115 The large scale production of organic acids by micrwrganisms Overview 5.1 Introduction 5.2 Metabolic pathways and metabolic control mechanisms 5.3 The industrial production of atric acid 5.4 The production of other TCA cycle intermediates 5.5 The industrial production of itaconic acid 5.6 The industrial production of gluconoladone and gluconic acid Summary and objectives 116 116 120 125 137 138 142 146 116 Chapter 5 The large scale production of organic acids by m icro-org an isms Overview In this Chapter we shall look at the use of mim~~rganisms to produce organic acids of commercial importance. Although all of the examples to be mentioned are relatively simple chemically, they are interesting in that they are metabolically diverse. Some are genuine end products of metabolism, while others are compounds considered to be central metabolites in all living cells. These central metabolites are normally present in relatively small, constant amounts. However, some micro-organisms can be "persuaded" to produce enormous yields of these metabolites. The first part of this Chapter, the Introduction, will identdy some of the organic acids produced by micro-organisms and highlight those which are of commercial interest. The products consided in this chapter are metabolites of the tricarboxylic acid UCA) cycle or oxidative derivatives of glucose. Since most of the biological coITLmerical processes involve interference with the metabolism of micro-organisms, we present a section discussing relevant pathways together with the control mechanisms involved. The industrial production of the most commercially important organic acid, citric acid, is then considered in depth. Finally, we outline the biochemistry, formation and downstream processing of other TCA cycle intermediates (malic acid, fumaric acid) and oxidation products of glucose (itaconic acid, gluconic acid and its derivatives). 5.1 Introduction organic acid definmon We must first indicate exactly what is meant by the tern 'organic acid' in the context of this Chapter. Inevitably it will be far more restricted in scope than the literal definition, which essentially means 'any organic compound which is acidic'. n acids which are produced by living cells? 1) Acids of non-carbohydrate origin which are produced by all living systems. 2) Adds continuously produced by all living systems. 3) Acids of carbohydrate origin which are constantly produced by living systems and are not considered as waste products. 4) Acidic examples of continuously produced waste products. Amptable answers to part 1) include amino acids and fitly acids or specific examples of each, such as glycine or stearic acid mpectively. The obvious answer for part 2) is the central metabolite pyruvate, though all of the acids of the TCA cycle would be appropriate. Answers to part 3) include the principal acid of the hexose monophosphate Can you give one or more examples in each of the categories below of organic The large scale production of organic acids by micro-organisms 117 pathway, 6-phosphogluconate, and the acid intermediate of glycolysis, 1,Wphosphoglycerate. Answers to part 4) include an enomus number of acids since all living systems produce acid end products. However, in the context of this chapter the waste products produced by bacteria growing anaerobically are particularly relevant These include: lactate, produced in great quantity by the lactic acid bacteria; lactate, acetate, formate and succinate produced by the EnterobacteriaCaae; butyrate and acetate produced by Clostridium species. There are many more correct answers to each part of the question. To simplify matters, all of the answers are compounds which are acidic because they contain the carboxyl group (-COOH). This chapter does not consider any organic acids which are organic compounds made acidic by the presence of, for example, phosphate or sulphate groups. Further, to warrant discussion in this Chapter, an organic acid has to stisfy the following criteria: 0 there has to be a micrmrganisms which will produce it in commercially significant quanti ties; 0 there has to be a demand for the compound industrially; 0 the overall costs of producing and extracting the acid have to be economic. In metabolic terms there are three clearly distinguishable types of compound to deal with. Firstly, compounds which are obviously waste products - end products of one or more pathways which would normally be excreted from the cell (for example lactic acid). Secondly, compounds which are end products of pathways but which are not waste products and whose synthesis is normally very carefully controlled (for example amino acids). Thirdly, compounds which are intermediates of pathways and hence not normally considered as end products or wastes at all (for example citric acid). Production of large quantities of organic acids by micrmrganisms would, on the face of it, seem easier if we are dealing with acids which are genuine waste products rather than non-waste compounds such as central metabolites. It is merely a technical problem to encourage certain bacteria to produce a waste product such as lactate as this compound is normally excreted into the surrounding medium. The removal of spent medium regularly and harvesting of lactate could allow continuous production of lactate. However, production of metabolites such as amino acids is more complex and to obtain sufficient quantities of intermediate compounds such as citric acid is even more of a problem. The key problem is how to encourage a micrmrganism to produce a vast excess of an organic acid whose synthesis is normally controlled very efficiently at relatively low concentration. A detailed discussion of this problem will occur later, but we can generalis here and identify the four main ways in which metabolic by altering the environmental conditions, eg temperature, pH, medium composition (especially the elimination of ions and cofactors considered essential for particular enzymes); by disrupting a pathway using substrate analogues; by mutation - giving rise to mutant organisms which may only use part of a metabolic pathway or regulatory mutants; 0 by genetic engineering. It should be noted that apart from a passing reference to natural selection of wild-types with enhanced specific properties, the genetics of organic acid producing micmrganisms is beyond the scope of this chapter. end Pro*& and intermediates manipulation of memblic pathways pathways can be manipulated: 118 Chapter 5 5.1.1 Generalised scheme for fermentation There are several stages common to most fermentation processes but before identifying these it is appropriate to define some terms which will appear during this Chapter. Let us first distinguish between primary and secondary metabolites. primmy metaboIites are compounds which are essential to the growth and well being of the cell and, during the growth phase, are produced continuously. Secondmy metabdites are those compounds not essential to the life of the cell and not produced continuously; often but not always, they are produced during non-growth phases of the cell. The growth phase where primary metabolites are produced is sometimes refend to as the traphophase, whereas the phase during which secondary metabolites are formed (usually the stationary phase) is termed the idiophase. All of the compounds we shall study in this Chapter are primary metabolites though both phases of growth will be studied. For example, as we shall see, citric acid is produced continuously at low levels during trophophase but only accumulates at high concentration during idiophase. Finally the word 'fermentation' will be used in its industrial sense, that is a commercially viable process in which a miawrganism produces a r+ product or change. Let us now consider the essentials of the fermentation process, largely as a revisionary exercise, before looking at the individual examples in detail. Study the dabelled block diagram, and then replace the question marks with the words and phrases to give a generalised scheme of an industrial fermentation. Assume in this example that the product is excreted from the microbial cells. primary and -ndarY metaboms mphophs idophas The large scale produdion of organic acids by microorganisms 119 You should note that the figure in SAQ 5.1 is a simple outline as fermentations generally have more steps than indicated; for example many have a multiple purification step. If the product were the whole cell (for example in *e cell protein processes) then purification of the cell biomass would be necessary. If the required product were an intracellular compound then some stage of cell breakage would be essential. 