffective to consult with a specialist vendor on the issue, where the main function of thecitric acid producers’ process engineer is the accurate definition of the unit requirements.

11.1.2 Chemical plant design

Although citric acid is a fermentation product, it is still a bulk commodity chemical. At theoutset it would therefore be appropriate to consider some similarities in designing a citricacid facility and a chemical plant:

• Isolation steps utilize unit operations standard to the chemical industry.

• The standard variable cost of production is a key economic factor.

• Plant capacities are increasing as new investors attempt to get maximum benefit from theeconomy-of-scale principle.

• Transport cost of raw material or product can be a major consideration.

11.2 Data required

The design process will start with some level of research and/or process development to bedone (Figure 11.1). Following on the R&D phase, the process design team needs certaininformation, before setting the design basis. This can be grouped as marketing inputs thatwill affect the design and technical data.


11.2.1 Customer and marketing information

• Would there be any seasonality to the product demand? If so, this will impact on thesizing of units.

• What is the required product specification, including the crystal size distribution?

• Would the plant be required to produce both anhydrous and monohydrate grades?

• What type and size packaging is required?

Figure 11.1 Schematic representation of the project design sequence


• Are there constraints imposed on the packaging material due to environmental legislation?(see Livingstone and Sparks (1994) for a discussion on the effect of new packaginglaws).

• Is the final product application known? For example, will it be used in dry formulations,where a specific crystal size distribution is important, or will it be dissolved and used insolution?

• Are there any other specific customer requirements?

11.2.2 Technical data

This includes all data that would ultimately be required for the design of the plant (see Table11.1). It is recommended to compile a process data book during the initial phases of thedesign and thereafter use it as a standard. The plant location has not necessarily beendetermined yet, therefore aspects such as available steam pressure and other site-relatedissues are omitted at this point.

11.3 Design basis

This stage is also referred to as the conceptual or preliminary design phase. At this point theprocess evaluation has been done with regard to Reisman (1988, p. 52):

• site selection;

• a comparison of yields and productivity with different strains and substrates;

• alternative processing routes; and

• plant capacity and utilization.

A process description and material balance quantifies aspects such as effluents, by-productsand site storage requirements for raw materials and products. Usually such a mass balanceis calculated backwards—i.e. starting with the stated customer requirement. The approachto process development has also been fixed at this stage. In other words: To

Table 11.1 Technical data required

Figu

re 1

1.2

Sche

mat

ic fl

ow d

iagr

am fo

r th

e pr

oduc

tion

of c

itric

aci

d


what extent will licensed technology be sought and which aspects might be developed fullyin-house. The process flow diagram (PFD) with the mass balance will now form the baselinefor more detail design.

11.3.1 New technology

In deciding on a processing route, the design team would have to consider new developmentsin technology, which might offer a competitive advantage. The driving force to considersuch technology, in spite of possible increased risk, would be due to one or more of thefollowing factors:

• lower capital outlay required for the process;

• cost competitive standard variable cost (SVC);

• environmental pressure to reduce effluents; or

• cost or availability of substrate.

The production of citric acid is a fairly mature technology, and it is unlikely to expect aradical breakthrough. More likely would be incremental advances (see Roussel et al. (1991,p. 54) for the context of the terms radical and incremental) in technology, as major producersfocus R&D efforts on staying competitive. Thus the processing of citric acid can be expectedto remain within the scheme as set out in Figure 11.2. Two areas where new technologymight impact, will be on fermenter design and direct crystallization routes. The formerrefers to the probable phasing out of mechanically stirred vessels, while the latter includesall processes aimed at recovering citric acid without a precipitation sequence. This wouldinclude:

• membrane applications;

• novel ion exchange resins;

• solvent extraction;

• electrodialysis; and

• chromatography.

11.4 Scope definition

The project scope definition is rather like a questionnaire or checklist that prompts thedesign team to ensure that nothing has been overlooked. Once a detail scope definition hasbeen documented, the elements required for the process package fall naturally into place.Depending on the process engineering company and client, the actual format may vary, butthe aspects listed in Table 11.2 will always be addressed.

11.5 Process package

Having documented the project scope, the process engineer can proceed with the detailedprocess package(s). Ideally it would not be an iterative process (Figure 11.1), but seldomwould enough cost data be available at the scope definition stage to obtain final project


Table 11.2 Details to demarcate with a formal project scope definition


approval. At this stage the design concepts and capacities are frozen, so that the focus is onthe detail design of the individual units. The plant battery limits have also been set, and thevarious interfaces are clearly defined. In parallel to the detail unit design, the drawing officecan start on plant layout options.

11.5.1 Process flowsheet

In terms of the process flow, two general rules apply:

• Limit the number of unit operations.

• Simplify the flow sheet.

The above stems from the fact that for each additional piece of equipment it is not only theunit cost to be considered, but the installed cost, which includes the associated civil work,piping, valves, instrumentation and electrical requirement. Hence one additional operation,such as a ‘polishing’ filter, should carefully be scrutinized from an economical point ofview. Often it is cost effective to increase the specification level on the primary operation.Following is a discussion on the design of typical unit operations for the production of citricacid. While a significant percentage of the world’s demand is still produced via surfacefermentation, this is not covered. It is unlikely that further new plants utilizing this technologywill be purpose-built. A typical PFD for the classical process is also not duplicated: theseare available in standard references (Reisman, 1988) and for the purposes of this sectionFigure 11.2 will suffice.

11.6 Raw material

The main design concerns with regards to raw material are the logistics involved andstorage volume required. Molasses is a seasonal product and if the intention is to operatethe citric acid plant throughout the year, this must be taken into account. In the worstcase, the citric acid producer might have to provide several months’ storage capacity onsite. At the other extreme the producer would be situated adjacent to a corn starch producer,where the substrate would be available year round, with minimal on-site storage required.Obviously such a scenario would offer significant cost advantages. The logistical issuesto be addressed include:

• Transport by road, rail or shipping?

