Optimisation of medium and culture conditions for the production of antifungal substances to Colletotrichum musae by Trametes elegans SR06

An endophytic fungus SR06 was isolated from a leaf of Amomum villosum Lour., which had a high antagonistic effect on Colletotrichum musae with an inhibition ratio of 41.20%. The antifungal substances could be secreted into fermentation broth, which had a high inhibitory activity. Strain SR06 was identified as Trametes elegans according to internal transcribed spacer sequence analysis. Response surface methodology (RSM) was used to optimise the process parameters of antifungal substances production. Using the Plackett–Burman design, three variables (glucose, yeast extract and MgSO₄·7H₂O) exerted significant effects on antifungal substances production. Then RSM experiments were conducted to further optimise the three variables. The optimal medium components were 26.45?g/L glucose, 10?g/L peptone, 14.96?g/L yeast extract and 1.49?g/L MgSO₄·7H₂O, and the optimal initial pH was 6.0, with a culture temperature of 28°C and a shaking speed of 180?rpm. Under the optimised conditions, a significant improvement in the production of antifungal substances by T. elegans SR06 was accomplished, and the inhibition zone diameter was up to 29.2?mm after culturing for 7d. The average control efficacy of the fermentation supernatant of SR06 against C. musae was 51.29% on banana fruits, which was significantly higher than that of the fungicide carbendazim.     

Factors Influencing Formation of Sclerotia in Grifola umbellate （Pers.） Pilaet Under Artificial Conditions

The effects of substrate composition and temperature on myceilal growth and sclerotium production in Grlfola umbellate （Pers.） Pilaet were Investigated In the present study. The Induction of sclerotla of G. umbellate was affected greatly by the type of medium, as well as the type of carbon source. Malt-extract agar was able to induce the production of sclerotia. The production of sclerotia was also observed when the carbon source in the GPC agar medium （glucose 20 g/L, peptone 6 g/L, corn steep liquor 10 g/L, and agar 15 g/L） was replaced with glycerol or mannitol. Altering the composition of the GPC medium with milk powder, thiamine hydrochlorlde, extract of Armlllarla mellea, active clay, dlatomite, kaolin, or arginlne did not induce the production of sclerotla. A temperature range of 18-25 ℃ was suitable for both mycellai growth and sclerotium formation. Glycerol significantly Induced slerotium formation on nutrient supplemented with sawdust substrates In bottle culture. 24S-Polyporusterone A and polyporusterone B were assayed In samples of natural and cultured sclerotla. Both natural and cultured sclerotla contained 24S- polyporusterone A and polyporusterone B.     

High-yield spore production from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> by solid state fermentation

Bacillus licheniformis was grown for 48 h at 37°C in solid state fermentation; a maximum of 1.7 × 10¹¹ spores/g dry substrate were obtained using rice straw powder (300 g/kg) and wheat bran (700 g/kg) supplemented with glucose (40 g/kg), peptone (20 g/kg), yeast extract (20 g/kg), KH₂PO₄ (10 g/kg) and CaO (5 g/kg) with an initial moisture content of 65%.     

Use of response surface methodology to optimize culture medium for production of lovastatin by Monascus ruber

Response surface methodology (RSM) was employed to study the effect of culture medium on the production of lovastatin in mixed solid-liquid state (or submerged) cultures by Monascus ruber. The maximal lovastatin yield (131 mg/L, average of three repeats) appeared at the region where the respective concentrations of rice powder, peptone, glycerin, and glucose were around 34.4 g/L, 10.8 g/L, 26.4 ml/L, and 129.2 g/L, respectively. The optimized medium resulted in a significant increase of lovastatin yield, as compared with that obtained by the fermentation of many other M. ruber species.     

Optimization of culture conditions for 1,3-propanediol production from crude glycerol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using response surface methodology

To produce 1,3-propanediol (1,3-PD) from crude glycerol, cultivation conditions were optimized by response surface methodology (RSM) based on a 2⁵ factorial central composite design (CCD). RSM was adopted to derive a statistical model for the individual and interactive effects of crude glycerol, (NH₄)₂SO₄, pH, cultivation time and temperature on the production of 1,3-PD. Optimal conditions for maximum 1,3-PD production were as follows: crude glycerol, 35 g/L; (NH₄)₂SO₄, 8 g/L; pH, 7.37; cultivation time, 10.8 h; temperature, 36.88°C. Under these optimal conditions, the design expert presented the maximal numerical solution with a predicted 1,3-PD production level of up to 13.74 g/L. The experimental production of 1,3-PD yielded 13.8 g/L, which was in close agreement with the model prediction.     

