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.     

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.     

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.     

The influence of energy deficiency-imposing conditions on the capacities ofAcetobacter methanolicus to oxidize glucose and to produce gluconic acid

The presence of dinitrophenol (DNP) during the chemostatic growth of Acetobacter methanolicuson glucose led to i) no significant increase in glucose dehydrogenase, ii) a decrease in the capacity to oxidize glucose by about 30%, not depending on the DNP concentration, and iii) a gradual decrease in the capacity to form gluconic acid, depending on the concentration of the uncoupler. Similar effects of DNP on Acetobacter methanolicuswere observed during the gluconic acid formation process. The loss in the capacity to form gluconic acid is indicated by a drop in the ATP concentration of the cells which cannot be counteracted by the ATP syntheses originating from the oxidation of gluconic acid and of glucose. ATP could be necessary for pumping protons out of the cells that were taken up together with gluconic acid and by the effect of DNP.     

Prebiotic origin of glycolytic metabolism: histidine and cysteine can produce acetyl CoA from glucose via reactions homologous to non-phosphorylated Entner-Doudoroff pathway

Conversion of glucose to pyruvate via reactions homologous to the non-phosphorylated Entner-Doudoroff (non-P ED) pathway could be achieved in the presence of two amino acid catalysts, cysteine and histidine: cystine oxidizes glucose to gluconic acid by the reaction homologous to glucose dehydrogenase and histidine changes gluconic acid to 2-keto-3-deoxy gluconic acid, then to pyruvate by the reaction homologous to gluconic acid dehydratase and 2-keto-3-deoxy gluconate aldolase, respectively. Pyruvate can be converted to acetyl CoA by the reaction with CoA, TPP and FAD in the presence of cysteine and histidine, which resembles pyruvate dehydrogenase reaction. It was found that gluconic acid dehydration alone is non-specific, in contrast to other reactions. The non-P ED pathway is used by some extreme thermophiles in bacteria and archaea, usually thought as the oldest among the contemporary organisms. This study suggests the possible contribution of amino acid to the origin of the glycolytic pathway.     

Optimization of gluconic acid synthesis by removing limitations and inhibitions

The optimization task was performed using the gluconic acid synthesis by the Acetobacter methanolicusMB 58 strain. The microorganisms were grown continuously on methanol as the growth substrate. After finishing the growth process by the deficiency of N and P, the gluconic acid synthesis was started by adding glucose. The synthesis process was performed continuously. The oxygen transfer rate depended on the gluconic acid concentration. During the growth process, the oxygen transfer rate reached a value of about 13 g O₂ · kg^?1 · h^?1using a 30-l glass fermenter equipped with a 6 blade stirrer and fully baffled. This rate declined to a value of between 2 and 5 g O₂ · kg^?1 · h^?1 in the presence of gluconic acid concentrations above 150 g gluconic acid · kg^?1medium. The yield (g gluconic acid · g^?1glucose) depended on the gluconic acid concentration and amounted to y = 0.7 in relation to 150 g gluconic acid · kg^?1medium and y = 0.8 in relation to 200 g · kg^?1medium, respectively. The fermenters were coupled with ultrafiltration moduls (Fa. ROMICON and Fa. SARTORIUS). The biomass concentrations amounted from 5 to 40 g dry mass kg^?1medium. The ultrafiltration modules retained the biomass within the fermentation system. A glucose solution (30 to 50 weight percent glucose) was continuously dosed into the fermenter. The retention time was chosen between 2 and 30 h. The gluconic acid synthesis rate reached values of up to 32 g gluconic acid · kg^?1 · h^?1. Within a range of up to 250 g gluconic acid · kg^?1medium, the acid concentration had no influence on the enzyme activity.     

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.     