• Off-loading facilities required and metering of quantities.

• Site access.

• Will delivery be a 24-hour, seven-day-a-week operation, or can it be planned as a dayshift activity, Monday to Friday?

11.7 Substrate preparation

Prior to fermentation, the substrate must be sterilized and the concentration adjusted to therequired sugar loading. On any large-scale operation, sterilization will be done continuouslyand not on a batch basis. One exception would be the seed fermenters, where sterilization


can still be done in situ. One of the reasons to avoid batch sterilization of media on theproduction scale, is the possible formation of complexes such as hydroxymethyl-furfural(HMF). Formed from the reaction between glucose and ammonium and nitrogen compoundsat elevated temperatures, HMF is a strong respiratory inhibitor. Due to the long heating andcooling cycle of large scale batch sterilization, HMF produced might inhibit the subsequentfermentation to non-optimal productivity levels. In discussing fermentation design, Söderberg(1983) lists the advantages of continuous sterilization and Wallhäuser (1985) offers acomprehensive treatment of media and vessel sterilization.

11.8 Fermentation

The mechanically stirred tank reactor (STR) has been the standard in bioprocessing for atleast 40 years. Although the picture is now changing, fewer scale-up studies (especially onthe larger scale) have been done on airlift reactors, while scale-up and mixing in the STRhas been extensively researched and several design correlations are available. The reader isreferred to a review article by Berovic (1991) where he discusses advances in reactor design.

11.8.1 Scale-up and design

Bioreactor design usually involves some experimentation on a scale smaller than theproduction scale. Various approaches have been followed in designing the production scalereactor. These include:

• rules of thumb;

• scaling according to one specific parameter;

• geometric similarity; and

• scale-down method.

Rule of thumb guidelines, based on historical data and conventions followed with previoussuccessful designs, provide the starting point for further detail calculations. The followingset (Sections 11.8.2–11.8.3) is not intended as a complete list, but as a summary of typicalaspects which would be included.

11.8.2 Heat transfer

• Heat production during aerobic fermentations is proportional to the oxygen consumptionrate.

• The heat liberated in the fermenter increases proportional to the volume (aDR

3), whilethe available surface area for heat transfer increases proportional to the square of thetank diameter.

• In order to improve the heat transfer, the engineer has three choices:

– Increase the available DT to the reactor.

– Improve the heat transfer coefficient. Unfortunately, no practical approach toaccomplish this has been proposed yet. The heat transfer coefficient is a function of


the mixing power input into the vessel, but only to an exponent of 0.2 to 0.3 (Oldshue,1985). As the mechanical energy input is dissipated as thermal energy, it does nothelp to increase agitator power input in order to improve heat transfer. Indications arethat the presence of dispersed air bubbles at the heat transfer surfaces increases thecoefficient (De Maerteleire, 1982). This is possibly due to a scouring action on thesurface. Effective utilization of mixing energy input to ensure complete gas dispersion,would thus be an important consideration.

– Increase the available heat transfer surface.

11.8.3 Mass transfer

• It is generally taken that the rate-limiting step in oxygen transfer is the gas-to-liquidinterface transfer. Further it is assumed that the concentrations within the gas bubble arehomogeneous and that the overall reaction is not limited by the cell oxygen uptake rate.A special case applies with pellet morphology, where diffusion effects into the biomasscluster might come into play.

• The oxygen transfer coefficient (kLa) is a positive rising function of the superficial gas

velocity and specific power input. Hence increasing either of these parameters will enhancek

La.

• Mixing becomes less ideal as the scale increases. Even with increased specific powerinput, mixing times still increase with scale. This is an important factor to bear in mindwith regard to the existence of local areas within the reactor of substrate limitation(Oldshue, 1989).

11.9 Design of a stirred tank reactor

When designing the bioreactor, the engineer has to specify the vessel geometry, the powerinput and aeration requirements of the system. The oxygen uptake rate would normally bedetermined experimentally as a function of the specific growth rate, while the requiredpower input is dependent on the system rheology. At the same time the vessel geometryaffects the superficial gas velocity and hence the required power input for a given oxygentransfer rate. Therefore these aspects are not arbitrary, but interrelated.

11.9.1 Non-aerated power input

The non-aerated power input, P0 is important in terms of correctly sizing the agitator motorfor start-up operation. It also gives the maximum loading that will occur in case of acompressor failure. The relevant equation is:

(11.1)

In the laminar flow range the power number declines linearly from an initial maximumvalue, while it is independent of the Reynolds number (and constant) in the turbulent flowregime. The power number varies according to the impeller geometry, but shows the sameprofile for a wide range of impellers (Mockel and Wollechensky, 1990).


11.9.2 Aerated systems

Gas hold-up during aeration decreases the effective density of the medium and hence theagitator power demand. The correlation used to predict the aerated power demand is:

(11.2)

which can be applied for non-Newtonian media (Taguchi and Miyamoto, 1966). In equation(11.2), the constant, C1, is dependent on system geometry.

11.9.3 Correlation of kLa

The commonly used form for correlating kLa to superficial gas velocity, ug, and power inputper unit volume, P/V, is:

(11.3)

The constant C2 and exponents a, ß and d are system specific (i.e. scale dependent andfunction of geometry) and must be determined experimentally. However, the followinggeneralizations can be stated:

• For Newtonian fluids the value of d is usually small and in the region of 0.10–0.14.Hence errors in the viscosity term do not drastically influence the accuracy of the result.Applying the concept of an apparent viscosity, this term can also be used in correlatingdata for non-Newtonian rheology. The wall viscosity, µ

w, can be taken to be equal to the

viscosity at zero biomass concentration.

