Optimization Of Fermentation Medium Research Articles

Optimization of critical medium components using response surface methodology for phenazine-1-carboxylic acid production by Pseudomonas sp. M-18Q

The optimal flask-shaking batch fermentation medium for phenazine-1-carboxylic acid (PCA) production by Pseudomonas sp. M-18Q, a qscR chromosomal inactivated mutant of the strain M18 was studied using statistical experimental design and analysis. The Plackett-Burman design (PBD) was used to evaluate the effects of eight medium components on the production of PCA, which showed that glucose and soytone were the most significant ingredients (P<0.05). The steepest ascent experiment was adopted to determine the optimal region of the medium composition. The optimum composition of the fermentation medium for maximum PCA yield, as determined on the basis of a five-level two-factor central composite design (CCD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum PCA yield of 1240 mg/l was obtained at 17.81 g/l glucose and 11.47 g/l soytone, and the production was increased by 65.3% compared with that using the original medium, which was at 750 mg/l.

Optimization of the Production of Total Carotenoids by Sporidiobolus salmonicolor (CBS 2636) Using Response Surface Technique

This work aimed at evaluating the conditions of growth and the recovery of total carotenoids produced by Sporidiobolus salmonicolor (CBS 2636). Optimization of carotenoid production was achieved by experimental design technique. A Plackett–Burman design was used, followed by a complete second-order design, to optimize the concentration of total carotenoids in a conventional medium. Maximum concentration of 1,019 μg l−1 of total carotenoids was obtained in a medium containing 40 g l−1 glucose, 10 g l−1 malt extract, and 14 g l−1 peptone, at 180 rpm, 25 °C, and initial pH of 4.0. So far, no previous systematic study using microorganisms of the genus Sporobolomyces (formerly Sporidiobolus) for production of carotenoids has been reported. In this study, very good yields of carotenoids (1 mg l−1) could be obtained after optimization of fermentation medium and operation conditions.

Biocatalytic conversion of CO to acetic acid by Clostridium aceticum—Medium optimization using response surface methodology (RSM)

Acetic acid is an important industrial feedstock which can be produced from renewable resources such as carbon monoxide (CO) for an infinite supply for future usage. The biological route of acetic acid production from CO through fermentation by acetogenic bacteria, Clostridium aceticum appeared to be potential substitution for current process technology. The fermentation medium which is responsible for sustaining and maintaining the biocatalyst growth usually accounts for 60–70% of the production cost. Therefore, efforts in creating cost effective process have been carried out by optimizing the nutrient requirements of NH 4Cl and yeast extract compositions which can still attain satisfactory responses during acetic acid fermentation. In this study, medium composition of NH 4Cl (studied ranges: 0.0–1.50 g/L) was optimized in the early stage and then followed by optimizing the yeast extract component (studied ranges: 0.0–2.00 g/L). The high-end concentration selected for each medium composition was based on the concentration consisted in the DSMZ growth medium 1496 as a guideline. Response surface methodology (RSM) which consisted of 2-D contour plot and 3-D surface plot was used to determine the optimum concentrations of NH 4Cl and yeast extract that enabled the maximum acetic acid production with high CO conversion in an actively growing culture. Fermentation when launched in medium of 0.2 g/L of NH 4Cl coupled with 1.5 g/L of yeast extract and operated for 72 h cultivation time resulted in 1.01 g/L of acetic acid production and 100% CO conversion. Optimum fermentation medium was defined as 0.2 and 1.5 g/L of NH 4Cl and yeast extract respectively in this experiment based on the 67% of response desirability achieved through RSM.

Medium optimization for enhanced production of cytosine-substituted mildiomycin analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119.

