In this article we will discuss about the production of citric acid by fermentation.
The functioning of the citric acid cycle (TCA cycle) in fungi has been well documented, and citric acid is produced as an overflow product due to the faulty operation of the TCA cycle. The presence of enzymes of the TCA cycle has also been demonstrated in A. niger.
Studies on enzyme content of A. nigerin relation to citrate accumulation and incorporation of 14C from labelled substrates into citrate have indicated the central role of this cycle in this fermentation. Two key enzymes that have been examined in detail in relation to citric acid fermentation are aconitase and isocitrate dehydrogenase.
The activities of these enzymes have been shown to decrease to very low levels during the period of citric acid accumulation while the activity of the condensing enzyme has been found to increase. The final step in the synthesis of citric acid is the condensation of acetyl CoA and oxaloacetate and this condensation of C2 and C4 components is the major route of citrate synthesis.
Direct carboxylation of pyruvate catalyzed by the malic enzyme provides malate which is readily used for citric acid synthesis after being converted into oxaloacetate through malic dehydrogenase. The incorporation of radioactive CO2 into citrate has been determined and the fixation involves two different enzyme systems. One system involves the condensation of CO2 with pyruvate catalyzed by pyruvate carboxylase.
The second system appears to be identical to the phosphoenol pyruvate carboxylase kinase system found in plants and yeasts. It utilizes phosphoenol pyruvate and CO2 but does not require ATP.
The glyoxylate cycle acts as another source of oxaloacetate for citrate synthesis. Acetyl CoA condenses with glyoxylate and the reaction is catalyzed by malate synthetase.
The glyoxylate required for the synthetase reaction is supplied by the isocitritase reaction as shown:
Aconitase, one of the two key enzymes in citric acid production, is sensitive to a high concentration of metallic ions such as Fe++. Restricting the activity of this enzyme during the production stage appears to be the key to success in citric acid production.
Citric acid production and excretion are apparently two different phenomena. Mutants with altered aconitase need not excrete citric acid. There is in fact evidence to suggest this although direct experimental proof is lacking. A mutant of A. niger which is not sensitive to trace metals but sporulates normally, yet produces citric acid.
The overall success in citric acid fermentation using fungi depends to a large extent on the regulation and functioning of the TCA cycle. The TCA cycle is complex and involves several enzymes. The regulation of synthesis if each one of these enzymes and their activities is perhaps under various control mechanisms that are known for controlling enzyme synthesis and unction. We have little information about the control of these enzymes except that the activity of some, such as aconitase, can be regulated by on-trolling trace element concentration.
Yeasts such as Candida are highly aerobic organisms and have a well-developed TCA cycle. The production of citric acid by Candida is generally accompanied by a simultaneous accumulation of isocitric acid. According to Tabuchi et al. (1973), the activities of aconitase and NAD- or NADP-linked isocitrate dehydrogenase do not decrease much throughout the period of citrate fermentation when C. lipolytica is grown in a medium containing glucose or hexadecane.
As in A. niger, addition of Fe increases the aconitase activity with decreased citrate and increased isocitrate accumulation. Addition of inhibitors of aconitase such as fluoroacetate leads to iron deprivation and to citrate accumulation. Also, in monofluoroacetate sensitive mutants an increase in the accumulation of citrate and a decrease in the accumulation of isocitrate have been reported.
The activity of isocitrate dehydrogenase in this organism also determines the amount of isocitrate and citrate produced. Addition of potassium ferrocyanide or thiamin deficiency in a glucose medium leads to a decrease in isocitrate dehydrogenase activity and to an increased citrate accumulation.
Recently, Marchal et al. (1977) have also examined the regulation of the central metabolism in relation to citric acid and isocitric acid production in Saccharomycopsis lipolytica (Candida lipolytica). In this organism, when grown on n-paraffins, citric and isocitric acid accumulation begins when growth ceases due to nitrogen limitation. At the same time, the concentrations of AMP and ADP decrease to a low level.
The activity of NAD- dependent isocitric dehydrogenase which requires AMP for its activity also decreases significantly. This prevents the oxidation of citrate while isocitrate lyase activity is not inhibited. Citric and isocitric acid accumulation occurs since the inhibition of citrate synthase by ATP is inadequate to stop n-paraffin degradation. One can, therefore, conclude that the excretion of citric and isocitric acids probably occurs due to other physiological changes brought about by nitrogen limitation and also by the alteration of cell permeability to these acids.
The involvement of the glyoxylate cycle and CO2 fixation has also been demonstrated in C. lipolytica during citrate accumulation. In this yeast, it appears that isocitrate lyase and malate synthetase, the key enzymes of the glyoxylate cycle, are inducible.
When n-paraffins and acetate are used as carbon sources, oxaloacetate for citrate synthesis is supplied through the glyoxylate cycle. While growing in a glucose-containing medium, oxaloacetate is generated by the CO2 fixation reaction. During active citric acid synthesis, the activity of citrate synthetase is high as compared to other enzymes of the TCA cycle.
Tabuchi and Hara (1974) have reported the production of a large amount of threo-Ds-methyl isocitric acid and trace amounts of 1-methyl citric acid and 2-methyl-cis-aconitic acid by a mutant of C. lipolytica when grown in a mixture of n-alkanes. Methylisocitric acid is produced mainly from the odd carbon alkanes from the terminal C3-residues left after successive removal of C2-fragments by β-oxidation.
Tabuchi et al. (1974) have proposed a hypothetical pathway for partial oxidation of propionyl CoA (formed during β-oxidation of odd carbon alkanes) to pyruvate via C7-tricarboxylic acids in yeast. This proposed hypothetical pathway (methylcitric acid cycle) for citric acid accumulation is given in Fig. 16.4.
According to this scheme, propionyl-CoA formed through the β-oxidation of n-alkanes condenses with oxaloacetate to yield methyl citric acid. This is then isomerized to methyl-isocitric acid. This is further cleaved to yield pyruvate and succinate. Succinate is then oxidized through the TCA cycle to yield oxaloacetate which is then recycled.
A methyl citrate condensing enzyme and a methyl isocitrate cleaving enzyme have been demonstrated in C. lipolytica. On the basis of the methyl citric acid cycle, the glyoxylate pathway, and the CO2 fixation reaction, the hypothetical pathway for citric acid production from n-alkanes as proposed by Tabuchi and Serizawa (1975) is shown in Fig. 16.5.
According to this pathway, 1 mole of odd carbon alkane having (2n + 1) carbon atoms yields (n – 1) moles of acetyl-CoA and 1 mole of propionyl CoA through β-oxidation. Propionyl CoA is then oxidized to pyruvate via the methyl citric acid cycle. Carboxylation of pyruvate to oxaloacetate and condensation with acetyl CoA yields citric acid.
Also, acetyl CoA formed by p-oxidation enters the glyoxylate cycle to yield oxaloacetate, which on condensation with acetyl CoA again yields citrate. Thus 1 mole of n-alkane having (2n + 1) carbon atoms gives rise to (2n + 1)/6 moles of citrate.