5.7.2 Organic acids relevant to this Chapter Table 5.1 shows the organic acids relevant to this Chapter together with the usual substrate, the miao-organisms employed to produce them and finally the potential end uses. Such acids are often grouped into two broad categories, those which are members of or related to the TCA cycle and secondly, those which are oxidation pducts of glucose. The carbon source for the latter group is usually quite specific, either glucose itself or a polysaccharide which yields glucose easily. The carbon source for the former group can be much more diverse and/or complex, for example: glucose or its polymers (molasses, starches); byproducts of industry (methanol, methane); waste products of industry (sulphite waste liquor from paper manufacture) or plant waste (lignins, cellulose derivatives). Acetate, lactate and some amino acids however do not readily fit into either of the groups. organic acid substrate producer end use(s) micro-organism citric acid sugar@) Aspergillus niger malic acid glucose Lactobacillus brevis fumaric acid glucose Rhizopus delemr itaconic acid glucose Aspergillus terreus gluconic acid glucose Aspegillus nger other Aspergillus spp Gluconobacter suboxidans acetic acid ethanol Acetobacter aceti lactic acid lactose Lactic acid bacteria Flavouring for beverages and confectionery. Pharmaceutical food syrups, resins, dye mordants, antifoaming agents, sequestering agents. Food and drink manufacture Used in the plastics industry and, to a lesser extent, in the food industry Intermediate for organic syntheses, eg acrylic resins Pharmaceutical industry and as a washing and softening agent preventing a build up of scale. Retards setting of building materials. Vinegar, food industry Dairy industry as a presewativenlavour enhancer Table 5.1 Example of organic acids produced commercially by micro-organisms; organic acids considered in this chapter are labelled with *. A related acid, a-oxoglutaric acid, is easy to produce microbiologically but has no current end use; succinic acid is produced chemically. Amino acids are beyond the scope of this chapter. A detailed study of the amino acids is beyond the scope of this Chapter. However, industrial production of amino acids is considered in Chapter 8 of this text. 120 Chapter 5 5.2 Metabolic pathways and metabolic control mechanisms 5.2.1 Revision of the reactions of the tricarboxylic acid (TCA) cycle A detailed revision of the TCA cycle is necessary to ensure an understanding of the mechanisms and reasons governing the choice of process conditions for encouraging production of any selected TCA cycle intermediate or related compound. Descriptions of the cycle can be found in many text books, for example in the open learning BIOTOL text entitled 'Principles of Cell Energetics'. Chapter 7 of that text describe in great detail the TCA and glyoxylate cycles, both of which are relevant to this Chapter. The most relevant parts of the cycle are the control mechanisms and processes involved in the intermediary metabolism; these are influenced and exploited in efforts to upset the balance of normal metabolism leading to overproduction of the desired organic acid. Let us first examine Figure 5.1. It is worth spending some time on this in order to understand the rationale of the remainder of this Chapter. Glycolysis (the Embden Meyerhof pathway) is a ten enzyme pathway which is summarised in Figure 5.1. During the course of this pathway, glucose is cleaved to two pyruvate molecules - a process involving the utilisation of two ATP (generating two ADP) but later there is formation of four ATP from four ADP. Thus a net yield of two ATP is achieved. Another consequence of glycolysis is the reduction of two molecules of nicotinamide adenine dinucleotide (NAD') to NADH i H'. Although the exact mechanism of the individual reactions of glycolysis is not really necessary for this Chapter, we must bear in mind the fact that during the process energy and reducing power are formed. The two reactions indicated by * are two further enzymatic steps which, along with three reactions of the TCA cycle, constitute the glyoxylate cycle. In the reaction that bridges glycolysis and the TCA cycle, for each pyruvate degraded to acetyl &A, one COZ is released and a further NAD+ is reduced to NADH + H+. gtycobsis A variety of starting materials other than glucose or its derivatives is possible for use by some micro-organisms; the four shown in Figure 5.1 are all initially converted to acetyl CoA for entry into the central metabolic pathways. The large scale production of organic acids by micro-organisms 121 ~~ Figure 5.1 A simplified diagram of glycolysis and the triirboxylic acid (TCA) cycle showing the entry points for various substrates. indicates the two reactions specific to the glyoxylate cycle. Compounds in boxes are potential substrates for entry into the TCA cycle, via acetyl CoA. 5.2.2 The relationship between anabolism and catabolism At this point we need to consider the two halves of metabolism -anabolism and catabolism - and in particular the metabolic control involved. Catabolism is the process by which intra- or extracellular molecules are degraded to yield smaller ones which are either waste products or building blocks for biosynthesis. Jhmg catabolism reducing power in the form of NADH + H', FADH2 or NADPH + H' is generated. Subsequent reoxidation of these cofactors (particularly NADH) by aerobic cells releases energy which is converted to ATP, the mapr short term energy storage cumncy. The mechanisms involved in this ATP formation are the electron transport chain and oxidative phosphorylation. These processes are intimately linked - a bit like two parts of a zip fastener - in that when oxidation of NADH takes place, the formation of 3 ATP from 3 ADP usually takes place. catabolism 122 Chapter 5 anabolism albsteric enzymes Anabolism is the building up or biosynthesis, of complex molecules such as protein, nucleic acids and polysaccharides, from raw materials originating from intra- or extracellular sources. The biosyntheses are energy (ATP) requiring processes. Catabolism and anabolism have to be carefully regulated and are inevitably intimately linked. What are the three areas where the processes of catabolism and anabolism am n linked? Catabolism produces ATP, reducing power and intermediates. Anabolism requires all three, thus these are the three main links. No living cells can store large amounts of ATP. There is a hite amount of 'adenine' distributed between AMP, ADP and ATP. Thus if the cell has a relatively high concentration of ATP, the concentrations of AMP and/or ADP must be lowered. The balance alters like a "see-saw", as one goes up the other must come down. In addition the total amount of NAD+/NADH and NADP+/NADPH in the cell is constant. What is the advantage of the see-saw type of change to the ratio of the n concentrations? The answer is that such a system is far more sensitive to small changes in concentration of the respective compounds. The cell is recognising a change of the ratio of compounds, rather than the rise or fall of a single compound. Although cells cannot store ATP, they must always have a minimum amount available to keep them alive. Thus a constant level of ATP must be maintained indicating that catabolism and anabolism occur constantly under normal conditions. One compound controls the overall metabolism of the cell regulating and n balancing anabolism and catabolism. Can you name it? The answer in practice is ATP though you would have been theoretically corred if you had said ADP and AMP. Indirectly, NAD+ or NADH are also compounds which regulate the anabolic/catabolic balance. The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through noncovalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). Thus ATP is the effective controller of metabolism but because AMP + ADP + ATP is constant, it is really the ratio of adenine nucleotides which is important. This ratio is termed the adenylate charge or energy charge and is expressed as: 0.5 [ADP] + [ATP] [AMP] + [ADP] + [ATPI Energy charge = The large scale production of organic acids by micro-organisms 123 The theoretical limits are 1.0 (all ATP) and 0 (all AMP) with a nod working range of 0.75 to 0.9. The involvement of energy charge in the integration and regulation of metabolism is considered further in the BIOTOL text entitled 'Biosynthesis and the Integration of Cell Metabolism'. After revising the TCA cycle reactions in more detail we shall return to the subject of metabolic control by ATP. Figure 5.2 shows a detailed version of the TCA cycle indicating cofactor changes and the individual intermediates. Figure 5.2 The tricarboxylic acid cycle. Enzymes: a) citrate synthase; b) aconitase; c) ismitrate dehydrogenase; d) a-oxoglutarate dehydrogenase; e) succinyl CoA synthetase; f) succinate dehydrogenase; g) fumarase; h) malate dehydrogenase; i) nucleoside diphosphokinase. 