Cytosine-substituted mildiomycin analogue (MIL-C) was produced effectively by supplementing cytosine into the culture of Streptoverticillium rimofaciens. In order to improve the yield of MIL-C, statistically-based experimental designs were applied to optimize the fermentation medium for S. rimofaciens ZJU 5119. Fifteen culture conditions were examined for their significances on MIL-C production using Plackett-Burman design. The Plackett-Burman design and one-variable-at-a-time design indicated that glucose and rice meal as the complex carbon sources, and peanut cake meal and NH4NO3 as the complex nitrogen sources were beneficial for MIL-C production in S. rimofaciens ZJU 5119. The results of further central composition design (CCD) showed that the optimal concentration of glucose, rice meal and peanut cake meal were 18.7 g/L, 64.8 g/L and 65.1 g/L, respectively. By using this optimal fermentation medium, the MIL-C concentration was increased up to 1336.5 mg/L, an approximate 3.8-fold improvement over the previous concentration (350.0 mg/L) with un-optimized medium. This work will be very helpful to the large-scale production of MIL-C in the future.

Bioconversion of palm oil mill effluent for citric acid production: statistical optimization of fermentation media and time by central composite design

A laboratory-scale study was conducted to evaluate the feasibility of using palm oil mill effluent (POME) as a major substrate and other nutrients for maximum production of citric acid using the potential fungal strain Aspergillus niger (A103). Statistical optimization of medium composition (substrate–POME, co-substrates–wheat flour and glucose, and nitrogen source–ammonium nitrate) and fermentation time was carried out by central composite design (CCD) to develop a polynomial regression model through the effects of linear, quadratic, and interaction of the factors. The statistical analysis of the results showed that, in the range studied, ammonium nitrate had no significant effect whereas substrate, co-substrates and fermentation time had significant effects on citric acid production. The optimized medium containing 2% (w/w) of substrate concentration (POME), 4% (w/w) of wheat flour concentration, 4% (w/w) of glucose concentration, 0% (w/v) of ammonium nitrate and 5 days fermentation time gave the maximum predicted citric acid of 5.37 g/l which was found to be 1.5 g/l in the experimental run. The determination of coefficient (R2) from the analysis observed was 0.964, indicating a satisfactory adjustment of the model with the response. The analysis showed that the major substrate POME (P < 0.05), glucose (P < 0.01), nutrient (P < 0.05), and fermentation time (P < 0.01) was more significant for citric acid production. The bioconversion of POME for citric acid production using optimal conditions showed the higher removal of chemical oxygen demand (82%) with the production of citric acid (5.2 g/l) on the final day of fermentation process (7 days). The pH and biosolids accumulation were observed during the bioconversion process.

Response surface methodology for optimizing the fermentation medium of alpha-galactosidase in solid-state fermentation

Alpha-galactosidase is applied in food and feed industries for hydrolysing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to develop an optimal culture medium for the production of alpha-galactosidase in solid-state fermentation (SSF) by a mutant strain Aspergillus foetidus. Response surface methodology (RSM) was applied to evaluate the effects of variables, namely the concentrations of wheat bran, soybean meal, KH(2)PO(4), MnSO(4).H(2)O and CuSO(4).5H(2)O on alpha-galactosidase production in the solid substrate. A fractional factorial design (FFD) was firstly used to isolate the main factors that affected the production of alpha-galactosidase and the central composite experimental design (CCD) was then adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 8.2137 g wheat bran, 1.7843 g soybean meal, 0.001 g MnSO(4).H(2)O and 0.001 g CuSO(4).5H(2)O in 10 g dry matter fermentation medium. After incubating 96 h in the optimum fermentation medium, alpha-galactosidase activity was predicted to be 2210.76 U g(-1) dry matter in 250 ml shake flask. In the present study, alpha-galactosidase activity reached 2207.19 U g(-1) dry matter. Optimization of the solid substrate was a very important measure to increase enzyme activity and realize industrial production of alpha-galactosidase. The process of alpha-galactosidase production in laboratory scale may have the potential to scale-up.

Optimization of polyhydroxybutyrate production by Bacillus sp. CFR 256 with corn steep liquor as a nitrogen source.

