The large scale production of organic acids by micro-organisms 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 NADH H must be accompanied by oxidative hosphorylation, the process by which atP is produced from ADP. When the cell has high energy charge(high ATP concentration and low IADP AMP] concentrations) en 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 itrate of ADP (and AMP). Thus the inhibition of citrate synthase is gradually ed. In a synthase 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 phospho- thin glycolysis, the main allosteric control is exercised by phosphoru fructokinase complicated enzyme unusual in that its activity is stimulated by one of i (ADP)and inhibited by one of its substrates (aTP). One further point about which will be important to us later: in Aspergillus spp. elevated levels of ions relieve phosphofructokinase of inhibition by citrate a further way in which metabolic control may be exercised is the artificial deprivation aconitase exami 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 world demand The world demand for citric acid around 1900 amounted to some 10,000 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 process and as such the price of citric acid was very high spergillus A major breakthrough in the fermentation process came in 1916-1920 when it was nger found that Aspergillus niger grew well at pH values below 3.5, producing citric acid in days rather than weeks. The faster incubation and highly acid conditions(often below pH20)also served to minimise potential problems caused by contamination
The large scale production of organic acids by micro-organisms 125 atrate synthase Ph=fJbfnrctDkinase 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
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 sing 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 1930s over 80% of the worlds citric acid was produced by fermentation. At present virtually all of the world production comes from this process. By 1981 over industry in the United Kingdom at that time being worth some 20 million per annum, one ten th of the worlds 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 micro-organisn yeasts was always an Aspergillus spp. In the early 1930s it was found that yeasts would produce citric acid from acetate. Since then a variety of yeasts, principally Candida spp has been shown to convert glucose, n-alkanes or ethanol to citric acid with great n-alkanes The realisation that yeasts would produce citric acid from n-paraffins was very attractive in the late 1960s 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 Candida spp and to harvest two useful end products rather than just one. The process has not bee petroleum prices rose sharply and have in fact continued to rise, the n-paraffins are no longer a cheap substrate In summary the majority of the world citric acid production is still via microbial fermentation of carbohydrate substrates( derived from plants) using Aspergillus niger. 5.3.2 Current uses of citric acid Citric acid, being an intermediate of the tca cycle is considered to be non- toxc and ery 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. Increasingly, less pure grades of citric acid are being used to produce citric acid esters which are used as plasticisers in the plastics industry. citric acid is used freely as a builder in detergents and is being used in laundries because it has the antage of being totally 5.3.3 The biochemistry of citric acid production ly we can say that molasses which is converted via problems which have to be overcome What, briefly, are the problems which have to be overcome?
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?