Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Medium optimization for keratinase production in hair substrate by a new <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KD-N2 using response surface methodology

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO₄ and K₂HPO₄ were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO₄ and K₂HPO₄ concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl₂ 0.05, KH₂PO₄ 0.7, sucrose 3, MgSO₄ 0.91, K₂HPO₄ 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.     

Design and optimization of fermentation medium for enhanced bacteriocin production by probiotic bacterium Enterococcus faecium MC13

Statistics-based experimental designs were used to develop a cost-effective medium for enhanced production of viable cells and bacteriocin by probiotic Enterococcus faecium MC13. Carbon, nitrogen, and mineral sources were first screened by one-variable-at-a-time (OVAT) methods. In order to increase yield production, the selected variables were further statistically optimized using response-surface methodology (RSM) with central composite design (CCD). The maximum and minimum levels of the selected variables were determined and a set of 34 experimental runs was performed. The optimum concentrations of the tested variables for production of viable cells (12.24 log CFU mL(-1)) and bacteriocin activity (25,600 AU mL(-1)) were tryptone (10.0 g/L), peptone (6.0 g/L), maltose (3.0 g/L), glucose (9.0 g/L), NaCl (15.0 g/L), sodium citrate (2.5 g/L), sodium acetate (1.0 g/L), and dipotassium PO(4) (0.1 g/L). Threefold increased yield of bacteriocin was achieved in optimized medium compared to the unoptimized counterpart, and this was two times less cost than commercial MRS medium.     

内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化

【目的】提高内生解淀粉芽孢杆菌CC09发酵产抗菌脂肽Iturin A的产量。【方法】首先采用单因子实验研究了碳源、氮源、NaCl浓度、pH、温度、转速和装液量等因子对CC09产Iturin A能力的影响,然后对其中显著性因子:氮源浓度、pH、温度及装液量4个因素进行正交实验,进一步优化发酵条件。【结果】优化培养基组成及发酵条件可以提高CC09菌株的生长速度及产Iturin A的量,其中可溶性淀粉以及一定比例的蛋白胨和酵母粉是CC09菌株产Iturin A的良好碳源和氮源;培养温度、装液量、培养液pH等也对CC09菌株产Iturin A有显著影响。优化后的培养基成分:可溶性淀粉(碳源)5 g/L、比例为3:1的胰蛋白胨酵母粉混合氮源15 g/L、NaCl 1 g/L;最佳培养条件:pH 6.0、28°C、摇床转速120 r/min、培养瓶装液量20%。【结论】在此条件下,Iturin A的产量可达到690 mg/L,较优化前的138 mg/L提高了4倍。     

Effect of medium components on bacteriocin production by Lactobacillus plantarum strains ST23LD and ST341LD, isolated from spoiled olive brine

Bacteriocin ST23LD levels of 2930AU/OD were recorded in MRS broth (pH of 6.5) and in the presence of tryptone and yeast extract as sole nitrogen sources. Growth in MRS broth at an initial pH of 6.0 yielded only 1460AU/OD bacteriocin ST23LD. Activities of 5861AU/OD were recorded with maltose (20, 30 and 40 g/l) as sole carbon source and 9036AU/OD with the addition of 2.0-10.0 g/l KH2PO4. Bacteriocin ST341LD levels of 2850 and 2841AU/OD were recorded in MRS broth at an initial pH of 6.0 or 5.5, respectively. Only 709AU/OD was recorded in the same medium with an initial pH of 6.5. Bacteriocin ST341LD production was stimulated by the presence of tryptone. However, glucose at 10 and 40 g/l, or the presence of 5.0 or 10.0 g/l K2HPO4, resulted in a 50% reduction of bacteriocin activity. Glycerol in the growth medium repressed bacteriocin production. No increased bacteriocin production was recorded in medium supplemented with vitamins.     

植物乳杆菌ZJ316生产细菌素

[目的]研究植物乳杆菌ZJ316生长和产细菌素的最佳培养基成分和发酵条件,以提高该菌产plantaricin ZJ316的能力.[方法]改变培养基成份和发酵条件,考察不同氮源、碳源等培养基成分和不同的发酵温度等条件对ZJ316生长和产细菌素的影响.[结果]最佳培养基为MRS培养基;优化后的培养基配方为葡萄糖10 g/L,麦芽糖10 g/L,酵母提取物10 g/L,蛋白胨10 g/L,柠檬酸三铵2 g/L,吐温80为1 Ml/L,K2HPO4·3H2O 6 g/L,乙酸钠5 g/L,硫酸镁0.2 g/L,硫酸锰0.05 g/L.培养基初始Ph6.5,30℃静置培养24 h.[结论]通过培养基成分和发酵条件的优化,细菌素产量提高了2.3倍,为进一步研究和规模化生产奠定基础.     

