Statistical optimization of conditions for protease production fromBacillus sp. and its scale-up in a bioreactor

A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained through the method of testing the parameters one at a time were starch, soybean meal, CaCl₂, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production, yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization of a large number of variables and provides profound insight into the interactive effect of various parameters involved in protease production.     

Statistical Optimization for Succinic Acid Production from E. Coli in a Cost-Effective Medium

Response surface methodology (RSM) was employed for optimization of medium components and cultural parameters in cost effective cane molasses based medium for attaining high yield of succinic acid. The important factors obtained by “one-variable-at-a-time-approach” (cane molasses, corn steep liquor, sodium carbonate, and inoculum density) were further optimized by RSM. The optimum values of the parameters obtained through RSM (cane molasses 12.5%, corn steep liquor 7.5%, and sodium carbonate 25 mM) led to almost double yield of succinic acid (15.2 g/l in 36 h) as against “one-variable-at-a-time-approach” (7.1 g/l in 36 h) in 500-ml anaerobic bottles containing 300-ml cane molasses based medium. Subsequently, in 10-l bioreactor succinic acid production from Escherichia coli was further improved to 26.2 g/l in 30 h under conditions optimized through RSM. This fermentation-derived succinic acid will definitely help in replacing existing environmentally hazardous and cost-intensive chemical methods for the production of succinic acid.     

Medium Optimization Based on Statistical Methodologies for Pristinamycins Production by <Emphasis Type="Italic">Streptomyces pristinaespiralis</Emphasis>

The optimization of nutrient levels for the production of pristinamycins by Streptomyces pristinaespiralis CGMCC 0957 in submerged fermentation was carried out using the statistical methodologies based on the Plackett–Burman design, the steepest ascent method, and the central composite design (CCD). First, the Plackett–Burman design was applied to evaluate the influence of related nutrients in the medium. Soluble starch and MgSO₄·7H₂O were then identified as the most significant nutrients with a confidence level of 99%. Subsequently, the concentrations of the two nutrients were further optimized using response surface methodology of CCD, together with the steepest ascent method. Accordingly, a second-order polynomial regression model was finally fitted to the experimental data. By solving the regression equation from the model and analyzing the response surface, the optimal levels for soluble starch and MgSO₄·7H₂O were determined as 20.95 and 5.67g/L, respectively. Under the optimized medium, the yield of pristinamycins in the shake flask and 5-L bioreactor could reach 1.30 and 1.01g/L, respectively, which is the highest yield reported in literature to date.     

Lignin peroxidase production by Streptomyces viridosporus T7A

Lignin peroxidase (LiP) production cost should be reduced to justify its use in the control of environmental pollution. In this work, we studied the enzyme production by Streptomyces viridosporus T7A using glucose or corn oil as a carbon source having 0.65% yeast extract as a nitrogen source. Enzyme activity, observed using either 0.65% glucose or corn oil at 0.1, 0.5, and 1.0% concentration, was 300, 150, 300, and 200 U/L, respectively. Although higher enzyme activity was obtained in both media containing 0.65% glucose and 0.5% corn oil, the use of corn oil resulted in a better LiP stability. When combined carbon sources were used, higher values of enzyme activity (360, 350, and 225 U/L) were observed in media with 0.65% glucose and supplemented with 0.1, 0.5, and 1.0% corn oil, respectively. Although the presence of both glucose and 0.5% corn oil is favorable for LiP production, satisfactory results in terms of enzyme production and stability could be also observed using 0.5% corn oil as a sole carbon source, which may lead to reduced production costs of the LiP enzyme.     

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20°C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH₄ ⁺:NO₃ ⁻ ratio (total nitrogen concentration of 60 mM) and PO₄ ³⁻ did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH₄ ⁺:NO₃ ⁻ ratio, and PO₄ ³⁻) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.     

响应面法优化黄芪多糖的提取工艺研究

为优化对黄芪多糖提取工艺.根据单因素实验结果,选取实验因素与水平,根据Box - Benhnken的试验设计原理,采用三因素三水平的响应面分析法,以获得多元二次线性回归方程,以多糖得率为响应值的响应面和等高线.结果表明,提取黄芪多糖最佳工艺条件:料水比1:13,提取温度94 ℃,提取时间64 min.在此条件下,黄芪多...     

High-productivity continuous biofilm reactor for butanol production

Corn steep liquor (CSL), a byproduct of the corn wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 h⁻¹. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(L·h), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation. Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Optimization of inulinase production by Kluyveromyces marxianus using factorial design

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2^5–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K₂HPO₄), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K₂HPO₄ had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K₂HPO₄. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.     