Process optimization of continuous gluconic acid fermentation by isolated yeast-like strains of Aureobasidium pullulans

This study was focused on the optimization of a new fermentation process for continuous gluconic acid production by the isolated yeast-like strain Aureobasidium pullulans DSM 7085 (isolate 70). Operational fermentation parameters were optimized in chemostat cultures, using a defined glucose medium. Different optima were found for growth and gluconic acid production for each set of operation parameters. Highest productivity was recorded at pH values between 6.5 and 7.0 and temperatures between 29 and 31 degrees C. A gluconic acid concentration higher than 230 g/L was continuously produced at residence times of 12 h. A steady state extracellular gluconic acid concentration of 234 g/L was measured at pH 6.5. 122% air saturation yielded the highest volumetric productivity and product concentration. The biomass-specific productivity increased steadily upon raising air saturation. An intracellular gluconic acid concentration of about 159 g/L (0.83 mol) was determined at 31 degrees C. This is to be compared with an extracellular concentration of 223 g/L (1.16 mol), which indicates the possible existence of an active transport system for gluconic acid secretion, or the presence of extracellular glucose oxidizing enzymes. The new process provides significant advantages over the traditional discontinuous fungi operations. The process control becomes easier, thus offering stable product quality and quantity.     

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     

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.     

Numerical simulation of a glucose sensitive composite membrane closed-loop insulin delivery system

Closed-loop insulin delivery system works on pH modulation by gluconic acid production from glucose, which in turn allows regulation of insulin release across membrane. Typically, the concentration variation of gluconic acid can be numerically modeled by a set of non-linear, non-steady state reaction diffusion equations. Here, we report a simpler numerical approach to time and position dependent diffusivity of species using finite difference and differential quadrature (DQ) method. The results are comparable to that obtained by analytical method. The membrane thickness directly determines the concentrations of the glucose and oxygen in the system, and inversely to the gluconic acid. The advantage with the DQ method is that its parameter values need not be altered throughout the analysis to obtain the concentration profiles of the glucose, oxygen and gluconic acid. Our work would be useful for modeling diabetes and other systems governed by such non-linear and non-steady state reaction diffusion equations.     

Gluconic acid production by Aspergillus niger mutant ORS-4.410 in submerged and solid state surface fermentation

Aspergillus niger ORS-4.410, a mutant of Aspergillus niger ORS-4 was produced by repeated irradiation with UV rays. Treatments with chemical mutagnes also resulted into mutant strains. The mutants differed from the parent strain morphologically and in gluconic acid production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maximum frequency of positive mutants (25%) was obtained along with a conidial survival of 59% after second stage of UV irradiation. Comparison of gluconic acid production of the parent and mutant ORS-4.410 strain showed a significant increase in gluconic acid production that was 87% higher than the wild type strain. ORS-4.410 strain when transferred every 15 days and monitored for gluconic acid levels for a total period of ten months appeared stable. Mutant ORS-4.410 at 12% substrate concentration resulted into significantly higher i.e. 85-87 and 94-97% yields of gluconic acid under submerged and solid state surface conditions respectively. Further increase in substrate concentration appeared inhibitory. Maximum yield of gluconic acid was obtained after 6 days under submerged condition and decreased on further cultivation. Solid state surface culture condition on the other hand resulted into higher yield after 12 days of cultivation and similar levels of yields continued thereafter.     

Evaluation of agro-food byproducts for gluconic acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ORS-4.410

Certain cost-effective carbohydrate sources in crude as well as after purification were utilized as the sole sources of carbon for gluconic acid production using Aspergillus niger ORS-4.410 under submerged fermentation. Crude grape must (GM) and banana-must (BM) resulted into significant levels of gluconic acid production i.e. 62.6 and 54.6 g/l, respectively. The purification of grape and banana-must led to a 20–21% increase in gluconic acid yield. Molasses as such did not favour gluconate production (12.0 g/l) but a significant increase in production (60.3 g/l) was observed following hexacyanoferrate (HCF) treatment of the molasses. Rectified grape must (RGM) appeared to be best suitable substrate which after 144 h resulted in 73.2 g of gluconic acid/l with 80.6% yield followed by the yield obtained from the rectified banana must (RBM) (72.4%) and treated cane molasses (TM) (61.3%). Abundant growth of mould A. niger ORS-4.410 was observed with crude grape (0.131 g/l/h) and banana must (0.132 g/l/h).     