• a and ß are positive and generally in the range, 0.25 < a, ß < 0.9.

11.9.4 Scale-up according to geometric similarity

With this approach, the geometry of a reference vessel on the smaller scale is used as the basisfor the specification of the large scale vessel. Several ratios (such as aspect ratio; impeller totank diameter, etc.) are determined and then held constant. In terms of the process requirements,this approach does not yield an equivalent micro-environment. This method should not beused for large scale design purposes, but can be useful when scaling at the laboratory scale.

11.9.5 Scale-up according to one specific parameter

The two parameters that are commonly used with this approach are:

• Constant specific power input.

• Constant kLa.

Scale-up with constant specific power input

This method would be recommended when scaling from the laboratory scale of 20 l to abench scale unit of, say 200–300 l. Using this approach when scaling directly to a productionvolume, leads to an uneconomical power consumption.


Scale-up with constant kLa

When transferring scale to production volumes, this is the preferred approach. However,the direct application of equation (11.3) can lead to errors, as the parameters a, ß and dcould be scale-dependent. For example, the influence of viscosity effects are more pronouncedon the larger scale. In practice the validity of parameters on the larger scale should thereforebe confirmed.

Setting kLa | scale 1 = kLa | scale 2 implies:

• The aspect ratio usually increases with scale. Using the definition of superficial gasvelocity (u

g = Q/A), it follows that at constant VVM, the velocity, u

g, increases. Thus, the

specific power input required decreases.

• If Pg/V is held constant, a lower aeration rate is required on the larger scale.

• In cases where viscosity effects are negligible, it can be stated directly that: (P

g/V)a(u

g)ß |

scale 1 = (P

g/V)a(u

g)ß |

scale2 (11.4)

11.9.6 Design constraints

Parameters such as Pg/V and ug cannot be chosen arbitrarily; at the lower limit ofpower input in the STR is the required impeller speed for complete gas dispersion.Gas dispersing capacity varies between axial and radial type impellers and must beconfirmed in each case. Axial impellers will handle a higher gas loading beforeflooding (at the same speed) than the Rushton impeller (McFarlane et al., 1995). ForRushton turbines the minimum speed required for gas dispersion was confirmed as(Hudcova et al., 1989):

(FlG)F = 30(D/T)3.5(Fr)F (11.5)

while a suggested working agitator speed is given by:

(11.6)

This is between the flooding point and point of gross recirculation, with FlG the aerationnumber and Fr the Froude number. These correlations are applied with regard to the bottomimpeller only; as long as the bottom impeller is not flooded, dispersion at the higher impellerswill not be a constraint. At the upper limit, the maximum impeller speed is set by the shearrate that can be allowed in the system. In practice this constraint leads to tip speeds in therange of 4–7 m s-1 for production scale vessels.

11.9.7 Regime analysis and scale-down

The application of regime analysis together with the scale-down technique was essentiallydeveloped at the Technical University of Delft (Oosterhuis, 1984; Sweere et al., 1987). Theconcept is applied in four parts:

• Regime analysis of the process at the production scale.

• Simulation of certain (rate-limiting) mechanisms at laboratory scale.

• Process optimization and modelling at laboratory scale.

• Translation of optimized conditions back to the production scale.


The purpose of doing the initial regime analysis is to establish which mechanisms are ratedetermining. This is done by comparing the order of magnitude of the different characteristictimes. If the small scale study is to be representative, the relative time-constants of rate-limiting steps must remain in the same order. It should be noted that the method is only anorder of magnitude comparison and not an exact procedure. Thus if the time constants fortwo processes (such as mixing time and oxygen transfer) are similar, the mechanisms shouldbe investigated further.

11.10 Airlift and bubble column reactors

Developments during the last decade indicate that pneumatically agitated vessels willeventually replace mechanically agitated reactors. This is due to several advantages of sucha design, as pointed out by Mashelkar (1970) and Söderberg (1983):

• No need to maintain sterility around an agitator shaft entry point.

• No mechanical constraints due to agitator shaft length or motor and gearbox size.

• Lower heat load, as the agitator power input can contribute as much as 30 per cent to thetotal energy input.

• Lower fabrication cost for the vessel.

• Lower cost in terms of structural steel.

• Lower maintenance cost.

• The vessel functions as a variable mixing power unit simply through controlling theaeration.

Airlift reactor refers to configurations where a draft tube is employed to set up a liquidcirculation pattern in the vessel. Such a draft tube can be internal or an external loop. Wherethe vessel does not have a draft tube, the term bubble column is used, rather than towerreactor. Scale-up and design of these reactors are done with empirical correlations establishedin terms of the macroscopic parameters such as pressure drop, gas hold-up, liquid velocitydistribution and mixing properties.

11.10.1 Approaches to design

Three methods can be recommended for the design of an airlift or bubble column reactor:

• Using the scale-down technique (Choi, 1990) as discussed in Section 11.9.7.

• Scale-up with constant superficial gas velocity.

• Maintaining constant kLa on scaling up.

Understandably, the critical parameter in these types of reactor is the superficial gas velocity:not only the power input, but also gas hold-up (f) and effective viscosity (heff) can becorrelated to ug. In the case of airlift fermenters, the important geometrical consideration isthe ratio of downcomer to riser area and the corresponding liquid velocities.