Polyhydroxyalkanotes (PHAs), the eco-friendly biopolymers produced by many bacteria, are gaining importance in curtailing the environmental pollution by replacing the non-biodegradable plastics derived from petroleum. The present study was carried out to economize the polyhydroxybutyrate (PHB) production by optimizing the fermentation medium using corn steep liquor (CSL), a by-product of starch processing industry, as a cheap nitrogen source, by Bacillus sp. CFR 256. Response surface methodology (RSM) was used to optimize the fermentation medium using the variables such as corn steep liquor (5-25 g l(-1)), Na(2)HPO(4) 2H(2)O (2.2-6.2 g l(-1)), KH(2)PO(4) (0.5-2.5 g l(-1)), sucrose (5-55 g l(-1)) and inoculum concentration (1-25 ml l(-1)). Central composite rotatable design (CCRD) experiments were carried out to study the complex interactions of the variables.The optimum conditions for maximum PHB production were (g l(-1)): CSL-25, Na(2)HPO(4) 2H(2)O-2.2, KH(2)PO(4) - 0.5, sucrose - 55 and inoculum - 10 (ml l(-1)). After 72 h of fermentation, the amount of PHA produced was 8.20 g l(-1) (51.20% of dry cell biomass). It is the first report on optimization of fermentation medium using CSL as a nitrogen source, for PHB production by Bacillus sp.

Enhanced Biosynthesis of Sulfide Oxidase by Arthrobacter Species Using Response Surface Methodology

AbstractResponse surface methodology was used to develop a fermentation medium for the enhanced biosynthesis of a novel sulfide oxidase by Arthrobacter species strain FR‐3. The interactive effect of the medium components – such as glucose as the carbon source, and tryptone and yeast extract as the nitrogen source – was evaluated by a 23‐factorial central composite statistical design. Glucose and yeast extract were found to be the more influential medium constituents compared to tryptone since they had lower coefficients of linear effect, P‐values (&lt; 0.02). The optimal fermentation medium components for the enhanced production of sulfide oxidase were recorded as glucose (8.98 g/L), tryptone (10.62 g/L) and yeast extract (7.3 g/L). Optimization of the medium constituents increased the experimental enzyme yield by 54 % compared to the unoptimized medium. This is the first report on the overproduction of sulfide oxidase by using response surface methodology.

Optimization of the Fermentation Medium for Alpha‐Galactosidase Production from Aspergillus foetidus ZU‐G1 Using Response Surface Methodology

The optimization of fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was investigated in shaker flask fermentation. A one-factor-at-a-time experiment was used to screen the preferable nutriment (carbon sources, nitrogen sources, and essential elements) for alpha-galactosidase production. A fractional factorial design was used to screen the main 5 factors, soybean meal, wheat bran, KH2PO4, FeSO4 x 7 H2O, and the medium initial pH, that affected the production of alpha-galactosidase. The central composite experimental design was further adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 3.2% soybean meal (w/v), 2% wheat bran (w/v), 0.1% KH2PO4 (w/v), and 0.05% FeSO4 x 7 H2O (w/v); initial medium pH was 6.31. The results further predicted that alpha-galactosidase activity reached 64.75 U/mL after 96-h incubation in this medium, which was approximately 7 times higher than that incubated in the nonoptimized medium. The time course of alpha-galactosidase production in the optimized medium composition was also carried out to validate the model.

Mixture design as a first step for optimization of fermentation medium for cutinase production from Colletotrichum lindemuthianum

Cutinase enzymes from fungi have found diverse applications in industry. However, most of the available literature on cutinase production is related to the cultivation of genetically engineered bacteria or yeast cells. In the present study, we use mixture design experiments to evaluate the influence of six nutrient elements on production of cutinase from the fungus Colletotrichum lindemuthianum. The nutritional elements were starch, glucose, ammonium sulfate, yeast extract, magnesium sulfate, and potassium phosphate. In the experimental design, we imposed the constraints that exactly one factor must be omitted in each set of experiments and no factor can account for more than one third of the mixture. Thirty different sets of experiments were designed. Results obtained showed that while starch is found to have negative influence on the production of the enzyme, yeast extract and potassium phosphate have a strong positive influence. Magnesium sulfate, ammonium sulfate, and glucose have low positive influence on the enzyme production. Contour plots have also been created to obtain information concerning the interaction effects of the media components on enzyme production.

