Citric Acid Production Rate Research Articles

Citric acid production by Aspergillus spp. through submerged fermentation using different production mediums containing agroindustrial residues

The production of secondary metabolites of high added value by microorganisms has been extensively studied, mainly in the formulation of medium containing agro-industrial residues. Citric acid is a tricarboxylic organic acid obtained through submerged fermentation processes. The Aspergillus genus is considered an excellent producer of bioactive substances of industrial interest. This study aims to determine the rate of citric acid production using four strains of the microorganism (UCP 1099, 1356, 1357, and 1463) in alternative culture mediums containing citrus pomace (pineapple, orange and lemon) from the food industries in the state of Pernambuco, and to compare the results with the production obtained with the standard production medium using computational statistical tools. Assays were carried out with four samples of Aspergillus isolated from Caatinga in three conventional mediums, for 144 h, 37°C, and 180 rpm. The pH variation, sugar consumption, and citric acid production were analyzed. The results showed that the medium-called two e and the sample UCP1357, presented a citric acid production of 7.22g.L-1. After selecting the medium and the best producer microorganisms, production tests were carried out with a medium formulated with citrus fruit residues. The results revealed that all the residues produced citric acid, with the lemon peel being the best of the residues tested (6.91 g.L-1). It appears that there is a possibility of using residues in the formulation of means of production, thus contributing to the reuse of the present nutrients and the reduction of the environmental impact.

Fine regulation of the starch liquefaction process and its application in the production of citric acid

Citric acid (CA), generally fermented from the starchy material, has attracted intense attention because of its wide applications in many aspects. However, the traditional starchy-liquefaction process based on the dextrose equivalent value and iodine-testing method is inappropriate as it hinders the subsequent CA fermentations. Here, a novel method of evaluating the starch liquefaction in the CA production process was established. Firstly, dextrin samples with the molecular weight (Mw) narrow distribution were prepared by alcohol fractional precipitation. Glucoamylase (GM) from the culture of CA-producing strain Aspergillus niger was purified. Then, the structure-activity relationship between the dextrin and GM was analyzed. Results demonstrated that the Mw of liquefied components aggregated in the range of 1.4–1.9 kDa could improve the efficiency of simultaneous saccharification and fermentation process. CA production rate and total sugar uptake rate were evidently improved with residual total sugar decreasing by 10.8% and fermentation efficiency enhanced by 21.1% in 9 h shorter fermentation time. All these results confirmed that fine regulation of the starch liquefaction based on the Mw characteristics was feasible and effective to improve the CA fermentation. Our proposed strategy could also be useful for other fermentations and sugar industry involving the starchy material.

Optimization of Citric Acid Production by Immobilized Cells of Novel Yeast Isolates

Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30–40 g/L) was greater than that of freely suspended cells (8–19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ∼52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 °C, with NH4Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 °C with (NH4)2 SO4 as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.

Techno-economic analysis and environmental impact assessment of citric acid production through different recovery methods

Evaluating the technical feasibility and economic viability of citric acid production at the commercial scale is challenging because it depends on several factors including type of feedstock, process parameters and production routes. In this research, techno-economic and environmental impacts of citric acid production from corn are comparatively evaluated for three scenarios. In the production process Aspergillus niger is used for corn fermentation in a commercial scale of 6400 kg/h citric acid production rate. The scenarios are defined based on application of three recovery methods, including calcium precipitation, solvent extraction, and ion exchange. From three investigated scenarios, the lowest selling price of citric acid is achieved in a scenario using solvent extraction recovery method, followed by the scenario with calcium precipitation recovery method. Sensitivity analysis shows that the minimum selling price of citric acid is highly sensitive to electricity cost, by-product credit, corn supply cost, fermentor cost and wastewater treatment cost. Environmental impacts of citric acid production in three scenarios are assessed using the life cycle assessment approach. Citric acid production with ion exchange recovery method is the most environment-friendly scenario. The results of this research are useful for the development of citric acid production in commercial scale.