The bubble column pressure drop is simply the sum of the sparger pressure drop and thehydrostatic head in the reactor:

DPtotal = DPsparger + DPhead (11.7)


while the energy input can be calculated from (Deckwer, 1985):

Pg = Qrg[RT ln(1 + a) + 1/2(ug)2] (11.8)

This is the sum of the gas kinetic energy and the compression energy to overcome thepressure drop. The parameter, a, is the ratio of the maximum hydrostatic head, to the pressureat the liquid surface:

a = rL(1 - f)gL/PT. (11.9)

Gas hold-up

The gas residence time in the reactor is determined primarily by the liquid circulation velocityand the bubble swarm rise velocity. As the liquid circulation velocity increases, the degreeof back-mixing in an airlift reactor and the fractional hold-up increases. This means moreefficient utilization of the available oxygen than in an STR of similar geometry. Thereforeairlift reactors typically employ aspect ratios as high as 10, to develop high liquid circulationvelocity (Onken and Weiland, 1983). Hold-up has been correlated as being directlyproportional to ug, i.e.

f = C3ug (11.10)

while Mashelkar (1970) proposes:

f = (ug/rL)/(30 + 2ug)(72/s)-1/3 (11.11)

which is applicable for Newtonian fluids in the range 5 < ug < 12 cm s-1. For non-Newtonianfluids, Barker and Worgan (1981) correlated hold-up data to the consistency index, K,according to:

f = 3.09 + 4.5K - 4.51K2 (11.12)

Effective viscosity and shear rate

Aspergillus niger fermentation broths exhibit deviation from Newtonian fluid behaviour,often correlated with the Power Law model. It has been shown that the non-Newtonianbehaviour can also be observed with pellet morphology and is due to the presence ofthe biomass (Mitard and Riba, 1988; Allen and Robinson, 1990). Apparent viscosity iscalculated from the average shear rate in the vessel, which in an STR is directlyproportional to the impeller speed. For bubble columns, Popovic and Robinson (1989)suggest a direct relationship to ug: g = ßug where ß is a constant [m-1]. At the samespecific power input, the shear rate in a bubble column is one order of magnitude lowerthan in an STR. It has also been established that at a specific power input in the range1–5 W/kg, µa does not impact on kLa for µa < 1.0 Pa.s. At higher viscosity (and the samespecific power input), kLa decreases by two orders of magnitude, as µa increases to 100Pa.s. This is explained in terms of reduced interfacial area per unit volume available formass transfer. Heat transfer

The correlation put forward by Mashelkar (1970) for calculation of the process side filmheat transfer coefficient in bubble columns is:


hf = 1380(ug)0.22/(Pr)0.5 (11.13)

In the case of airlift reactors, Chisti (1989) quotes two equations:

hf = 8.71(Ar/Ad)0.25(ug)0.22/(Pr)0.5 (11.14)

and

hf = 13.34(1 + Ar/Ad)-0.7(ug)0.275 (11.15)

11.10.2 Mass transfer correlations

Oxygen transfer as a function of ug

Several researchers (Mashelkar, 1970; Barker and Worgan, 1981; Deckwer, 1985) havefound a direct dependence of kLa on superficial gas velocity. These correlations are of theform:

kLaa(ug)n (11.16)

or:kLaa(ug)n/(µa)b (11.17)

with the coefficient, n, typically in the range 0.7–0.8. For airlift fermenters, Popovic andRobinson (1989) found:

(11.18)

which can be reduced to equation (11.16) or (11.17). In this case Al is proportionality constant,and refers to the sparger hole diameter [m]. The exponents a1, b1, d1, e1, f1, g1 and h1 mustbe determined experimentally. Dimensional analysis approach

The general equation proposed for design purposes (Deckwer, 1985), incorporates severaldimensionless groups:

kLa = C4(D/DR2)Scb1 Bob2 Gab3 Fr(1 + C5De

m)-b4 (11.19)

Again C4 and C5 are constants and b1–4 must be determined experimentally. Depending onthe system under investigation, not all groups would necessarily be relevant. The Froudenumber, Fr, is often omitted where vortex formation is not a factor, while the Deborah, De,number accounts for the elastic properties of the broth. A number of correlations of theformat of equation (11.18) and (11.19) are summarized by Chisti (1989).

11.11 Product isolation

In schematic form, the isolation sequence is presented in Figure 11.2 as: biomass removal;purification; and crystallization. Irrespective of the technology employed, the first stepremains the separation of cell mass from the fermentation broth. Thereafter the sequenceand unit operations during purification will depend on the specific technology used. Finally,the crystallization section is again generic to bulk producers.


11.12 Cell removal

Conventionally, this is a filtration operation: unlike in the baker’s yeast industry, centrifugesare not the preferred units for harvesting biomass. This is due mainly to the higher capital,operating and maintenance cost of centrifuges in comparison to filtration operations. Theproduct of value in this case is also the filtrate and not the biomass. Suitable filter typescould include:

• Rotary vacuum drum filters.

• Plate and frame filter press.

• Continuous belt filter.

• Disc filters.

The design of the filter is based on the application of the Poiseuille equation (Boss, 1983).The equation can be written in various forms (Coulson and Richardson, 1983, p. 323), butalways relates the rate of filtration, dV/dq, to the pressure drop across the filter, DP, filtrationarea, A, liquid viscosity, µ, resistance to filtration, r, and cake compressibility, d:

(11.20)

where a is the average specific cake resistance, which is a function of the pressure applied andthe cake compressibility d: a = a’DPd, where a’ is a constant related to the size of the particlesin the cake. A value of d = 0 corresponds to an incompressible filter cake, while d = 1 wouldbe a gelatinous protein sludge. From equation (11.20) it follows that for such cases, the rate offiltration is independent of the applied pressure. The resistance of the filter medium, r, is oftenexpressed as an additional cake resistance, with a fictitious thickness, to simplify the handlingof experimental data. On a (batch) laboratory apparatus, the rate of filtration is measuredversus the applied pressure. The parameters of the equation are then determined throughcurve fitting of the data. Scaling up the filter design, is in essence the specification of a requiredfilter area to achieve a certain rate of filtration, at the allowable pressure drop.

