Application of response surface methodology in medium optimization for pyruvic acid production of Torulopsis glabrata TP19 in batch fermentation

Response surface methodology (RSM) was used to optimize the fermentation medium for enhancing pyruvic acid production by Torulopsis glabrata TP19. In the first step of optimization, with Plackett-Burman design, ammonium sulfate, glucose and nicotinic acid were found to be the important factors affecting pyruvic acid production significantly. In the second step, a 23 full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components were obtained as follows: ammonium sulfate 0.7498 (10.75 g/L), glucose 0.9383 (109.38 g/L) and nicotinic acid 0.3633 (7.86 mg/L) with a predicted value of maximum pyruvic acid production of 42.2 g/L. Under the optimal conditions, the practical pyruvic acid production was 42.4 g/L. The determination coefficient (R2) was 0.9483, which ensures adequate credibility of the model. By scaling up fermentation from flask to jar fermentor, we obtained promising results.

Medium Optimization for Improved Ethanol Production in Very High Gravity Fermentation

An optimal medium （300 g·L^-1 initial glucose） comprising 6.3 mmol·L^-1 Mg2＋, 5.0 mmol·L^-1 Ca2＋, 15.0 g·L^-1 peptone and 21.5 g·L^-1 yeast extract was determined by uniform design to improve very high gravity （VHG） ethanol fermentation, showing over 30% increase in final ethanol （from 13.1% to 17.1%, by volume）, 29% decrease in fermentation time （from 84 to 60 h）, 80% increase in biomass formation and 26% increase in glucose utilization. Experiments also revealed physiological aspects linked to the fermentation enhancements. Compared to the control, trehalose in the cells grown in optimal fermentation medium increased 17.9-, 2.8-, 1.9-, 1.8- and 1.9-fold at the fermentation time of 12, 24, 36, 48 and 60 h, respectively. Its sharp rise at the early stage of fermentation when there was a considerable osmotic stress suggested that trehalose played an important role in promoting fermentation. Meanwhile, at the identical five fermentation time, the plasma membrane ATPase activity of the cells grown in optimal medium was 2.3, 1.8, 1.6, 1.5 and 1.3 times that of the control, respectively. Their disparities in enzymatic activity became wider when the glucose levels were dramatically changed for ethanol production, suggesting this enzyme also contributed to the fermentation improvements. Thus, medium optimization for VHG ethanol fermentation was found to trigger the increased yeast trehalose accumulation and plasma membrane ATPase activity.

Improvement of spergualin production by mutation and medium optimization

In order to screen a high-yield spergualin-production strain and an optimal fermentation medium, large numbers of isolates were selected after ultraviolet （UV） mutation and self-tolerant mutation of Bacillus laterosporus A7. A high-yield strain A-94-7 was obtained and the spergualin productivity was 4. 1-fold of the parent strain. The genetic stability of the high-yield strain Bacillus laterosporus A-94-7 was very good. Aider fermentation medium optimization, Bacillus laterosporus A-94-7 was able to produce 380 mg· L^-1 The spergualin productivity in Bacillus laterosporus A7 was increased 4.75-fold by mutation and medium optimization.

A New Pullulan-Producing Yeast and Medium Optimization for Its Exopolysaccharide Production

Yeast strain Y68 producing high level of pullulan was isolated from the phyton collected in Toulouse, France. This strain was identified to be Rhodotorula bacarum by BIOLOG analysis. This is the first report that pullulan was produced by Rhodotorula bacarum. The optimal medium (g L -1) for pullulan production by this strain was 80 glucose, 20 soybean cake hydrolysate, 5 K 2HPO 4, 1 NaCl, 0.2 MgSO 4·7H 2O, 0.6 (NH 4) 2SO 4, pH 7.0. Under this condition, 54 g L -1 pullulan was produced within 60 h at 30 ℃. Pullulan is a better starting material for producing marine prodrugs.

