Permeabilization and inhibition of the germination of spores of Aspergillus niger for gluconic acid production from glucose

In this study, the role of citral to permeabilize the spores of Aspergillus niger and replace sodium azide in the bioconversion medium was studied. Further, characterization of glucose oxidase of spores was carried out by exposing both permeabilized and unpermeabilized spores to different pressures (1, 2, 2.7 kb) and temperatures (60, 70, 80, 90 degrees C). Unpermeabilized spores after exposure to high temperatures were permeabilized by freezing before using as catalyst in the bioconversion reaction. Results showed that citral permeabilized the spores and could inhibit spore germination in the bioconversion medium. Rate of reaction was significantly increased from 1.5 to 4.35 g/Lh which was higher than the commercial glucose oxidase 2g/Lh). Glucose oxidase activity of A. niger was resistant to pressure. However, pressure treatment could not permeabilize them. Behaviour of fresh and permeabilized spores to temperature varied significantly. Glucose oxidase activity of fresh spores exposed to high temperature was unaffected at 70 degrees C till 15 min and 84% of relative activity was retained even after 1h at 70 degrees C while permeabilized spore got inactivated at 70 degrees C for 15 min, which followed the same pattern as commercial glucose oxidase. Cellular membrane integrity was lost due to permeabilization by freezing which resulted in heat-inactivation of glucose oxidase when spores were permeabilized before heat treatment. Thus, glucose oxidase of spore remains heat stable when unpermeabilized and active while permeabilized and its reaction rate is higher than the commercial glucose oxidase.     

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.     

Gluconate production by spores of Aspergillus niger

Spores of Aspergillus niger obtained by solid state fermentation on buckwheat seeds produced gluconic acid from glucose with a high yield, near 1.06 g gluconic acid/g glucose, close to the stoichiometric value. The reaction itself could be carried out either with purified biocatalyst or with the whole buckwheat medium resulting from spore production process. 200 g gluconic acid/L were obtained in 200 h with sequential feedings of glucose up to 190 g/L.     

Solid-state fermentation for gluconic acid production from sugarcane molasses by Aspergillus niger ARNU-4 employing tea waste as the novel solid support

Solid-state fermentation (SSF) was evaluated to produce gluconic acid by metal resistant Aspergillus niger (ARNU-4) strain using tea waste as solid support and with molasses based fermentation medium. Various crucial parameters such as moisture content, temperature, aeration and inoculum size were derived; 70% moisture level, 30 degrees C temperature, 3% inoculum size and an aeration volume of 2.5l min(-1) was suited for maximal (76.3 gl(-1)) gluconic acid production. Non-clarified molasses based fermentation media was utilized by strain ARNU-4 and maximum gluconic acid production was observed following 8-12 days of fermentation cycle. Different concentrations of additives viz. oil cake, soya oil, jaggary, yeast extract, cheese whey and mustard oil were supplemented for further enhancement of the production ability of microorganism. Addition of yeast extract (0.5%) was observed inducive for enhanced (82.2 gl(-1)) gluconic acid production.     

Continuous gluconic acid production by isolated yeast-like mould strains of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

By extensive microbial screening, about 50 strains with the ability to secrete gluconic acid were isolated from wild flowers. The strains belong to the yeast-like mould Aureobasidium pullulans (de Bary) Arnaud. In shake flask experiments, gluconic acid concentrations between 23 and 140 g/l were produced within 2 days using a mineral medium. In batch experiments, various important fermentation parameters influencing gluconic acid production by A. pullulans isolate 70 (DSM 7085) were identified. Continuous production of gluconic acid with free-growing cells of the isolated yeast-like microorganisms was studied. About 260 g/l gluconic acid at total glucose conversion could be achieved using continuous stirred tank reactors in defined media with residence times (RT) of about 26 h. The highest space-time-yield of 19.3 g l(-1) x h(-1)) with a gluconic acid concentration of 207.5 g/l was achieved with a RT of 10.8 h. The possibility of gluconic acid production with biomass retention by immobilised cells on porous sinter glass is discussed. The new continuous gluconate fermentation process provides significant advantages over traditional discontinuous operation employing Aspergillus niger. The aim of this work was the development of a continuous fermentation process for the production of gluconic acid. Process control becomes easier, offering constant product quality and quantity.     

