Optimization of culture medium for lactosucrose ( G-beta-D-galactosylsucrose) Production by Sterigmatomyces elviae mutant using statistical analysis

In this study, the optimization of culture medium using a Sterigmatomyces elviae mutant was investigated using statistical analysis to increase the cell mass and lactosucrose ((4)G-beta-D-galactosylsucrose) production. In basal medium, the cell mass and lactosucrose production were 4.12 g/l and 140.91 g/l, respectively. However, because of the low cell mass and lactosucrose production, optimization of culture medium was carried out to increase the cell mass and lactosucrose production. Culture media were optimized by the S. elviae mutant using analysis of variance (ANOVA) and response surface methodology (RSM). Central composite designs using RSM were utilized in this investigation. Quadratic models were obtained for cell mass and lactosucrose production. In the case of cell mass, optimal components of the medium were as follows: sucrose 1.13%, yeast extract 0.99%, bactopeptone 2.96%, and ammonium sulfate 0.40%. The predicted maximum value of cell mass was about 5.20 g/l and its experimental value was 5.08 g/l. In the case of lactosucrose production, optimal components of the medium were as follows: sucrose 0.96%, yeast extract 1.2%, bactopeptone 3.0%, and ammonium sulfate 0.48%. Then, the predicted maximum value of lactosucrose production was about 194.12 g/l and the corresponding experimental value was about 183.78 g/l. Therefore, by culturing using predicted conditions, the real cell mass and lactosucrose production increased to 23.3% and 30.42%, respectively.     

Medium optimization for hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods

Statistics-based experimental design was used to investigate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl2.2H2O) on hen's egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2(5-1) fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other factors were not important within the levels tested. The method of steepest ascent was used to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g L-1, peptone 34 g L-1, ammonium sulfate 11.9 g L-1, yeast extract 0.5 g L-1, and CaCl2.2H2O 0.5 g L-1. This medium was projected to produce, theoretically, 212 mg L-1 lysozyme. Using this medium, an experimental maximum lysozyme concentration of 209+/-18 mg L-1 verified the applied methodology.     

Optimization of batch fermentation conditions for dextran production

The nutrient medium (containing sucrose, yeast extract and K₂HPO₄), temperature and initial pH conditions were optimised for batch dextran production in shake flask fermentations using a strain of Leuconostoc mesenteroides NRRL B 512 (F). A 2⁵⁻¹ fractional factorial central composite experimental design was attempted. Multistage Monte Carlo optimization program was used to maximize the multiple regression equation obtained. The optimal values of tested variables for maximal dextran production were found to be: sucrose, 300 g/l; yeast extract, 10 g/l; K₂HPO₄, 30 g/l; temperature, 23°C and initial pH 8.3 with a predicted dextran yield of 154 g/l.     

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.     

Use of coconut coir fibers as an inert solid support for production of cyclosporin A

In the present study, coconut coir was evaluated as an inert support for the production of cyclosporin A (CyA) using Tolypocladium inflatum MTCC 557 by solid state fermentation. Initially, four different inert supports such as coconut coir, polyurethane foam, polystyrene beads, and sugarcane baggase were screened using different production media as moistening agents for the maximum production of CyA. Different parameters such as fermentation time, carbon sources, moisture content, pH, and inoculum size were optimized. It was observed that coconut coir impregnated with medium modified with glycerol as carbon source, pH 6, at 80% moisture content, and inoculum size of 2.5 mL/2.5 g support produced 2641 mg/kg of CyA after 12 days as compared to 998 mg/kg before optimization. The yields were further increased to 3597 mg/kg substrate with addition of combination of amino acids after 48 h of fermentation.     

Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94-0.97 g/g of starch or sugars associated with 4-5 g/l of fungal biomass produced, while 17-19 g/l fungal biomass with a lactic acid yield of 0.65-0.76 g/g was produced by the R. oryzae 2062 in 36-48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8-15% lactic acid yield and 10-20% fungal biomass.     

