Medium Composition For Production Research Articles

Optimization of medium composition for production of a simulated urine sample containing Enterococcus faecalis

Optimization of medium composition for production of a simulated urine sample containing Enterococcus faecalis

Statistical optimization and comparative study of lipopeptides produced by Bacillus amyloliquefaciens SAS-1 and Bacillus subtilis BR-15

Two Bacillus sp. namely B. amyloliquefaciens SAS-1 and B. subtilis BR-15 isolated from two different sites were employed in biosurfactant production. The current work describes the statistical optimization of biosurfactant production and comparative characteristics study of lipopeptides produced by these microbial cultures . The composition of production medium and culture conditions were optimized to enhance the lipopeptide yield using response surface methodology (RSM). For each strain, the experiments were conducted in two sets as per designed by Central Composite Rotatable Designs (CCRD). The surface quadratic models obtained with these experimental results were validated by analysis of variance (ANOVA) and were found to be significant with F-test. Under optimized conditions, lipopeptides yield was increased from 1.13 and 1.72 g/L to 2.08 and 2.40 g/L respectively with strains SAS-1 andBR-15. The lipopeptides produced by both strains were equally efficient to emulsify (~68.0%) crude oil, nevertheless better than chemical surfactants. Though, the lipopeptides of strain BR-15 were more effective in terms of yield and crude oil displacement (2.40 g/L and 36.298 ± 2.548 cm 2 ) as compared to those of strain SAS-1 (2.08 g/L and 30.183 ± 3.781 cm 2 ). The characteristics difference can be attributed to the production of different isoforms of surfactin (lipopeptides) by B. amyloliquefaciens SAS-1 and B. subtilis BR-15. Further, the emulsification and oil displacement efficiency of lipopeptides highly recommend these as potentially applicable in bioremediation, oil recovery enhancement or as emulsifiers. • Lipopeptide production was optimized statistically with two Bacillus sp . • Lipopeptide yield was increased two folds after optimization with both strains. • The lipopeptides of strain BR15 were having higher yield and oil displacement efficiency. • The lipopeptides illustrated higher emulsification than the chemical surfactants. • The produced lipopeptides have potential application in bioremediation.

Optimization of medium composition for production of chitin-glucan complex and mannose-containing polysaccharides by the yeast Komagataella pastoris

Komagataella pastoris was recently proposed as a source of valuable polysaccharides, namely, the co-polymer chitin-glucan complex (CGC) and mannose-containing polymers (mannans), that are extracted from its cell-wall. In this study, a novel cultivation medium, Medium K, was developed envisaging the simultaneous production of both types of cell-wall polysaccharides. The use of Medium K for the cultivation of K. pastoris resulted in high contents of CGC (19 wt%) and mannans (21 wt%) in the biomass, corresponding to significantly higher products’ volumetric productivities (17.5 and 19.2 g/L day, respectively) compared to previous studies. The produced CGC had a chitin:β-glucan molar ratio of 12:88, similarly to previously reported values for K. pastoris CGC (11:89–19:81), while the mannans were mainly composed of mannose units, with a protein content of 10 wt%. These results demonstrated that the developed optimized medium was suitable for cultivation of K. pastoris and the production of both CGC and mannans. It comprised fewer components with considerably reduced salts content, thus representing a significant simplification of the bioprocess with no precipitation problems, without impacting on the polymers’ composition.

Production and characterization of collagenase from a new Amazonian Bacillus cereus strain

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39 ± 5.15 U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45 °C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2–11.0 and 25–50 °C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76 kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.

Optimization of cultivation medium composition for production of bioactive compounds effective against Penicillium sp.

Biological control is one of the best alternatives to pesticides as it avoids their weak points in plant disease control. In this study, the composition of cultivation medium for production of bioactive compounds by Bacillus subtilis ATCC 6633 was optimized. The produced bioactive compounds were tested against a phytopathogenic Penicillium sp. known for infesting different agricultural products and causing substantial crop losses. Antimicrobial activity assaying was carried out using the diffusion-disc method, and inhibition zone diameters were measured as direct indicators of antifungal activity. The response surface methodology (RSM) was used to evaluate the effects of different contents of initial nutrients (glycerol, NaNO2 and K2HPO4) in cultivation medium on inhibition zone diameter. Optimization was carried out using the desirability function method in order to maximize bioactive compounds yield and to minimize residual nutrients contents. The optimized concentrations of the selected nutrients in cultivation medium for production of bioactive compounds were: glycerol 20 g/l, NaNO2 1 g/l and K2HPO4 15 g/l.