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).     

Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Statistical optimization of lovastatin production by Omphalotus olearius (DC.) singer in submerged fermentation

In this study, culture conditions were optimized to improve lovastatin production by Omphalotus olearius, isolate OBCC 2002, using statistical experimental designs. The Plackett–Burman design was used to select important variables affecting lovastatin production. Accordingly, glucose, peptone, and agitation speed were determined as the variables that have influence on lovastatin production. In a further experiment, these variables were optimized with a Box–Behnken design and applied in a submerged process; this resulted in 12.51 mg/L lovastatin production on a medium containing glucose (10 g/L), peptone (5 g/L), thiamine (1 mg/L), and NaCl (0.4 g/L) under static conditions. This level of lovastatin production is eight times higher than that produced under unoptimized media and growth conditions by Omphalotus olearius. To the best of our knowledge, this is the first attempt to optimize submerged fermentation process for lovastatin production by Omphalotus olearius.     

Improving probiotic spore yield using rice straw hydrolysate

Spore-forming Bacillus sp. has been extensively studied for their probiotic properties. In this study, an acid-treated rice straw hydrolysate was used as carbon source to produce the spores of Bacillus coagulans. The results showed that this hydrolysate significantly improved the spore yield compared with other carbon sources such as glucose. Three significant medium components including rice straw hydrolysate, MnSO₄ and yeast extract were screened by Plackett–Burman design. These significant variables were further optimized by response surface methodology (RSM). The optimal values of the medium components were rice straw hydolysate of 27% (v/v), MnSO₄ of 0·78 g l⁻¹ and yeast extract of 1·2 g l⁻¹. The optimized medium and RSM model for spore production were validated in a 5 l bioreactor. Overall, this sporulation medium containing acid-treated rice straw hydrolysate has a potential to be used in the production of B. coagulans spores.     

一株高产洛伐他汀红曲霉的筛选与液态发酵条件优化

【背景】洛伐他汀(lovastatin)是红曲霉的次生代谢产物,是重要的临床用降血脂药物。在液态发酵条件下,红曲霉的洛伐他汀产量较低,难以满足工业化生产的要求。【目的】筛选获得一株高产洛伐他汀的红曲霉株,并通过优化液态发酵条件提高洛伐他汀的产量。【方法】从红曲米中筛选获得一株高产洛伐他汀的红曲霉株,依据形态学特征、生理生化特性及18S rRNA基因序列分析对分离菌株进行鉴定;通过响应面法对其产洛伐他汀的液态发酵条件进行优化。【结果】获得一株产洛伐他汀的紫红曲霉(Monascus purpureus M4),该菌在甘油57.80g/L、酵母浸粉5.52 g/L、接种量为6.90%条件下,洛伐他汀产量(173.60 mg/L)较优化前提高了4.8倍。【结论】菌株M4产洛伐他汀最优液态发酵条件的建立,为洛伐他汀的大规模生产及该菌株的工业化应用提供了技术支撑。     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Optimization of submerged fermentation medium for citrinin-free monascin production by Monascus

Microbial fermentation of citrinin-free Monascus pigments is in favor in the development of food industry. This study investigated the influences of carbon source, nitrogen source, and mineral salts on the cell growth, monascin (MS), and citrinin (CT) production in Monascus M9. A culture medium composition was established for maximizing the production of citrinin-free MS in submerged culture, as follows: 50?g/L Japonica rice powder, 20?g/L NH₄NO₃, 3?g/L NaNO₃, 1.5?g/L KH₂PO₄, 1?g/L MgSO₄?·?7H₂O, 0.2?g/L MnSO₄. Under these conditions, no CT was detectable by high performance liquid chromatography. The yield of MS reached 14.11?mg/g, improving approximately 30% compared with before optimization.     

Enhanced biosynthesis of phenazine-1-carboxamide by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> strains using statistical experimental designs

Phenazine-1-carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and  9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett–Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p?<?0.05). Response surface methodology (RSM) based on the central composite design (CCD) was adopted to determine these factors optimal levels and their interactive effects between culture components for PCN production. The predicted maximum PCN production was 9002 mg/L, whereas an actual PCN production of 9174 mg/L was recorded in the validation experiments using the optimal medium containing glycerol 37.08 mL/L, tryptone 20.00 g/L, and soy peptone 25.03 g/L, which was nearly threefolds higher than without optimization and 20-folds higher than the wild-type strain. In conclusion, the results revealed that P. chlororaphis display a high potential for industrial-scale production for phenazine biopesticides.     

Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology

Optimization of the fermentation medium for maximization of actinorhodin production by Streptomyces coelicolor A3(2) was carried out. Response surface methodology (RSM) was applied to optimize the medium constituents. A 2⁴ full-factorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. sucrose, glucose, yeast extract (YE) and peptone, and to design a minimum number of experiments. The P-values of the coefficients for linear, quadratic and cross-product effect of sucrose and glucose concentration were <0.0001, suggesting that these were critical variables having the greatest effect on the production of actinorhodin in the complex medium. The optimized medium consisting of 339 g/l sucrose, 1 g/l glucose, 1.95 g/l YE and 2.72 g/l peptone predicted 195 mg/l of actinorhodin which was 32% higher than that of the unoptimized medium. The amounts of glucose, YE and peptone required were also reduced with RSM.     

STATISTICAL OPTIMIZATION OF PIGMENT PRODUCTION BY Monascus sanguineus UNDER STRESS CONDITION

Natural pigments are produced by the Monascus sp., which are used for coloring food substances. The intent of this study was to optimize the pigment yield and biomass produced from the unexplored Monascus sanguineus in submerged culture under stress conditions. For inducing thermal stress, the spores were incubated at various temperatures at higher ranges. For inducing osmotic stress, varied concentrations of NaCl, glycerol, and peptone were used. The medium components were optimized by response surface methodology (RSM). The combined effects of the four medium constituents mentioned were studied using a 2⁴ full factorial central composite design (CCD). The relationships between the predicted values and actual values, independent variable, and the response were calculated according to a second-order quadratic model. It was deduced that the variable with the leading effect was the linear effect of glycerol concentration. Furthermore, the quadratic effects of peptone and the interactive effects of temperature and glycerol were more noteworthy than other factors. The optimum values for the test variables in coded factors were found to be spores treated with 70°C for temperature, 0.25 M for glycerol, 0.51% (w/v) for peptone, and 1.25% (w/v) for NaCl, corresponding to a maximum red pigment yield of 55.67 color value units (CVU)/mL. With optimized conditions, the pigment yield was almost three times the yield observed with the control.     

A statistical approach for the enhanced production of thermostable alkaline protease showing detergent compatibility activity from Bacillus circulans

Abstract

Response surface methodology (RSM) was employed to enhance the production of a thermostable alkaline protease from Bacillus circulans. Significant influences of peptone, yeast extract, and glucose on protease production were noted with a one-variable-at-a-time optimization strategy. Then, a full factorial central composite design (CCD) was applied to study the effects of glucose, peptone, and yeast extract to determine the optimal concentrations of these compounds for protease production by B. circulans under shake flask fermentation conditions. The statistical reliability and significance of the model was validated by an F-test for analysis of variance (ANOVA); enzyme production was improved significantly under optimized conditions. The enzyme was purified by ammonium sulphate fractionation, and gel filtration chromatography. Maximum enzyme activity was observed at 60°C temperature, and at pH 10. Alkaline protease from B. circulans showed excellent compatibility and stability in the presence of commercial detergents like Ariel, Surf Excel, Tide, Rin, Nirma, Wheel, and Doctor and showed excellent blood destaining effectiveness with commercial detergents.     

Optimizing the medium compositions for accumulation of the novel FR-008/Candicidin derivatives CS101 by a mutant of Streptomyces sp. using statistical experimental methods

Medium compositions for the production of the novel derivatives of FR-008/Candicidin which was produced by a mutant of Streptomyces sp. FR-008 were optimized using two statistical methods including Plackett–Burman design (P–B), which was applied to find the key ingredients for the best medium composition, and response surface methodology (RSM), which was used to determine the optimal concentrations of these components. Results indicated that peptone, copper sulfate and glycerol had significant effects on the production. Under the proposed optimized conditions, the CS101 experimental yield (191.259 mg/L) closely matched the yield (203.536 mg/L) predicted by the statistical model. The optimization of the medium contributed to 10-fold higher antibiotic production than that of the control. It was first revealed that copper could stimulate FR-008/Candicidin and their derivatives synthesis at an optimal concentration in this paper, moreover, the basis of this phenomenon was also explained by investigating the enhancement of the enzymatic pathways.