Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

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.     

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.     

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.     

Application of carbon balance to submerged citric acid production by Aspergillus niger

Summary In an air-lift fermenter, the following production was obtained from 125 g sucrose in mineral medium at pH 2.5 : 15.76 g mycelium dry wt, 107.2 g citric acid anhydrous and 0.594 mol CO₂ within 138 h (run I) and 13.72 g mycelium dry wt, 114.28 g citric acid and 0.516 mol CO₂ within 144 h (run II). Initially, the carbon content of consumed sugar and products did not balance. At the end of fermentation, the carbon content of the products was 0.9%–5.5% higher than that of the consumed sugar. For the purpose of the calculations the carbon content in mycelium was accepted as 0.462.The work was a part of Project No 04.11 CPBP, topic No 2.24     

Improved reproducibility of citric acid production in Aspergillus niger

Summary We report the derivation of Aspergillus niger strains having a lower standard deviation in citric acid production by the use of ⁶⁰Co gamma radiation and by the parasexual cycle. On the basis of the results obtained by isolating diploids and segregants, diploidization seems to be sufficient for reducing variability. The implications of these results in terms of improvement of industrial strains are discussed.     

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.     

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.     

Galactose inhibition of citric acid production from glucose by Aspergillus niger

Summary Previous work in this laboratory has demonstrated that although Aspergillus niger can readily utilize galactose, no citric acid is produced from this carbon source (Hossain et al. 1984). Experiments were now conducted where galactose was added at various concentrations to synthetic growth medium containing glucose as carbon source, so that the effect of galactose on citric acid production from glucose could be observed. The results showed that the presence of galactose or a product of galactose metabolism caused inhibition of citric acid production, and also reduced the rate of glucose utilization. Enzyme analyses using mycelial cell-free extracts indicated that galactose interfered with the glucose-repression of the key enzyme 2-oxoglutarate dehydrogenase.     

Application of kaolin to improve citric acid production by a thermophilic Aspergillus niger

Citric acid production by a thermophilic strain of the filamentous fungus Aspergillus niger IIB-6 in a medium containing blackstrap cane molasses was improved by the addition of kaolin to the fermentation medium. The fermentation was run in a 7.5-l stirred bioreactor (60% working volume). The optimal sugar concentration was found to be 150 g/l. Kaolin (1.0 ml) was added to the fermentation medium to enhance volumetric production. The best results in terms of product formation were observed when 15 parts per million (ppm) kaolin was added 24 h after inoculation. With added kaolin, citric acid production was enhanced 2.34-fold, compared to a control fermentation without added kaolin. The length of incubation to attain this product yield was shortened from 168 to 96 h. The comparison of kinetic parameters showed improved citrate synthase activity of the culture (Y (p/x)=7.046 g/g). When the culture grown at various kaolin concentrations was monitored for Q (p), Q (s), and q (p), there was significant improvement in these variables over the control. Specific production by the culture (q (p)=0.073 g/g cells/h) was improved several fold. The addition of kaolin substantially improved the enthalpy (DeltaH (D)=74.5 kJ/mol) and entropy of activation (DeltaS=-174 J/mol/K) for citric acid production, free energies for transition state formation, and substrate binding for sucrose hydrolysis. The performance of fuzzy logic control of the bioreactor was found to be very promising for an improvement ( approximately 4.2-fold) in the production of citric acid (96.88 g/l), which is of value in commercial applications.     

Regulatory aspects of carbohydrate metabolism in relation to citric acid accumulation by aspergillus niger

Fermentative production by the fungus Aspergillus niger is the world's exclusive source of citric acid to be used for the food, pharmaceutical and detergents' industry. The extraordinary high yields and the delicate nutrient requirements of this process have attracted numerous investigations on its mechanism. This review highlights recent results on the regulation of its biosynthesis, emphazising especially (a) the role of pH, (b) the role of type and concentration of the carbon source, and (c) the role of anaplerotic carbon dioxide fixation in the accumulation of citric acid.     

Morphology and citric acid production of Aspergillus niger PM 1

Summary Aspergillus niger PM 1 was grown in a tubular loop and a stirred tank bioreactor. Batch fermentations were performed under various agitation conditions and pH. Citric acid, oxalic acid, extracellular polysaccharides and proteins were assayed. The following morphological parameters were measured: mean perimeter of clumps, mean perimeter of the central core of clumps, mean length of filaments and mean diameter of filaments. Citric acid production and morphology in both reactors were dependent on agitation intensity and pH. The length of the filaments was shown to be the only parameter that could be related to citric acid production in both reactors: the shorter the filaments the more citric acid was produced. However, for the same amount of citric acid produced the morphology of the organism grown in the stirred tank differed considerably from that grown in the loop reactor.     

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.     

Improvement in citric acid production by haploidization of Aspergillus niger diploid strains

Segregation of diploid strains by a haploidizing agent was used to improve citric acid producing strains of Aspergillus niger. Stable diploid strains were obtained via protoplast fusion between two citric acid-producing strains from different genealogies, one for shaking culture and the other for solid culture. Diploid strains were treated by benomyl as a haploidizing agent, and many segregants were obtained. Prototrophic segregants were selected and their haploidy was confirmed by their conidial size and DNA contents. The prototrophic segregants were very variable in their citric acid productivities, some of them better either in shaking culture or in solid culture than both the parental strains. The presence of methanol stimulated citric acid production by the parental and the diploid strains. However, all prototrophic segregants derived from one diploid strain had higher productivities in solid cultures without methanol than in those with methanol.