Optimization of critical medium components for the maximal production of gentamicin by Micromonospora echinospora ATCC 15838 using response surface methodology

Optimization of the fermentation medium components for maximum gentamicin production by Micromonospora echinospora ATCC 15838 was carried out. Response surface methodology was applied to optimize the medium constituents. A 2⁴full-factorial central composite design was chosen to explain the combined effects of the four medium constituents, viz. starch, soyabean meal, K₂HPO₄, and CoCl₂ and to design a minimum number of experiments. A second order model was developed and fitted using least square method. The R ² value of the model was 0.9723, which shows that model is best fit for the present studies. The results of analysis of variance and regression of a second order model showed that the linear effects of starch (p<0.001697) and CoCl₂(p<7.99E-13), and cross product effects of starch and soyabean meal (p<0.029876) and soyabean meal and CoCl₂ (p<0.008909) were more significant, suggesting that these were critical variables having the greatest effect on the production of gentamicin in the production medium. The optimized medium consisting of 9 g/L starch, 3 g/L soyabean meal, 0.9 g/L K₂HPO₄, and 0.01 g/L CoCL₂ predicted 850 mg/L of gentamicin which was almost 110% higher than that of the unoptimized medium. The amounts of starch, soyabean meal, and K₂HPO₄ required were also reduced with RSM.     

Evaluation of optimization techniques for an extractive alcoholic fermentation process

The mathematical optimization of a continuous alcoholic fermentation process combined with a flash column under vacuum was studied. The objective was to maximize % yield and productivity in the fermentor. The results using surface response analysis combined with modeling and simulation were compared withy those obtained when the problem was written as a nonlinear programming problem and was solved with a successive quadratic programming (SQP) technique. Two process models were evaluated when the process was optimized using the SQP technique. The first one is a deterministic model, whose kinetic parameters were experimentally determined as functions of the temperature, and the second is a statistical model obtained using the factorial design technique combined with simulation. Although the best result was the one obtained using the rigorous model, the values for productivity and % yield obtained using the simplified model are acceptable, and these models can be used when the development of a rigorous model is excessively difficult, slow, or expensive.     

Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

Stepwise optimization approach for improving LC‐MS/MS analysis of zwitterionic antiepileptic drugs with implementation of experimental design

In this article, a step‐by‐step optimization procedure for improving analyte response with implementation of experimental design is described. Zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, were chosen as model compounds to undergo chloroformate‐mediated derivatization followed by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) analysis. Application of a planned stepwise optimization procedure allowed responses of analytes, expressed as areas and signal‐to‐noise ratios, to be improved, enabling achievement of lower limit of detection values. Results from the current study demonstrate that optimization of parameters such as scan time, geometry of ion source, sheath and auxiliary gas pressure, capillary temperature, collision pressure and mobile phase composition can have a positive impact on sensitivity of LC‐MS/MS methods. Optimization of LC and MS parameters led to a total increment of 53.9%, 83.3% and 95.7% in areas of derivatized vigabatrin, pregabalin and gabapentin, respectively, while for signal‐to‐noise values, an improvement of 140.0%, 93.6% and 124.0% was achieved, compared to autotune settings. After defining the final optimal conditions, a time‐segmented method was validated for the determination of mentioned drugs in plasma. The method proved to be accurate and precise with excellent linearity for the tested concentration range (40.0 ng ml^?1–10.0 × 10³ ng ml^?1). Copyright © 2013 John Wiley & Sons, Ltd.     

Response Surface Optimization of the Critical Medium Components for Pullulan Production by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> FB-1

Culture conditions for pullulan production by Aureobasidium pullulans were optimized using response surface methodology at shake flask level without pH control. In the present investigation, a five-level with five-factor central composite rotatable design of experiments was employed to optimize the levels of five factors significantly affecting the pullulan production, biomass production, and sugar utilization in submerged cultivation. The selected factors included concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride. Using this methodology, the optimal values for concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride were 5.31%, 0.11%, 0.07%, 0.05%, and 0.15% (w/v), respectively. This optimized medium has projected a theoretically production of pullulan of 4.44%, biomass yield of 1.03%, and sugar utilization of 97.12%. The multiple correlation coefficient ‘R’ was 0.9976, 0.9761 and 0.9919 for pullulan production, biomass production, and sugar utilization, respectively. The value of R being very close to one justifies an excellent correlation between the predicted and the experimental data.     

