Citric acid fermentation from starch and dextrose syrups by a trace metal resistant mutant of Aspergillus niger

Potato starch and both untreated and decationized dextrose syrups were used as substrates for submerged citric acid biosynthesis using a mutant of Aspergillus niger. The same yield of product (80%) was achieved with both syrups and the starch despite having different trace metals content. The obtained mutant was more sensitive than the parent to Cd²⁺, Mo²⁺, and As³⁺, with decreasing yields of citric acid at 10 mg of ions l^–1. Fe²⁺, Mn²⁺, V²⁺ below 50 mg l^–1 and Cr³⁺, Ni²⁺, Cu²⁺ up to 100 mg l^–1, did not significantly inhibit citric acid production.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger

 Mutants having impaired protein synthesis, that is cycloheximide-sensitive mutants of a citric-acid-hyper-accumulating strain, were induced from Aspergillus niger WU-2223L. Selection was on the basis of a presumption that the mutants should be more sensitive to cycloheximide than WU-2223L. In shake culture without methanol as a promotor substance, seven mutants accumulated approximately 1.8–3.5 times as much citric acid as WU-2223L. The best mutant, CHM I-C3, accumulated 69.4 mg citric acid/ml from 120 mg glucose/ml in shake culture without methanol, this amount being 1.1 times the amount accumulated by WU-2223L with methanol. Furthermore, under the conditions without methanol the mutants appeared to be more efficient than WU-2223L in employing the consumed glucose for the accumulation of citric acid. It was also confirmed that CHM I-C3 exhibited a significantly increased level of intracellular NH⁺ ₄ accumulation. The addition of 2% (v/v) methanol or 20 μg cycloheximide/ml to the medium caused a remarkable increase of citric acid accumulation by WU-2223L: about 3.1 and 2.4 times respectively. However, the addition of these substances produced negative effects on citric acid accumulation by the mutants. With 2% (v/v) methanol, WU-2223L showed a remarkably decreased level of protein accumulation but a substantially increased level of intracellular NH⁺ ₄ accumulation. However, these phenomena were also observed in CHM I-C3 without methanol. These results indicate that the intracellular circumstances of the cycloheximide-sensitive mutants without methanol were similar to those of WU-2223L with methanol, and that the impairment of protein synthesis contributed to increased citric acid accumulation by the mutants in the absence of methanol. Received: 21 November 1994 / Received last revision: 10 July 1995 / Accepted: 26 July 1995     

Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger

Summary Many mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glycerol as a carbon source were induced from Aspergillus niger WU-2223L, a citric acid-producing strain. The mutant strains were classifiable into two types according to their growth characteristics. On the agar plates containing glucose as a sole carbon source, mutant strains of the first type showed good growth irrespective of the presence or absence of DG. When cultivated in shake cultures, some strains of the first type, such as DGR1–2, showed faster glucose consumption and growth than strain WU-2223L. The period for citric acid production shortened from 9 days for strain WU-2223L to 6–7 days for these mutant strains. The levels and yields of citric acid production of the mutant strains were almost the same as those of strain WU-2223L. The mutant strains of the second type, however, showed very slow or no growth on both the agar plates containing glucose and fructose as sole carbon sources. In shake cultures, mutant strains such as DGR2-8 showed decreased glucose consumption rates, resulting in very low production of citric acid.     

Citric and gluconic acid production from fig by Aspergillus niger using solid-state fermentation

The production of citric and gluconic acids from fig by Aspergillus niger ATCC 10577 in solid-state fermentation was investigated. The maximal citric and gluconic acids concentration (64 and 490 g/kg dry figs, respectively), citric acid yield (8%), and gluconic acid yield (63%) were obtained at a moisture level of 75%, initial pH 7.0, temperature 30°C, and fermentation time in 15 days. However, the highest biomass dry weight (40 g/kg wet substrate) and sugar utilization (90%) were obtained in cultures grown at 35°C. The addition of 6% (w/w) methanol into substrate increased the concentration of citric and gluconic acid from 64 and 490 to 96 and 685 g/kg dry fig, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 298–304. Received 15 April 2000/ Accepted in revised form 11 August 2000     

Citric acid production by Aspergillus niger ATCC 9142 from a treated ethanol fermentation co-product using solid-state fermentation

