Optimizing medium constituents and fermentation conditions for citric acid production from palmyra jaggery using response surface method

The quantitative effects of pH, temperature, time of fermentation, sugar concentration, nitrogen concentration and potassium ferrocyanide on citric acid production were investigated using a statistical experimental design. It was found that palmyra jaggery (sugar syrup from the palmyra palm) is a suitable substrate for increasing the yield of citric acid using Aspergillus niger MTCC 281 by submerged fermentation. Regression equations were used to model the fermentation in order to determine optimum fermentation conditions. Higher yields were obtained after optimizing media components and conditions of fermentation. Maximum citric acid production was obtained at pH 5.35, 29.76 °C, 5.7 days of fermentation with 221.66 g of substrate/l, 0.479 g of ammonium nitrate/l and 2.33 g of potassium ferrocyanide/l.     

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 citric acid fermentation by <Emphasis Type="Italic">Candida oleophila</Emphasis> under nitrogen limitation at constant C/N ratio

Summary The central aspect of this work was to investigate the influence of nitrogen feed rate at constant C/N ratio on continuous citric acid fermentation by Candida oleophila ATCC 20177. Medium ammonia nitrogen and glucose concentrations influenced growth and production. Space-time yield (STY) meaning volumetric productivity, biomass specific productivity (BSP), product concentration, product selectivity and citrate/isocitrate ratio increased with increasing residence time (RT). BSP increased in an exponential mode lowering nitrogen feed rates. Highest BSP for citric acid of 0.13 g/(g h) was achieved at lowest NH₄Cl concentration of 1.5 g/l and highest STY (1.2 g/l h) with 3 g NH₄Cl/l at a RT of 25 h. Citric acid 74.2 g/l were produced at 58 h RT and 6 g NH₄Cl/l. Glucose uptake rate seems to be strictly controlled by growth rate of the yeast cells. Optimum nitrogen concentration and adapted C/N ratio are essential for successful continuous citric acid fermentation. The biomass-specific nitrogen feed rate is the most important factor influencing continuous citric acid production by yeasts. Numerous chemostat experiments showed the feasibility of continuous citrate production by yeasts.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

Accumulation and partial re-consumption of polyols during citric acid fermentation by Aspergillus niger

Summary Quantitative balances have been made for sugar and oxygen uptake rates during citric acid accumulation by Aspergillus niger: during the first phase of citric acid accumulation (up to 130 h) more sugar is taken up than the production of biomass, CO₂ and citric acid account for. In contrast, during later phases of fermentation more citric acid, CO₂ and biomass are formed than sugar uptake would theoretically allow. A similar pattern is obtained for oxygen uptake, where less uptake occurs during the early phase of fermentation than needed for complete balance, and the reverse is observed during the late stage of fermentation. It could subsequently be shown that this is caused by the intermediate accumulation and partial re-consumption of a number of polyhydric alcohols (glycerol, arabitol, erythritol and mannitol) during citric acid fermentation.Dedicated to Professor H. J. Rehm on the occasion of his 60th birthday with kind regards     

Enhanced solid‐state citric acid bio‐production using apple pomace waste through surface response methodology

Aims: To evaluate the potential of apple pomace (AP) supplemented with rice husk for hyper citric acid production through solid‐state fermentation by Aspergillus niger NRRL‐567. Optimization of two key parameters, such as moisture content and inducer (ethanol and methanol) concentration was carried out by response surface methodology. Methods and Results: In this study, the effect of two crucial process parameters for solid‐state citric acid fermentation by A. niger using AP waste supplemented with rice husk were thoroughly investigated in Erlenmeyer flasks through response surface methodology. Moisture and methanol had significant positive effect on citric acid production by A. niger grown on AP (P < 0·05). Higher values of citric acid on AP by A. niger (342·41 g kg^?1 and 248·42 g kg^?1 dry substrate) were obtained with 75% (v/w) moisture along with two inducers [3% (v/w) methanol and 3% (v/w) ethanol] with fermentation efficiency of 93·90% and 66·42%, respectively depending upon the total carbon utilized after 144 h of incubation period. With the same optimized parameters, conventional tray fermentation was conducted. The citric acid concentration of 187·96 g kg^?1 dry substrate with 3% (v/w) ethanol and 303·34 g kg^?1 dry substrate with 3% (v/w) methanol were achieved representing fermentation efficiency of 50·80% and 82·89% in tray fermentation depending upon carbon utilization after 120 h of incubation period. Conclusions: Apple pomace proved to be the promising substrate for the hyper production of citric acid through solid‐state tray fermentation, which is an economical technique and does not require any sophisticated instrumentation. Significance and Impact of the Study: The study established that the utilization of agro‐industrial wastes have positive repercussions on the economy and will help to meet the increasing demands of citric acid and moreover will help to alleviate the environmental problems resulting from the disposal of agro‐industrial wastes.     

Characterization of metabolically active developmental stage of Aspergillus niger cells immobilized in polyacrylamide gel

Summary The in-situ development of Aspergillus niger entrapped in polyacrylamide gel from spores and the gel surface characteristics were studied during the repeated shake flask batch citric acid fermentation. A marked increase in the rate of citric acid production was observed with the periodic replacement of culture with fresh media at an interval of 6 days reducing the fermentation time nearly to half. The metabolically active A. niger cells for citric acid production were characterized by the appearance of thick and bulbous hyphae scattered in and on the gel surface.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

Evidence for nitrogen catabolite repression during citric acid production by Aspergillus niger under phosphate-limited growth conditions

Experiments performed in batch fermentation under phosphate-limited growth conditions showed that the citric acid yield was inversely related to the excess nitrogen concentration in the medium. Results from chemostat culture confirmed a negative relationship between the citric acid yield and both the specific growth rate and the nitrogen consumption rate. This is evidence for nitrogen catabolite repression. A fed-batch fermentation performed under dual phosphate/nitrogen limitation produced results very similar to those from a culture limited by nitrogen alone. There is no advantage in maintaining an excess of phosphate during citric acid production and the process will therefore be more economic when operated under dual limitation conditions.     

