Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn²⁺ ions has been investigated using high citric acid-yielding, Mn²⁺ ion-sensitive as well as Mn²⁺ ion-tolerant mutant strains of A. niger. In the presence of Mn²⁺ (1.5 mg/l), citric acid production by the Mn²⁺ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn²⁺ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn²⁺ ion-requiring NADP⁺-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn²⁺ ions during fermentation seem to be responsible for the Mn²⁺ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn²⁺.     

Cyclic AMP and citric acid accumulation by Aspergillus niger

Aspergillus niger accumulated citric acid in the medium under certain conditions. Cyclic AMP concentrations of the order of 10^?6M and higher caused an increase in the rate of citrate synthesis. Adenosine, ATP, and cyclic GMP at 10^?3M also stimulated, but were ineffective at 10^?4M. 5′-AMP had no effect while 5′-GMP and guanosine inhibited slightly. ADP showed a 42% inhibition. Theophylline enhanced the cyclic AMP effect. It is proposed that citric acid accumulation by Aspergillus niger may result from abnormal cyclic AMP metabolism.     

Oxalate accumulation from citrate by Aspergillus niger

Carbon-14 was incorporated from citrate-1,5-¹⁴C, glyoxylate-¹⁴C(U), or glyoxylate-1-¹⁴C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-¹⁴C(U) was superior to glyoxylate-1-¹⁴C and citrate-1,5-¹⁴C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC 3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC 1.1.3.1; glyoxylate oxidase, EC 1.2.3.5; NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC 1.2.1.17) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.Abbreviations Used DCPIP 2,6-dichlorophenolindophenol     

The role of the tricarboxylic acid cycle in citric acid accumulation by Aspergillus niger

Summary Determinations of the momentary levels of various intermediates related to the activity of the tricarboxylic acid cycle have been made during citric acid production in high-accumulating (manganese deficient) and lowaccumulating (manganese supplemented) mycelia of Aspergillus niger. During the growth period the levels of almost all TCA cycle acids, with the exception of 2-oxo-acids, were unusually high; during the induction phase of citrate accumulation malate, fumarate, and isocitrate decreased, whereas pyruvate, oxalacetate, and citrate increased. The presence of succinate could not be demonstrated. The interrelations of the momentary concentrations of the intermediates mainly demonstrate a lack in activity of 2-oxoglutarate dehydrogenase, representing a block in the TCA cycle concomitant with a strongly operating glycolysis as a prerequisite for citrate accumulation. Inhibition studies with crude enzyme preparations suggest that an inhibition of malate dehydrogenase by citrate and also inhibition of isocitrate dehydrogenase by citrate and 2-oxoglutarate occur during the production phase as additional factors.     

Aspergillus niger cyclic AMP levels are not influenced by manganese deficiency and do not correlate with citric acid accumulation

Summary The role of intracellular levels of cyclic AMP in the control of citic acid accumulation by Aspergillus niger has been investigated. For this purpose, A. niger was grown in media containing either high (14%, w/v) or low (2%, w/v) concentrations of sucrose, supplemented with 10 M Mn²⁺ (manganese-sufficient) or not (manganese-deficient), to obtain conditions leading to variable citrate accumulation. Citric acid accumulation was only observed in high-sugar, manganese-deficient medium. Intracellular levels of cyclic AMP were significantly higher in mycelia grown on low-sugar media, but were not significantly influenced by the absence of manganese ions. When sucrose in the high-sugar medium was substituted by other mono- or disaccharides, similar intracellular concentrations of cyclic AMP were observed. However, citric acid accumulation was only significant with sucrose, glucose and fructose. It is thus concluded that the intracellular level of cyclic AMP is not causally related to the accumulation of citric acid by the fungus, and —noteworthy — is not affected by manganese deficiency (despite adenylate cyclase reputed to be a manganese-requiring enzyme).Offprint requests to: C. P. Kubicek     

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP⁺) [threo-d_s-isocitrate:NADP⁺ oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH₂COO^?→acetylCoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml^?1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP⁺) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.     

Aconitase and citric acid fermentation by Aspergillus niger.

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.     

Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: II. Sensitivity analysis

Steady state sensitivity analysis of a model of carbohydrate metabolism and anaplerotic synthesis of oxalacetate were, in Aspergillus niger under conditions of citric acid accumulation, carried out. The flux and metabolite concentration control structure of the system obtained shows that the hexokinase/substrate transport step is the main controlling step of the pathway. The quantitative contribution of the other enzyme catalyzed or transport steps are also discussed. These results allow the design of a proper strategy of biotechnological manipulation aimed at improvement of the process. (c) 1994 John Wiley & Sons, Inc.     

Production of citric acid with immobilized Aspergillus niger

Summary The spores of Aspergillus niger were entrapped in calcium-alginate beads and precultivated in growth media with various amounts of nitrogen. During the following citric acid production in shaking cultures an optimum of acid formation and yield was observed after the precultivation with 100–200 mg/l NH₄NO₃. The productivity of the immobilized Aspergillus was found to be 1.5 times higher than in the case of free pellets. The outgrowth of free mycelia into the medium could be provided by increasing the ratio particle-volume: medium volume, using a 1-l air-lift fermenter, by which means the productivity was increased twice as much as obtained in shaking culture.     

Aconitase and citric acid fermentation by Aspergillus niger

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.     