5.2.3 The control of metabolism In section 5.2.2 we considered a simple equation expressing the energy charge of the cell in terms of the ratio of adenine nucleotides. Figure 5.3 summarises the principal allosteric enzymes of glycolysis and the TCA cycle and indicates how the individual adenine nucleotides influence the activity of a variety of enzymes. The enzymes to the right of the glucose to pyruvate pathway are those involved in glycolysis; those to the left are involved in gluconeogenesis, ie the synthesis of glucose from pyruvate- 1 24 Chapter 5 Figure 5.3 Major control points of glycolysis and the TCA cycle. Enzymes: I, hexokinase; 11, phosphofructokinase; 111, pyruvate kinase; IV, pyruvate dehydrogenase; V, citrate synthase; VI, aconitase; VII, isocitrate dehydrogenase; VIII, u-oxoglutarate dehydrogenase. The large scale production of organic acids by micro-organisms 125 atrate synthase Ph=fJb- fnrctDkinase amniase world demand Aspergillus rfiger Let us now consider how the control system operates. Overall the TCA cycle produces large amounts of reducing power in the form of NADH + H'. As we noted earlier, subsequent reoxidation of NADH + H' must be accompanied by oxidative phosphorylation, the process by which ATP is produced from ADP. When the cell has a high energy charge (high ATP concentration and low [ADP + AMP] concentrations) then temporarily no more ATP is required by the cell. Thus the logical thing to do is to stop or 'switch off' the TCA cycle. The first reaction of the cycle is the most appropriate one to inhibit and we know that citrate synthase is inhibited by high ATP concentration. As the ATP is used by the cell, its level will fall with a concomitant increase in the level of ADP (and AMP). Thus the inhibition of citrate synthase is gradually removed. In a more positive sense, the TCA cycle is stimulated at this stage because the next two enzymes of the cycle, aconitase and isocitrate dehydrogenase are stimulated by increased concentrations of both ADP and AMP. Within glycolysis, the main allosteric control is exercised by phosphofructokinase, a complicated enzyme unusual in that its activity is stimulated by one of its products (ADP) and inhibited by one of its substrates (ATP). One further point about this enzyme which will be important to us later: in Aspergillus spp., elevated levels of ammonium ions relieve phosphofructokinase of inhibition by citrate. A further way in which metabolic control may be exercised is the artificial deprivation of requid ions and cofactors, for exampleaconitase must have ferrous ions for activity. Conversely, addition of toxic ions is possible, for example aconitase is inhibited by cupric ions. Finally the use of metabolic analogues is possible. If monofluoroacetate is added to cells then monofluorocitrate is produced by citrate synthase and this compound inhibits the activity of aconitase. Great care has to be taken when using metabolic analogues, however, they are often less than 100% specific and may have unexpected and unwanted serious side effects. 5.3 The industrial production of citric acid 5.3.1 Historical introduction Historically the production of citrate has been an important development in the pioneering of fermenter technology. It was shown back in 1893 by Wehmer that a fungus, Citromyces (now reclassified as a Penicillium spp.) would accumulate citric acid in liquid culture. Wehmer in fact tried to scale up the process to an industrial level but there were two main problems. Firstly, the duration of the process under his conditions took far too long: of the order of several weeks. Secondly, a problem was caused by Wehmer's incorrect belief that citric acid only accumulated around neutral pH and lengthy incubation at this pH inevitably leads to contamination. The world demand for citric acid around 1900 amounted to some 10,OOO tonnes per annum. This was realised by pressing citrus fruits and precipitation of the citric acid as calcium citrate. An Italian, government-led cartel had virtual monopoly of this pmss and as such the price of citric acid was very high. A major breakthrough in the fermentation process came in 1916 - 1920 when it was found that AspersitIus niger grew well at pH values below 35, producing citric acid in days rather than weeks. The faster incubation and highly acid conditions (often below pH 2.0) also served to minimise potential problems caused by contamination. 126 Chapter 5 Industrial production began in Belgium in 1919 followed by America in 1923 and England in 1927. In these early processes, high sugar concentrations were employed using pure ingredients. Newer materials were continually being tried, for example sugar beet molasses which was used commercially for the first time in 1928. By the mid 1930's over 80% of the world's citric add was produced by fermentation. At present virtually all of the world production comes from this process. By 1981 over 20,000 tonnes were produced annually (possibly as high as 300,000 tonnes); the industry in the United Kingdom at that time being worth some E20 million per annum, one tenth of the world's turnover. One of the more recent innovative approaches was to look for new micro-organisms and novel carbohydrate substrates. The early fermentations used sugar beet or cane molasses, various syrups, sweet potato starch or glucose itself and the miaxwrganism was always an Aspergdllus spp. In the early 1930's it was found that yeasts would produce citric acid from acetate. Since then a variety of yeasts, principally Cmrdida spp., has been shown to convert glucose, n-alkanes or ethanol to citric acid with great efficiency. The realisation that yeasts would produce citric acid from n-paraffins was very attractive in the late 19f3I's. Petroleum byproducts were plentiful and very cheap and there was detailed knowledge available on these processes because the use of hydrocarbon-utilising yeasts for single cell protein was well developed. The strategy was to use n-alkane to produce high yields of citric acid-producing crmdida spp. and to harvest two useful end products rather than just one. The process has not been commercially successful however. Crmdida spp. produce mixtures of citric acid and isocitric acid and the latter is not a useful product. In addition, since 1973 when petroleum prices rose sharply and have in fact continued to rise, the n-paraffins are no longer a cheap substrate. In summary the maprity of the world citric acid production is still via microbial fermentation of carbohydrate substrates (derived from plants) using AspergiZZus niger. 5.3.2 Current uses of citric acid Citric acid, being an intermediate of the TCA cycle, is considered to be non-toxic and very safe for human consumption. As such it has long since had unlimited approval by the World Health Organisation Expert Committee as a food additive. Over 60% of the production is used by the food industry, particularly in soft drinks, jams, jellies, sweets and wines. Around 10% of the production is used in the pharmaceutical industry and in cosmetics. Inmasingly, less pure grades of citric acid are beii used to produce citric acid esters which are used as plastiasers in the plastics industry. Citric acid is used freely as a builder in detergents and is being used in laundries because it has the advantage of being totally biodegradable. 