Equation (11.20) can be extended to account for applications such as a rotary drumfilter, where the total filter area is not continuously submerged in the slurry (Peters andTimmerhaus, 1968, p. 487), by defining an effective area: ADyf. If AD is the total filter areaand yf the fraction immersed in the slurry, then:

(11.21)

where VR is the volume of filtrate per revolution of the filter.Final points to consider in selecting a suitable filter include:

• Will washing of the filter cake be necessary to recover the maximum citric acid?

• What is the desired or acceptable moisture content of the cake?

• Can a filter aid such as diatomaceous earth be used, or is this undesirable? If so, is thisfrom an economical point, or because it impacts on the fodder value of the biomass?

• Can gypsum be used as a filter aid?

• Is there an additional time constraint on the required rate of filtration? This applies incases where the operation must be completed within a short time (usually less than 16hours) after the end of fermentation, before complete cell lysis occurs.


11.13 Purification

11.13.1 Membrane applications

Microfiltration, ultrafiltration and nanofiltration have been investigated for application incitric acid processes. Microfiltration, as with lactic acid production, offers possibilities withregard to cell retention in continuous citric acid fermentation systems (Enzminger and Asenjo,1986; Daniel and Brauer, 1994; Rubbico et al., 1996).

Where the aim is to remove proteins or enzymes from the fermentation broth, ultrafiltration(UF) or nanofiltration (NF) is employed (Bohdziewicz and Bodzek, 1994). With NF it isalso possible to remove residual sugars from the process liquor (Raman et al., 1994) at lowpH. This is due to the structure of the NF membrane, where the active membrane layertypically consists of negatively charged groups. Thus salts are rejected due to electrostaticinteraction between the ions and the membrane while sugars are rejected on molecular size.At low pH values, the citric acid is un-dissociated and permeates the membrane in spite ofsugars (with a similar molecular weight) being rejected. Conceivably it is therefore possibleto put together a process scheme which would eliminate the lime precipitation step byremoving, not only proteins, but also a significant percentage of residual sugars, throughNF.

Industrial exploitation of this concept has been hindered by:

• Developing membrane material that offers long-term stability at low (±2) pH.

• Membrane fouling.

• Cost of membranes.

• Energy requirements due to high trans-membrane pressures.

These factors are being resolved with continued research in the field: developments such asceramic membranes offer mechanically rigid filters resistant to chemical attack. Onefavourable aspect of the membrane applications is that scale-up can be done through modularduplication of pilot plant units. Hence it is possible to predict accurately the performance ofa full-scale unit from a series of laboratory or pilot plant tests. As with standard filteroperations, it is however of prime importance to use a representative sample in doingexperiments.

The application areas of UF and NF overlap to some extent, but can be grouped accordingto molecular cut-off point (Gyure, 1992). Mathematically, ultrafiltration can be modelled withthe Hagen–Poiseuille equation (Kula, 1985). Analogous to equation (11.20), the flux, J isrelated to the trans-membrane pressure applied, dynamic viscosity and membrane properties:

(11.22)

Table 11.3 Classification of ultrafiltration and nanofiltration


Equation (11.22) applies in the ideal case, where the membrane pores are of uniformdistribution and size and fouling of the membrane or concentration polarization can beneglected. The latter effect occurs due to an accumulation of the retained solute at themembrane surface, resulting in a concentration higher than the bulk concentration. Suchconcentration of the retained species means that the component will diffuse back to thebulk flow conditions. At a sufficiently high concentration of the retained species,saturation concentrations at the membrane surface lead to the formation of a gel layer,which then offers an additional filtration resistance. Once such gel polarization isestablished, the flux becomes independent of the pressure: increased pressure forms athicker gel layer, which in turn offers increased resistance and hence the flux does notincrease.

In practice, the UF process is better described by the mass transfer limited (i.e. diffusionlimited) models. For a comprehensive treatment of the topic, the reader is referred to theUltrafiltration Handbook (Cheryan, 1986); Reisman (1988) also presents some comparativedata on capital and operating costs of membrane units.

11.13.2 Colour removal

The function of the colour removal step is to yield an aesthetically acceptable (i.e. white)food grade product. While colour can be removed with resin applications, the norm is stillto use activated carbon for this purpose.

Specifying the required carbon loading per volume of citric acid process liquor, requiressome laboratory experiments. This is done to quantify the carbon/acid ratio and give anindication of the volume of process liquor that can be treated, as well as the kinetics ofcolour removal. Treatment can either be in a fixed bed system, or by simply adding fineactivated carbon directly to the process liquor. After a calculated residence time in contactwith the activated carbon, the latter is then removed by filtration. This method offers theadvantage that a simple stirred batch tank, sized for the calculated residence time, willsuffice. A disadvantage is the filtration step required afterwards and the disposal cost ofspent carbon. In a fixed bed system, the spent carbon can be regenerated with steam. Thesecolumns are usually installed as two parallel units: while one is in operation the other isregenerated.

For laminar flow through the column, the pressure drop at a specific superficial fluidvelocity can be calculated from the Carman–Kozeny equation (Coulson and Richardson,1983):

DP = -(u h µ)/B (11.23)

where:

B = [1/K²][e3(S2(1 - e)2)] (11.24)

B, is the bed permeability coefficient, while the Kozeny constant, K², is generally assumedto be »5. This constant is a function of particle shape and porosity.

It should be noted that the equation was derived on the basis of the bed consisting ofuniformly sized, spherical particles. Where significant deviation occurs from this situation,some corrections have to be taken into account (Coulson and Richardson, 1983). Equation(11.24) can also be applied to calculate the pressure drop across an ion-exchange columnresin bed. In such a case it is to be expected that the particles will be of uniform size andspherical.