Effect of the nutrient solution in the microbial production of citric acid from sugarcane bagasse and vinasse

Abstract The process known as “solid-state cultivation” (SSC) refers to microbial growth on solid supports under limited water conditions. Citric acid is a bioproduct with several industrial applications, and its production is traditionally microbial. The present work evaluated the production of citric acid by microbial consortium of Aspergillus niger and Trichoderma reesei in sugarcane bagasse with vinasse and ethanol as inducer of the metabolic production of this organic acid. The results indicate that the highest yields of glucose in citric acid, productivity and specific production rate for the conditions having vinasse and vinasse/ethanol as nutrient solution. In addition, the kinetic parameters for the citric acid and glucose profiles indicate that only vinasse as a nutrient solution, has a maximum citric acid concentration of 734 mg L−1 in 48 h.

Transcriptomic analysis of Aspergillus niger strains reveals the mechanism underlying high citric acid productivity

BackgroundAspergillus niger is a highly important industrial microorganism because of its amazing capacity to produce citric acid (CA). To explore the metabolic mechanism and physiological phenotype associated with high CA productivity, the transcriptomes of high CA-producing A. niger YX-1217 and degenerative strain YX-1217G were investigated using A. niger ATCC1015 as a control.ResultsThese strains showed distinct transcriptional differences in CA production. By contrast, the genes encoding glycoside hydrolases, aspartyl endoproteases, and carboxypeptidases were unusually upregulated in CA-producing strain YX-1217, which involved the carbohydrate hydrolysis and polypeptide degradation pathways, and should be related to its powerful capacity to utilize cornmeal fluidified liquid as raw material for the production of CA. In central metabolism of YX-1217, gene 9.735.1, which encodes glyceraldehyde 3-phosphate dehydrogenase, and two transcriptionally outstanding genes, 6000119 (An15g01920) and 3.2152.1 (An08g10920) that encode citrate synthase, were upregulated, thereby ensuring CA accumulation. In addition, a relatively strong electron transport chain, a regeneration system for NAD+/NADP+, and an efficient resistance mechanism may have contributed to the high CA production rate of YX-1217.ConclusionsThese comparisons have shed light on the mechanism underlying high CA yield in A. niger YX-1217 as well as provide insights into the development of novel strains that produce other organic acids.

Optimization of citric acid production from a carrot juice-based medium by Yarrowia lipolytica using response surface methodology

In this study, diluted and fortified carrot juice was used for modelling and optimization of citric acid production by a new mutant strain, Yarrowia lipolytica K-168. Protein concentrate obtained from fine flour -a byproduct of semolina production- was used as a nitrogen source in the fermentation medium. Interactive effects of selected independent variables, initial total sugar concentration, initial pH, initial concentration of protein concentrate obtained from fine flour of semolina and temperature, on the growth and citric acid production of the yeast were investigated. An experimental design including 30 experiments was conducted by using the method of central composite design. Modelling the effects of these independent variables on maximum citric acid concentration, maximum citric acid production rate, citric acid yield, the ratio of maximum citric acid concentration to maximum isocitric acid concentration and specific growth rate were performed by response surface methodology. The variations of all of the responses with the independent variables were defined by a quadratic model. Numeric optimization was performed by using the desireability function. The conditions with 190.83 g/L initial sugar concentration, 5.90 initial pH, 0.07 g/L initial concentration of fine flour protein concentrate and 27.86 °C were determined as optimal conditions for citric acid production. The maximum citric acid concentration reached to 80.53 g/L in optimal conditions.

Effect of Biomass and Sugar in Citric Acid Production by Aspergillus niger Using Molasses and Jackfruit as Substrates

