Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

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.     

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.     

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

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.     

High cell density culture of Yarrowia lipolytica using a one-step feeding process

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.     

Statistical optimization of culture media and conditions for production of mannan by Saccharomyces cerevisiae

In view of the increase in Saccharomyces cerevisiae mannan content, the culture medium and condition for S. cerevisiae were optimized in this study. The influence of culture medium ingredients such as carbon and nitrogen sources, inorganic ion, and enzyme activator on mannan production were evaluated using factional design. The mathematical model was established by the quadratic rotary combination design through response surface analysis. The optimized concentrations of culture medium were determined as follows: 4.98 g/100 mL, sucrose; 4.39 g/100 mL, soybean peptone; 3.10 g/100 mL, yeast extract; and 2.21 g/100 mL, glycerol. The optimized culture medium increased mannan production from 82.7 ± 3.4 mg/100 mL to 162.53 ± 3.47 mg/100 mL. The influence of original pH, inoculum size, temperature, and media volume on mannan production was evaluated and confirmed by orthogonale experimental design, with the order of effect as follows: media volume > temperature > initial pH > inoculation size. The optimized culture condition was pH, 5; inoculum size, 5 ml; temperature, 32°C; and media volume, 40 mL. The maximum mannan production increased to 258.5 ± 9.1 mg/100 mL at the optimum culture condition. It was evident that the mannan production was affected significantly by culture medium and condition optimization (p < 0.01).     

Improvement of Bioinsecticides Production by Sporeless <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strains in Response to Various Stresses in Low Cost Medium

The use of bioinsecticides, particularly those produced by sporeless Bacillus thuringiensis strains, has been shown to be a good alternative in pest management. Two types of sporeless mutants were distinguished. The asporogenic mutants which completely lack spores produce a regular bipyramidal crystal inclusion. The oligosporogenic mutants kept the ability to produce insecticidal crystal proteins. However, sporulation in such mutants was not totally blocked and very few of them could still produce spores. In order to improve bioinsecticides production, adaptation of sporeless strains to heat shock and osmotic stress was investigated. Delta-endotoxin production by 78% of sporeless mutants was significantly improved by osmotic stress with an overproduction of about 17%, compared to the wild strain BNS3. However, toxin production was improved by only 21% of mutants after heat shock, in low cost medium. The statistical analysis proved that delta-endotoxin production, cell growth, and spore formation of asporogenic and oligosporogenic mutants depended on the type of applied stress. Each strain has an important potential when applying the adequate stress. Moreover, adaptation of sporeless mutants to NaCl may allow the substitution of all minerals of the medium by diluted sea water which appeared to be a good alternative for the economic production of bioinsecticides at industrial scale which is of great importance from the practical point of view.     

Optimization of fermentation conditions for production of anti-TMV extracellular ribonuclease by Bacillus cereus using response surface methodology

Bacillus cereus ZH14 was previously found to produce a new type of antiviral ribonuclease, which was secreted into medium and active against tobacco mosaic virus. In order to enhance the ribonuclease production, in this study the optimization of culture conditions using response surface methodology was done. The fermentation variables including culture temperature, initial pH, inoculum size, sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃ were considered for selection of significant ones by using the Plackett–Burman design, and four significant variables (sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃) were further optimized by a 2⁴ factorial central composite design. The optimal combination of the medium constituents for maximum ribonuclease production was determined as 8.50 g/l sucrose, 9.30 g/l yeast extract, 2.00 g/l MgSO₄·7H₂O, and 0.62 g/l KNO₃. The enzyme activity was increased by 60%. This study will be helpful to the future commercial development of the new bacteria-based antiviral ribonuclease fermentation process.     

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.     

Mitochondrial activity of 2,6-diaminopurine in Saccharomyces cerevisiae.

Summary 2,6-diaminpurine (DAP) selectively inhibited mitochondrial protein synthesis in yeast cells with concomitant failure of cells to grow in non-fermentable (yeast extract, glycerol) medium. The selectivity was pronounced in all strains tested (15) nearly all of which were able to grow in yeast extract, glucose medium containing 5 mg/ml DAP (maximum solubility) whereas growth was arrested in all strains at 250–500 g/ml DAP in the glycerol medium. The inhibition was reversed by further addition of adenine to the culture medium. RNA synthesis in rat liver mitochondria was depressed by DAP suggesting that the analogue affected RNA polymerase activity.There was no evidence of nuclear mutagenicity by DAP but resistance to the antibiotics chloramphenicol and oligomycin was induced by the drug. Genetic evidence, although limited, indicated that the resistance mutations were cytoplasmic. The mitochondrial petite mutation was also induced by DAP but only at comparatively high concentrations. The mutagenic effects were seen only in the glycerol medium.     