Aims:  To investigate the ability of the citric acid-producing strain Aspergillus niger ATCC 9142 to utilize the ethanol fermentation co-product corn distillers dried grains with solubles for citric acid production following various treatments.
Methods and Results:  The ability of A. niger ATCC 9142 to produce citric acid and biomass on the grains was examined using an enzyme assay and a gravimetric method, respectively. Fungal citric acid production after 240 h was higher on untreated grains than on autoclaved grains or acid-hydrolysed grains. Fungal biomass production was enhanced after autoclaving and acid-hydrolysis of the grains. Phosphate supplementation to the grains slightly stimulated citric acid production while methanol addition decreased its synthesis. Using the phosphate-supplemented grains, the optimal incubation temperature, initial moisture content of the grains and the length of fermentation time for ATCC 9142 citric acid production were determined to be 25°C, 82% and 240 h, respectively.
Conclusions:  A. niger ATCC 9142 synthesized citric acid on corn distillers dried grains with solubles. The phosphate-treated grains increased citric acid production by the strain.
Significance and Impact of the Study:  The ethanol fermentation co-product corn distillers dried grains with solubles could be useful commercially as a substrate for A. niger citric acid production.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Optimisation of fermentation conditions for gluconic acid production by a mutant of Aspergillus niger

Aspergillus niger ORS-4, isolated from the sugarcane industry waste materials was found to produce notable level of gluconic acid. From this strain, a mutant Aspergillus niger ORS-4.410 having remarkable increase in gluconic acid production was isolated and compared for fermentation properties. Among the various substrates used, glucose resulted into maximum production of gluconic acid (78.04 g/L). 12% concentration led to maximum production. Effect of spore age and inoculum level on fermentation indicated an inoculum level of 2% of the 4-7 days old spores were best suited for gluconic acid production. Maximum gluconate production could be achieved after 10-12 days of the fermentation at 30 degrees C and at a pH of 5.5. Kinetic analysis of production indicated that growth of the mutant was favoured during initial stages of the fermentation (4-8 days) and production increased during the subsequent 8-12 days of the fermentation. CaCO3 and varying concentrations of different nutrients affected the production of gluconic acid. Analysis of variance for the factors evaluated the significant difference in the production levels.     

Effect of ammonium and nitrate ratio on glucose oxidase activity during gluconic acid fermentation by a mutant strain of Aspergillus niger

Of the factors tested, the source and concentration of carbon and nitrogen in the medium exerted maximum effect on growth and acid production. Glucose (15%) and urea (0.14%) induced glucose oxidase synthesis and optimum yield of calcium gluconate. Potassium dihydrogen phosphate (0.2%) and magnesium sulphate (0.06%) stimulated glucose oxidase activity and calcium gluconate production. Borax at a concentration of 1.5 g/L induced maximum glucose oxidase and calcium gluconate production with increased glucose utilization.     

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.     

Semi-pilot scale studies on citric acid fermentation by a gamma-ray induced mutant of Aspergillus niger

Summary Semi-pilot scale production of citric acid was investigated with a gamma-ray induced mutant (HB3) of Aspergillus niger using 500, 1000 and 1500 ml medium in 51 fermentation jars. Yield of citric acid was found to be seven-fold higher compared to the parent in 1000 ml medium and the corresponding increase was two-fold in the 500 ml medium. With 1500 ml/fermentation jar the yield was low with both the parent and the mutant strain though the mutant gave higher yield compared to the parent.     

Citric acid production in chemically defined media by Aspergillus niger

Summary The effect of changing the composition of a chemically defined medium on citric acid production by Aspergillus niger was investigated. High and reproducible amounts of citric acid were obtained with deionized commercial sugar solutions, proper phosphate concentrations, low initial pH values and suitable amounts of copper as growth inhibiting agent.Comparison of high and low yielding process parameters showed that under high yielding conditions, (deionized sugar, Cu⁺⁺ addition) besides more citric acid, less mycelium and less mycelial lipids were formed; the consumption of sugar, nitrogen and phosphorus was related to the amount of biomass.Partly presented at the XII. International Congress of Microbiology; München, September 3–8, 1978     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Citric acid production by the diploid strains of Aspergillus niger obtained by protoplast fusion

Summary Diploid strains were obtained following protoplast fusion between two citric acid producers of Aspergillus niger, one for the solid culture and the other for the shaking culture. In the shaking culture, all the diploid strains exhibited lower productivities than one parental strain. However, in the solid culture, some diploid strains exhibited higher productivities than either parental strain; the best diploid strain produced 1.2 times as much citric acid as the parental strain in solid culture.     

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.     

Citric acid production by Aspergillus niger on wet corn distillers grains

AIMS: To determine which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. METHODS AND RESULTS: Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC 9029 and ATCC 201122. CONCLUSIONS: It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger was ATCC 9142. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.     

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.