Under surface culture condition, a number of cheap indigenous raw materials like cane molasses and jackfruits were selected for citric acid fermentation and gamma ray induced mutant strains of 14/20 and 79/20 of Aspergillus niger were used. The rate of citric acid production was also influenced by biomass production and residual sugar used. In both cases on the day 13 without presence of Prescott salt for the mutant strain of Aspergillus niger 14/20 the citric acid production was highest. The biomass production was found different in various fermentation medium. It was found to increase with the increase of fermentation period and maximum biomass was found on day 13. It is also found that biomass production was higher in mutant strain of A. niger 14/20 than 79/20. In addition to this, wet-weight biomass was higher than dry weight biomass. Without presence of Prescott salt, wet weight of mycelial body of the fungus Aspergillus niger 14/20 was found highest on day 13 in mixed substrates which was 160.15. Mixed substrate prepared with molasses and jackfruit media was proved to be the best and potential for biomass production. Sugar utilization rate was also different in various media during citric acid production by A. niger 14/20 and 79/20. Prescott salt was also found to have remarkable effect on sugar utilization for citric acid production except on days 0, 3 and 5. In presence of Prescott salt, sugar utilization was lower than without presence of Prescott salt in the molasses, jackfruit and mixed fermentation media during production of citric acid. With the increase of fermentation period, sugar concentration in the medium was reduced and maximum reduction was found on day 13. Without presence of Prescott salt and in mixed fermentation medium the highest production of citric acid was found by further mutated Aspergillus niger 14/20 which was about 16.16mg/ml.

EFFECT OF AGITATION AND AERATION ON THE CITRIC ACID PRODUCTION BY Yarrowia lipolytica GROWN ON GLYCEROL

The effects of agitation rates from 400 to 900 rpm and aeration rates ranging from 0.18 to 0.6 vvm on biomass and citric acid production on glycerol media by acetate-negative mutants of Yarrowia lipolytica, Wratislavia 1.31 and Wratislavia AWG7, in batch culture were studied. The agitation rates of 800 and 900 rpm (at a constant aeration rate of 0.36 vvm) and aeration rates within the range of 0.24–0.48 vvm (at a constant agitation rate of 800 rpm), which generated dissolved oxygen concentration (DO) higher than 40%, were found the best for citric acid biosynthesis from glycerol. An increase in agitation rate (higher than 800 rpm) and aeration rate (higher than 0.36 vvm) had no impact on DO and citric acid production. The highest citric acid concentration (92.8 g/L) and yield (0.63 g/g) were obtained with Wratislavia 1.31 strain at 0.24 vvm. The highest volumetric citric acid production rate (1.15 g/Lh) and specific citric acid production rate (0.071 g/gh) were reached at 0.48 vvm.

Utilization of different agro-industrial wastes for sustainable bioproduction of citric acid by Aspergillus niger

In view of ever growing demand of citric acid, there is an urgent need to look for inexpensive and novel substrates for feasible production of citric acid. In this context, the present study was carried out to evaluate the potential of different agro-industrial wastes for hyper production of citric acid through solid-state and submerged fermentation by Aspergillus niger NRRL 567 and NRRL 2001. It was found that among all the solid substrates utilized, apple pomace with 66.0 ± 1.9 g/kg of dry substrate proved to be an excellent substrate for citric acid production by A. niger NRRL 567 at 72 h of incubation. A. niger NRRL 2001 resulted in slightly lower citric acid concentration of 61.0 ± 1.9 g/kg of dry substrate at the same incubation time. APS-1 (apple pomace ultrafiltration sludge-1) gave highest citric acid production rate of 9.0 ± 0.3 g/l and 8.9 ± 0.3 g/l of substrate by A. niger NRRL 567 and NRRL 2001 by submerged fermentation, respectively. Further study with apple pomace and apple pomace ultrafiltration sludge-1 by A. niger NRRL 567 was carried out. Addition of 3% (v/w) ethanol and 4% (v/w) methanol to apple pomace gave significantly higher citric acid values of 127.9 ± 4.3 g/kg and 115.8 ± 3.8 g/kg of dry substrate by A. niger NRRL 567 by solid-state fermentation. Higher citric acid values of 18.2 ± 0.4 g/l and 13.9 ± 0.4 g/l of apple pomace ultrafiltration sludge-1 were attained after addition of 3% (v/v) ethanol and 4% (v/v) methanol, respectively by A. niger NRRL 567. Apple pomace solid waste and apple pomace ultrafiltration sludge-1 thus proved to be an excellent source for citric acid production, of the different substrates chosen.