5.3.3 The biochemistry of citric acid production Broadly we can say that molasses are converted to glucose which is converted via glycolysis to pyruvate. Citric acid is then produced via acetyl CoA. Although this statement is largely correct, we must develop the details because them are two main problems which have to be ovemme. matkanes bod hdusby n What, briefly, are the problems which have to be overcome? The large scale production of organic acids by micro-organisms 1 27 biochemical problems ferrous ions isocitrate dehydrogenases OroxoglUSrate dehydrogenase If you are in doubt as to one or both of them, consider the following question -how is citrate normaI2y produced and what would normally happen to it? We know that citric acid is formed from acetyl CoA and oxaloacetate by the reaction: acetyl CoA + oxaloacetate -+ citrate The first problem is that if citric acid is removed, there is apparently no way of regenerating oxaloacetate. The second problem is that to accumulate citric acid, aconitase has to be blocked to avoid citric acid being converted to aconitate. Examine Figure 5.1 again. There is an apparent conflict; if citric acid accumulates there would appear to be no way in which the TCA cycle could continue to regenerate oxaloacetate. On the other hand continuation of the TCA cycle would regenerate oxaloacetate but there will be correspondingly less citric acid accumulated. In fact the aconitase enzyme in A. niger is active even when citric acid is accumulating. This aconitase, if allowed to come to equilibrium, yields 90% citrate, 3% cis-aconitate and 7% isocitrate. To lower the activity of the enzyme, femus ions (essential for activity) are not added to the medium and may have to be removed from complex sources of carbon (such as molasses) before they are used. The fact that isocitrate is being produced means that some way of bloclung the next enzyme, isocitrate dehydrogenase would be desirable. This enzyme or rather these enzymes, because A. niger has four of them (they differ in cofactor requirements), are largely inhibited by higher-than-average concentrations of citric acid. Thus there is little, if any, transfer of isocitrate to a-oxoglutarate. Finally, a comment about a-oxoglutarate dehydrogenase. Synthesis of the enzyme in A. nip is repressed by glucose and by increased ammonium ion concentration. In citric acid fermentation the latter is above physiological levels; thus a-oxoglutarate dehydrogenase is inactive. Draw your own version of the reactions from pyruvate onwards to incorporate your understanding of the TCA cycle in A. nip during citric acid formation. The diagram looks very promising in terms of citric acid formation in that a-oxoglutarate dehydrogenase is inactive, isocitrate dehydrogenase has very low activity and aconitase equilibrates 90% towards citric acid. What problem would you expect the organism to have if the scheme shown in n the diagram (response to SAQ 5.2) was operating? The answer is that not only is thew no apparent regeneration of oxaloacetate; there is seemingly no way of producing any of the TCA cycle intermediates from succinyl CoA through to oxaloacetate. Ori@y it was thought that the glyoxylate cycle was important in xegenerating at least some oxaloacetate in A. nip. However it is now known that there is a particularly active pyruvate carboxylase which operates in glucose grown A. nip. ?Iris enzyme carboxylates pyruvate to oxaloacetate and is not subject to metabolic regulation (it is a constitutive enzyme), therefore: carboxyhse 128 Chapter 5 The oxaloacetate and acetyl CoA can both be generated if necessary in a 1 to 1 ratio to produce citrate without loss of carbon dioxide. n pyruvate to produce oxaloacetate? The answer is that there is now a mechanism by which all of the other TCA cycle intermediates from oxaloacetate to succinyl CoA can be produced (all of these reactions are reversible). There are several apparent problems which we still need to resolve in this section before we have a better understanding of citric acid production. Let us consider Figure 5.3 again. Both pyruvate kinase and citrate synthase (enzymes III and V) are inhibited by elevated ATP concentrations. During citric acid production ATP concentrations are likely to arise (ATP produced in glycolysis) and either of these enzymes could, if inhibited, slow down the process. In fact all of the evidence suggests that both enzymes are modified or controlled in some way such that they are insensitive to other cellular metabolites during citric acid production. We have indicated earlier that phosphofructokinase is the major regulator of glycolysis. It is inhibited by high citrate and ATP concentrations and stimulated by high AMP and ADP concentrations. During citric acid production the cell would normally have low AMP + ADP and high citrate plus ATP levels. Phosphofructokinase should, therefore, under ‘normal’ conditions be inhibited during citric acid production. However, research has shown that this enzyme becomes insensitive to these cellular metabolites when elevated levels of ammonium ions are present. Such conditions are brought about during manganese deficiency. We have not mentioned manganese (h4n2’) before in this Chapter but its presence, or rather its absence, is crucial to successful citric acid production. Manganese deficiency in A. nip causes: lowering of the TCA cycle enzymes (synthesis of enzymes repressed); a severe limitation of protein turnover; elevated ammonium ion levels; changes to the cell surface - altering permeability and also the shape of the hyphae. Look at the answer to SAQ 5.2. Can you see another advantage of carboxylating citrate SYnhse pyruvate kinase phosphofruct- kinase manganese hfiamCY The large scale production of organic acids by rnicro-organisrns 129 Figure 5.4 summarises the changes occuring in A. nip in citric acid production mode when compared to conventional metabolism. It is worth studying the Figure for som time because it explains some of the features necessary for a successful fermentation p'ocess. Figure 5.4 Changes in certain allosteric enzyme sensitivities of A. nber in citric acid producing mode compared to general metabolism. Conditions required for citric acid accumulation: manganese deficiency; ferrous ion deficiency; low pH; high concentration of glucose. There is one, final, major problem to be addressed. Compare Figures 5.3 and 5.4. n Can you identify exactly what still has to be resolved? 130 Chapter 5 The answer is that many of the enzymes are sensitive to elevated NADH concentrations. Glycolysis and the bridging reaction will produce NADH in a relatively high quantity. If we suggested that a 'simple' answer is - "that all of the relevant enzymes are insensitive to NADH levels during citric acid production" - would this solve the problem? rI Looking at Figure 5.3 in isolation it would appear to be a satisfactory solution. However in practice it is not sufficient. Remember that there is only a finite amount of NAD+ and NADH in cells. Therefore the NADH has to be continually reoxidised to NAD+ making the latter available for continued operation of glycolysis. Can you remember the name of the two processes involved in the oxidation of n NADH? What is the relationship between these two processes? The two processes are electron transport and oxidative phosphorylation. NADH is mxidised by the process of electron transport using the electron transport chain and the energy released from this process is harnessed by oxidative phosphorylation to generate ATP. We noted earlier that the two processes are intimately linked or coupled. Normally one cannot proceed without the other. During citric acid production there is massive generation of NADH but little demand for ATP. Thus the situation could quickly arise where there is no further ADP available for oxidative phosphorylation within the cells. This means that the electron transport chain cannot operate and no further oxidation of NADH can OCCUT. It has been shown however that A. niger whilst producing citric acid is unusual in that it operates two separate electron transport chains (Figure 5.5). One is very conventional, similar to that in other organisms; it is tightly coupled to oxidative phosphorylation and inhibited by antimycin and cyanide. The other, still not completely understood, is not linked to oxidative phosphorylation and is inhibited by salicyl-hydroxamic acid (SHAM). Thus oxidation of NADH can occur independently of ATP synthesis; the energy generated is not conserved but is lost as heat energy. ma ekdm mvrt *ains Figure 5.5 Respiratory chains in A. niger. SHAM = salicyl-hydroxamic acid; Fp = flavoprotein; - = inhibition; x and y are unidentified components. The overall reaction for the metabolic conversion of glucose to citric acid is: glucose + 05 02 + citric acid + 2H20 The large scale production of organic acids by micmrganisms 131 Thus, in theury at least, there are two substrates for citric acid production: glucase and oxygen. The latter has to be supplied at all times because if oxygen supply is interrupted, even for a few minutes, then citric acid production drops dramatically and does not recover, even after a rapid resumption of the oxygen supply. Intemhgly, pwth is unaffected by a transient cessation of