Optimization of alkaline protease production by Bacillus sp. using taguchi methodology

Optimization of alkaline protease production parameters by Bacillus sp. was investigated using Taguchi methodology. The pH of the medium was observed to be the most significant factor among all selected optimization parameters at an individual level. The combinatorial influence of least significant factors, inoculum level and salt solution concentration (at the individual level), resulted in an interacting severity index of 76%, suggesting their interactive role in the regulation of protease production in this microbial species. Protease production could be improved more than 100% with Taguchi’s optimized conditions of the medium composition by this microorganism.     

Statistical Optimization of Medium Components for Improved Product Ion of Recombinant Hyperthermophilic Esterase

Introduction Hyperthermophilicorganismsthriveattempera turesnearandevenabove100℃[1—3].Inrecent years,muchinteresthasbeenfocusedonthecharacter izationofthesemicroorganisms,andinparticularon theirenzymes.Identifyingthebasesofproteinadapta tiontohightemper…     

Screening and analysis of biologically active compounds inAngelica sinensis by molecular biochromatography

Summary A novel strategy for the screening and analysis of biologically active compounds in traditional Chinese medicine by molecular biochromatography is proposed. Molecular biochromatography with human serum albumin (HSA) immobilized on silica as stationary phase was used to screen and analyse the bioactive compounds in the typical Chinese medicine ofAngelica sinensis (Oliv.) Diels. Ten peaks showed retention on this column, which is based on their affinity for HSA. Ferulic acid and liguistilide were identified as the principal active components, which agrees very well with the results in the literature. A quality control method was also developed based on the simultaneous determination the concentrations of ferulic acid and liguistilide in solutions ofAngelica sinensis (Oliv.) Diels extracted with water and methanol. It was observed that the concentrations of ferulic acid and liguistilide in solution extracted with methanol were 2 and 53 times higher, respectively, than those with water. It was shown that molecular biochromatography is an effective way of analysing and screening biologically active compounds in traditional Chinese medicine.     

Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugard-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design. Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinantZ. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a “benchmark” yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5–2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.     

Enhanced adsorption performance of a novel Fe‐Mn‐Zr metal oxide nanocomposite adsorbent for anionic dyes from binary dye mix: Response surface optimization and neural network modeling

A novel adsorbent, Fe‐Mn‐Zr metal oxide nanocomposite was synthesized and investigated for removal of methyl orange (MO) and eosin yellow (EY) dyes from binary dye solution. The magnetic nanocomposite has shown surface area of 143.01 m²/g and saturation magnetization of 15.29 emu/g. Optimization was carried out via response surface methodology (RSM) for optimizing process variables, and optimum dye removal of 99.26% and 99.55% were obtained for MO and EY dye, respectively with contact time 62 min, adsorbent dose 0.45 g/l, initial MO concentration 11.0 mg/l, and initial EY concentration 25.0 mg/l. A feed forward back propagation neural network model has shown better prediction ability than RSM model for predicting MO and EY dye removal (%). Adsorption process strictly follows Langmuir isotherm model, and enhanced adsorption capacities of 196.07 and 175.43 mg/g were observed for MO and EY dye, respectively due to synergistic effects of physicochemical properties of trimetal oxides. Surface adsorption and pore diffusions are the mechanisms involved in the adsorption as revealed from kinetic studies.     

Application of micellar enhanced ultrafiltration (MEUF) for arsenic (v) removal from aqueous solutions and process optimization

In this study, the removal of arsenic was evaluated using micellar-enhanced ultrafiltration process. Response surface methodology and Box-Behnken matrix methods were also applied to design the experiments and determine the optimum conditions. Therefore, the main operational parameters including pH (4–10), initial concentration of arsenic (20–80?µg/L), and concentration of Cetyltrimethylammonium bromide (CTAB) (1–3?mM) were investigated. The results of analysis of variance revealed a good agreement between experimental data and the built model with the determination coefficient (R²) of 0.99. Accordingly, the removal efficiency obtained was about 94.8% at the optimal condition (pH?=?6.73, the initial concentration of arsenic?=?29.44?µg/L, and CTAB concentration of 2.83?mM).     

Culture medium optimization for acetic acid production by a persimmon vinegar-derived bacterium

A new acetic acid-producing microorganism, Acetobacter sp. RKY4, was isolated from Korean traditional persimmon vinegar, and we optimized the culture medium for acetic acid production from ethanol using the newly isolated Acetobacter sp. RKY4. The optimized culture medium for acetic acid production using this microorganism was found to be 40 g/L ethanol, 10 g/L glycerol, 10 g/L corn steep liquor, 0.5 g/L MgSO₄·7H₂O, and 1.0 g/L (NH₄H₂PO₄. Acetobacter sp. RKY4 produced 47.1 g/L of acetic acid after 48 h of fermentation in a 250 mL Erlenmeyer flask containing 50 mL of the optimized medium.