11.13.3 Ion-exchange

Ion exchange involves the interchange of any ion between the process liquor and the polymerresin. The essential points (Dechow, 1983) are that ion exchange reactions are:

• stoichiometric;

• reversible;

• possible with any ionizable compound;

• a function of the resin selectivity and reaction kinetics;

• subject to the usual chemistry kinetic behaviour with regard to concentration andtemperature.

Typically, resins are polymers based on the cross-linking of polystyrene with divinylbenzene.Other possibilities are the cross-linking of divinyl-benzene with an acrylate or acrylonitrile,as well as phenol-formaldehyde and polyalkylamine resins. Three types of resin exchangereaction can be found in the production of citric acid:

• Demineralization.

• Metathesis, which is the conversion of salts of citric acid to the acid.

• Adsorptive purification.

The metathesis reaction can be represented as:

[resin]-H+ + Na+-[citrate]- « [resin]-Na+ + H+-[citrate]-

with equilibrium constant, K, defined as the concentration of products divided by reagents(at equilibrium). A large K-value indicates a high affinity of the resin for sodium ions andmeans that an excess of strong acid will be required to regenerate the resin. The polymerstructure and composition determine the resin selectivity for a specific ion, but at ambienttemperatures, two generalizations apply to dilute aqueous solutions:

• the exchange potential increases with increasing ion valence; and

• at the same ion valence, the exchange potential increases with atomic number.

Thus, in increasing order of exchange potential:

Na+ < Ca++ < Al+++ andLi+ < Na+ < K+ or Mg++ < Ca++ < Sr++ < Ba++

Similarly, for anions: F- < Cl- < Br-

These principles are important in monitoring the ion exchange column effluent: it followsthat the monovalent ions would be expected to ‘break through’ first as the resin reachescapacity loading. As the resin reactions are stoichiometric, the quantity of resin materialrequired to remove a certain concentration of cations or anions, can easily be calculated.The resin capacity is expressed as equivalents per kilogram (on a dry basis) or per litre on awet basis (eq/l). The equivalents number is simply an indication of the number of activesites available for adsorption and can be obtained from the resin supplier. Analysis of theprocess liquor will then determine the quantity of resin to be used for the required throughput.Because the exchange process is an equilibrium reaction, the resin is utilized at a level wellbelow the theoretical capacity, thereby shifting the equilibrium in the desired direction (LeChâtelier’s principle). This does not significantly increase costs, as the resin cost is typicallyonly 10 per cent of the unit cost (Dechow, 1983).


Specifying the ion-exchange unit

Once the required ion loading to be removed and the specific resin capacity are known, theunit can be specified. Three configurations are employed: batch stirred tank, batch columnand continuous operation. The most common installation is the batch column, either as twounits in parallel to allow uninterrupted operation, or with an adsorption– regeneration cycleon a single column. Because the resin cost is not a major factor, it is sound design practiceto allow for some excess capacity in specifying the column size. As an example, considerspecifying a column of diameter d1 and a unit with diameter d2 = 1.5d1. Assuming the bedheight in both columns to be h, the increase in resin volume is:

V2/V1 = (1.5)2 = 2.25 (11.25)

Thus the resin volume and hence column capacity more than doubles, but the capital cost tofabricate and install the unit does not increase by the same factor. The calculation of thepressure drop for a specific column geometry can be done with equation (11.23).

Adsorptive purification

Possibly a commercially viable direct crystallization route, this process is already employedfor the recovery of lysine. In some cases, adsorption is done directly from the fermentation broth(Van Walsem et al., 1997) thereby simplifying the flowsheet considerably. A scheme proposedfor the recovery of citric acid (Ernst and McQuigg, 1992) uses temperature swing adsorption(TSA) to purify the citric acid solution. In this case the regeneration is done by utilizing thedifference in resin capacity for citric acid as a function of temperature. Thus citric acid is adsorbedat ambient temperatures and desorbed with hot water. Resin capacity in excess of 155 g citricacid per litre resin, with a 96 per cent reduction in RCS values were reported.

While such a process eliminates the lime precipitation route and associated by-productdisposal dilemma, the environmental focus might shift to the actual resin in this case. Theadsorptive resins are structured from poly-vinylpyridine-co-divinylbenzene, the productionprocess of which generates some environmental concerns in itself. Although the effluents(pyridine compounds) can be treated, it ultimately becomes a cost which is passed on to theend user in the pricing of the resin.

11.13.4 Electrodialysis

Recovering lactic acid by electrodialysis has been researched and several processes patentedduring the last 20 years (Nomura et al., 1987; Siebold, et al., 1995). Although it is activelybeing researched (Karklins et al., 1996; Moresi and Sappino, 1996), it is this author’s opinionthat employing electrodialysis for citric acid recovery is not at the point of commercialexploitation yet. The technology is proven, but the energy consumption does not yet offer acompetitive advantage (Novalic and Kulbe, 1996). Membrane filtration routes, solventextraction and adsorptive ion exchange seem more likely to succeed on a cost competitivebasis.

11.13.5 Solvent extraction

As a direct crystallization route, the application of solvent extraction seems to offer interestingpossibilities. Similar to membrane processes, this might necessitate the use of more refined


substrates such as glucose syrups, to avoid the extraction of impurities in beet and canemolasses. Once again, with increasing environmental pressure on reducing effluents, thismight become an economically viable alternative to the classical lime precipitation route.Presenting a discussion of solvent extraction principles is beyond the scope of this text.Suffice it to say that processes utilizing butan-2-ol tributyl phosphate plus kerosene andtertiary amines have been published (Melsom and Meers, 1985). A further simplification ofthe flow sheet would be direct extraction from the fermentation broth (Stuckey, 1997),which is currently being researched.