oxygen supply. It has been shown that the conventional electron transport chain is associated with normal growth mechanisms but the second, special electron transport chain, is specifically involved with citric acid production. Insert the missing words from the list below into the following paragraph, which describes the mechanism of citric acid accumulation in A. niger. A large intracellular pool of a) ions (caused by a negative effect of sevexe limitation of b) on protein turnover) and an inmased respiratory activity, which in part is not coupled to rl synthesis, stimulates metabolic flux through glycolysis without significant metabolic control. This, together with d) pyruvate carboxylase and the peculiarities in the operation of the TCA-cycle, results in elevated cellular concentrations of _el. This in turn enhances citric acid accumulation by inhibiting R dehydrogenase. Word list: isocitrate, constitutive, ammonium, ATP, manganese, citrate. effedofa srarvation For each of the following enzymes in A. nip, select metabolites from the list provided that either inhibit or stimulate activity during a) balanced growth and b) during citric acid accumulation (caused by Mn2' deficiency). enzyme balanced growth citric acid accumulation phosphofructokinase pyruvate kinase pyruvate carboxylase pyruvate decarboxylase citrate synthase isocitrate dehydrogenase metabolltes: inhibits stimulates inhibits stimulates ATP ADP AMP GTP citrate glucose-6- phosphate In summary, and regarded at its simplest, A. nip degrades suitable carbon sources by glycolysis which is occurring at an uncontrolled rate. There is inevitably an overflow at the pyruvate level which is converted to citric acid. 1 32 Chapter 5 5.3.4 Medium requirements and environmental factors involved in citric acid production An understanding and awareness of the biochemistry of citric acid production has given us the opportunity to predict suitable medium and environmental conditions. The substrate must obviously be either glucose, sucrose or a mixture of simple compounds such as molasses. It is important that the A. niger can obtain glucose readily and rapidly. Many polysaccharides are not suitable here because the rate at which they are degraded becomes limiting. The optimal concentration of sugars in the medium seems to be of the order of 15 - 25%. One of the possible problems of using the complex mixtures is the presence of unwanted materials such as certain metal ions, particularly manganese and iron. In addition, inhibitory compounds such as acetic acid may be present. AU need to be removed before fermentation can proceed. Nitrogen is normally supplied as an ammonium compound in citric acid fermentations and suffiaent has to be supplied to enable the effect of manganese deficiency (increased levels of ammonium in the metabolic pool) to occur. Remember that ineased metabolic pool ammonium has the effect of releasing the allosteric controls exerted on phosphofructokinase. In the presence of sufficient metal ions such as zinc, phosphate deficiency is known to inhibit growth and increase yields of citric acid. However, phosphate is added not only as a source of phosphorus but also as phosphoric acid to acidify the medium. Restricted growth but good citric acid yield is also achieved by maintaining iron and zinc deficiency; hence low phosphate levels are not necessary. Trace metals have to be removed, notably manganese, ferrous ions and zinc. This is often accomplished using the compound potassium hexacyanoferrate which precipitates or complexes the metals and, in excess, acts to inhibit pwth and indiredy promotes citric acid production. The amount of potassium hexacyanofemte quid is variable depending on the nature of the ion content of the carbon source. Finally, aeration levels have to be carefully monitored and maintained continuously. Remember that it is particularly important that oxygen supply is not intempted, not even temporarily. List the main ingredients and properties of a possible growth medium together with any comments regarding optimal environmental conditions. Try to give brief reasons for your choices. 5.3.5 The fermentation process Currently citric acid is still manufactured almost entirely using A. nip growing on carbohydrate substrates. Three types of fermentation process have been used: the Japanese Koji process; the surface culture process; the submerged culture process. The Koji process The use of the Koji process is very restricted and apart from this paragraph, it will not be discussed further. The substrates are either starch or the residue of sweet potatoes which are placed in shallow trays. Water is added to a 70% weight ratio. Steaming of glucose unwanted meta'ions nitrogen phosphate potassium hexacyam- lerrate bji process The large scale production of organic acids by micro-organisms 133 the mixture yields a pasteurised paste which is inoculated with A. niger. The initial pH is 45 and an incubation temperature of 30°C is used. The pH drops to 2.0 during citric acid production and the process is completed, optimally, in four days. Citric acid is obtained as an aqueous solution from crushed mycelial/substrate residue and purified by conventional means, as will be discussed shortly. The advantage is that the process time is short and local resources can be used. However, the yield is low and it has proved difficult to remove effectively the trace metals from the substrate economically. Reliance therefore has been placed on obtaining A. niger strains which accumulate citric acid in the presence of such elements - so far with limited success. The surface culture process A flow diagram of the process is presented in Figure 5.6. The surface culture process is so called because the fermentation takes place in shallow trays. The trays are conventionally stacked up to ten high and several stacks may be pIaced together in the same room. The requirements for the plant are simple: limited cleaning of the room to produce aseptic conditions at the beginning and a facility to pass warm, moist air over the trays. surface culture P~~~ Figure 5.6 The surface culture process. The raw substrate, usually molasses at 20 to 25%, together with added nutrients is acidified and heated to reduce the level of contamination in the medium Sufficient amounts of potassium hexacyanoferrate are added to precipitate or chelate the trace 134 Chapter 5 metals, together with a controlled excess to function as a metabolic inhibitor. Care is nquired since a vast excess will cause unwanted effects in the fungus and dramatically lower the yields. A prior knowledge of the trace metal content of the starting material is required because the amount of potassium hexacyanoferrate to be added will depend upon it. The trays are charged with the hot medium and, on cooling to N0C, the incxulum is added. Two days are normally required for germination and growth and towards the end of this period the pH drops. These first two days are crucial in a microbiological sense because it is here that problems of contamination are most likely to occur. The process is usually completed within eight days after which a yield of 210 to 250 kg m-3 of citric acid may be obtained, assuming a conversion ratio of 100 g glucose to 75 g citric acid. The submerged culture process The process (outlined in Figure 5.7) is more sophisticated in terms of initial plant requirements and is also more difficult and costly to operate. However, the volumes which can be processed exceed by far those used in the surface culture method. In addition, the relative space required is much less per cubic metre of medium. submwed culture process Figure 5.7 The submerged culture process. The large scale production of organic acids by micro-organisms 135 The carbohydrate (again often molasses, 15 - 25%) and added nutrients are pH-adjusted to below 4.0 and, for this process, have to be sterilised. It is necessary to add potassium hexacyanoferrate but greater care is xequired in this process compand to surface culture. The A. nip seems to be more sensitive to and more easily inhibited by hexacyanoferrate in submerged culture. It is essential however to lower the ferrous and manganese concentrations, probably below 200 and 5 pg 1" respectively, to optimise the performance of A. nip. Conventional stirred reactors with working volumes of 50 to 150 m3 have beenpsed routinely for citric acid production whereas tower bioreactors, currently 200 m and larger (greater than 600 m3) are envisaged. Inoculation is by conidia of A. nip or alternatively using pre-cultured mycelial