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from Bacillus sphaericus PAD-91 in Escherichia coli.

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD+ and exhibited diaphorase activity. The molecular weight of enzyme was about 50kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30°C and pH 8.5. K m and V max values with NADH were estimated to be 0.025mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10g/L sucrose, 25g/L yeast extract, 5g/L NaCl and 0.25g/L MgSO4. At these concentrations, the actual diaphorase activity was calculated to be 345.0±4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3±5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.

Optimization of the medium composition for production of antimicrobial substances by bacillus subtilis ATCC 6633

In the effort to overcome the increase in antimicrobial resistance of different pathogens, natural products from microbial sources appear to be the most favorable alternative to current antibiotics. Production of antimicrobial compounds is highly dependent on the nutritional conditions. Hence, in order to achieve high product yields, selection of the media constituents and optimization of their concentrations are required. In this research, the possibility of antimicrobial substances production using Bacillus subtilis ATCC 6633 was investigated. Also, optimization of the cultivation medium composition in terms of contents of glycerol, sodium nitrite and phosphates was done. Response surface methodology and the method of desirability function were applied for determination of optimal values of the examined factors. The developed model predicts that the maximum inhibition zone diameters for Bacillus cereus ATCC 10876 (33.50 mm) and Pseudomonas aeruginosa ATCC 27853 (12.00 mm) are achieved when the initial contents of glycerol, sodium nitrite and phosphates were 43.72 g/L, 1.93 g/L and 5.64 g/L, respectively. The results of these experiments suggest that further research should include the utilization of crude glycerol as a carbon source and optimization of composition of such media and cultivation conditions in order to improve production of antimicrobial substances using Bacillus subtilis ATCC 6633.

Construction and application of recombinant strain for the production of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, Apr, from Bacillus licheniformis 2709 was cloned into an expression vector pET - 28b (+), to yield the recombinant plasmid pET-28b (+) - Apr. The pET-28b (+) - Apr was expressed in a high expression strain E. coli BL21. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 2709. Sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE) was used to access the protein expression. SDS-PAGE analysis indicated a protein of Mr of 38.8 kDa. The medium components and condition of incubation were optimized for the growth state of a recombinant strain. The optimal composition of production medium was composed of glucose 8 g/L, peptone 8 g/L and salt solution 10 mL. The samples were incubated on a rotary shaker of 180 r/min at 37°C for 24 h.

Spore production of entomopathogenic fungus Beauveria bassiana BNBCRC for biocontrol: Response surface optimization of medium using decanter cake from palm oil mill

Beauveria bassiana is an efficient entomopathogenic fungus for biological control. Optimization of medium composition for production of B. bassiana BNBCRC spores in decanter cake (DC)-based solid culture was performed using response surface methodology (RSM). Effects of inorganic and organic nitrogen sources, mineral salt, and incubation temperature in a DC-based composition were experimentally studied in two stages, first using a Taguchi design. Among the decision variables tested, urea, peptone, and MgSO4 significantly affected the spore concentration, and the maximal 1.89×108 spores g−1 was found at 30°C incubation temperature. In the second stage, these three selected decision variables were optimized by RSM, using a factorial central composite design. The maximum average spore concentration of B. bassiana BNBCRC increased to 4.80×108 spores g−1 in the optimal DC-based medium containing 0.80 g L−1 urea, 2.10 g L−1 peptone, and 0.75 g L−1 MgSO4. The coefficient of determination was R2=0.97 for the RSM model fit to data. Compared to the original DC-based medium, the performed optimization increased the spore production of B. bassiana BNBCRC by 2.5 fold. After optimization of moisture content, the DC -based medium containing 60% moisture gave the highest spore concentration of 4.71×108 spores g−1.

Screening of Marine Bacterial Producers of Polyunsaturated Fatty Acids and Optimisation of Production

Water samples from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea were screened in order to isolate new polyunsaturated fatty acids (PUFAs) bacterial producers especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Two hundred and fifty-one isolates were screened for PUFA production and among them the highest number of producers was isolated from the Mid-Atlantic Ridge followed by the Red Sea while no producers were found in the Mediterranean Sea samples. The screening strategy included a simple colourimetric method followed by a confirmation via GC/MS. Among the tested producers, an isolate named 66 was found to be a potentially high PUFA producer producing relatively high levels of EPA in particular. A Plackett-Burman statistical design of experiments was applied to screen a wide number of media components identifying glycerol and whey as components of a production medium. The potential low-cost production medium was optimised by applying a response surface methodology to obtain the highest productivity converting industrial by-products into value-added products. The maximum achieved productivity of EPA was 20 mg/g, 45 mg/l, representing 11% of the total fatty acids, which is approximately five times more than the amount produced prior to optimisation. The production medium composition was 10.79 g/l whey and 6.87 g/l glycerol. To our knowledge, this is the first investigation of potential bacteria PUFA producers from Mediterranean and Red Seas providing an evaluation of a colourimetric screening method as means of rapid screening of a large number of isolates.