Optimization of Culturing Condition and Medium Composition for the Production of Alginate Lyase by a Marine Vibrio sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the produc- tion of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL–1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had nega- tive effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had posi- tive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of algi- nate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

Improved elastase production by Bacillus sp. EL31410—further optimization and kinetics studies of culture medium for batch fermentation

An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method. Central composite design (CCD) and response surface method-ology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K2HPO4, MgSO47H2O. The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K2HPO4 0.206 g/100 ml and MgSO47H2O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also affected elastase production and cell growth.

Fuel ethanol production using novel carbon sources and fermentation medium optimization with response surface methodology

In this study,ethanol production abilities of the novel carbon sources:sodium and calcium gluconate in different minimal and rich media were compared with glucose using Escherichia coli KO11.The strain produced higher ethanol yield in the rich medium Luria-Bertani(LB)than the other two minimal media:corn steep liquor(CSL)and M9 for two substrates(sodium and calcium gluconate).Additionally,higher ethanol yields were achieved when the strain was grown in LB and M9 medium with calcium gluconate than sodium gluconate,while the ethanol yields were similar when both sodium and calcium gluconate were added into CSL medium respectively.Response surface methodology was used to optimize the fermentation medium components for enhancing ethanol production using strain E.coli KO11 in CSL medium with calcium gluconate as the substrate in batch culture.The concentration of the potassium phosphate buffer is the only significant factor among five factors considered.A quadratic model was developed to describe the relationship between ethanol production and the factors.The optimal conditions predicted for five factors were 14.38 g/L CSL,0.0398 g/L FeCl_(3)·6H2O,1.12 g/L MgSO_(4)·6H_(2)O,15.41 g/L(NH4)_(2)SO_(4),and 1.58/1.26 g/L KH_(2)PO_(4)/K_(2)HPO_(4)(2:1 molar ratio).The highest ethanol concentration under optimal conditions was 31.5 g/L,which was 5.6 g/L higher than that from the same fermentation concentration of calcium gluconate in LB media.The high correlation between the predicted and experimental values confirmed the validity of the model.

Optimization of Medium Composition for Production of Recombinant Calf Chymosin from Kluyveromyces lactis in Submerged Fermentation

Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799.The previous data indicated that the most suitable carbon source,nitrogen source,salt and vitamin were glucose,yeast extract,KH2PO4 and Ca D-Pantothenate,respectively.The concentration of four media components were optimized by using central composite design of response surface methodology.The optimum medium composition for recombinant calf chymosin production was found to contain glucose 29.84 g· L-1,yeast extract 19.85 g·L-1,KH2PO4 0.1 g·L-1 and Ca D-Pantothenate 4.49 mg·L-1.The enzyme activity of recombinant calf chymosin was 722 U· mL-1,which was in an excellent agreement with the predicted value（723 U·mL-1）.The production of recombinant calf chymosin from Kluyveromyces lactis GG799 was effectively increased by response surface methodology.

Optimization of Fermentation Medium for Epothilones Production with Sequential Statistical Approach

A sequential statistical approach was applied to optimizing the fermentation medium of epothilones（Epos） production by means of a mutant which was obtained by treating polyangium cellulosum ATCC 15384 with nitrite and ultraviolet. The effects of different carbon sources and nitrogen sources on the fermentation medium were tested, and the suitable ones were selected. Then a uniform design was employed to design the experiments. A linear model was developed for identifying the significant components in fermentation medium, while a third degree polynomial model was used for studying the relationship between the concentration of the components in fermentation medium and the yield of Epos（YEPs）. A pattern search method was used for searching the optimum fermentation medium in the test space, which was as follows（g/L）： potassium nitrate 8.00, soybean peptone 17.60, potassium hydrogen phos- phate 1.00, beef extraction 6.46, yeast extraction 1.00, calcium chloride 0.25, sodium chloride 1.00 and ferric chloride 0.02. The optimum fermentation medium was expected to result in a yield of Epos（YEPs） of 2.48 mg/L. The validation experiments with the optimum medium were performed in triplicate and the average yield of Epos was 2.45 mg/L which was 7.78 times higher than that of Epos prepared without optimization.