pellets. Broken mycelial masses are slow to grow initially and are unsuitable here. Two to three days are required for germination during which heat input to maintain 30°C is mpired. During citric acid formation, cooling is necessary. The process is complete after 10 - 12 days with yields of up to 125 kg m-3. working vd~~mes Can you think of a reason why heat generation during citric acid accumulation is n relatively high? The reason for this is that reoxidation of NADH via the alternative electron transport chain {not coupled to oxidative phosphorylation) liberates heat. In summary, the Koji process serves as a small scale, relatively localised and specialid process designed largely to take advantage of the available carbohydrate source. The surface culture technique is undoubtedly easier to perform and cheaper to install. The restricted volumes of the system are, however, a disadvantage. The submerged culture process continues to increase in terms of the percentage of citric acid produced compared to that produced by the surface culture method. Tower bioreactors are preferred over stirred reactors because they cost less, there is less risk of contamination and they are less limited by sue. Continuous culture is not considered suitable for citric acid production; the requirement for a multi-tank system to separate growth and product formation would make the process uneconomic. 5.3.6 Downstream processing After fermentation, large volumes of spent medium containing citric acid and mycelium remain. comparison of pro^^^ Where do you think the citric acid will be located in the liquid culture? The correct answer here is in both the medium and the mycelium. It is known that some 15% may remain in the mycelium immediately after harvesting. Usually the citric acid outweighs the biomass by a ratio of 5 : 1. The initial task is to remove the mycelium from the medium, a process usually camed out by rotary filtration. The wet biomass is crushed and rewashed to obtain most of the 15% citric acid contained within and the washings are added to the spent medium. The next stage is to precipitate the soluble citric acid as insoluble calcium citrate using mWfih~m calcium hYdmXj* calcium hydroxide. 136 Chapter 5 Care has to be taken to ensure that the calcium hydroxide is very low in n magnesium salts. Can you think of a reason for this? The answer is that the objective is to precipitate out all of the citric acid as insoluble calaum citrate. Magnesium citrate is very soluble and would, therefore, be lost in the aqueous phase during the next separation. The insoluble calaum citrate is dissolved in sulphuric acid yielding soluble citric acid and insoluble calcium sulphate and other calcium salts. The solution is then evaporated, aystallised as necessary to pudy it, centrifuged, washed and dried; this leaves purified citric acid. Downstream processing recovers up to 95% of citric acid in the culture. The process is The final product is marketed as an anhydrous crystalline or monohydrate crystalline compound available as a powder or in granular form. percentage r-erY summarised in Figure 5.8. ~ ~ ~ ~ ~ ~ ~ ~~~ Figure 5.8 Downstream processing in citric acid production. The large scale production of organic acids by micro-organisms 1 37 Match each of the following statements with a process for citric acid formation using A. niger. Statements 1) The process produces the highest yields of citric acid. 2) The process is complete within four days. 3) The starting pH of the medium is less than 4.0. 4) Nutrients are sterilised prior to inoculation. 5) The substrate is starch or the residue of sweet potatoes. 6) The medium is acidified with phosphoric acid to pH 6.0. 7) Cooling required during citric acid accumulation. Processes The surface culture process. The Koji process. The submerged culture process. 5.4 The production of other TCA cycle intermediates In addition to citrate, there are other acidic intermediates of &e TCA cycle and several of these are used in industry. Even though some have been produced by fermentation, production is currently from other, cheaper sources. 5.4.1 Fumaric acid Fumaric acid is used in the plastics industry, in the food industry and as a source of malic acid. Although demand has inmased rapidly over the last 30 years its production from fermentation has been totally replad by a chemical method. It is now produced far more cheaply by the catalytic oxidation of hydrocarbons, particularly benzene. With the continuing uncertainties concerning the availability and cost of petroleum, however, fermentation may yet be a viable alternative. Fumaric acid can be produced in high yield by several genera of fmg, notably AspergzZZus and Rhizopus, using glucose, corn steep liquor and other substrates. Large amounts of calcium carbonate have to be added to neutralise the fumaric acid. Several Crmdida spp. will metabolise n-hydrocarbons to produce fumaric acid; the exact process is not fully worked out although glycolysis and reverse TCA are central features of the biochemistry of the process. 5.4.2 Malic acid Malic acid has a limited use in tkie food industry as an acidlfylng agent where it is an alternative to citric acid. In nature, only L(-) malic acid is found whereas the relatively cheap, chemical synthetic methods yield D/L mixtures. The favoured industrial way to produce the L(-) acid is by enzymic transformation from fumaric acid. Either whole cells or isolated and immobilised enzymes can be used, with high conversion efficiencies. fumaric acid malic acid 138 I SAQ5-7 Chapter 5 In the late 1970's attention again turned to microbial fermentation processes. Several Candida spp. were shown to produce high yields of malic acid from n-paraffins. The problem is that large amounts of both malic acid and succinic acid are produced, and only malic acid is desired as SUCC~M~~ can be produced very cheaply by chemical means. Careful adjustment of media conditions has enabled cmrdida hmpfii to produce 0.88 g malic acid per g of n-paraffin, yielding 25 g per litre. Growth occurs over two days followed by a four day product formation phase. Biochemically, the glyoxylate cycle is very important during malic acid production, channelling isocitrate and acetyl CoA to two malate molecules in the presence of a much reduced complete TCA cycle. Draw a simple sketch diagram (refer to Figure 5.2 if you need help) and then, by using equations, show that the following statement is correct: 'the glyoxylate cycle and part of the TCA cycle are important in malic acid production'. 1 5.5 The industrial production of itaconic acid 5.5.1 Introduction Itaconic acid was discovered in 1929 as a metabolite of an Aspergillus species which was subsequently named Aspergillus itaconicus. A short time later A. fmeus was shown to accumulate the acid and one particular strain of this, superior to all others, is still the current industrial producer of itaconic acid. The compound itaconic acid - or methylene succinate - is a substituted methacrylic acid having the structural formula: CH2 = C - COOH I CH2 I COOH It is used largely in the plastics and paints industries and, when used at the 5% level in acrylic resins, it imparts the ability to hold printing inks. It is sold as the free acid in two grades: industrial grade or the purer, more expensive, refined grade. 5.5.2 The biochemistry of the process Originally itaconic acid was produced chemically by the pyrolysis of citric acid. The treatment results in water loss converting citric acid to aconitate. Subsequent decarboxylation of the latter gives two isomers, itaconic acid and citraconic acid. The p'ocess was not very successful, partly because succinate and itatartaric acids were also It was originally thought that microbial fermentation by AspersllZus terreus followed a similar pattern in that citric acid - produced by glycolysis plus the bridging reaction and the first reaction of the TCA cycle - was converted to aconitic acid. This compound was then decarboxylated to itaconic acid. This pathway is the one to the right of Figure 5.9 (alternative B). However, further studies of the process showed that the left hand scheme (alternative A) was also a possible metabolic route to itaconic acid. In the latter the presence of a condensing enzyme which combines pyruvate and acetyl CoA to yield citramalic acid has been confirmed. Subsequent conversion to itaconic acid via citraconic acid and itatartaric acid has been demonstrated. produced. The large scale production of organic acids by rnicro-organisrns 1 39 Figure 5.9 Two routes of formation of itaconic acid. The effects of copper ions on the process were elucidated as follows. It was known that copper ions inhibit aconitase activity. copper ions What would be the effect on itaconic acid yield of adding excess copper ions if 1) scheme B was the mapr pathway and 2) scheme A was the major pathway (see Figure 5.9)? 