Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.     

A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production

In this study, an integrated process coupling citric acid and methane fermentations was proposed to solve severe wastewater pollution problem in cassava-based citric acid production. The accumulation patterns of the potential and major inhibitors in this process, including organic compounds, volatile fatty acids (VFAs), total ions and pigments were investigated. Both simulation and experimental results indicated that these inhibitors could reach their equilibrium levels after 3–7 fermentation runs when reutilizing the treated citric acid wastewater. As a result, the proposed citric acid fermentation process by recycling the wastewater treated in methane fermentation could be stably operated for more than 15 runs, which could save a large amount of fresh water and relieve the severe wastewater pollution in citric acid production potentially.     

Influence of calcium on fungal growth and citric acid production during fermentation of a sugarcane molasses-based medium by a strain of Aspergillus niger

The effect of CaCl₂ on the growth, morphology and citric acid production from sugarcane molasses by Aspergillus niger 419 was studied. The addition of 0.5g CaCl₂/l to the fermentation medium induced a loose pelleted form of growth, reduced the biomass concentration and increased the volumetric productivity (g citric acid/h) and the specific production (g citric acid/g biomass dw) from 0.02 and 0.37 to 0.13 and 3.72, respectively.     

Influence of nitrilotriacetic acid and 8-hydroxyquinoline on the production of citric acid from molasses using Aspergillus niger

The effect of nitriloacetic acid (NTA) and 8-hydroxyquinoline on the production of citric acid by Aspergillus niger was investigated. The complexing agents showed an effect only during inoculation of the microorganism. Subsequent addition after inoculation did not produce any significant increase in citric acid yield. When 200 ppm of NTA was added during inoculation, an increase of 10.9 g·dm⁻³ citric acid over that produced by the control culture was observed. 8-Hydroxyquinoline, on the other hand, produced a higher concentration of citric acid which was 34 g·dm⁻³ over that of the control culture. The use of 8-hydroxyquinoline is therefore suggested for the production of citric acid from molasses using Aspergillus niger.     

Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose

Citric acid production by Aspergillus niger NCIM 548 and Candida lipolytica NCIM 3472 has been studied in shake culture using glucose and molasses as carbon sources. Methanol addition (3% v/v) at 40 h of fermentation enhanced the production of citric acid by Aspergillus niger whereas a reduction in citric acid production by Candida lipolytica was observed with addition of methanol. Maximum citric acid concentration of 12 kg/m³ was obtained with Aspergillus niger using molasses in the presence of methanol, while maximum citric acid concentration of 8.4 kg/m³ was obtained with Candida lipolytica using glucose without methanol. It appears that product formation by Aspergillus niger is either non-growth associated or partially growth associated depending on the substrate. Methanol addition changes the nature of product formation in case of Candida lipolytica.     

Redox potential in submerged citric acid fermentation

Summary The role and importance of the redox potential phenomena in submerged citric acid production are discussed. The redox potential of the fermentation broth is the result of oxydo-reduction processes where the metabolic activity of the microorganism Aspergillus niger plays the most significant role. The course of the redox curve for a good yielding citric acid production is presented and interpreted. The experiments of submerged citric acid production were carried out on beet molasses treated with potassium hexacyanoferrate and inoculated with A. niger spores.     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Repeated batch production of citric acid from sugarcane molasses using recycled solid-state surface culture ofAspergillus niger

Summary The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.     

Isolation of 2-Phenylacetamide as a Plant Growth Regulator Produced by Actinomyces

Isolation and screening tests were carried out in order to find microorganisms which were able to produce citric acid directly from blackstrap molasses. Some strains were obtained which accumulate considerable quantities of citric acid. Certain temperature changes during the course of incubation were found to increase the yield of citric acid.

The present investigation was undertaken to see if a simple method could be found to improve the yield of citric acid from blackstrap molasses, and we could obtain the yield of more than 70% from the untreated molasses using a newly isolated strain of Asp. niger.     

The effect of pH on production of citric and gluconic acid from beet molasses using continuous culture

Summary The effect of pH on the production of citric and gluconic acid, from beet molasses byAspergillus niger, was studied using continuous culture. At pH values above 2.5 gluconic acid was the major product, citric acid being the predominant product at low pH values. The optimum specific activities of citrate synthase, aconitase, NAD-linked isocitrate dehydrogenase, and NADP-linked isocitrate dehydrogenase occurred at pH 4 and of glucose oxidase at pH 5.     

Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium

Natural oils with high unsaturated fatty acids content when added at concentrations of 2% and 4% (v/v) to beet molasses (BM) medium caused a considerable increase in citric acid yield from Aspergillus niger. The fermentation capacities were also examined for production of citric acid using BM-oil media under different fermentation conditions. Maximum citric acid yield was achieved in surface culture in the presence of 4% olive oil after 12 days incubation.     

Nutrients and oxygen alter reservoir biochemical characters and enhance oil recovery during biostimulation

Biostimulation of petroleum reservoir to improve oil recovery has been conducted in a large number of oilfields. However, the roles and linkages of organic nutrients, inorganic salts and oxygen content during biostimulation have not been effectively elucidated. Therefore, we investigated the relationships between carbon source, nitrogen source, phosphorus source, oxygen content, and microbial stimulation, oil emulsification, and oil degradation. The organic nutrients (molasses) accelerated microbial growth, and promoted oil emulsification under aerobic conditions. The added molasses also promoted metabolites production (CO₂, CH₄ and acetic acid) and microbial anaerobic hydrocarbon degradation under anaerobic conditions. (NH₄)₂HPO₄ improved gases production by neutralizing the acidic production and molasses. NaNO₃ could also improve gases production by inhibiting sulfate-reducing bacteria to adjust pH value. Oxygen supply was necessary for oil emulsification, but bountiful supply of oxygen aggravated oil degradation, leading the entire ranges of alkanes and some aromatic hydrocarbons were degraded. Core-flooding experiments showed an oil displacement efficiency of 13.81 % in test with air package injected, 8.56 % without air package injection, and 4.77 % in control test with air package injection and 3.61 % without air package injection. The results suggest that the combined effect of organic nutrients, inorganic salts and oxygen content determines microbial growth, while production of metabolites, oil emulsification and biodegradation alter the reservoir biochemical characters and influence oil recovery during stimulation.     

Continuous production of citric acid from sugarcane molasses using a combination of submerged immobilized and surface stabilized cultures of Aspergillus niger KCU 520

Summary A high performance fermentation process for the continuous production of citric acid from sugarcane molasses by using the combination of submerged calcium alginate-immobilized and surface-stabilized cultures of Aspergillus niger KCU 520 in a continuous flow horizontal bioreactor is described. The citric acid productivity was dependent on the dilution rate with an optimum value of 0.015/h. Presaturation of fermentation medium with sterile air, in addition to surface aeration, before feeding into the bioreactor enhanced the citric acid productivity. The highest productivity, citric acid product concentration and yield obtained were 1.7 kg M^–3h^–1, 110kg M^–3 and 91% respectively. The cultures were continuously used for 30 days without any apparent loss in citric acid productivity.     

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.     

柠檬酸,葡萄糖和有机质对植物磷素吸收和土壤磷素形态的影响

研究了柠檬酸、葡萄糖和有机质（栎树类凋落物和三叶草茎叶）对几种栽培和野生植物磷素吸收以及高度风化老成土中磷素形态的影响。在未加入无机磷的情况下,连续加入柠檬酸溶液增加了温室盆栽大豆（Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒｒ．）和高粱（Ｓｏｒｐｈｕｍｂｉｃｏｌｏｒ）对磷素的吸收。这表明,柠檬酸可以溶解土壤中被铁、铝氧化物固定的磷。当无机磷和柠檬酸溶液同时加入后。由于有机配位体能够阻止土壤对磷素的吸附和固定,植物对磷的吸收总量明显高于仅仅加入无机磷的对照。不论是否加入无机磷,葡萄糖均没有增加植物对磷素的吸收,但却改变了土壤中磷素的形态。该试验的结果还表明,Ｈｅｄｌｄｙ等人提出的土壤磷素分级方法不适于有机质含量很低的酸性土壤。在另一种性质极为相似的酸性土壤中施入粉碎的栎树（Ｑｕｅｒｃｕｓｓｐｐ．）凋落物和三叶草（ＴｒｉｆｏｌｉｕｍＰｒａｔｅｎｓｅ）后,野生商陆（Ｐｈｙｔｏｌａｃｃａａｍｅｒｉｃａｎａ）吸收磷素的能力增强。通过进一步对土壤中的磷素进行化学分级,结果表明,这些有机物质可以改变土壤中磷素存在的形态。本文还就有机质分解过程中的中间产物对土壤磷素有效性的影响机理进行了探讨。     