Enhancement of polymyxin B1 production by an artificial microbial consortium of Paenibacillus polymyxa and recombinant Corynebacterium glutamicum producing precursor amino acids

Polymyxin B,produced by Paenibacillus polymyxa,is used as the last line of defense clinically.In this study,exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P.polymyxa CJX518-AC(PPAC)from 0.15 g/L and 61.8%to 0.33 g/L and 79.9%,respectively.The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01,which produces high levels of threonine,leucine,and isoleucine,increased polymyxin B1 production to 0.64 g/L.When strains PPAC and C.glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone,polymyxin B1 production was increased to 0.97 g/L.Furthermore,the polymyxin B1 production in the co-culture of strains PPAC and C.glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone.This study provides a new strategy to improve polymyxin B1 production.

Optimization of Callus Induction Conditions from Immature Embryos of Maize under Stress

The embryos of maize(Zea mays L.)inbred lines GS02,GS07,GS08,GS11 and GS15 were used as receptor materials to optimize the receptor system from the aspects of genotype,medium components and stress(PEG6000,mannitol,salt and low phosphorus).The results showed that GS07 had the highest induction rate(95.2%).Orthogonal test analysis showed that the best combination of medium components in induction was A2B3C1D3(2),namely,the concentration of 2,4-dichlorophenoxy acetic acid(2,4-D)was 4 mg·mL^(-1),the concentration of L-Proline(L-Pro)was 0.8 mg·mL^(-1),and the concentration of silver nitrate(AgNO3)was 10 mg·mL^(-1)(or 5 mg·mL).Interestingly,we found that the optimal medium supplemented with 30 g·L^(-1)PEG6000 or 80 g·L^(-1)mannitol was suitable for antioxidant enzyme activity and malondialdehyde(MDA)content in GS07 callus.Exogenous 10 mmol·L^(-1)Ca^(2+)in the medium components with 100 mmol·L^(-1)sodium chloride(NaCl)could improve the activity of antioxidant enzymes in GS07 callus.Callus of GS07 could divide normally and grow well in medium components with 27 mg·L^(-1)KH_(2)PO_(4).This study enhanced the adaptability of maize callus to stress and optimized the culture conditions.

Improvement of xylanase production by Aspergillus niger XY-1 using response surface methodology for optimizing the medium composition

Objective: To study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF). Methods: Statistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield. Results: Firstly, NaNO3, yeast extract, urea, Na2CO3, MgSO4, peptone and (NH4)2SO4 were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization. Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each sig-nificant variable, which included urea, Na2CO3 and MgSO4. Subsequently a second-order polynomial was determined by multiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x1 (urea)=0.163 (41.63 g/L), x2 (Na2CO3)=?1.68 (2.64 g/L), x3 (MgSO4)=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask. Conclusion: By using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.

Accelerating the menaquinone‑7 production in Bacillus amyloliquefaciens by optimization of the biosynthetic pathway and medium components

Menaquinone-7(MK-7)has an important role in preventing diseases such as cardiovascular disease and osteoporosis.In this study,a combination strategy of strain improvement and medium optimization is investigated to increase MK-7 production in Bacillus amyloliquefaciens.Conventional breeding method was first used to modify the biosynthetic pathway to construct a MK-7 high-producing strain by atmospheric and room temperature plasma mutagenesis and protoplast fusion.The resulted strain Ba-4 with resistance to sulfaguanidine,1-hydroxy-2-naphthoic acid,menadione,2-deoxy-d-glucose and rifampicin as well as sensitive toβ-fluoropyruvate produced 73.57±1.61 mg/L of MK-7,which was 1.36 times more than that of the parent strain H.β.D.R.-5(i.e.,31.12±1.40 mg/L).Subsequently,single-factor optimization and response surface method-ology(RSM)were used to optimize the medium components for increasing MK-7 production by strain Ba-4.Strain Ba-4 produced 90.43±1.32 mg/L of MK-7 under the single-factor optimized medium.Moreover,the results of response surface methodology indicated that glycerol,soy peptone and Tween-80 had significant effects on MK-7 production,and the highest MK-7 production(i.e.,95.03±1.01 mg/L)was obtained under the optimized medium,which was 0.29 times higher than that of the initial medium.These results confirmed that the conventional breeding methods and fermenter control system are effective strategies in improving MK-7 production by B.amyloliquefaciens.