Fate and role of ammonium ions during fermentation of citric acid by Aspergillus niger

Stoichiometric modeling of the early stages of the citric acid fermentation process by Aspergillus niger revealed that ammonium ions combine with a carbon-containing metabolite inside the cell, in a ratio 1:1, to form a nitrogen compound which is then excreted by the mycelium. High-performance liquid chromatography analysis identified glucosamine as the product of the relationship between glucose and ammonium during the early stages of the citric acid fermentation process. Slightly acidic internal pHs, extremely low ammonium ion concentrations inside the cell, and glucosamine synthesis come into direct contradiction with the earlier theory of the ammonium pool inside the cell, regarded as responsible for inhibition of the enzyme phosphofructokinase. At later fermentation stages, when the mycelium is involved in a process of fragmentation and regrowth, the addition of ammonium sulfate leads to a series of events: the formation and secretion of glucosamine in elevated amounts, the short inhibition of citrate synthesis, growth enhancement, the utilization of glucosamine, and finally, the enhancement of citric acid production rates. Obviously, the enzymatic processes underlining the phenomena need to be reexamined. As a by-product of the citric acid fermentation, glucosamine is reported for the first time here. Suitable process manipulations of the system described in this work could lead to successful glucosamine recovery at the point of its highest yield before degradation by the fungus occurs.     

Influence of manganese on enzyme synthesis and citric acid accumulation inAspergillus niger

Summary A comparison of citric acid fermentations in manganese-deficient and manganese-containing media showed that manganese strongly influences idiophase metabolism. In the presence of manganese, cell growth increases, sugar consumption is diminished and acidogenesis decreases drastically. An investigation of the key enzymes of glycolysis, the pentosephosphate pathway, TCA-cycle, nitrogen metabolism, and gluconeogenesis indicated that manganese deficiency was accompanied by a repression of anabolic and TCA-cycle-enzymes with the exception of citrate synthase. The activity of this enzyme and the enzymes of glycolysis paralleled the sugar consumption rate. In the presence of manganese, no repression of enzyme synthesis was observed. Activities of 2-oxoglutarate dehydrogenase and isocitrate lyase could not be detected in either case. The results support the hypothesis that manganese deficiency mainly affects the operation of biosynthetic reactions inAspergillus niger, thus leading to an overflow of citric acid as an end product of glycolysis.     

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.     

Relationship between citric acid production and accumulation of phytate-degrading enzymes in Aspergillus niger mycelia.

The ability of eight strains of Aspergillus niger to produce citric acid by the solid surface method were found to correlate with their capabilities to synthesize intracellular enzymes which degrade phytates (phytase and acid phosphatase). Another high correlation was observed between phytase and acid phosphatase activities bound to the cell walls of mycelia.     

Production of citric acid from starch-hydrolysate by Aspergillus niger

The present investigation explored the possible use of a rarely used agro-industrial by-product, maize starch-hydrolysate, for economic production of citric acid. To achieve this, seventeen strains of Aspergillus niger were screened for their capacity to produce citric acid using starch-hydrolysate as a substrate. The most efficient strain, ITCC-605 was selected for further improvement in citric acid content by mutation. Mutants developed by treatment with EMS and UV, singly and in combination, produced citric acid in the range of 0.51-64.7 g kg(-1) of glucose consumed. The mutant UE-1 produced the maximum citric acid which was about 130 times more than that produced by the parent strain, ITCC-605. For further increase in citric acid production from this substrate, the cultural conditions were optimized: concentration of starch-hydrolysate, 15% (glucose equivalent); ammonium nitrate, 0.25%; KH2PO4, 0.15%; nicotinic acid, 0.0001% and initial pH of 2.0. Under these conditions, the mutant strain UE-1 yielded 490 g citric acid kg(-1) of glucose consumed in 8 days of incubation at 30 degrees C. The productivity of 341 mgl(-1)h(-1) corresponded to 49% substrate conversion to citric acid.     

Parasexual hybridisation and citric acid production by Aspergillus niger

Summary In this investigation hybridisation experiments were performed with two auxotrophic mutants of Aspergillus niger. A heterozygous diploid was derived from them and it produced segregants including parental haploids and a recombinant. Their yield characters were studied.     

A drop of intracellular pH stimulates citric acid accumulation by some strains of Aspergillus niger

By comparing kinetic parameters of plasma membrane proton pumps from two Aspergillus niger strains, significant differences in specific activities were observed. In low citric acid producing A158 strain the H+ -ATPase activity was about four-fold higher than in a high yielding A60 strain. Previously pH homeostasis was reported in A158 strain while in A60 strain spontaneous drop of intracellular pH was observed. During the growth in the medium with ammonium ions more rapid drop of extracellular pH was recorded with A158 strain and not so fast proton accumulation in the medium with A60 strain, indicating that proton pumps from later strain perhaps can not extrude all the protons that are released in the cytosol after the assimilation of ammonium ions. Vanadium ions were found to be potent inhibitors of both H+ -ATPases. By adding sodium vanadate in millimolar concentrations to the chemically defined medium that induces citric acid accumulation by A. niger, reduced pHi and increased rate of acid production was observed in A158 strain while in A60 strain intracellular pH decreased below 6.5 and concomitantly citric acid overflow was suppressed. The presented results suggest that one of the mechanisms stimulating citric acid accumulation by A. niger could be also a slight cytoplasmic acidification.