The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-Al74

Burkholderia cepacia CC-Al74 with a high ability for solubilizing tricalcium phosphate (TCP) was used to study the P-solubilization mechanism. We collected filtrates able to solubilize TCP from the cultures of strain CC-Al74 and demonstrated that the P-solubilization increased from 0 microg ml(-1) to 200 microg ml(-1) during exponential growth, when the pH decreased from 8 to 3. HPLC-analysis revealed that the solubilization of TCP was mainly caused by the release of 16.3 mM gluconic acid. At this concentration, gluconic acid was capable of solubilizing 376 microg ml(-1) of TCP whereas water at pH 3 only solubilized 35 microg ml(-1). The difference is due to the final H+ concentrations which were 13.5 mM and 1 mM in 16.3 mM gluconic acid and deionized water, respectively at pH 3.     

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     

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.     

Gluconic acid production byPenicillium puberulum

Twenty-five Penicillium species isolated from Egyptian soil were examined for their ability to produce gluconic acid in surface culture. Of the eight species capable of producing gluconic acid, Penicillium puberulum gave the maximum yield (91% gluconic acid from glucose after 7 days of fermentation with 3% CaCO3). Peptone was the best nitrogen source for acid fermentation and glucose was superior to sucrose. Addition of low concentrations of KH2PO4 and MgSO4 - 7 H2O stimulated acid production. An initial pH of 6.1 was most favourable for acid accumulation and addition of CaCO3 was necessary for maximum acid production.     

Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant.

The production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants has been evaluated. Optimization of stirrer speed, time cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. Three hundred rpm appeared to be optimum to ensure good growth and best glucose oxidase production, but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Significant increase of dissolved oxygen concentration in culture (16-21%) and extracellular catalase activity were obtained when the traditional aeration was employed together with automatic dosed hydrogen peroxide.     

Gluconic Acid Fermentation by Pullularia pullulans

During the study on the sugar metabolism of molds, several strains of Pullularia pullulans were found to produce large amounts of gluconic acid from glucose. Thirty seven strains of P. pullulans were then tested for their acid-producing abilities. Seven strains did not produce any amount of gluconic acid. However, all of the other strains were shown to be capable of producing this acid. The superior strains produced yiclds of gluconic acid as high as about 90%, based on glucose available, in shaking cultures at 30°C after 2 days. The yields were increased up to approximately 100% during later stages. In addition to high yields, gluconic acid was produced exclusively by these strains. Glutamic acid and inorganic ammonium salts, such as (NH₄)₂SO₄, NH₄Cl and (NH₄)₂HPO₄, were favorable nitrogen sources for acid production. In the case of (NH₄)₂SO₄, the optimum concentration was 0.05%. The addition of CaCO₃ was essential for gluconic acid production by P. pullulans and a 3% concentration of CaC0₃ appeared to be desirable for the maximum conversion to gluconic acid in a medium containing 10% glucose.     

Critical analysis of the effect of metal ions on gluconic acid production by Aspergillus niger using a treated Indian cane molasses

Gluconic acid fermentation by Aspergillus niger has been investigated using untreated and treated Indian cane molasses. The yield of gluconic acid was found to be reduced using an untreated molasses medium compared to a defined medium. Hence, molasses was subjected to various pretreatment techniques. Pretreatment reduced the levels of various cations and anions. As the synthesis of gluconic acid has been observed to be influenced more by cations than anions, the effect of various metal ions, viz., copper, iron, zinc, manganese, calcium, and magnesium on the yield of gluconic acid has been critically examined in both untreated and treated cane molasses. These results have been compared with a defined medium. The yield of gluconic acid was influenced more by a combination of metal ions rather than individual ions. Potassium ferrocyanide treatment gave the most promising results compared to other treatment techniques.