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.     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.     

Gluconic acid production by Aspergillus niger with oxygen supply by hydrogen peroxide

The production of gluconic acid was carried out with high catalase containing Aspergillus niger mutant. This osmofil strain enables to convert the concentrated solutions of D-glucose (300g/l) to D-gluconic acid without gasing using hydrogen peroxide as oxygen source. A controlled addition of hydrogen peroxide based on the pO₂ measurement was performed. The conversion of 300g/l glucose solution was achieved with 7 hours and triple conversion (with biomass recycling) within 27 hours with yield with regard to the substrate over 98%. Kinetics of inactivation of glucose oxidasecatalase complex as a whole was examined. Some general factors influencing the inactivation of glucose oxidase and catalase in mycelium are discussed.     

Production of vanillin from waste residue of rice bran oil by Aspergillus niger and Pycnoporus cinnabarinus

A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic acid were compared, and the highest yield reached 2.2 g l(-1) of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l(-1). The filtrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the filtrate was bio-converted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l(-1) when 5 g l(-1) of glucose and 25 g of HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that delta13C(PDB) of vanillin prepared was much different from chemically synthesized vanillin.     

The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis

Summary The production of sorbitol and gluconic acid by toluene-treated, permeabilized cells of Zymomonas mobilis has been evaluated. From a 60% total sugar solution (300 g/l glucose and 300 g/l fructose), a sorbitol concentration of 290 g/l and a gluconic acid concentration of 283 g/l were achieved after 15 h in a batch process using free toluene-treated cells. A continuous process with immobilized cells was developed and only a small loss of enzyme activity (less than 5%) was evident after 120 h. With a strongly basic anion exchange resin and an eluent of 0.11 M Na₂B₄O₇/0.11 M H₃BO₃, good separation of sorbitol and gluconic acid was achieved.     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

Bioconversion of waste office paper to gluconic acid in a turbine blade reactor by the filamentous fungus Aspergillus niger

Gluconic acid production was investigated using an enzymatic hydrolysate of waste office automation paper in a culture of Aspergillus niger. In repeated batch cultures using flasks, saccharified solution medium (SM) did not show any inhibitory effects on gluconic acid production compared to glucose medium (GM). The average gluconic acid yields were 92% (SM) and 80% (GM). In repeated batch cultures using SM in a turbine blade reactor (TBR), the gluconic acid yields were 60% (SM) and 67% (GM) with 80-100 g/l of gluconic acid. When pure oxygen was supplied the production rate increased to four times higher than when supplying air. Remarkable differences in the morphology of A. niger and dry cell weight between SM and GM were observed. The difference in morphology may have caused a reduction of oxygen transfer, resulting in a decrease in gluconic acid production rate in SM.     

Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass

To investigate the production of cellulases and hemicellulases from Aspergillus niger KK2, solid state fermentation (SSF) was performed by using different ratios of rice straw and wheat bran. When A. niger KK2 was grown on rice straw alone as a solid support in SSF, the maximum FPase activity was 19.5 IU g(-1) in 4 days. Also, CMCase (129 IU g(-1)), beta-glucosidase (100 IU g(-1)), xylanase (5070 IU g(-1)) and beta-xylosidase (193 IU g(-1)) activities were concurrently obtained after 5-6 days of fermentation. The higher enzyme activities produced by A. niger KK2 is a significant advantage from the viewpoint of practical saccharification reaction. Cellulases and hemicellulases produced by A. niger KK2 might be applied to pulp and paper industry, feed industry and chemical industry.     

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.     