Synergistic effect of Candida maltosa HY-35 and Enterobacter aerogenes W-23 on hydrogen production

An aciduric high-yielding hydrogen yeast named Candida maltosa HY-35 was screened, which can grow and produce hydrogen at pH 1.3. Further research was carried out batchwise to measure the hydrogen-producing ability of a mixed culture of C. maltosa HY-35 and a facultative anaerobe Enterobacter aerogenes W-23. In this method, with the mixed culture of these two strains at 35 ∘ C for 28 h, the hydrogen yield was 1735 ml/l, which was 17.15% and 119.90% higher than those of the monoculture of E. aerogenes W-23 and C. maltosa HY-35, respectively. Meanwhile, the average hydrogen production rate with the mixed culture was 261.1 ml/h/l, which was 7.85% and 146.23% higher than those of the monoculture of E. aerogenes W-23 and C. maltosa HY-35, respectively. The results suggested that mixed culture of these two strains had the synergistic effect on hydrogen production. The optimum fermentation medium and operation conditions for hydrogen production with mixed culture were also investigated.

Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans

The effect of in situ immobilization of Gluconobacter oxydans on a novel carrier material in a repeated-fed-batch operated packed-bed bubble-column bioreactor for the production of the fine chemical dihydroxyacetone was investigated experimentally. The carrier material were biocompatible, durable, coated Ralu-rings. The coating was a porous silicone matrix with satisfactory wetting characteristics. Settling of cells was relatively rapid. The cells were protected from abrasion caused by shear forces. A sufficiently high oxygen supply rate to the immobilized cells was provided due to the high oxygen permeability of the silicone matrix. The immobilized biomass was estimated to be about 65% of the total biomass contained in the bioreactor after 18 days of operation. The observed space-time yield was approx. 76% higher compared to a similar process which was performed without an optimized fermentation medium. Compared to previous experiments with a trickle-bed bioreactor, the space-time yield was approx. 3.7 times higher. A typical time course of the immobilization process was observed: after an induction phase, a transition phase followed which later on gave way to a nearly linear accumulation phase. A stationary phase with regard to the amount of immobilized active cells, however, was not reached. Hence, a higher bioreactor performance than observed could be expected at longer operation times.

Optimization of fermentation media for exopolysaccharide production from Lactobacillus plantarum using artificial intelligence-based techniques

A Lactobacillus strain was isolated from the fermented Eleusine coracana. This strain was characterized as Lactobacillus plantarum and was found to produce an exopolysaccharide (EPS) in quantitative amounts. The objective of the present paper is to determine optimum media composition and inoculum volume for the stated fermentative production of the EPS. A hybrid methodology comprising the Plackett–Burman (PB) design method , artificial neural networks (ANN) and genetic algorithms (GA) was utilized. Specifically, the PB, ANN and GA formalisms were used for identifying influential media components, modeling non-linear process and optimizing the process, respectively. More specifically, the PB method was used to determine those media components, which significantly influence the EPS yield. By ignoring the less influential media components, the dimensionality of the input space of the process model could be reduced significantly. Out of the five media components only three were found influential namely, lactose, casein hydrolysate and triammonium citrate. Next, an ANN-based process model was developed for approximating the non-linear relationship between the fermentation operating variables and the EPS yield. The average % error and correlation coefficient for the developed ANN model were 4.8 and 0.999, respectively. The input parameters of ANN model were subsequently optimized using the GA formalism for obtaining maximum EPS yield in batch fermentation. The optimized media composition has predicted the yield of 7.01 g/l. The GA-optimized solution comprising media composition and inoculum volume was verified experimentally and it comes out be 7.14 g/l.

