Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.     

beta-Galactosidase from Aspergillus niger. Separation and characterization of three multiple forms.

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.     

Citric acid production in submerged and solid state culture of Aspergillus niger

In this work, the citric acid production in solid state culture was performed, evaluating the isolated effect and interactions of particle size and liquid phase employed, by means of the factorial design of first order. The results indicate that the particle size is the most determinant variable. An analysis comparing submerged and solid state in optimal conditions was performed. When solid state culture was used, the productivity of citric acid was doubled, reducing the fermentation time from 14 to 6 days, compared to the submerged culture, obtaining a maximum citric acid concentration of 21.24 g/l.List of Symbols B _s , B _v main effects - B _sv crossed effects - s cm particle size - S coded particle size - v ml liquid phase volume - V coded liquid phase volume     

Citric acid production by Aspergillus niger in surface culture on inulin

Aspergillus niger produces citric acid during surface fermentation on inulin, a reserve carbohydrate of plant tubers. Citric acid yields can be improved by airflow over the surface of the fermentation but yields from inulin are 20–30% lower than from sucrose, the traditional commercial substrate.     

Conidiation of Aspergillus niger in continuous culture

Summary Conidiation of Aspergillus niger was studied in carbon-limited and nitrogen-limited chemostat culture. Under citrate-limitation conidiation intensity varied inversely with dilution rate. Conidiophores were less complex than in aerial conidiation and at high dilution rates conidia occasionally developed from modified hyphal tips. Conidiation was difficult to achieve under glucose-limitation. At the low dilution rates that allowed limited conidiation steady state could not be maintained due to onset of autolysis. At higher dilution rates when steady state was readily obtained conidiation did not occur. The maximum yield constants under citrate-limitation and glucose-limitation were respectively 0.145 and 0.4 mg dry weight/mg substrate, while the relative specific maintenance values were 0.045 and 0.018 mg substrate/mg dry weight/h. Under ammonium-limitation with citrate as the carbon source there was no conidiation. When nitrate became the limiting nitrogen source conidiophore initiation occurred but biomass production was low and wash-out occurred at D=0.034 h^-1.     

Protoplast fusion between Aspergillus oryzae and Aspergillus niger in relation to their multinuclear nature

Protoplasts of cycloheximide-resistant strains from Aspergillus oryzae IFO 5239 were fused with those of kabicidin-resistant strains from Aspergillus niger IFO 4407. By nuclear staining in conidia, it appeared that all of the fusant conidia had two kinds of nuclei. Small nuclei seemed to be derived from A. oryzae and large nuclei seemed to be derived from A. niger. However, three types of antibiotic resistance were shown among the conidia of fusants. Almost all were kabicidin resistant. Conidia of fusants were multinuclear and had various DNA contents and various sizes. By the comparison with the growth rates of parental strains, the growth rates of A. niger were superior to those of A. oryzae. The inclination in the distribution of antibiotic resistance of fusant conidia seemed to owe more to the differences of growth rates between parental strains than the influence of the multinucleate nature of a parental strain.     

Formation of autodiploid strains in Aspergillus niger and their application to citric acid production from starch

Autodiploid strains were induced by colchicine treatment of Aspergillus niger WU-2223L, a citric acid-producing strain. In shaking culture, a representative autodiploid strain, L-d1, yielded higher citric acid than the parental strain, WU-2223L. When glucose was used as a carbon source, L-d1 and WU-2223L produced 67.2 g/l and 62.0 g/l of citric acid, respectively, from 120 g/l of glucose in 9 d-cultivation. Furthermore, the autodiploid strain L-d1 produced 49.6 g/l of citric acid, 1.4 times as much as that produced by WU-2223L from 120 g/l of soluble starch. During the whole period of cultivation with starch, the extracellular glucoamylase activity of L-d1 was on the same level as that of WU-2223L, but the extracellular acid-protease activity of L-d1 was much higher. The addition of pepstatin, an inhibitor of acid protease, to the culture broth at 2 d greatly increased the extracellular glucoamylase activity, and citric acid production by L-d1 reached a level of 59.0 g/l. During several subcultivations on both minimal and complete agar media, the autodiploid strains were genetically stable since they formed diploid conidia in their uniform colonies without producing sectors, and maintained citric acid productivity. However, when cultivated on minimal and complete agar media containing benomyl as a haploidizing reagent, the autodiploid strains readily formed sectors of haploid segregants. The properties of the haploid strains obtained by the benomyl treatment of the autodiploid strains were similar in morphology and citric acid productivity to those of the parental strain, WU-2223L. These results indicated that the enhanced production of citric acid from soluble starch by the autodiploid strains was due to autodiploid formation but not to gene mutation caused by the colchicine treatment.     

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.     

Effect of cultivation and storage pH on the production of multiple forms of polygalacturonase by Aspergillus niger

Summary The production of multiple forms of extracellular polygalacturonase by Aspergillus niger is directly dependent on the pH of maintenance media. The pH of cultivation medium as well as the carbon-source are only secondary factors with limited influence on this production.     

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

Esterase activity of Aspergillus niger in continuous culture

In citrate limiting medium the esterase activity of Aspergillus niger had a maximum value at the lowest dilution rate (D=0.013 h^-1) and at all higher dilution rates progressively decreased in activity. In glucose limiting medium the esterase activity values were always lower than in citrate limiting medium and did not show much variation with varying dilution rate. Electrophoresis of cell free extracts from all dilution rates revealed a multimolecular esterase profile only at D=0.013 h^-1 in citrate limiting medium, which was also the only dilution rate to support good conidiation. The increase in esterase activity at D=0.013 h^-1 was observed cytochemically to occur in the phialides. No cytochemical esterase staining occurred in the vegetative cultures at all other dilution rates.     

Solid-state culture of Aspergillus niger on support

The growth kinetics of Aspergillus niger on a solid support i.e. sugarcane bagasse, impregnated with a liquid glucose medium, were investigated under different culture conditions. The water activity of the medium, the amount of spore inoculum and the support particle size were shown to be critical factors for mold growth. The elevated rates of growth observed with high substrate concentration media demonstrated the feasibility of the method for culturing filamentous fungi.     

Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger

The production of extracellular glucose oxidase in a submerged culture by a number of auxotrophic, 2-deoxy-D-glucose resistant and protease-less mutants of Aspergillus niger was evaluated. Among the auxotrophic strains, no evident dependence was found between the kind of the nutritional requirements and the level of the glucose oxidase activity. However, the majority of auxotrophs, requiring serine or niacin, showed a higher enzyme activity (from 16 to 680%) than the parent strain. The dynamics of the glucose oxidase synthesis by the free and immobilized mycelium of the most active niacin^? mutant of A. niger was also investigated.     

Stimulation of the gibberellic acid synthesis by Aspergillus niger in submerged culture using a precursor

Stimulation of Aspergillus niger in submerged culture using a commonly known precursor, mevalonic acid (MVA), was investigated in terms of growth and gibberellic acid production. Increasing concentrations of MVA up to 60 M enhanced product and growth yields. Above this amount, gibberellic acid yields and growth were gradually decreased.