다약제내성균에 대한 항균 활성을 가지는 Bacillus subtilis MP56 균주의 분리 및 특성분석

최근 국내외로 MRSA, VRE 등의 다약제 내성 균주의 분리빈도가 매년 급증하고 있고, 시판하고 있는 항생제 중 가장 효능이 강력한 제품들도 다약제 내성균 출현율이 증가함에 따라 이를 극복할 새로운 항생제의 개발이 요구되고 있다. 다약제 내성균에 유효한 항생제의 예로 vancomycin, teicoplanin, linezolid가 다약제 내성균에 유효한 의약품으로 쓰이고 있으며 방선균이나 Bacillus 등의 배양액에서 다약제 내성균에 유효한 물질의 발굴에 대한 연구가 활발히 진행되고 있다. 특히 넓은 항균범위를 가지고, 체내 축척이 적은 항생물질에 개발에 대한 연구가 활발히 진행되고 있다. 본 연구진은 국내 서해안의 갯벌에서 Bacillus 속 미생물을 수종 분리하였으며, 다약제 내성균에 항균 활성을 지니는 MP56 균주를 선발하였고, 균주 동정 결과 Bacillus subtilis 와 99.93%의 16S rRNA 상동성을 나타냄을 본 연구에서 확인하였다. 국내외 연구진에 의하여 다수의 Bacillus 속 미생물이 분리된 바가 있으며, 항균 물질에 대한 연구도 보고된 바 있다. 그러나 국내 갯벌에서 분리된 Bacillus 속 균주 배양액의 임상균주에 대한 항균 효능 평가에 관한 연구는 보고된 바가 없다. 본 연구에서 Bacillus 속 균주 MP56 배양액이 국내에서 분리한 임상 균주인 MRSA, IMP 균주에 대해 우수한 항균 활성을 나타내어 최적 발효조건과 항균범위를 본 논문을 통해 보고하였으며, 추후 MP56 균주 배양액에서 항균물질의 분리, 정제 및 구조 규명을 위한 후속 연구를 수행 할 예정이다. A new Bacillus strain designated as MP56 producing antimicrobial substance has been isolated from the mud flat of Korea. The strain MP56 was found to exhibit broad spectrum of antimicrobial activity against Gram-positive pathogenic microorganisms and MDR (multi drug resistant) strains. The 16S rRNA sequence revealed that the MP56 was closely related to Bacillus subtilis with 99.93% homology. The optimal medium composition for production of antimicrobial substance in the B. subtilis MP56 were 1% mannitol, 1% oat meal, 0.01% <TEX>$CaCl_2$</TEX>. Antimicrobial activity of the culture broth against different pathogenic strains was assessed using the antimicrobial spectrum. The result suggests that Bacillus strain MP56 produces high quality antimicrobial substance that might be very useful to control varieties of pathogenic microbial growth.

Optimization of extracellular lipase production by Debaryomyces hansenii isolates from dry-salted olives using response surface methodology

The lipase production by Debaryomyces hansenii strains isolated from dry-salted olives cv. Thassos was investigated. Glucose, olive oil and pH were essential to obtain a high lipase yield. Optimization of the medium components which enhance lipase production by the strain D. hansenii YLL29 was achieved with the aid of response surface methodology. The composition of the optimized medium to enhance lipase production by D. hansenii is as follows (g/L): yeast extract 5.0, peptone 10, K2HPO4 4.0, MgSO4·7H2O 1.0, glucose 13.1, olive oil 19, Tween 80 3.8, and pH 6.4. Practical validation of the optimum medium gave lipase activity 7.44U/mL, which was 2.28-fold higher than the unoptimized conditions. Under the optimized conditions the twenty D. hansenii isolates showed increased lipase activity fluctuating between 6.00 and 7.44U/mL. The results corroborated the validity and the effectiveness of the model, as the statistical approaches proved to be suitable in predicting the optimum production medium composition for maximum extracellular lipase yield. The high lipolytic activity of D. hansenii YLL29 (7.44U/mL) indicates the possible commercial importance of this isolate.

Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min−1 ml−1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X1); peptone (X2) and soy bean flour (X3) were examined, each at three different levels coded −1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min−1 ml−1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl2 0.02, Y.E. 2.5, K2HPO4 1.0, MgSO4.7H2O 0.2 and Fe2 (SO4)3 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min−1 ml−1.

Production of tyrosinase by Auricularia auricula using low cost fermentation medium

Low cost fermentation media using agricultural by-products (wheat bran extract, rice bran extract and soybean meal extract) as a major nutrient source, were evaluated for the production of tyrosinase from the fungus Auricularia auricula in submerged culture. In single-factor experiments, three components (wheat bran extract, casein and CuSO4) were chosen to further optimize medium composition using response surface methodology (RSM). The central composite experimental results showed the following optimum medium composition: wheat bran extract 36.0 %, casein 1.1 g/l and CuSO4 0.13 g/l. Under these conditions, the highest tyrosinase activity was 17.22 U/ml, which was 2.1 fold higher than that obtained using the non-optimized medium. The present study is the first to report the statistical optimization of medium composition for production of tyrosinase by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of tyrosinase.

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K2HPO4/KH2PO4) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5g/l glucose; 3% (v/v) liquid paraffin; 1.5g/l yeast extract; 3.36g/l NH4Cl and15g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07g/l.

Optimization of medium composition for production of antifungal active substance from Streptomyces hygroscopicus BS-112

Streptomyces hygroscopicus BS-112 was isolated from forest land on Mountain Tai, Shandong Province, China. This bacterium showed broad-spectrum antifungal and antibacterial activities. The medium that was necessary for the production of antifungal active substances from S. hygroscopicus BS-112 was optimized by the Plackett-Burman design (PBD) and response surface methodology (RSM). Initially, glucose, soybean powder and KH2PO4 were tested using the PBD and were found to be important factors that affected the production of active substances. The steepest ascent method was then used to determine the optimal region of each factor to include in the medium. The concentrations of important factors were subsequently optimized by using a central composite design (CCD). The results obtained demonstrated that the optimal media was composed of the following factors (g/l): corn meal, 5.00; glucose, 18.61; soybean powder, 31.15; KH2PO4, 0.38 and MgSO4, 0.85. This optimization strategy enhanced the production of active substances nearly 2.8 times (4956.28 µg/ml) compared to the yield of active substances that were produced using the basal medium. The antifungal active substances showed strong antimicrobial activity against Aspergillus flavus with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 3.13 and 6.25 µg/ml, respectively. Key word: Streptomyces hygroscopicus BS-112, antifungal active substances, Plackett-Burman design, response surface methodology, central composite design.

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. 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.

Enhanced Production of Laccase from Coriolus versicolor NCIM 996 by Nutrient Optimization Using Response Surface Methodology

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO(4), MgSO(4) x 7H(2)O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett-Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO(4), 0.002; MgSO(4) x 7H(2)O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.

Optimisation of the medium composition for production of protease and soybean peptides by Bacillus subtilis SHZ using response surface methodology

SummaryResponses surface methodology was employed to enhance the production of protease and soybean peptides by Bacillus subtilis SHZ. For screening of medium composition significantly influencing protease and soybean peptides yield, the two‐level Plackett–Burman design was used. Among thirteen variables tested; KH2PO4, glucose and defatted soybean flour (DSF) were selected based on their high significant effect on both protease activity and soybean peptides yield. Then, a three‐level Box–Behnken design was employed to optimise the medium composition for the production of the protease and soybean peptides in submerged fermentation. Mathematical models were then developed to show the effect of each medium composition and their interactions on the production of protease and soybean peptides. The model estimated that, the maximal protease activity (320 ± 1 U mL−1) could be obtained when the concentrations of glucose, KH2PO4, DSF were set at 8–9 g L−1, 2–3 g L−1, 55–65 g L−1, respectively; while a maximal yield of soybean peptides (8.5 ± 0.1 g L−1) could be achieved when the concentrations of glucose, KH2PO4, DSF were set at 7–9 g L−1, 3–4 g L−1 and 55–58 g L−1, respectively. These predicted values were also verified by validation experiments.

Optimization of nutrients for gellan gum production by Sphingomonas paucimobilis ATCC-31461 in molasses based medium using response surface methodology

A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket–Burman design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 variables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Placket–Burman design. Most suitable medium composition for production of gellan was (g/l): molasses—112.5; tryptone—1; casaminoacid—1; disodium hydrogen orthophosphate—1; manganese chloride—0.947 and the optimum gellan production was 13.814 g/l.