11.14 Crystallization stages

Unit operations included in this section are the evaporator, crystallizer, centrifuge and dryer.These units require specialist vendor input and will rarely be designed in-house.

11.14.1 Evaporation

At the evaporation stage, the process liquor will contain 15 to 20 per cent citric acid insolution. It is an energy intensive operation and the efficient utilization of energy is animportant design consideration. The norm is to specify multiple effect evaporators, wherevapour from one effect is condensed in the subsequent unit re-boiler, with the process sideoperated at a lower pressure. Mechanical vapour recompression can also be considered anddepending on the relative steam/electricity cost, is often economical at large capacities. Thetypes of evaporators employed vary, but forced circulation and falling film types have beenused successfully for a number of years. In planning the energy integration, care must betaken to ensure that the citric acid will not be discoloured through exposure to hightemperature. Especially if a direct crystallization route is considered, trace amounts of residualsugar are still present at the evaporation stage. This means that the temperature in the firsteffect evaporator, where steam is used as the heating medium, must be limited to well below100°C. Such a constraint necessitates the use of low-pressure steam and also dictates thevacuum required in subsequent stages.

Removing colour from the concentrated citric acid solution after the evaporator stagepresents practical problems and should be avoided where possible. As the solution is closeto saturation, the prevention of blockages due to crystals settling in a unit such as an activatedcarbon column is cumbersome.

11.14.2 Crystallization

Typically this is a two-stage operation, where the first stage is the final purification step.The second crystallization must yield the correct crystal size distribution, according to thespecified customer requirement. Usually continuous forced circulation crystallizers, in linewith pusher centrifuges will be used. The mother liquor produced from the crystallizers canbe recycled through an adsorptive ion exchange unit, or utilized for the production of sodiumcitrate. Alternatively, the acid can be recovered with the lime precipitation route.

In discussing the unit specification with a vendor, it will be necessary to stipulate if theoption of anhydrous and monohydrate acid is required. As the crystallization temperature ofmonohydrate citric acid is lower, this impacts on the capacity of the vacuum ejectors/pumps.


11.14.3 Product drying

Again a specialist vendor can consult on a suitable type of dryer. This might typically be amoving bed/fluidized bed unit. From the centrifuge, the wet cake should not contain morethan 5 per cent moisture; a simple energy balance will therefore determine the dryer heatload. Where high humidity conditions exist, the unit will have to incorporate an air-dryingsequence to obtain product according to specification.

11.15 Product packaging

The process engineer’s responsibility here lies in correctly transferring the customer requirementinto a technical specification. Before this is done, it should be decided in principle if productpackaging is to be done during one, two or three shifts. Where volumes or specific market needswarrant, one grade of product might have a dedicated packaging line. These issues also set thestorage hopper volume required. In planning this area, cognisance should be taken of the timerequired for final product quality approval. This usually implies that product is kept in an areaimmediately adjacent to the bagging area, while quality control analysis is being done. Thereforesufficient space should be allocated for unhindered flow of material in the area.

11.16 Effluent and by-products

The main out-flows produced during the process are:

• biomass from the cell removal stage;

• excess water;

• gypsum—if recovery is done along the classical route; and

• retentate from membrane filtration steps.

As a protein source, the biomass does have some value: a recent study (Szoltysek et al.,1996) reported on an investigation into using the mycelium as a component of chickenfeed. Aqueous effluent from the plant can be expected to have a relatively high BOD: in theregion of 12 000–14 000 mg/l, or even higher where molasses is used as substrate. Whiletechnically this does not pose a problem, it does have a cost implication to reduce theselevels. Similarly, the disposal of gypsum could be a significant cost factor, if the plant is notlocated in a region where there is a demand from industries in the construction sector.Volumes of retentate are small relative to the other effluents and disposal does not seem tobe a problem. For example, this could be mixed as a protein source with animal fodder.

11.17 In conclusion

The preceding paragraphs bear out the fact that plant design is largely a discipline genericto the chemical engineering industry. However, it should also be stressed that fermentationplants require unambiguous communication between the chemical engineer andmicrobiologist. Provided the process engineer can correctly interpret the sometimes unusualrequirements of a ‘living system’, the application of sound engineering practice will ensurea successful design.


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absorption 142absorptive purification 181activated carbon 139acyl CoA synthetase 38–9adenine nucleotides 43adsorption 142aerated systems 172aeration 5agitation effects 70air-lift bioreactor 129, 131, 174alcohol dehydrogenase 37alcohols 37aldehyde dehydrogenase 37aliphatic alcohols 141alkali metal salts 138alkanes 6, 157 uptake 35alkylsuphoxides 141alternative oxidase 123ammonia 20, 158anion exchange resins 142approaches to design 174arabinose 151aspergillus model 107available electron balance 130axial impellers 173

beet molasses 151–3 microelements 152vitamins 153 amino acid content 152

betaine 151bipolar membranes 144blackstrap 153bubble column reactor 174butanol 139

cane molasses 153–5 vitamins 153composition 154

caramel 151carbon balance 130carbon content of substrates 150Carmen-Kozeny equation 179

cation exchange 138, 155cell removal 177cellulose 158centrifugation 139chemical plant design 163citrate synthase 41–4citrate-free recovery 139 flowsheet 140citric acid applications 8 biochemistry 11

biosynthetic pathway 12–14 chemicalmethod 2 continuous production 5 kojiprocess 7, 58 microbial 2 overproduction129 regulation 19–21 solid state process 7submerged process 4, 58 surface method 3synthetic 2 transport 24–5, 44–6, 49–50uses 7, 8 yeast based processes 6 yeastsynthesis from alkanes 35–46 yeastsynthesis from glucose 46–50

cloned genes 16coagulating agents 139colour removal 179compartmentation 42constant specific power input 172copper 81counter current flow 143crystallisation 136, 138, 182crystallization stages 181cube root growth model 106, 107customer information in design 164customer requirements in design 163cyclic AMP 21cytochrome P-450 hydroxylase 36

date syrup 156design of an industrial plant 163design basis of an industrial plant 165–7design constraints 173di-calcium citrate 136, 137dilution rate 111dimensional analysis 176dissolved oxygen 4, 23