Chemostat study of citric acid production from glycerol by Yarrowia lipolytica

The aim of the study was to examine how the dilution rate and the chemical composition of the production medium impacts on the synthesis of citric acid by the Yarrowia lipolytica strain Wratislavia AWG7 from glycerol in a chemostat culture. The yeast Y. lipolytica Wratislavia AWG7, an acetate (acet −) and morphological (fil −) mutant, was cultured in a nitrogen- and phosphorus-limited medium at the dilution rate of 0.009–0.031 h −1 in the chemostat. Under steady-state conditions, the increase in the dilution rate was paralleled by the decrease in citric acid concentration (from 86.5 to 51.2 g L −1), as well as by the increase in the volumetric rate (from 0.78 to 1.59 g L −1 h −1) and specific rate (from 0.05 to 0.18 g g −1 h −1) of citric acid production. The yield of the production process varied from 0.59 to 0.67 g g −1. In a 550-h continuous culture of the yeast test, at a dilution rate of 0.01 h −1, in a medium with enhanced concentrations of carbon, nitrogen and phosphorus sources, the concentration of citric acid, the concentration of biomass and the volumetric rate of citric acid production were 97.8 g L −1, 22.2 g L −1 and 0.98 g L −1 h −1, respectively. The yield of the process decreased to 0.49 g g −1. The number of dead cells did not exceed 1% while that of the budding cells accounted for about 20%. Owing to the low content of isocitric acid and polyols, the fermentation process was characterized by a high purity. This study has produced the following finding: the double mutant Y. lipolytica AWG7 is an effective citric acid producer, with the ability to preserve its properties unchanged during the long run of the continuous chemostat process. This is a valued technological feature of such mutants.

Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes

Yarrowia lipolytica A-101-1.22 produces high citric acid (112 g l(-1)) with a yield of 0.6 g g(-1) and a productivity of 0.71 g l(-1) h(-1) during batch cultivation in the medium with glycerol-containing waste of biodiesel industry. However, it was observed that the specific citric acid production rate, which was maximal at the beginning of the biosynthesis, gradually decreases in the late production phase and it makes continuation of the process over 100 h pointless. The cell recycle and the repeated batch regimes were performed as ways for prolongation of citric acid synthesis by yeast. Using cell recycle, the active citric acid biosynthesis (96-107 g l(-1)) with a yield of 0.64 g g(-1) and a productivity of 1.42 g l(-1) h(-1) was prolongated up to 300 h. Repeated batch culture remained stable for over 1000 h; the RB variant of 30% feed every 3 days showed the best results: 124.2 g l(-1) citric acid with a yield of 0.77 g g(-1) and a productivity of 0.85 g l(-1) h(-1).

Comparison of citric acid production from glycerol and glucose by different strains of Yarrowia lipolytica

A wild type strain A-101 of Y. lipolytica and its three acetate-negative mutants (Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1) were compared for the production of citric acid from glucose and glycerol (pure and crude) in batch cultures. The substrates were used either as single carbon sources or as mixtures of glucose and pure or crude glycerol. The kinetic parameters, i.e., the volumetric citric acid production rate and yield obtained in the study show that the Wratislavia 1.31 and Wratislavia AWG7 strains produced the highest amount of citric acid from glycerol, with a yield from 0.40 to 0.53gg(-1). This substrate was found to be a better carbon source for the biosynthesis of citric acid than glucose. The results obtained with the same strains have shown low content of isocitric acid and polyols, such as erythritol and mannitol. Y. lipolytica A-101 strain produced the highest amount of isocitric acid, from 13.8 to 21% isocitric acid in the sum of citric acids. However, the highest concentrations of erythritol were found in cultures with Y. lipolytica Wratislavia K1, from 18.1 to 30gl(-1), for glucose and pure glycerol, respectively.

Optimization of Trace Metals Concentration on Citric Acid Production by Aspergillus niger NRRL 2001

Citric acid is nowadays produced by submerged fermentation of Aspergillus niger. The process yield depends on the composition of the medium, as well as on the microorganism strain. In this work, the effect of Fe+3, Zn+2, and Mn+2 on citric acid production by A. niger NRRL 2001 is presented. The culture medium composition was glucose (120 g/L) KH2PO4 (1.0 g/L); K2HPO4 (1.0 g/L), MgSO4.7H2O (0.5 g/L), (NH4)2SO4 (3.0 g/L). The ions Fe+3, Zn+2, and Mn+2 had their concentrations changed according to an experimental design. The experiments were carried out in an orbital shaker at 200 rpm and 30°C. The strain produced an extracellular polysaccharide that was also quantified. The optimum experimental condition was found using 7.0 mg/L of Fe+3 and 6.5 mg/L of Zn+2 in absence of Mn+2. No oxalic acid formation was observed using this experimental condition. Metal contents were not significant for the production of the polysaccharide. The highest production rate (2.95 g L−1 day−1) was reached after 10 days of fermentation. After this period, the productivity decreased slightly. In 20 days, the citric acid production rate (2.44 g L−1 day−1) was 82% of the highest productivity. The conversion into citric acid increased continuously, yielding 45.8% in 20 days of fermentation.

