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).     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Continuous 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.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

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.     

Batch xylitol production by<Emphasis Type="Italic"> Candida guilliermondii</Emphasis> FTI 20037 from sugarcane bagasse hemicellulosic hydrolyzate at controlled pH values

Batch fermentation of sugarcane bagasse hemicellulosic hydrolyzate by the yeast Candida guilliermondii FTI 20037 was performed using controlled pH values (3.5, 5.5, 7.5). The maximum values of xylitol volumetric productivity (Q _p=0.76 g/l h) and xylose volumetric consumption (Q _s=1.19 g/l h) were attained at pH 5.5. At pH 3.5 and 7.5 the Q _p value decreased by 66 and 72%, respectively. Independently of the pH value, Y _x/s decreased with the increase in Y _p/s suggesting that the xylitol bioconversion improves when the cellular growth is limited. At the highest pH value (7.5), the maximum specific xylitol production value was the lowest (q _pmax=0.085 g/l h.), indicating that the xylose metabolism of the yeast was diverted from xylitol formation to cell growth.List of symbols P _max xylitol concentration (g/l) - Q _x volumetric cell production rate (g/l h) - Q _s volumetric xylose uptake rate (g/l h) - Q _p volumetric xylitol production rate (g/l h) - q _pmax specific xylitol production (g/g h) - q _smax specific xylose uptake rate (g/g h) - _max specific cell growth rate (h^–1) - Y _p/s xylitol yield coefficient, g xylitol per g xylose consumed (g/g) - Y _p/x xylitol yield coefficient, g xylitol per g dry cell mass produced (g/g) - Y _x/s cell yield coefficient, g dry cell mass per g xylose consumed (g/g) - _cell percentage of the cell yield from the theoretical value (%) - _xylitol percentage of xylitol yield from the theoretical value (%)     

A High Performance Fermentation Process for Citric Acid Production by Aspergillus niger NGGCB-101, Using Vermiculite as an Additive in a Stirred Bioreactor

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG^GCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levels of vermiculite were added as an additive into the fermentation medium. The best results were observed when 0.20 g/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citric acid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelial pellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at different vermiculite levels was monitored for Q _p , Q _s and q _p , there was a significant enhancement (P 0.05) in these variables over the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basic parameters for high performance citric acid fermentation.     

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca⁺⁺ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl₂ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl₂ at a level of 2.0 M, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NG^GCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 using molasses medium was carried out in a jar fermentor. When molasses was subjected to H₂SO₄-heat treatment, the maximum BC concentration increased to 76% more than that achieved using untreated molasses, and the specific growth rate increased 2-fold. When the initial sugar concentrations in the H₂SO₄-heat treated molasses were varied from 23 g/l to 72 g/l, BC concentration, production rate, and yield were maximum at sugar concentrations of 23 g/l and 37 g/l, and production of by-products, such as polysaccharides and CO₂, was lower than at sugar concentrations of 48 g/l and 72 g/l, indicating that maintaining a lower molasses concentration is essential for efficient BC production in jar fermentors, this being due mainly to the complex nature of molasses. Molasses has a clear advantage over pure sugars as a carbon source from an economic viewpoint.     

Ethanol production from sulphuric acid wood hydrolysate ofPinus radiata using free and immobilized cells ofPichia stipitis

Summary Ethanol was produced by a strain ofPichia stipitis adapted to an inhibitory acid wood hydrolysate ofPinus radiata. The best ethanol productivity for batch cultures was 0.21 g/l h at 0.7% ethanol. Varying culture conditions increased ethanol concentration to 0.76%, however the productivity decreased to 0.18 g/l h. A decrease in ethanol concentration in the culture fluid was noted late in the batch which suggested ethanol catabolism. Values of kinetic parameters (K _m,K _s, _max, andV _max) were evaluated for this system. The use of calcium alginate immobilized cells in a continuous-flow stirred tank reactor lead to enhanced fermentative performance, namely a maximum productivity of 0.27 g/l h and 1.13% ethanol yield. The immobilized cells in continuous flow reactors represent an attractive option for fermenting sugars released by sulphuric acid hydrolysis ofP. radiata wood.     

