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.     

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.     

Biotechnological production of gluconic acid: future implications

Gluconic acid (GA) is a multifunctional carbonic acid regarded as a bulk chemical in the food, feed, beverage, textile, pharmaceutical, and construction industries. The favored production process is submerged fermentation by Aspergillus niger utilizing glucose as a major carbohydrate source, which accompanied product yield of 98%. However, use of GA and its derivatives is currently restricted because of high prices: about US$ 1.20–8.50/kg. Advancements in biotechnology such as screening of microorganisms, immobilization techniques, and modifications in fermentation process for continuous fermentation, including genetic engineering programmes, could lead to cost-effective production of GA. Among alternative carbohydrate sources, sugarcane molasses, grape must show highest GA yield of 95.8%, and banana must may assist reducing the overall cost of GA production. These methodologies would open new markets and increase applications of GA. Authors’ contributions OVS and RK are the sole contributors of this original review article. This review is based upon the published research in the area of gluconic acid fermentation.     

Utilization of grape must and concentrated rectified grape must to produce gluconic acid by Aspergillus niger,in batch fermentations

Summary Grape must and concentrated rectified grape must were used for the gluconic acid synthesis using Aspergillus niger batch cultures. The latter substrate was the better, with a production, at 72 h, of 67.43 g/l and a yield (calculated on converted glucose) of 0.96. Citric acid was also observed as a by-product. In order to decrease the residual fructose content, at the end of the gluconate production cycle, an experimental model of sequential fermentation A. niger — Rhizopus arrhizus was proposed for the synthesis of gluconic and fumaric acid. The use of Glucose-isomerase (EC 5.3.1.5) to convert fructose to glucose was also tested.     

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.     

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.     

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H₂SO₄-pretreated black strap molasses (containing 150 g sugars/l) in Vogel's medium. On the basis of comparison of kinetic parameters, namely the volumetric substrate uptake rate (Q _s), and specific substrate uptake rate (q _s), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and had the ability to overproduce citric acid.     

Production of mycelial protein and cellulolytic enzymes from food wastes

Summary Extracted grape waster material and pressed apple pulp were tested as carbon sources forPenicillium funiculosum 515,Myrothecium verrucaria 9095 andAspergillus niger TMF-15. They were good growth substrates, especially forA. niger. When cultivated on mixed substrate in optimized nutrient medium,A. niger accumulated a product of 35% crude protein with a maximum productivity of 0.117 g protein/1/h and cellulose consumption of 90.92%.A. niger also produced the highest levels of cellulase activity. Maximum carboxymethyl cellulase and activity against filter paper were 494 units/l and 97 units/l, respectively.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

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     

Production of thuringiensin from Bacillus thuringiensis using a net-draft-tube,modified airlift reactor

A net-draft-tube, modified airlift reactor and a stirred-tank reactor were used for thuringiensin production by Bacillus thuringiensis subsp. darmstadiensis growing with various concentrations of molasses. The optimum concentration of molasses for thuringiensin production in both reactors was 15 g/l. There was a 6 h delay in sporulation in the modified airlift reactor compared with that in the stirred-tank reactor. Thuringiensin yield in the modified airlift reactor (2.2 g/l) was consequently higher than that in the stirred-tank reactor (1.1 g/l).     

Production of carotenoids by strains of Rhodotorula glutinis cultured in raw materials of agro-industrial origin

The production of carotenoids by strains of Rhodotorula glutinis on different raw materials of agro-industrial origin (grape must, glucose syrup, beet molasses, soybean flour extract, maize flour extract) was investigated. Maximum yield (5.95 mg/l of total carotenoids culture fluid, 630 μg/g dry cell weight) was obtained with a particular strain of Rhodotorula glutinis after a batch culture of 120 h in a substrate containing concentrated rectified grape must as the sole carbohydrate source. In all experiments, the major pigments forming carotenoids (β-carotene, torulene, torularhodin) were quantified.     

Optimization of the growth parameters of Aspergillus foetidus

The batch production of gluconic acid in the presence of glucose, sucrose and molasses was investigated using free mycelia of Aspergillus foetidus NRRL 337 in shake flasks. Eight growth parameters were chosen as independent variables. The temperature, pH, substrate type and initial concentrations, inoculum percentage and shake rate directly affected the specific microorganism growth and gluconic acid production rates. The optimum temperature and initial pH values were found to be 33°C and five to six, respectively. The maximum specific growth and gluconic acid production rates were established as 57 g/dm³ of glucose, 75 g/dm³ of sucrose and 150 g/dm³ of molasses. The optimum values of the shake rate, inoculum percentage and initial ammonium nitrate concentration were determined as 100 1/min, 0.5% and 1.5 g/dm³, respectively. The maximum gluconic acid concentrations corresponding to these initial substrate concentrations were observed to be 8.3 g/dm³, 17.4 g/dm³ 37.0 g/dm³, respectively. The optimum specific microbial growth and gluconic acid production rates were found as 0.0145 1/h and 0.0375 g/g × h, respectively, for the fermentation conditions of S_Go = 57 g/dm³, T = 28°C, initial pH = 6.5, N = 84 1/min, A = 0.5 g/dm³ and I = 0.5%.     

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.     

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.     

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.     

Ram horn peptone as a source of citric acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis>, with a process

The present study deals with the production of citric acid from a ram horn peptone (RHP) by Aspergillus niger NRRL 330. A medium from RHP and a control medium (CM) were compared for citric acid production using A. niger in a batch culture. For this purpose, first, RHP was produced. Ram horns were hydrolyzed by treatment with acids (6 N H₂SO₄, 6 N HCl) and neutralizing solutions. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined. RHP was compared with peptones with a bacto-tryptone from casein and other peptones. The results from RHP were similar to those of standard peptones. The optimal concentration of RHP for the production of citric acid was found to be 4% (w/w). A medium prepared from 4% RHP was termed ram horn peptone medium (RHPM). In comparison with CM, the content of citric acid in RHPM broth (84 g/l) over 6 days was 35% higher than that in CM broth (62 g/l). These results show that citric acid can be produced efficiently by A. niger from ram horn.     

Batch and continuous cultures of<Emphasis Type="Italic"> Mannheimia succiniciproducens</Emphasis> MBEL55E for the production of succinic acid from whey and corn steep liquor

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce a large amount of succinic acid in a medium containing glucose, peptone, and yeast extract. In order to reduce the cost of the medium, whey and corn steep liquor (CSL) were used as substrates for the production of succinic acid by M. succiniciproducens MBEL55E. Anaerobic batch cultures of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in the production of succinic acid with a yield of 71% and productivity of 1.18 g/l/h, which are similar to those obtained in a whey-based medium containing yeast extract (72% and 1.21 g/l/h). Anaerobic continuous culture of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in a succinic acid yield of 69% and a succinic acid productivity as high as 3.90 g/l/h. These results show that succinic acid can be produced efficiently and economically by M. succiniciproducens MBEL55E from whey and CSL.     

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.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.