Solid Culture of Lactarius deliciosus

The research explored the effects of carbon, nitrogen and inorganic salt nutritional factors on Lactarius deliciosus hyphae as per solid culture. The optimal recipe of culture medium was selected by observing hypha density and computing the growth index, which is the most suitable for hypha growth. The results indicated that glucose and peptone were optimal carbon and nitrogen for Lactarius deliciosus;MgSO4, CuSO4 and VB2 promoted hypha growth; FeSO4 and CoCl2 had inhibition effects. The orthogonal test concluded the optimal recipe contained glucose of 20 g/L, peptone of 1 g/L, KH2PO4 of 0.3 g/L, MgSO4 of 0.3 g/L, VB2 of 2 mg/L, and agar of 20 g/L, with original pH value adopted. After optimization, growth index of hyphae cultured at 28 ℃ was 4 055.9.

Enhanced Production of Hybrid Extracellular β-Glucanase by Re-combinant Escherichia coli Using Experimental Design Method

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid ex-tracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those ob-tained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.

A marked enhancement in production of amylase by Bacillus amyloliquefaciens in flask fermentation using statistical methods

A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was identified as Bacillus sp. based on morphological and physiochemical characterization. According to 16S rRNA gene sequencing data, the closest phylogenetic neighbor of strain YL was Bacillus amyloliquefaciens （99.54%）. After that, an optimization of culture conditions was carried out for the improvement of a-amylase production. Response surface methodology （RSM） was applied to evaluate the effect of medium components including wheat bran, cottonseed extract, yeast extract, starch, NaC1 and CaCl2. Three variables （wheat bran, cottonseed extract, and starch）, which were identified to significantly affect amylase production by Plackett-Burman design were further optimized using response surface methodology of Box-Behnken design （BBD）. The optimal concentrations estimated for each variable related to the maximum of amylase activity （86 kU/mL） were 10.80 g/L wheat bran, 9.90 g/L cottonseed extract, 0.5 g/L starch, 2.0 g/L yeast extract, 5.00 g/L NaCl and 2.00 g/L CaC12. The fermentation using optimized culture medium allowed a significant increase in amylase production （by 3-fold）. The improvement in the a-amylase production after optimization process can be considered adequate for large-scale applications.

Study on the submerged fermentation （SMF） of termitomyces eurrhizus

The influence of KH\-2PO\-4, peptone and brown sugar on \%Termitomyces fuliginosu's\% mycelium formation was studied with the application of the design of quadratic rotation general combination based on determining the growth curve. A quadratic regression model of biomass to the doses of the above three factors was established. The model fit well and therefore the optimum fermentation condition was obtained. Responses of biomass to the three single factors and to their interactions were discussed. Thus, the highest level of biomass, 21.1 g/L, appeared under the optimized conditions when the initial KH\-2PO\-4, peptone and brown sugar were 1 g/L, 4 g/L and 72 g/L respectively.

Biodecoloration of Reactive Black 5 by the methylotrophic yeast Candida boidinii MM 4035

Azo dyes are extensively used in textile dyeing and other industries. Effluents of dying industries are specially colored and could cause severe damage to the environment. The anaerobic treatment of textile dying effluents is nowadays the preferred option, but it could generate carcinogenic aromatic amines. Recently, yeasts have become a promising altemative, combining unicellular growth with oxidative mechanisms. This work reports the characterization of the first methylotrophic yeast with dye decolorizing ability, Candida boidinii MM 4035 and some insights into its decoloration mechanism. The analysis of two selected media revealed a possible two stages mechanism of Reactive Black 5 decoloration. In glucose poor media, decoloration is incomplete and only the first stage proceeds, leading to the accumulation of a purple compound. In media with higher glucose concentrations, the yeast is able to decolorize totally an initial concentration of 200 mg/L. The entire process is co-metabolic, being largely dependent on glucose concentration but being able to proceed with several nitrogen sources. Manganese dependent peroxidase but not laccase activity could be detected during decoloration. Aromatic amines do not accumulate in culture media, supporting an oxidative decoloration mechanism of unknown ecophysiological relevance.