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

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

Evaluation of agro-food byproducts for gluconic acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ORS-4.410

Certain cost-effective carbohydrate sources in crude as well as after purification were utilized as the sole sources of carbon for gluconic acid production using Aspergillus niger ORS-4.410 under submerged fermentation. Crude grape must (GM) and banana-must (BM) resulted into significant levels of gluconic acid production i.e. 62.6 and 54.6 g/l, respectively. The purification of grape and banana-must led to a 20–21% increase in gluconic acid yield. Molasses as such did not favour gluconate production (12.0 g/l) but a significant increase in production (60.3 g/l) was observed following hexacyanoferrate (HCF) treatment of the molasses. Rectified grape must (RGM) appeared to be best suitable substrate which after 144 h resulted in 73.2 g of gluconic acid/l with 80.6% yield followed by the yield obtained from the rectified banana must (RBM) (72.4%) and treated cane molasses (TM) (61.3%). Abundant growth of mould A. niger ORS-4.410 was observed with crude grape (0.131 g/l/h) and banana must (0.132 g/l/h).     

Fermentation ofd-xylose,d-glucose andl-arabinose mixture byPichia stipitis Y 7124: sugar tolerance

Summary The effect of substrate concentration (S ₀) on the fermentation parameters of a sugar mixture byPichia stipitis Y 7124 was investigated under anaerobic and microaerobic conditions. Under microaerobiosisP. stipitis maintained high ethanol yield and productivity when initial substrate concentration did not exceed 150 g/l; ethanol yield of about 0.40 g/g and volumetric productivity up to 0.39 g/l per hour were obtained. Optimal specific ethanol productivity (0.2 g/g per hour) was observed withS ₀=110 g/l. Under anaerobic conditionsP. stipitis exhibited the highest fermentative performances atS ₀=20 g/l; it produced ethanol with a yield of 0.42 g/g, with a specific rate of 1.1 g/g per day. When the initial substrate level increased, specific ethanol productivity declined gradually and ethanol yield was dependent on the degree of utilization of each sugar in the mixture.Abbreviations E _m maximum produced ethanol (g/l) - E ₀ initial ethanol (g/l) - E _v evaporated ethanol (g/l) - Q _p volumetric productivity of ethanol (g ethanol/l per hour or g/l per day) - q _p specific productivity of ethanol (g ethanol/g cells per hour) - q _pm maximum specific productivity of ethanol (g/l per hour) - S ₀ initial substrate concentration (g/l) - t _f time at which produced ethanol is maximum (h) - Y _p/s ethanol yield (g ethanol produced/g substrate utilized) - Y _x/s cell yeild (g cells produced/g substrate utilized) - Y _xo/xy xylitol yield (g xylitol produced/g xylose utilized) - probability coefficient - specific growth rate coefficient (h^-1 or d^-1)     

Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process

Alcoholic fermentation by a commercial baker's yeast in a fed-batch process with cell recycling and high-test molasses as substrate was strongly inhibited by Lactobacillus fermentum CCT 1407 after a few recycles. When total acidity (mainly lactic acid) exceeded 4.8 g/l broth it seriously interfered with yeast bud formation and viability and above 6.0 g/l it decreased alcoholic efficiency.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

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 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.     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Production of 2,3-butanediol from corncob molasses, a waste by-product in xylitol production

Corncob molasses, a waste by-product in xylitol production, contains high concentrations of mixed sugars. In the present study, corncob molasses was used to produce 2,3-butanediol (BD) using Klebsiella pneumoniae SDM. This was the first report on the use of corncob molasses to produce bulk chemicals. Our results indicated that K. pneumoniae SDM can utilize various sugars contained in the corncob molasses in a preferential manner: glucose > arabinose > xylose. It was shown that high sugars concentration had an inhibitory effect on the cells growth and BD production. The maximum concentration of BD was 78.9 g/l after 61 h of fed-batch fermentation, giving a BD productivity of 1.3 g/l h and a yield of 81.4%. The present study suggests that the low-cost corncob molasses could be used as an alternative substrate for the production of BD by K. pneumoniae SDM, as well as a potential carbon source for production of other high-value chemicals.