Continuous gluconic acid production by the yeast-like Aureobasidium pullulans in a cascading operation of two bioreactors

The application of a new developed process for the continuous production of gluconic acid using a cascade of two bioreactors in a continuous process is shown reaching the highest concentration of gluconic acid described in the literature for continuous culture fermentation. Very high gluconic acid concentrations of 272-308 g/l have been achieved under continuous cultivation of free-growing cells of Aureobasidium pullulans in the first bioreactor at residence times (RT) between 19.5 and 24 h with formation rates for the generic product between 12.7 and 13.9 g/(l h). Gluconic acid, 350-370 g/l, was continuously reached in the second bioreactor at a total RT of 30.8-37 h with R (j) of 9.2-12 g/(l h). The highest specific gluconic acid production (m (p)) of 3.6 g/(g h) was found in the first bioreactor at the lowest RT of 19.5 h. The highest selectivity of 93.6% was determined in the first bioreactor as well. Complete glucose consumption was obtained at 37 h total residence time in the second bioreactor. Gluconic acid, 433 g/l, was continuously produced in the second bioreactor at a total RT of 37 h.     

Synthesis of 2-keto-L-gulonic acid from gluconic acid by co-immobilized Gluconobacter oxydans and Corynebacterium sp.

2-Keto-L-gulonic acid was produced from gluconic acid using co-immobilized cells of Gluconobacter oxydans and Corynebacterium sp. with 2,5-diketo-D-gluconic acid. Gluconobacter oxydans and Corynebacterium sp. were entrapped together with polyvinylalcohol and alginate. 50 g/l glucose, 50 g/l gluconic acid, and the mixture of equal volume of 50 g/l glucose and 50 g/l gluconic acid were used as substrates. When the ratio of two cells was 1 to 1 with 100 mg cells/ml, the conversion of 2-KLG from gluconic acid was 38% (g/g). © Rapid Science Ltd. 1998     

Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase

Gluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose-fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross-linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin-immobilized inulinase in alginate beads. The optimum amounts of both chitin-immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h(-1) and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed-bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h(-1) and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed-bed reactor without any loss of activity, while half-life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.     

Statistical optimization of sulfite pretreatment of corncob residues for high concentration ethanol production

In this study, a central composite design of response surface method was used to optimize sulfite pretreatment of corncob residues, in respect to sulfite charge (5-10%), treatment time (1-2h), liquid/solid (l/s) ratio (6:1-10:1) and temperature (150-180°C) for maximizing glucose production in enzymatic hydrolysis process. The relative optimum condition was obtained as follows: sulfite charge 7.1%, l/s ratio 7.6:1, temperature 156°C for 1.4h, corresponding to 79.3% total glucan converted to glucose+cellobiose. In the subsequent simultaneous saccharification and fermentation (SSF) experiments using 15% glucan substrates pretreated under this kind of conditions, 60.8 g ethanol l(-1) with 72.2% theoretical yield was obtained.     

三角酵母整细胞酶促转化头孢菌素C为戊二酰—7—氨基头孢烷酸

研究了利用含D－氨基酸氧化酶(D-amino acid oxidase,DAO EC1.4.3.3）的透性化三角酶母多倍体FA10（Trigonopsis variabilis FA10）细胞酶促转化头孢菌素C（cephalosporin C,CPC）为戊二酰－7－氨基头孢烷酸（Glutaryl-7-ACA,GL-7ACA）的反应过程和细胞中同时存在的过氧化氢酶（Catalase,CAT）通过水解H2O2而对转化反应产生的干扰作用及其对策。实验证明适量添加外源H2O2（6％）或在反应体系中加入过氧化氢酶抑制剂NaN3(0.13mg/mL )可使GL－7ACA生成率分别为73.0％和70.1％。如果将透性化的FA10细胞在pH10.5-11.0,20℃条件下保温30min,CAT被不可逆性完全钝化,以无过氧化氢酶的FA10细胞进行CPC的酶促转化反应GL－7ACA的生成率可达84％。     

Synthesis of glucose oxidase and catalase by Aspergillus niger in resting cell culture system

The synthesis of glucose oxidase and catalase by Aspergillus niger was investigated using a resting cell culture system without growth being established. Calcium carbonate induced the synthesis of both enzymes and calcium chloride inhibited it. The optimal pH for the biosynthesis of glucose oxidase and catalase was 6.0 and 5.7, respectively. The effects of other bivalent cations, reductive compounds and metabolic products on enzyme synthesis were also tested. The biosynthesis of glucose oxidase and catalase was promoted by MnCO3, thioglycolic acid, pyroracemic acid and gluconic acid.