1) With B as the mapr pathway the yield would fall dramatically if aconitase was totally inactivated. 2) With A as the major pathway, it is reasonable to assume that there would be no effect on itaconic acid production since aconitase is not involved. In fact it was shown that the presence of copper ions inmases the yield of itaconic acid by a factor of up to 3. These facts indicate that the mapr or only pathway involved in itaconic acid production is pathway A. The empirical formula for glucose is C6H1206 and for itaconic acid it is C5H604, so itaconic acid production is another ’aerobic fermentation’ and requires aeration. Cd-hfl6 + 1.502 + fiH60.t + COz + 3HzO Like in citric acid production relatively modest aeration rates are Tequired. Stopping aeration for literally a minute or so will permanently arrest product formation. n 140 Chapter 5 5.5.3 The fermentation process sugar beet molaasses 3 day fermentation filtration and acidification Much of the details of the process remains in the hands of the manufacturers but an overall description will be presented here. Conventional stock cultures of A. terreus are maintained and, when required, are germinated in large volumes of sugar beet molasses (15% sugar) medium. Continuous aeration for 18 hours and incubation at 33 - 37°C ensures germination into mycelia of a suitable biochemical type. By the time of inoculation of the fermenting vessel the pH will have dropped from 75 to around 4.0. Inoculation using one fifth of the volume together with maintenance of a higher temperature, 45"C, reveals a pH change of 4.1 down to 3.1 over the next 24 hours. Fermentation is allowed to continue for a further 2 days during which the pH will be brought back to pH 3.8 using either calcium hydroxide or ammonia. If ammonia is used, ammonium itaconate is found in the purified free acid product; this is an irritation rather than a problem and is preferable to addition of calcium hydroxide. The reason for this is that the resulting calcium itaconate, which is relatively insoluble, would stick to the mycelium and increase aeration problems. Throughout the fermentation vigorous aeration with agitation is required. 5.5.4 Downstream processing A simplified scheme is shown in Figure 5.10. The fermentation mixture is filtered to remove mycelium and suspended solids. The solution is treated with hot carbon and then filtered. During the process acidification is necessary to reverse the neutralisation by calcium hydroxide or ammonium hydroxide employed during fermentation. The large scale production of organic acids by micro-organisms 141 Figure 5.10 Downstream processing in itaconic acid production. Any of the four products can be esterified as required. The filtrate following carbon treatment is evaporated, allowed to cool and crystallised. This yields refined grade itaconic acid. The solution may be further evaporated bo yield a second sample - this time designated industrial grade. Industrial grade samples can also be made directly by evaporating the fermentative filtrate and thus avoiding the activated carbon step. Finally the mother liquor can be extracted by solvents (such as n-amyl alcohol) or passed through a suitable ion exchange resin. Elution is by mineral salts to yield a crude grade mixture. If itaconic acid esters are required, esterification of any of the grades can be camed out yielding esters of varying punty. Give three reasons why it is believed that the formation of itaconic add in A. terreus is via a route which does not involve TCA cycle intermediates. 142 Chapter 5 one-sbp and WfCStEtQ reactions submerged pro-= sodium gluconate sequastering aemt calcium and iron deficiencies 5.6 The industrial production of gluconolactone and gluconic acid 5.6.1 Introduction The production of gluconolactone and gluconic acid are, in a biochemical sense, the simplest of the processes studied in this Chapter. From glucose they are only one-step and two-step reactions respectively. Their production by bacteria was first detected in 1880 and gluconic acid was then demonstrated in fungal culture filtrates in 1920. Aspergzllus nip was found to produce large quantities of gluconic acid, particularly when the acid was neutralised by the addition of calcium carbonate. Investigation and use of surface cultures and various types of submerged cultures occurred. Finally, a submerged method using sodium hydroxide to neutralise the acid produced was developed. Currently only submerged processes are used commercially. It is interesting that even though the conversion is very simple, a chemical process is not favoured. In fact several different oxidising agents are available but the processes have proved to be too costly and less efficient. The ability of bacteria - particularly Pseudomonas spp. and Gluumobacfer spp. - to produce gluconolactone and gluconic acid has been exploited and the process is used commercially, mainly in the production of the lactone. Whilst world wide demand continues to increase, the requirement is far lower than that for citrate. Gluconic acid is commercially available as a 50% technical grade solution. In addition technical grade calcium and sodium salts of gluconic acid together with gluconolactone are produced. Quantitatively the greatest demand is for sodium gluconate which is a very efficient sequestering agent in neutral or alkaline environments. The hydroxyl groups of the gluconate bind di- and tri-valent metals (for example calcium, magnesium and iron) in soluble form, preventing precipitation. There is an industrial demand for sodium gluconate in the cleaning of glassware, for example bottles, particularly when automated washing equipment is used. In addition it is used for washing walls, metals and other surfaces to remove insoluble metal carbonates. Calcium gluconate is one of the relatively few soluble calcium salts and is used in the pharmaceutical industry as a source of calcium for patients with calcium deficiency. Many drugs are supplied as the gluconate derivatives. Other gluconates such as iron gluconate can be used, in this case to treat iron deficiency. The free acid is a mild aadulant and is used in a variety of foods. It is also used as a cleansing agent, for example in the dairy industry to prevent the build up of 'milk stone'. All of the gluconates can be used as a cement or plaster additive where they retard setting times whilst increasing the strength and water resistance of the materials. Finally gluconolactone - largely produd from Gluconobactm suboxydmrs is used in baking powder and bread mixes and other areas where its effervescent properties may be exploited. The large scale production of organic acids by mkrwrganisms 143 5.6.2 The biochemistry of the production of gluconic acid and derivatives Virtually all living systems have a degradative pathway called the hexose monophosphate pathway which has the initial reactions: glucose -+ glucose - 6 - phosphate -+ 6 - phosphogluconolactone -+ 6 - phosphogluconate The dehydrogenase catalysing the glucose6phosphate to 6-phosphogluconate step is specific for the phosphorylated derivative of gluconate. A similar but independent process involving non-phosphorylated derivatives is found in several fungi, notably AspmgzZZus and PeniciZZium sp and also bad notably Pseudomonas and GZuconobacter spp. In this pathway & glucose is converted to gluconolactone directly by the enzyme &D-glucose: Or oxidoreductase (glucose oxidase). D-glucose + Or > D-gluconolactone + &Or f3 - D - glucose: Or oxidoreductase Glucose oxidase is specific for &D-glucose and where a-D-glucm is the available substrate, prior conversion from the a to the form is required. In this reaction, hydrogen peroxide is produced which is toxic to cells and has to be removed quickly and efficiently. This is carried out by the enzyme catalase. The equation also indicates the need for molecular oxygen and the fermentation process needs a continuous supply of air. Conversion of the gluconolactone to gluconic acid occurs under certain conditions by spontaneous hydrolysis, though a rather specific lactonising enzyme is present in cells. The control of glucose oxidase production is still not fully elucidated, nor is the role of gluconic acid in cells. However, elevated levels of glucose oxidase are found in conditions of high glucose concentrations and above nod oxygen tension. 