Sclerotia formed by citric acid producing strains of Aspergillus niger: Induction and morphological analysis

Some strains of Aspergillus niger have been previously reported to produce sclerotia under certain conditions. Sclerotia are aggregations of hyphae which can act either as survival or as sexual structures in species related to A. niger. In this study, we were able to induce the formation of sclerotia in the progenitor of the industrial citric acid producing strains of A. niger, ATCC 1015, and in pyrG mutants derived from it. Sclerotia can be stably formed by ATCC 1015 on malt extract agar medium supplemented with raisins, showing a spatial differentiation of the fungus dependent on the addition and on the position of the fruits into the medium. On other media, including malt extract agar, pyrG auxotrophs also form abundant sclerotia, while the complementation of this gene reverses this phenotype. Additionally, a macro- and microscopical analysis of the sclerotia is reported. Our results show that the sclerotia formed by A. niger are similar to those formed by other fungi, not only in their morphology but also in their ability to germinate and regenerate the organism.     

Formation and location of glucose oxidase in citric acid producing mycelia of Aspergillus niger

Summary The formation and location of glucose oxidase was studied in Aspergillus niger, which was pregrown under citric acid producing conditions. Glucose oxidase could be de novo induced by a shift in pH from 1.7 to 5.5. The induction required the intracellular presence of either glucose or glucose-6-phosphate. Glucose oxidase so produced was rapidly secreted into the medium, which was not due to autolysis.     

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.     

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.     

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.     

Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger

ATP:citrate lyase (EC 4.1.3.8) has been identified in cell-free extracts from the filamentous fungus Aspergillus niger. The enzyme was located in the cytosol. It exhibits an activity at least ten times that of acetate-CoA-kinase (EC 6.2.1.1) during growth on carbohydrates as carbon sources, and is thus considered responsible for acetyl-CoA formation under these conditions. It is formed constitutively and its biosynthesis does not appear to be controlled by changes in the nitrogen or carbon source or type. ATP:citrate-lyase appears to be very labile during conventional purification procedures; a method involving fast protein liquid anion exchange chromatography was thus developed in order to obtain enzyme preparations sufficiently free of enzymes which could interfere with kinetic investigations. This preparation displays commonly known characteristics of ATP:citrate lyase with respect to substrate affinities and cofactor requirements, with the exception that the affinity for citrate is rather low (2.5 mM). No activator was found. The enzyme is inhibited by nucleoside diphosphates, nucleoside monophosphates and palmitoyl-CoA. Regulation of ATP:citrate lyase be the energy charge of the cytosol in relation to lipid or citric acid accumulation is discussed in view of these findings. Present address: Institut für Allgemeine Biochemie, Universität Wien, Währingerstrasse 38, A-1090 Wien, Austria     

Specific effects of mutagens onAspergillus niger producing citric acid

Aspergillus niger C was treated with UV and gamma irradiation (⁶⁰Co), nitrogen mustard and colchicine. Mutagenic treatments resulted in substrains with either increased or decreased citric acid production, while some were unchanged in this respect. Low-yielding strains predominated in all the experiments. Analysis of the superior substrains showed gamma irradiation to be most effective in producing the greatest number of superior mutants. This investigation was conducted in the Microbiology Laboratory, Bose Institute, Calcutta.     

Mathematical modelling and assessment of the pH homeostasis mechanisms in Aspergillus niger while in citric acid producing conditions

In this work we introduce an extended model of the Aspergillus niger metabolism while in citrate production conditions. The model includes many recent findings related to various transport processes. It now considers a new information about the fructose uptake system and the proton and amino acids carriers between cytoplasm and the external medium. It also accounts for recent information about both the malate-citrate antiport between mitochondria and cytoplasm and the dihydrogen citrate ion excretion symport with protons. Finally, the model also accounts for new information about the glycerol-3-phosphate shuttle and pH buffering systems. Provided with this updated representation and after having assessed its quality and dynamic behaviour, we were able to explain the observed pH homoeostasis found in A. niger while in citrate producing conditions. The model also serves to enhance our comprehension of the molecular mechanisms operating in order to keep homoeostasis of pH in A. niger and other fungi, bacteria and yeast of biotechnological relevance.     

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.     

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.     

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.     

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.     

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.     

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.