Citric acid production by solid state fermentation using sugarcane bagasse

A solid state fermentation (SSF) method was used to produce citric acid by Aspergillus niger DS 1 using sugarcane bagasse as a carrier and sucrose or molasses based medium as a moistening agent. Initially bagasse and wheat bran were compared as carrier. Bagasse was the most suitable carrier, as it did not show agglomeration after moistening with medium, resulting in better heat and mass transfer during fermentation and higher product yield. Different parameters such as moisture content, particle size, sugar level and methanol concentration of the medium were optimised and 75% moisture level, 31.8 g sugar/100 g dry solid, 4% (v/w) methanol and particles of the size between 1.2 and 1.6 mm were found to be optimal. Sucrose and clarified and non-clarified molasses medium were also tested as moistening agents for SSF and under optimised conditions, 20.2, 19.8 and 17.9 g citric acid /100 g of dry solid with yield of 69.6, 64.5 and 62.4% (based on sugar consumed) was obtained in sucrose, clarified and non-clarified molasses medium respectively, after 9 days of fermentation.     

Effects of various conditions on the production of citric acid during fermentation of molasses by Aspergillus niger

Citric acid production by Aspergillus niger is sensitive to pH, temperature and the concentration of carbohydrate in the medium. The maximum production of citric acid, 19.5% (w/v) is obtained at pH 5.4 in a 16% (w/v) molasses medium fermenting at 28°C.     

A marked enhancement in phytase production by a thermophilic mould Sporotrichum thermophile using statistical designs in a cost-effective cane molasses medium

AIMS: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermophile in a cost-effective cane molasses medium. METHODS AND RESULTS: Sporotrichum thermophile secreted phytase in cane molasses medium at 45 degrees C and 250 rev min(-1) after 5 days. The important factors identified by Plackett-Burman design (magnesium sulfate, Tween 80, ammonium sulfate and incubation period) were further optimized by response surface methodology (RSM). An overall 107% improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. When inorganic phosphate was reduced from the cane molasses medium by treatment with calcium chloride, the enzyme production increased. The phytase activity was not affected by the enzyme treatment with trypsin and pepsin. CONCLUSIONS: A twofold increase in phytase production was achieved due to optimization using statistical designs in a cost-effective cane molasses medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase production was doubled due to optimization. The enzyme, being resistant to trypsin and pepsin, thermostable and acid stable, can find application in animal feed industry for improving nutritional status of the feed and combating environmental phosphorus pollution.     

Occurrence of an organic inhibitor in cane molasses affecting citric acid production by Aspergillus niger

A highly potent inhibitor of citric acid fermentation by Aspergillus niger is shown to be present, in addition to minerals, in cane molasses. Sephadex G-10 fractionation of molasses shows the inhibitor to be present in the hexose fraction. This fraction, after concentration reduces the citric acid yield from a citric acid-producing system. The hexose fraction of the sulphitated syrup is much more inhibitory than the hexose fraction of unsulphitated syrup, indicating enhanced activity of the inhibitor at this step of cane-juice processing in sugar-mills.     

Effect of volume of culture medium on enhanced citric acid productivity by a mutant culture of Aspergillus niger in stirred fermentor

AIMS: The present study deals with the effect of volume of culture medium on enhanced citric acid productivity by a mutant culture of Aspergillus niger. METHODS AND RESULTS: A laboratory scale stirred fermentor of 15-l capacity was employed for all microbial cultivations. Blackstrap molasses, a by-product of sugar industries is easily and abundantly available for its exploitation as a carbon source in fermentation processes. The parental culture of A. niger was improved by mutation using ultraviolet radiations and N-methyl N-nitro N-nitroso guanidine, i.e. mutagen MNNG. Six MUV and eight MNNG-treated mutant strains were isolated after extensive screening and optimization. Mutant strain of A. niger MNNG-2 showed enhanced citric productivity (87.60 g l-1) over the parental strain BTL-45 (19.53 g l-1) and other mutant derivatives (49.85 g l-1 citric acid in case of mutant MUV-5 and 76.82 g l-1 in case of mutant MNNG-7). The optimal sugar level was found to be 150 g l-1 (optimum volume of the medium, 60%) after 6 days of inoculation, which is economically significant. Specific productivity of the mutant culture MNNG-2 (qp = 0.057 g/g cells h-1) was several folds higher than other strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study are of commercial level. All kinetic parameters including yield coefficients and volumetric rates revealed the hyper-producibility of citric acid by mutant MNNG-2 using blackstrap molasses as the basal medium in stirred fermentor.