5.6.3 The fermentation process The first culture technique, reported in 1927, to be attempted commercially was a surfaceculture, shallow-pan technique, though this method has not been used for many years. Relatively soon after this, in 1933, production using a submerged culture technique was reported and this method has been in use continuously since then. Various significant developments have been made, notably the addition of calcium carbonate to neutralise the acids produced in order to increase yields (1937) and the use of sodium hydroxide for neutralisation (1952). Write down the fermentation conditions which you think may encourage optimal n gluconic acid production. There are several conditions that you could have listed which have been mentioned so far in Section 5.6. For example: 1) Perhaps the most important of all is that there should be a high concentration of glucose, between 15 and 35% dependq on the type of fermentation. 144 Chapter 5 2) Secondly the concentrations of the nitrogen source should be carefully calculated and kept relatively low. Ideally the pmss requires minimal pwth and maximum product accumulation. HI& concentrations of nitrogen source would probably encourage far too much growth thus lowering the yield efficiency. 3) Sufficient quantities of other essentials such as minerals must be provided. 4) The pH has to be controlled - the acid which is produced has to be neutralised maintaining a pH in excess of 6.0. Below pH 3.0 the glucose oxidase is inactivated and in fungal systems low pH encourages citric acid production. 5) High aeration rates are required to stimulate glucose oxidase production and activation. Depending on which of the products, calcium gluconate, sodium gluconate or gluconic acid (fwe acid) is requwd, the fermentations have some basic differences. Calcium gluconate Either a spore suspension or mycelium can be used to inoculate the production vessel. The medium contains a maximum glucose concentration of 15%. The upper limit reflects the low solubility of calcium gluconate which is normally about 4% at 30"C, but it can form supersaturated solutions up to about 15% without risk of precipitation. Sterilised calcium carbonate slurry is added in increments to the sterilised medium to maintain pH. Addition of all the necessary calcium &~te at the beginning of the process would irreversibly and adversely affect the inoculum Fermentation usually occurs in a conventional stirred vessel at 3OoC (with cooling) and vigornus aeration. The process from start to finish can take as little as 24 hours thus absolute sterilisation is not crucial. However, several processes reuse the mycelium many times and in these circumstances clean conditions are a minimum requirement. low s'lublw SOdiUm @UCOMk The two main differences between calcium glu~~te and sodium gluconate production are that, in the latter, pH control is performed by addition of sodium hydroxide and the initial glucose concentration is different. pH control by sodium hydroxide is much easier and more precise than pH control using calcium carbonate slurry. Because the sodium gluconate is far more soluble (40% at WC) than calcium g~uconate, higher glucose concentrations can be used to give higher production yields. high glucose ooncentrations Medium containing up to 35% glucose is steam sterilised by a steam jacket around a conventional stirred tank reactor. pH adjustment starts as the dum cools and is maintained at 6.5. Vigorous aeration is again reguired. Inoculation is usually by using a mycelial suspension. Under these conditions a 30% glucose solution can be almost quantitatively converted to sodium gluconate within 36 hours. 5.6.4 Downstream processing Downstream processing to obtain calcium gl~c~nate is different to that employed to obtain either sodium gluconate or the free gluconic acid. The large scale production of organic acids by microarganisms 145 In calcium gluconate extraction, the mycelium and suspended solids are removed by filtration and the resulting filtrate is then usually decolourised by active carbon treatment. After further filtration to remove the carbon the solution is heated and evaporated to a 20% calcium gluconate concentration. On cooling, crystallisation - which may need encouraging by addition of seeding crystals -begins. After a short time the process is complete and the crystals arr removed by centrifugation and washed with cold water. The pure crystals are then dried at 80°C or converted to other end products. Sodium gluconate is easier to extract than the calcium salt. To obtain commercial grades of sodium gluconate the culture filtrate, after filtration to remove mycelium and suspended solids, is simply concentrated to 45% solids, pH-adjusted to 7.5 with sodium hydroxide and drum dried. Purer products may be obtained by active carbon treatment of the hot solution before drying or by carbon treatment and recrystallisation. Free gluconic acid and the gluconolactone may be obtained from a calcium glu~~~te fermentation or, most often, from a sodium gluconate fermentation. Calcium gluconate is treated with sulphuric acid and the calcium sulphate is subsequently remved by centrifugation to leave the free acid. Sodium gluconate is converted to the free acid by ion exchange. Aqueous solutions of gluconic acid are equdibrium mixtures of gluconic acid itself, together with the gamma and delta ladones. The relative concentrations of each of these can be profoundly influenced by the temperature at which crystals are allowed to separate out, a situation aided by seeding with the appropriate required uystal. At temperatures below SC, and particularly between 0 to 4"C, free gluconic acid predominates. Between 30 and 75°C the predominant crystals are mainly delta lactone and above 70°C the principal crystals are the gamma lactone. Identify each of the following statements as true or false; if false give a reason for your response. 1 ) Free gluconic acid can be obtained from calcium gluconate by treatment with 2) During sodium gluconate formation, pH control is by addition of sodium 3) Starting glucose concentrations in calcium gluconate fermentations are caicium @-a@ sodium @-a@ free gluconic add and g'conota*ne Bmma and deb'acroneS sulphuric acid. hydroxide. higher than those for sodium gluconate fermentations. 4) D-gluconolactone is an intermediate in the hexose monophophate pathway. 5) Production of D-glumnolactone from glucose in several fungi is a one-sbep 6) Glucose oxidase is specific for a-D-glucose. process catalysed by glucose oxidase. Draw a flow diagram of downstream ~"xessing for gluconic acid and its derivatives. Commence with "fermentation mixture " and end with the following five products: calcium gluconate, sodium glu~~~te, free gluconic acid, gum lactone and delta lactone. (Use the style of the flow diagram shown in Elgure 5.10). 146 Chapter 5 Summary and objectives This chapter has described the industrial production of organic acids that are intermediate in the TCA cycle (citric acid, malic acid, fumaric acid) or oxidative derivatives of glucose (itaconic acid, gluconic acid and its derivatives). We have seen how an understanding of metabolic pathway regulation and the physiology of mimrganisms can be exploited to 'overproduce' central metabolites. The approach involves careful consideration of medium design and culture conditions, rather than genetic manipulation. Subsequent downstream processing depends on the type of organic acid product and may involve precipitation, evaporation and crystallisation stages. Further purification stages give rise to one or more commercial grades of the desired organic acid or its derivatives. Now that you have completed this Chapter you should be able to: list organic acids from central metabolic pools that are in demand industrially and are produced commercially by micro-organisms; list the reactions of the TCA cycle leading to the formation of organic acids of commercial significance; discuss the mechanisms controlling glycolysis and the TCA cycle; describe in depth the metabolic pressures imposed on selected micro-organisms in order to obtain high yields of citric acid; compare and contrast types of citric acid fermentations; broadly describe the commercial production by fermentation of fumaric acid, malic acid, itaconic acid and various gluconic acid and gluconolactone derivatives. outline schemes for downstream processing of various organic acids.