High-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs

Medium composition was optimized for high-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs. Glucose and yeast extract were the most important factors affecting astaxanthin production. Glucose 3.89%, yeast extract 0.29%, KH2PO4 0.25%, MgSO4 0.05%, MnSO4 0.02%, and CaCl2 0.01% were optimum for high-level production of astaxanthin. Under optimized conditions, the maximum concentration of astaxanthin obtained after 7 d of cultivation was 36.06 mg/l. The concentration of astaxanthin predicted by a polynomial model was 36.16 mg/l.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Summary Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeastHansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures ofH. polymorpha, equivalent to 35–40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract. it became clear that the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8–9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinantH. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20–22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.Part of these results have been presented at the Deuxième Congrès de la Société Française de Microbiologie, Strasbourg, France, September 1989.     

Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media

Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).     

Statistical media optimization for the biomass production of postharvest biocontrol yeast Rhodosporidium paludigenum

A cane molasses-based medium for the biomass production of biocontrol agent Rhodosporidium paludigenum was statistically optimized. Molasses concentration (after pretreatment), yeast extract, and initial pH were identified by the Plackett-Burman design to show significant influence on the biomass production. The three factors were further optimized by central composite design and response-surface methodology. The statistical analysis indicated the optimum values of the variables were 89.98?g/L for cane molasses, 2.35?g/L for yeast extract and an initial pH of 8.48. The biomass yield at the optimal culture achieved 15.89?g/L in flask fermentation, which was 2.1 times higher than that at the initial NYDB medium. In a 10-L fermenter, 18.97?g/L of biomass was obtained after 36?hr of cultivation. Moreover, the biocontrol efficacy of the yeast was investigated after culture optimization. The results showed the yeast harvested in the optimal medium maintained its initial biocontrol properties by reducing the percentage of decayed apples to below 20%.     

Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone

Gluconobacter oxydans could be immobilized as a biocatalyst for the conversion of glycerol to dihydroxyacetone. To reduce the production cost, the cells were produced from agricultural byproducts. Corn meal hydrolysate and corn steep liquor were employed to replace of sorbitol and yeast extract as medium for G. oxydans cell production. The optimal medium contained 80 g/L reducing sugar, 25 g/L corn steep liquor, and 10 g/L glycerol. The cell mass was about 4.22 g/L and the glycerol dehydrogenase activity was about 5.23 U/mL. For comparison, the cell mass was about 4.0 g/L and the glycerol dehydrogenase activity was about 5.35 U/mL cultured in sorbitol and yeast extract medium. These studies shown the corn meal hydrolysate and corn steep liquor medium was similar in performance to a nutrient-rich medium, but the cost of production was only 15% of that cultured in sorbitol and yeast extract medium. It was an economical process for the production of G. oxydans cells as biocatalyst for the conversion of glycerol to dihydroxyacetone in industry.     

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.     

Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae

Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH(4)Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g.L(-1) glycerol, 5.7 g.L(-1) KCl, 13.8 g.L(-1) NH(4)Cl, 1.5 g.L(-1) CaCl(2) and 3.0 g.L(-1) yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L.h). Furthermore, 1,3-propanediol (6.7 g.L(-1)) was also obtained from the liquid medium as a by-product.     

Production of biomass, carotenoid and other lipid metabolites by several red yeast strains cultivated on waste glycerol from biofuel production – a comparative screening study

This study was focused on a comparison of growth and production properties of seven red yeast strains of the genus Rhodotorula, Sporobolomyces and Cystofilobasidium cultivated on glycerol substrate. Production of enriched yeast biomas and specific yeast metabolites (carotenoids, ergosterol, lipids) was evaluated on medium with glucose, pure technical glycerol and/or waste glycerol from biofuel production (40 g/L) and mixture of glycerol and glucose (1:3, 1:1, 3:1; C/N ratio 57 in all cultivations). All tested strains were able to utilize glycerol as the only carbon source. Production of biomass on waste glycerol was in most strains higher than in control as well as in medium with pure technical glycerol and reached 15.97–21.76 g/L. Production of carotenoids and ergosterol was better in glucose medium than in medium with glycerol only. Nevertheless, using glycerol medium with addition of glucose, higher yields of total carotenoids, beta-carotene and ergosterol were obtained than in control. The highest yields of total pigments were reached by Sporobolomyces roseus (3.60 mg/g cell dry weight (CDW); glycerol:glucose 1:3), Sporobolomyces salmonicolor (2.85 mg/g CDW; glycerol:glucose 1:3) and Rhodotorula glutinis (2.80 mg/g CDW; glycerol:glucose 3:1) In glucose medium, most tested strains except Cystofilobasidium capitatum (22.6 %) produced neutral lipids in the range of 11–15 %. Production of triacylglycerols in all strains was in 10–30 % better in glycerol medium, in which Rhodotorula aurantiaca and Sporobolomyces shibatanus also reached intracellular triacylglycerol concentrations up to 20 % of biomass. This study has shown that oleaginous red yeasts could have great potential for converting crude glycerol to valuable lipids and carotenoids in respect of efficient bioresources utilization.