Production of Bacterial Cellulose by &lt;i&gt;Gluconacetobacter&lt;/i&gt; sp. RKY5 Isolated From Persimmon Vinegar

The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K2HPO4, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.

574. Early Stage Product Development to Scale-Up: Use of Mini-Fermentation System for Screening and Process Development of Plasmid DNA Products

Top of pageAbstract Introduction: As the number of gene therapy studies and trials has increased in the past few years, an ever-increasing demand for plasmid DNA-based products exists. In this expanding field of plasmid production, emphasis has always been placed on the quality and safety of the plasmid product. Much of the focus has been placed on increasing the expression of these products, while few focus on the early-stage screening and development of efficient plasmid production. The typical transition of a plasmid from research to the manufacturing group begins with shaker flask culture productions, which often yield little information on the cell growth and yield characteristics. Scale-up of these shaker flask-derived products has been difficult to achieve, and there is a need to translate the results from these cultures to lab scale and pilot or production scale. Adequate information of growth and yield characteristics for various plasmid products is important in the early developmental stage of any product. Method: We have successfully demonstrated the use of the Sixfors|[trade]| mini-fermentation system for the efficient screening of early-stage development products along with process development of plasmid DNA production and the ability to scale up the process to more than 100L. The Sixfors|[trade]| mini-fermenters, with a maximum working volume of 500mL, have been configured to mimic a large-scale fermenter with efficient control of dissolved oxygen. These fermenters were used for process development of plasmid DNA production. Their use has been established in the screening of bacterial strains and the optimization of fermentation media and process parameters using statistical experimental design techniques. Results: Using the mini-fermenter system, we were able to achieve early-stage predictability of the use of bacterial strains and growth conditions for growing a particular plasmid along with the scalability of fermentation conditions. The use of statistical design techniques for optimizing media formulations and process conditions resulted in the optimum process conditions in a limited amount of time with excellent reproducibility. The studies conducted to demonstrate the successful scale-up of the fermentation process indicated that the data obtained from the mini-fermenters are reproducible even at larger scales of operation. The use of this mini-fermenter system resulted in overall reduction of the time required for the completion of plasmid DNA production. Conclusions: The use of Sixfors|[trade]| has been shown as an excellent tool for early stage process development, including plasmid DNA screening studies. This method is far superior to performing early experiments with shaker flasks and is an ideal replacement for using these cultures for screening studies. The process using this Sixfors|[trade]| system has been tested in the lab scale (10L) and the pilot scale (100L) fermenters and has been shown to scale-up with excellent reproducibility.

Response surface methodology for optimizing the fermentation medium of Clostridium butyricum.

Strains of Clostridium butyricum have been increasingly used as probiotics for both animals and humans. The aim of this study was to develop a growth medium for cultivating C. butyricum ZJUCB using a statistical methodology. Response surface methodology (RSM) was used to evaluate the effects of variables, namely the concentrations of the glucose, pectin, soyabean cake extract, casein, corn steep flour, ammonium sulphate, sodium bicarbonate and the medium initial pH. A fractional factorial design was applied to study the main factors that affected the growth of a probiotic strain of C. butyricum currently preserved in our lab and the central composite experimental design was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for the growth of C. butyricum was composed of 2% glucose (w/v), 0.5% pectin (w/v), 0.2% casein (w/v), 3.98% soyabean cake extract, 0.1% (NH4)2SO4 (w/v), 0.124% NaHCO3 (w/v), 0.37% corn steep flour (w/v), 0.02% MnSO4 H2O (w/v), 0.02% MgSO4 7H2O (w/v) and 0.002% CaCl2 (w/v) at pH 7.5. After incubating 24 h in the optimum fermentation medium, the populations of the viable organisms were estimated to be 10(9) CFU ml(-1). In the present study, we report the optimization of a growth medium that produced increased yields using statistical approach. The use of bacteria as a probiotic is showing increasing potential. The development of a growth medium that has a high yield is an obvious need, and the approach to optimizing a growth medium is innovative.

