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.     

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.     

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⁺.     

Production of citric acid by Aspergillus niger immobilized in polyacrylamide gels

Summary Living Aspergillus niger cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column bioreactors to produce citric acid from sucrose. With the replacement batch bioreactor, increase of citric acid was observed under conditions of higher aeration and of wider surface of immobilized cells. With the continuous bioreactor, the maximum citric acid yield was 39.1 mg/h per 40 g gels. The biocatalyst activity or half-life was 105 days.     

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.     

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.     

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.     

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.     

Citrate transport during the citric acid fermentation by Aspergillus niger

Summary Aspergillus niger SZ was grown under citric acid accumulation conditions in a 1l bioreactor. Radioactive citric acid was added during the fermentation to the culture medium. The accumulation of radioactive citric acid in the mycellium was measure by measuring radioactive CO₂ in the offgas. Evidence was obtained of significant turnover and metabolism of exogenous citric acid even during the phase of maximum accumulation into the medium.     

Production of citric acid by immobilized dried reactivatedAspergillus niger

Summary Immobilization followed by drying was fried as a new technique for obtaining small beads which might be more suitable for industrial fermentations and for the storage of the immobilized microorganisms.Aspergillus niger NRRL 2270 was used to produce citric acid in free, immobilized, and immobilized dried reactivated (IDR) forms. The productivity based on the bead volume used increased several folds with the use of IDR cells although the absolute level of citric acid did not increase.     

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.     

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.     

Production of thermostable acid protease by Aspergillus niger

Aspergillus niger F2078 produces high levels of extracellular thermostable acid protease within 96 h. Although glucose and peptone were the best carbon and nitrogen sources, respectively, sucrose and a cheap nitrogen source, corn steep liquor, also gave satisfactory enzyme yields. Supplementation of groundnut meal to the basal medium enhanced enzyme production. Temperature and pH optima of the enzyme activity were 60°C and 3.0–4.0, respectively. The enzyme was stable between pH 3.0 and 6.0 and at temperatures up to 60°C.     

Improved production of citric acid by a diploid strain of Aspergillus niger

Aspergillus niger strain CGU 87 was treated with UV radiation and some auxotrophic mutants were obtained. These mutants were less productive than CGU 87, which produced an average of 7.4% citric acid. All possible crosses in pair wise combinations were carried out between these auxotrophs, and three heterokaryons were synthesised. Finally, one heterozygous diploid was isolated from each of them. These heterokaryons and diploids showed improved productivity when compared with their component parents, but except in one diploid D5, all others produced less citric acid than CGU 87. The yield of D5 exceeded that of CGU 87 by 1.2 times and it produced 9% citric acid. This is a significant improvement and the increased productivity seems to be the result of successful adaptation of D5 to its fermentation environment.     

Effect of some metabolic inhibitors on citric acid production by Aspergillus niger

Stationary cultures of Aspergillus niger grown on a synthetic medium have been used to study the effect of some metabolic inhibitors on citric acid production. Addition of 0.05 to 1 mM sodium malonate or 0.01 to 0.1 mM potassium ferricyanide, iodoacetate, sodium azide, sodium arsenate or sodium fluoride stimulated citric acid production (3.6 to 45%), but not total titratable acids. Addition of higher concentrations (0.2 to 10 mM) of later inhibitors caused a marked inhibition of fungal growth and citric acid production. The implications of these preliminary findings are discussed.