Index

Index188

downstream processing 135–46

economics 1effective viscosity 175effluent 8, 183electrodialysis 138, 144, 181 scheme 145electron activity 87, 88elemental composition 124elementary balances 122energy balance 121energy consumption 144energy yield coefficients 128equilibrium distribution coefficient 141esters 141ethers 141ethylenediamineacetic acid 155evaporation 182exchange potential 180

fats 158fermentation aspects of design 170filter types 177fixed bed filter 142formaline 156four compartment cell 144Froude number 173fructose-2, 6-bisphosphate 19

gas hold-up 175genes 15–19, 44geometric similarity 172gluconic acid 12, 58glucose oxidase 12glucose uptake 20glutamate 49glycerol 22, 49glycolytic pathway 12, 58glyoxylate cycle 39growth kinetics 127

Hagen-Poiseuille equation 178heat removal 3heat transfer 170, 175hydrocarbons 149hydrol 157

idiophase 117, 119, 123immobilization 5, 145initial conditions model 113inoculum 4ion exchange 180–1, 138–9, 142iron 81isocitric acid 59isocitrate lyase 41

kestose 151ketones 141

kinetic modelling 105Kjærgaard equation 87K

La 172

Kozeny constant 179

lemon 1lime 136–7linear growth model 106–7, 118liquid membranes 143log growth model 106–7logistic growth equation 113Ludeking-Piret equation 107–8, 113, 119

magnesium 81manganese 21, 22, 25, 59–60, 81mannose 151market 2, 9marketing information 164mass balance 121, 126mass transfer 171mass transfer correlations 176mass yield coefficient 122mechanistic models 105melanoidines 151membrane applications 178metabolic control analysis 61–2, 106metabolic description of A. niger growth 123metathesis reaction 180methyl citrate 40–1methyl isocitrate 40–1microelements 158microfiltration 178microporous hollow fibres 143mitochondria 22mixed biomass 111modelling 105–19molasses 150–6 colour 151 microflora 154

non-volatile compounds 151 pH 153volatile compounds 151 nitrogencompounds 151, 153

Monod equation 106, 111monopotassium phosphate 158morphology 5, 23, 60 A. niger 71 dissolved

oxygen 81 effect of carbon source 75 effectof inoculum 82 effect of nutritional factors74 effect of pH 79 initial glucoseconcentration 75 nitrogen limitation 78phosphate level 78 trace metal levels 81

mutagenesis 56mycelium formation 126

NADH oxidation 23, 62–3NADH: ubiquinone oxidoreductase 63nanofiltration 145, 178neokestose 151Nernst equation 87, 98new technology in design 167nitrogen metabolism 49

Index 189

nitrogen source 158nomenclature 85, 121, 126, 161–3non-aerated power input 171non-ionic resins 142non-Newtonian behaviour 175

oils 158organophosphorus compounds 141oxalate biosynthesis 15oxalic acid 13, 58–9 removal 135oxidation potential 87, 8oxidative phosphorylation 124oxygen yield coefficient 128oxygenation 3

parasexual cycle 60pectinolytic enzymes 145pellets 5, 69pentose phosphate pathway 12peroxisomes 38–40pH 3, 6, 23, 24phase related model 117phosphofructokinase overexpression 64phosphorus source 158Poiseuille equation 177polyhydric alcohols 131polysulphone 145potassium ferricyanide 91potassium ferrocyanide 155power law 175precipitation 136 flow sheet 137pre-treatment 144 submerged fermentation

135 surface process 135process economics 149process flowsheet 169process package 167product drying 183 isolation 176 packaging

183purification 178pyruvate kinase overexpression 64

QSSA 125

raffinose 151, 153raw materials 169redox dyes 88redox electrodes 88 calibration 88redox potential 85 in citric acid fermentation

91–5 measurement 88 optimal 102regulation 95 significance 89 theory 87time course 92–3 dissolved oxygenrelationship 90 temperature change 94

redox regulation chemical 97 physical 97–100refined sucrose 156regime analysis 173regulatory enzymes 18regulatory network 14respiratory chain 62, 63

reverse osmosis 145Rushton impeller 173Rushton turbine 173

scale down 173scale up 101, 170, 172scope definition 167–8seeding 138shear 70shear rate 173, 175simple structured model for A. niger 107solvent extraction 139–41, 181starch 58, 157sterilisation 155stirred tank reactor 71, 170 design 171stoichiometry 124structured models 105submerged method 150substrate level phosphorylation 128substrate preparation 169substrates 149sucrose 151, 153sulphamide 156superficial gas velocity 176surface method 150syrups 156

technical data in design 165teratogenic effect 142three-compartment cell 144transcriptional regulation 15–16transport costs 150treatment of molasses 155therapies-6-phosphate 20, 64tricalcium citrate 136, 137tricarboxylate transporter 22tri-n-butylphosphate 141trioctylphosphine oxide 141trophophase 117–18, 123tubular loop reactor 71

ultrafiltration 139, 145–6, 178unstructured models 105

vacuum evaporation 138

wastes 136water 159water-soluble amines 141

xylose 151

yeast based models 113yeasts 6yield coefficients 106–7, 113, 125–9

zinc 81

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