Characterization of Yarrowia lipolytica and related species for citric acid production from glycerol

Twenty-seven Yarrowia lipolytica strains as well as five strains from three other species of the Yarrowia clade ( Aciculoconidium aculeatum, Candida hispaniensis and Candida bentonensis) were screened for citric acid production with pure glycerol as the carbon source. The cultures were grown under nitrogen-limited conditions. None of the non- Yarrowia strains produced citric acid, although they were able to grow on glycerol. All of the Y. lipolytica strains were able to produce citric acid in varying concentrations and, under the screening conditions used, the yields obtained formed a continuum from very low to among the highest reported for this substrate. The highest yielding strain, Y. lipolytica NRRL YB-423, produced 21.6 g/L citric acid from 40 g/L glycerol (54% yield). The citric acid to isocitric acid ratio produced by this strain in the initial screen was 11.3, while most of the strains produced ratios of between 2 and 6. Further work on medium optimization with this strain showed that the optimum carbon to nitrogen ratio for the rate of citric acid production was 172 while the best combination of rate and yield was obtained at a C/N ratio of 343. The citric acid to isocitric acid ratios produced reached an optimum at C/N ratios of 343–686.

Aspergillus niger ATCC 9142 세포에 의해 농축된 우유공장폐수로부터 구연산생산에 대한 동력학 연구

Aspegillus niger 세포에 의한 구연산 생산에 대한 발효 매체로서 우유공장폐수가 연구되었다. 발효매체에 <TEX>$Mn^{2+},\;Fe^{2+}\;and\;Cu^{2+}$</TEX>의 첨가가 구연산 생산을 일정하게 증가시켰다. 그러나 다른 금속이온인 <TEX>$Mg^{2+}$</TEX>의 첨가는 구연산 생산을 감소시켰다. 구연산의 농도는 우유공장폐수에서 환원당 농도의 농도가 50 g/l와 100 g/1로 Aspegillus niger ATCC 9142에 의한 batch bioreactor에서 각각 7.2 g/1과 16.5 g/l로 높게 표시되었다. 본 실험에서 적용한 수리적 모델식들이 세포성장의 예상, 구연산 생산, 기질소모 속도에 대해 잘 적용되는지를 알아보기 위해서 발효반응 모델식에 적용시켜 본 결과 실험치와 잘 부합되었다. The waste water from a milk factory was investigated for possibility of use to the production of citric acid by cells of Aspegillus niger ATCC 9142. The addition of <TEX>$Mn^{2+},\;Fe^{2+}\;and\;Cu^{2+}$</TEX> ions to waste increased citric acid production steadily, but addition of metal ion <TEX>$Mg^{2+}$</TEX> decreased the citric acid production. The amount of produced citric acid by Aspegillus niger ATCC 9142 with addition 50 g/1 and 100 g/1 of reducing sugar in milk factory waste water were 7.2 g/1 and 16.5 g/1 respectively. Mathematical model was simulated for their predictability of cell growth, citric acid production and substrate consumption rate and coincided with experimental data.

Optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy d-glucose resistant culture of Aspergillus niger NG d-280

The present investigation is concerned with the optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy d-glucose resistant culture of Aspergillus niger NG d-280 in a 15 l stirred tank bioreactor. Nutrients, especially nitrogen source have a marked influence on citrate productivity because it is an essential constituent of basal cell proteins. Citric acid has been known to be produced when the nitrogen source was the limiting factor. Ammonium nitrate was employed as a nitrogen source in the present study and batch culture experiments were carried out under various concentrations of ammonium nitrate. Specific growth rate was decreased and the biosynthesis of citric acid was delayed at higher concentrations of ammonium nitrate. Specific citric acid production rate was the highest when intracellular ammonium ion concentration was between 2.0 and 3.0 mmol g −1 cells. Citrate production was however, stopped when intracellular ammonium ion concentration decreased below 1.0 mmol g −1 cell.