Statistical media design for efficient polyhydroxyalkanoate production in Pseudomonas sp. MNNG-S

Polyhydroxyalkanoate (PHA) is a promising polymer for various biomedical applications. There is a high need to improve the production rate to achieve end use. When a cost-effective production was carried out with cheaper agricultural residues like molasses, traces of toxins were incorporated into the polymer, which makes it unfit for biomedical applications. On the other hand, there is an increase in the popularity of using chemically defined media for the production of compounds with biomedical applications. However, these media do not exhibit favorable characteristics such as efficient utilization at large scale compared to complex media. This article aims to determine the specific nutritional requirement of Pseudomonas sp. MNNG-S for efficient production of polyhydroxyalkanoate. Response surface methodology (RSM) was used in this study to statistically design for PHA production based on the interactive effect of five significant variables (sucrose; potassium dihydrogen phosphate; ammonium sulfate; magnesium sulfate; trace elements). The interactive effects of sucrose with ammonium sulfate, ammonium sulfate with combined potassium phosphate, and trace element with magnesium sulfate were found to be significant (p??1 to 4.56?g?L^?1).     

Production of glutaminase (E.C.3.2.1.5) from Zygosaccharomyces rouxii: statistical optimization using response surface methodology

A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of sucrose, yeast extract, sodium chloride, and glutamine, identified earlier by one-factor-at-a-time approach, on glutaminase production by Zygosaccharomyces rouxii. A significant influence of yeast extract on glutaminase production was noted. Response surface methodology (RSM) showed that a medium containing (g/l) sucrose, 17.8; yeast extract, 48.0; glutamine, 5.0 and sodium chloride, 55.6 to be optimum for the production of glutaminase. This medium was projected to produce, theoretically, an enzyme activity of 149.98 U/l and a specific activity of 0.488 U/mg protein. The applied methodology was validated using this optimized media and enzyme activity 155.89+/-1.68 U/l and specific activity of 0.468+/-0.088 U/mg protein was obtained. Further, this optimization strategy combined with an increase in inoculum enhanced the enzyme activity and specific activity by 2.94 and 3.58 fold, respectively, as compared to the unoptimized media.     

Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology

Optimization of the fermentation medium for maximization of actinorhodin production by Streptomyces coelicolor A3(2) was carried out. Response surface methodology (RSM) was applied to optimize the medium constituents. A 2⁴ full-factorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. sucrose, glucose, yeast extract (YE) and peptone, and to design a minimum number of experiments. The P-values of the coefficients for linear, quadratic and cross-product effect of sucrose and glucose concentration were <0.0001, suggesting that these were critical variables having the greatest effect on the production of actinorhodin in the complex medium. The optimized medium consisting of 339 g/l sucrose, 1 g/l glucose, 1.95 g/l YE and 2.72 g/l peptone predicted 195 mg/l of actinorhodin which was 32% higher than that of the unoptimized medium. The amounts of glucose, YE and peptone required were also reduced with RSM.     

Fatty acid and carotenoid production by Sporobolomyces ruberrimus when using technical glycerol and ammonium sulfate

The production of carotenoids, lipid content, and fatty acid composition were all studied in a strain of Sporobolomyces ruberrimus when using different concentrations of technical glycerol as the carbon source and ammonium sulfate as the nitrogen source. The total lipids represented an average of 13% of the dry weight, and the maximum lipids were obtained when using 65.5 g/l technical glycerol (133.63 mg/ g). The optimal conditions for fatty acid production were at 27 degrees C using 20 g of ammonium sulfate and a pH range from 6 to 7, which produced a fatty acid yield of 32.5+/-1 mg/g, including 1.27+/- 0.15 mg of linolenic acid (LNA), 7.50+/-0.45 mg of linoleic acid (LLA), 5.50+/-0.35 mg of palmitic acid (PA), 0.60+/-0.03 mg of palmitoleic acid (PAL), 1.28+/-0.11 mg of stearic acid (SA), 9.09+/-0.22 mg of oleic acid, 2.50+/-0.10 mg of erucic acid (EA), and 4.25+/-0.20 mg of lignoceric acid (LCA), where the palmitic, oleic, and linoleic acids combined formed about 37% of the total fatty acids. The concentration of total carotenoids was 2.80 mg/g when using 20 g of ammonium sulfate, and consisted of torularhodin (2.70 mg/g) and beta-carotene (0.10 mg/ g), at 23 degrees C and pH 6. However, the highest amount with the maximum specific growth rate was obtained (micromax=0.096 h(-1)) with an ammonium sulfate concentration of 30 g/l.     