Optimization of medium composition for the production of clavulanic acid by Streptomyces clavuligerus

The optimization of medium components for the production of clavulanic acid by Streptomyces clavuligerus was performed using both factional factorial design and response surface methodology. The factional factorial design experiment was initially employed for this purpose and it was found that soy meal powder, FeSO 4·7H 2O, and ornithine were the most important medium components (confidence levels above 85%). Response surface methodology for optimization was further investigated. These three composing elements showed significant effects on producing clavulanic acid. Their optimal concentrations were: soy meal powder 38.102 g/l, FeSO 4·7H 2O 0.395 g/l, and ornithine 1.177 g/l. The yield of clavulanic acid was increased by 50% by culturing strain S. clavuligerus in the nutritionally optimized fermentation medium.

Optimization of rifamycin B fermentation in shake flasks via a machine‐learning‐based approach

Rifamycin B is an important polyketide antibiotic used in the treatment of tuberculosis and leprosy. We present results on medium optimization for Rifamycin B production via a barbital insensitive mutant strain of Amycolatopsis mediterranei S699. Machine-learning approaches such as Genetic algorithm (GA), Neighborhood analysis (NA) and Decision Tree technique (DT) were explored for optimizing the medium composition. Genetic algorithm was applied as a global search algorithm while NA was used for a guided local search and to develop medium predictors. The fermentation medium for Rifamycin B consisted of nine components. A large number of distinct medium compositions are possible by variation of concentration of each component. This presents a large combinatorial search space. Optimization was achieved within five generations via GA as well as NA. These five generations consisted of 178 shake-flask experiments, which is a small fraction of the search space. We detected multiple optima in the form of 11 distinct medium combinations. These medium combinations provided over 600% improvement in Rifamycin B productivity. Genetic algorithm performed better in optimizing fermentation medium as compared to NA. The Decision Tree technique revealed the media-media interactions qualitatively in the form of sets of rules for medium composition that give high as well as low productivity.

The screening of culture condition and properties of xylanase by white-rot fungus Pleurotus ostreatus

Cultural conditions for xylanase production by Pleurotus ostreatus SYJ042 were screened in shake flask cultures. The results indicated that various factors including carbon and nitrogen source, air, pH, and inoculum size influence xylanase secretion. The optimum fermentation medium contained a carbon source (2.5% corn cob+2.5% wheat bran), nitrogen (0.8% peptone), inoculation level (four disks, 0.5 cm in diameter), aeration rate (1/3 fermentation liquid volume) and initial pH 6.0. Under these culture conditions, the maximum level of xylanase activity (24.98 U ml −1) was reached in 7 days. The enzyme was stable up to 40 °C, with 99.3% of the activity remaining after the enzyme had been incubated for 15 min. The enzyme appeared to be stable over a broad pH range (3.0–9.0) under assay conditions. The majority of the metal ions tested had little effect on enzyme activity with the exception of MnO 4 2− (strong inhibitor) and Ag + (modest inhibitor).

Optimization of media by evolutionary algorithms for production of polyols.

Biotransformation of sucrose-based medium to polyols has been reported for the first time using osmophilic yeast, Hansenula anomala. A new, real coded evolutionary algorithm was developed for optimization of fermentation medium in parallel shake-flask experiments. By iteratively employing the nature-inspired techniques of selection, crossover, and mutation for a fixed number of generations, the algorithm obtains the optimal values of important process variables, namely, inoculum size and sugar, yeast extract, urea, and MgSO4 concentrations. Maximum polyols yield of 76.43% has been achieved. The method is useful for reducing the overall development time to obtain an efficient fermentation process.