Influence of the glycolytic rate on production of citric acid and oxalic acid by Aspergillus niger in solid state fermentation

A study was performed to understand the physiology and biochemical mechanism of citric acid accumulation during solid state fermentation of sweet potato using Aspergillus niger Yang No.2. A low citrate-producing mutant was isolated followed by a comparative study of the fermentation process and selected physiological and biochemical parameters. In contrast with the parent strain, the mutant strain displayed lower concentrations, yields and production rates of citric acid, accompanied by higher concentrations, yields and production rates of oxalic acid. In addition, the mutant utilized starch at a lower rate although higher concentrations of free glucose accumulated in the cultures. Biochemical analyses revealed lower rates of glucose uptake and hexokinase activity of the mutant strain in comparison with the parent strain. It is proposed that, in common with submerged fermentation, over-production of citric acid in solid state fermentation is related to an increased glucose flux through glycolysis. At low glucose fluxes, oxalic acid is accumulated.     

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.     

碳源对固定化细胞生产柠檬酸的影响

柠檬酸是利用微生物代谢生产的一种极为重要的有机酸．广泛应用于食品、饮料、化工、冶金、印染等各个领域。在国外,近10年来,利用固定化细胞生产柠檬酸已获得较广泛的研究^〔1－6〕,国内也有学者指出,柠檬酸发酵的趋向是利用固定化细胞进行连续化生产⑺。而国内这方面的研究报道很少〔8,9〕。我们利用海藻酸钙凝胶包埋固定化黑曲霉细胞生产柠檬酸．探讨了碳源种类及其浓度对固定化细胞生产柠檬酸的影响。现将结果报道如下。     

Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: II. Sensitivity analysis

Steady state sensitivity analysis of a model of carbohydrate metabolism and anaplerotic synthesis of oxalacetate were, in Aspergillus niger under conditions of citric acid accumulation, carried out. The flux and metabolite concentration control structure of the system obtained shows that the hexokinase/substrate transport step is the main controlling step of the pathway. The quantitative contribution of the other enzyme catalyzed or transport steps are also discussed. These results allow the design of a proper strategy of biotechnological manipulation aimed at improvement of the process. (c) 1994 John Wiley & Sons, Inc.     

Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: I. Model definition and stability of the steady state

A model of the carbohydrate metabolism and the anaplerotic synthesis of oxalacetate in Aspergillus niger, under conditions of citric and accumulation, is presented. In this first article we set the stage for subsequent analysis within the framework of the biochemical system theory (BST): we formulate the model and develop the system representation in power law forms, showing that the steady state is locally stable. In the second article, the control structure of the system is described and a rationale for the optimization of the process is developed. (c) 1994 John Wiley & Sons, Inc.     

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.     

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.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

Transformation of Aspergillus alliaceus using the Aspergillus niger niaD gene encoding nitrate reductase

Abstract A heterologous transformation system for Aspergillus alliaceus based on the Aspergillus niger nitrate reductase structural gene ( niaD ) has been developed. Two mutants of A. alliaceus (M3 and M17), each carrying an niaD mutation were isolated by screening UV-irradiated cells for the inability to grow on nitrate as sole nitrogen source. Using plasmid pSTA 10, transformation frequencies of 4 and 200 per μg DNA respectively were obtained for these two strains. All the niaD ⁺ transformants tested were mitotically stable. Southern hybridisation analyses showed that the vector DNA sequences were present.     

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.     

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     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

Phenotypes of gene disruptants in relation to a putative mitochondrial malate–citrate shuttle protein in citric acid-producing Aspergillus niger

The mitochondrial citrate transport protein (CTP) functions as a malate–citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.     

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.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.     

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.     

Increases in Gene-Targeting Frequencies Due to Disruption of kueA as a ku80 Homolog in Citric Acid-Producing Aspergillus niger

Low efficiencies of gene targeting hamper functional genomics in industrially important strains of Aspergillus niger. To generate strains showing high gene-targeting frequencies in A. niger WU-2223L producing citric acid, disruption of kueA encoding Ku80 homolog was performed. Disruption of kueA increased gene-targeting frequencies to 70%, and had no effect on citric acid production.     

低功率He-Ne激光照射黑曲霉的方法与效应

目的:探索低功率He-Ne激光对柠檬酸生产菌黑曲霉诱变育种的简易方法。方法:应用带扩束镜的低功率He-Ne激光装置,在无菌条件下对柠檬酸生产菌黑曲霉的单孢子膜进行不同时间的垂直照射,无菌水洗脱,指示性平板筛选,液体培养,测定黑曲霉诱变菌株的柠檬酸产量。结果:不同时间的激光照射黑曲霉单孢子,其存活率与激光照射时间没有线性关系。激光照射6min,黑曲霉M2代产酸的正变率高达37.5%,而此时的存活率也高达40.0%。获得了黑曲霉柠檬酸产酸率提高了10%的突变菌株,为黑曲霉的遗传育种提供了材料。结论:这种方法便于在无菌条件下操作,保证了激光照射黑曲霉单孢子的均匀性,是一种简便易行的微生物诱变育种方法。     

苹果渣基质柠檬酸高产菌株选育

分别以固态苹果渣、苹果渣固态酶解物、苹果渣酶解液和10%葡萄糖溶液为发酵基质研究了17株野生黑曲霉的柠檬酸产生能力,并对获得的4株柠檬酸高产菌进行了诱变育种。结果表明:17株黑曲霉在4种发酵基质中均能良好生长并发酵产生柠檬酸,不同菌株在同一发酵基质中产酸能力间存在差异。FG17、FG23、FG26、FG30等4株菌苹果渣基质柠檬酸产生能力较高,且在4种不同发酵基质中产酸性能稳定。4株菌分别经紫外线和60Co-γ射线诱变后得到的正向突变株柠檬酸产率均显著提高,突变株中FG26-15-4(UV)发酵苹果渣后柠檬酸产率最高,达11.32%,FG23-13-3(γ)发酵苹果渣酶解液后柠檬酸产率最高,达2.73 mg/mL,均高于现有研究报道,可作为不同类型苹果渣基质柠檬酸发酵用菌种。