Medium optimization of carbon and nitrogen sources for the production of spores from Bacillus amyloliquefaciens B128 using response surface methodology

The production of spores from Bacillus amyloliquefaciens B128 was investigated in shaker-flask cultivation. Optimization of the culture medium was carried out by using a two-step approach. A quick identification of a suitable source of carbon and nitrogen was obtained by a simple screening experiment, which was followed by an application of response surface methodology (RSM) for further optimization design. A five-level four-factor central composite design was employed to determine the maximum spore yield at optimum levels for lactose, tapioca, ammonium sulfate and peptone. Tapioca and peptone showed a significant linear main effect, while lactose and ammonium sulfate had no significant linear effect. The spore production was also significantly affected by lactose–ammonium sulfate and lactose–peptone. Optimum cultivation parameters were (g/L): 12.7 of lactose, 16.7 of tapioca, 1.8 of ammonium sulfate and 8.0 of peptone. The prediction spore yield was 5.93 × 10⁸ (no/mL). The actual experimental results were in agreement with the prediction.     

Dynamics of l-valine in relation to the production of cyclosporin A by Tolypocladium inflatum

Summary We report the kinetics of endogenous l-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented l-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular l-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/1 l-valine. The final CyA titre under these conditions was 710 mg/1 compared to only 130 mg/1 attained without l-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in l-valine-supplemented medium. As a result, the conversion yield of CyA on l-valine remained constant during the stationary phase at 0.27 g CyA/g l-valine.     

Production of phytase by a marine yeast Kodamaea ohmeri BG3 in an oats medium: optimization by response surface methodology

Statistical experimental designs were applied for the optimization of phytase production by a marine yeast Kodamaea ohmeri BG3 in a cost-effective oats medium. Using Plackett-Burman design, oats, ammonium sulfate and initial pH were identified as significant factors and these factors were subsequently optimized using a central composite design (CCD). The optimum variables that supported maximum enzyme activity were oats 1.0%, ammonium sulfate 2.3%, glucose 2.0%, NaCl 2.0% and initial pH 6.3. The validity of the optimized variables was verified in shake-flasks level. An overall 9-fold enhancement in phytase activity (62.0-->575.5 U/ml) was attained due to the optimization.     

Statistical optimization of the medium composition by response surface methodology to enhance schizophyllan production by Schizophyllum commune

The response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the fermentation medium for the cell growth and schizophllan production by Schizophyllum commune CGMCC 5.113 in submerged culture at pH 6.5 and 26 degrees C. The four variables involved in this study were glucose, yeast extract, ammonium nitrate, and magnesium sulfate. The statistical analysis of the results showed that, in the range studied, glucose and yeast extract had a highly significant effect on schizophyllan production. The optimal medium for schizophyllan production calculated from the regression model of RSM was as follows: glucose, 18 g/l; yeast extract, 0.5 g/l; NH4NO3, 0.48 g/l; and MgSO4, 0.05 g/l, with a predicted maximum schizophyllan production of 11.74 g/l. These predicted values were experimentally validated. The excellent correlation between predicted and measured values justifies the validity of the response model. The results of bioreactor fermentation also show that the optimized medium enhanced schizophyllan production (12.80 g/l) by S. commune in a 5-1 fermenter.     

Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization

Sequential optimization strategy based on statistical experimental designs was employed to enhance glucan production by Leuconostoc dextranicum NRRL B-1146 in flask culture. A two-level Plackett–Burman design was employed first where 11 variables were studied for their influence on glucan production. Sucrose, peptone and yeast extract were the most significant variables improving glucan production. A three-level Box–Behnken factorial design was employed for maximizing the glucan production. A mathematical model was developed to show the effects of each medium component and their combinatorial interactions on glucan production. The optimal medium composition for maximum glucan production was sucrose 5.95%, peptone 0.52% and yeast extract 2.9%. This composition predicted 1063 mg/l glucan, the experimentally found glucan was 1015 ± 4.5 mg/l that showed a good agreement with the predicted value. The purified glucan was homogenous and its structural characteristics investigated by FT-IR, ¹H NMR and ¹³C NMR spectroscopic techniques showed that it contained α-(1 → 6) and α-(1 → 4) linkages.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

An excellent new medium was developed for the production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon. We investigated the effects of all the components of White's medium on the production of these derivatives. Nitrate, phosphate, copper, sulfate and sucrose had especially marked effects. With the new, M-9, medium produced from these studies the yield of shikonin derivatives was 1400 mg/l and the yield for dried cells was about 12%, whereas it was 120 mg/l, or about 2% with White's medium.     

Deoxynivalenol and 15-monoacetyl deoxynivalenol production by Fusarium graminearum R6576 in liquid media

Growth and toxigenesis by Fusarium graminearum R6576, were compared in four liquid media. Parameters monitored during the fermentation were deoxynivalenol (DON) and 15-acetyl deoxynivalenol (15-ADON) production, fungal mass, carbohydrate utilization, and pH. Factors which were varied included basal medium composition, corn steep liquor (CSL) concentration, sucrose concentration and ammonium tartrate concentration. Growth in modified Fries medium resulted in only low levels of DON (0.25 mg/ L) and 15-ADON (0.25 mg/ L) after 20 days. Addition of 4% CSL to modified Fries medium raised the 20 day DON yield to 16.5 mg/ l. Increasing the sucrose concentration in modified Fries medium amended with 4% CSL resulted in increased mycelial dry weight but decreased levels of DON. Concentrations of ammonium tartrate greater than 1% in modified Fries amended with 4% CSL greatly reduced DON yield. Use of glucose-yeast extract-peptone (GYEP) for toxin production resulted in higher yields of 15-ADON (14.0 mg/ L) than DON (5.5 mg/ L) after 20 days. However, supplementation of GYEP with 4% CSL resulted primarily in DON production (4.5 mg/ L) after 20 days. In general, qualitative and quantitative production of DON and 15-ADON by Fusarium graminearum R6576 were dependent on the composition of the complex liquid medium.     

Production of a novel transfructosylating enzyme from Bacillus macerans EG-6

A new bacterium producing a novel transfructosylating enzyme was isolated from soil and designated as Bacillus macerans EG-6. Various culture conditions for enzyme production were optimized in a flask culture. 1% (w/v) sucrose as a carbon source and a mixed nitrogen source (1% yeast extract, 1% polypeptone, and 0.5% ammonium chloride) gave the best enzyme production. Addition of phosphate and magnesium ion into the medium enhanced the enzyme yield. Optimum culture pH and temperature were 7.0 and 37?°C, respectively. Under optimal culture conditions, transfructosylating enzyme was rapidly produced in the early growth period, thereafter invertase activity was predominant as the culture proceeded. Using the culture filtrate, production of fructooligosaccharides from sucrose was preliminarily carried out. In a low sucrose concentration (200?g/l), transfructosylating activity competes with invertase activity in sucrose utilization. Subsequently, low fructooligosaccharide yield (20%) was achieved due to liberation of high amounts of glucose and fructose. The best oligosaccharide yield (43%) was achieved when 500?g/l sucrose was utilized.     

Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology

Response surface methodology was employed to optimize the composition of medium for the production of avermectin B1a by Streptomyces avermitilis 14-12A in shaker flask cultivation. Corn starch and yeast extract were found to have significant effects on avermectin B1a production by the Plackett–Burman design. The steepest ascent method was used to access the optimal region of the medium composition, followed by an application of response surface. The analysis revealed that the optimum values of the tested variables were 149.57 g/l corn starch and 8.92 g/l yeast extract. A production of 5128 mg/l, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of original level (3528 mg/l), 1.45-fold increase had been obtained.