Relation between Citric Acid Production and Respiration Rate of Aspergillus niger in Solid‐State Fermentation

AbstractAmong the organic acids produced industrially, citric acid is the most important in quantitative terms. Solid‐state fermentation (SSF) has been an alternative method for citric acid production using agro‐industrial residues such as cassava bagasse (CB). The use of CB as a substrate can avoid environmental problems caused by its disposal into the environment. This study was developed to verify the influence of the treated bagasse amount, and consequently, the influence of the gelatinization degree of CB starch on citric acid production by SSF in Erlenmeyer flasks, horizontal drums, and trays. The best results were obtained in a horizontal drum bioreactor using 100 % of treated CB. However, trays showed advantages and good perspectives for large‐scale citric acid production due to economic reasons such as energy costs. A kinetic study was also carried out in order to compare citric acid production in glass columns (laboratory scale) and horizontal drum bioreactors (semi‐pilot scale). This study was accomplished in order to follow the influence of aeration on citric acid accumulation. In addition, the production of CO2 was evaluated as an indirect method of biomass estimation. Citric acid production was higher in glass columns (309.70 g/kg of dry CB) than in HD bioreactors (268.94 g/kg of dry CB). Finally, it was possible to show that citric acid production was favored by a limited biomass production, which occurred with low aeration rates. Biomass production is related to CO2 production and as a result, a respirometry analysis could be used for biomass estimation.

Optimization of the citric acid production by Aspergillus niger through a metabolic flux balance model

Idiophase, the citric acid producing stage of Aspergillus niger was mathematically modeled to identify required genetic manipulations to optimize citric acid production rate. For this reason, a consistent picture of cell functioning had to be achieved. The transient idiophase nature was established by stoichiometric analysis. The main intracellular fluxes were computed by application of material and physiological constraints (ATP, reduction equivalents, proton motive force) at culture time 120 hours. The HMP pathway accounts for 16% of the glucose input (carbon basis), the Krebs cycle for 13% and the citric acid synthesis for the remaining 71%. This profile implies an operative glycerol-P shuttle. It recycles 93% of the cytosolic glycerol-P to cytosolic DHAP thus coupling the transformation of cytosolic NADH to mitochondrial FADH. A cellular maintenance energy of 3.7 mmol ATP/g·h was determined. It would be spent in fueling cytoplasmatic (1.4 mmol H + /g·h) and mitochondrial (1.8 mmol H + /g·h) H + -ATPase pumps with efficiencies of 0.65 and 1.2 mmol H + /mmol ATP respectively. The role and extent of the alternative respiration system activity and polyol excretion is accounted by the model as well. In addition, the significance of GABA shunt and futile NH 4 + /NH 3 cycle were rejected. According to the developed model, the specific citric productivity would be increased in 45% by an unique change if glucose influx were duplicated. Differences with predictions from other model that required many manipulations are also discussed.

Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production.

Using a combination of dye adsorption and affinity elution we purified Aspergillus niger citrate synthase to homogeneity using a single column and characterised the enzyme. An A. niger citrate synthase cDNA was isolated by immunological screening and used to clone the corresponding citA gene. The deduced amino acid sequence showed high similarity to other fungal citrate synthases. After processing upon mitochondrial import, the calculated M(r) of A. niger citrate synthase is 48501, which agrees well with the estimated molecular mass of the purified protein (48 kDa). In addition to an N-terminal mitochondrial import signal, a peroxisomal target sequence (AKL) was found at the C-terminus of the protein. Whether both signals are functional in vivo is not clear. Strains overexpressing citA were made by transformation and cultured under citric acid-producing conditions. Up to 11-fold overproduction of citrate synthase did not increase the rate of citric acid production by the fungus, suggesting that citrate synthase contributes little to flux control in the pathway involved in citric acid biosynthesis by a non-commercial strain.