Bioconversion of palm oil mill effluent for citric acid production: statistical optimization of fermentation media and time by central composite design

A laboratory-scale study was conducted to evaluate the feasibility of using palm oil mill effluent (POME) as a major substrate and other nutrients for maximum production of citric acid using the potential fungal strain Aspergillus niger (A103). Statistical optimization of medium composition (substrate–POME, co-substrates–wheat flour and glucose, and nitrogen source–ammonium nitrate) and fermentation time was carried out by central composite design (CCD) to develop a polynomial regression model through the effects of linear, quadratic, and interaction of the factors. The statistical analysis of the results showed that, in the range studied, ammonium nitrate had no significant effect whereas substrate, co-substrates and fermentation time had significant effects on citric acid production. The optimized medium containing 2% (w/w) of substrate concentration (POME), 4% (w/w) of wheat flour concentration, 4% (w/w) of glucose concentration, 0% (w/v) of ammonium nitrate and 5 days fermentation time gave the maximum predicted citric acid of 5.37 g/l which was found to be 1.5 g/l in the experimental run. The determination of coefficient (R ²) from the analysis observed was 0.964, indicating a satisfactory adjustment of the model with the response. The analysis showed that the major substrate POME (P < 0.05), glucose (P < 0.01), nutrient (P < 0.05), and fermentation time (P < 0.01) was more significant for citric acid production. The bioconversion of POME for citric acid production using optimal conditions showed the higher removal of chemical oxygen demand (82%) with the production of citric acid (5.2 g/l) on the final day of fermentation process (7 days). The pH and biosolids accumulation were observed during the bioconversion process.     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Fate and Role of Ammonium Ions during Fermentation of Citric Acid by Aspergillus niger

Stoichiometric modeling of the early stages of the citric acid fermentation process by Aspergillus niger revealed that ammonium ions combine with a carbon-containing metabolite inside the cell, in a ratio 1:1, to form a nitrogen compound which is then excreted by the mycelium. High-performance liquid chromatography analysis identified glucosamine as the product of the relationship between glucose and ammonium during the early stages of the citric acid fermentation process. Slightly acidic internal pHs, extremely low ammonium ion concentrations inside the cell, and glucosamine synthesis come into direct contradiction with the earlier theory of the ammonium pool inside the cell, regarded as responsible for inhibition of the enzyme phosphofructokinase. At later fermentation stages, when the mycelium is involved in a process of fragmentation and regrowth, the addition of ammonium sulfate leads to a series of events: the formation and secretion of glucosamine in elevated amounts, the short inhibition of citrate synthesis, growth enhancement, the utilization of glucosamine, and finally, the enhancement of citric acid production rates. Obviously, the enzymatic processes underlining the phenomena need to be reexamined. As a by-product of the citric acid fermentation, glucosamine is reported for the first time here. Suitable process manipulations of the system described in this work could lead to successful glucosamine recovery at the point of its highest yield before degradation by the fungus occurs.     

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.     

Studies on aspergilli xix. Role of carbon and nitrogen on citric acid production

Summary The effect of various carbon and nitrogen compounds on the production of citric acid byAspergillus luchuensis was studied under controlled conditions. Unhydrolysed sucrose was better than the hydrolysed one. The other carbon sources, viz., maltose, lactose, Gur, Ral and cane molasses were unsuitable for citric acid fermentation. With all these carbohydrates low yields were obtained. Sodium nitrate was found to be the best nitrogen source for the maximum yield of citric acid. It was closely followed by potassium nitrate. Ammonium compounds and amino acids, in general, were unsuitable for the production of citric acid.     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn²⁺ ions has been investigated using high citric acid-yielding, Mn²⁺ ion-sensitive as well as Mn²⁺ ion-tolerant mutant strains of A. niger. In the presence of Mn²⁺ (1.5 mg/l), citric acid production by the Mn²⁺ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn²⁺ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn²⁺ ion-requiring NADP⁺-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn²⁺ ions during fermentation seem to be responsible for the Mn²⁺ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn²⁺.     

Fate and role of ammonium ions during fermentation of citric acid by Aspergillus niger

Stoichiometric modeling of the early stages of the citric acid fermentation process by Aspergillus niger revealed that ammonium ions combine with a carbon-containing metabolite inside the cell, in a ratio 1:1, to form a nitrogen compound which is then excreted by the mycelium. High-performance liquid chromatography analysis identified glucosamine as the product of the relationship between glucose and ammonium during the early stages of the citric acid fermentation process. Slightly acidic internal pHs, extremely low ammonium ion concentrations inside the cell, and glucosamine synthesis come into direct contradiction with the earlier theory of the ammonium pool inside the cell, regarded as responsible for inhibition of the enzyme phosphofructokinase. At later fermentation stages, when the mycelium is involved in a process of fragmentation and regrowth, the addition of ammonium sulfate leads to a series of events: the formation and secretion of glucosamine in elevated amounts, the short inhibition of citrate synthesis, growth enhancement, the utilization of glucosamine, and finally, the enhancement of citric acid production rates. Obviously, the enzymatic processes underlining the phenomena need to be reexamined. As a by-product of the citric acid fermentation, glucosamine is reported for the first time here. Suitable process manipulations of the system described in this work could lead to successful glucosamine recovery at the point of its highest yield before degradation by the fungus occurs.