Optimization of culture conditions and medium composition for the production of micrococcin GO5 by Micrococcus sp. GO5

To enhance the production of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5, cultivation conditions and medium composition were optimized. The optimal initial pH and temperature for bacteriocin production were 7.0-9.0 and 37 degrees C, respectively. Micrococcus sp. GO5 displayed the highest micrococcin GO5 activity when grown in modified MRS medium that contained lactose or sucrose, rather than glucose, as a carbon source. The maximum bacteriocin activity was obtained in modified MRS medium containing 0.5% tryptone and 1.0% yeast extract as nitrogen sources instead of the other nitrogen sources present in MRS medium. Bacteriocin production was greatly affected by the concentration of K(2)HPO(4); strain GO5 produced eight-fold more bacteriocin in medium containing 2.0-2.5% K(2)HPO(4) than in medium containing 0.2% K(2)HPO(4). The optimal concentration of MgSO(4).7H(2)O for bacteriocin production was 0.5%. The production of micrococcin GO5 was increased 32-fold in shake flask culture and 16-fold in a bioreactor using the optimized medium (TY medium), compared with culturing in MRS medium.     

响应面法优化洋葱假单胞菌产脂肪酶液体发酵工艺

用响应面法对洋葱假单胞菌G-63液体发酵产脂肪酶条件进行了优化。首先运用单因子试验筛选出麦芽糖和豆粉水解液为最适碳源和氮源。在此基础上,通过Plackett-Burman设计试验,对影响产酶条件的11个相关因子进行评估并筛选出具有显著效应的3个因子:橄榄油、豆饼粉水解液以及初始pH值。在用最陡爬坡实验逼近以上3个因子的最大响应区域后,采用响应面分析法,确定出橄榄油、豆粉水解液的最佳浓度和最佳初始pH值分别为4.337%,1.956%和8.38。优化后液体发酵培养基中脂肪酶活力提高到44.39 U/mL,比初始酶活13.45 U/mL提高了3.3倍。     

Optimization of medium for the production of a novel aquaculture probiotic,<Emphasis Type="Italic">Micrococcus</Emphasis> MCCB 104 using central composite design

A marine isolate ofMicrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic toVibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen,Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L). ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L).     

STATISTICAL APPROACH FOR THE ENHANCED PRODUCTION OF COLD-ACTIVE β-GALACTOSIDASE FROM Thalassospira frigidphilosprofundus: A NOVEL MARINE PSYCHROPHILE FROM DEEP WATERS OF BAY OF BENGAL

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active β-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for β-galactosidase production. Under optimal conditions, the production of β-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. β-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active β-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active β-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.     

Statistical optimization for production of carboxymethylcellulase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> DL-3 by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/DL-3 from rice bran using response surface method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.     

Isolation and Characterization of Biosurfactant-and Bioemulsifier-Producing Bacteria from Petroleum Contaminated Sites in Western Canada

Biosurfactant-producing bacteria were isolated from two petroleum contaminated sites in western Canada. Seven potential biosurfactant/bioemulsifier-producing isolates were screened and characterized. All of the seven isolates were able to form emulsions. Emulsion-stabilizing capacity was also measured up to 48 hrs. Strain C-111-2 and C-203-2 would lead to highly reduced surface tension. For strain C-203-2, the optimum conditions that supported bacteria growth and production were investigated. The influences of carbon sources, medium pH values, and temperature were taken into account. The experimental results indicated that the crude oil and glucose were promising carbon sources for biosurfactants production; the isolated strains produced a maximum concentration of biosurfactant in a neutral pH environment and showed a higher surface activity under the temperature level of 35°C than that under 10°C. To further optimize the carbon and nitrogen source for biosurfactant production, response surface methodology (RSM) was applied to explore the favorable concentration of two carbon sources: glucose, crude oil, and one nitrogen source, NaNO₃. The optimal concentration of 8.1g/L, 4% and 3.9 g/L for glucose, crude oil, and NaNO₃, respectively, which can be obtained through RSM analysis.     

Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification

Cellulolytic enzymes produced by Trichoderma sp. have attracted interest in converting the biomass to simple sugars in the production of cellulosic ethanol. In this work, a novel cellulolytic strain M501 was isolated and identified as T. gamsii by sequencing the ITS rDNA region. The production of cellulase (CMCase) by T. gamsii M501 was enhanced by employing statistical methods. The strain grown in the optimized production medium composed of mineral salts, microcrystalline cellulose (13.7 g/l), tryptone (4.8 g/l) and trace elements (2 mL/l) at pH 5.5 and 28 °C for 72 h produced a maximum CMCase of 61.3 U/mL. The optimized production medium also showed the other enzyme activity of FPU (2.6 U/mL), β-glucosidase (2.1 U/mL), xylanase (681 U/mL) and β- xylosidase (0.6 U/mL). The crude cellulase cocktail produced by T. gamsii M501 efficiently hydrolyzed alkali pretreated sugarcane bagasse with glucose and xylose yield of 78 % and 74 % respectively at 10 % solid loading. This study is the first of its kind research on biomass saccharification using T. gamsii cellulase cocktail. Therefore, the novel strain T. gamsii M501 would be useful for further development of an enzyme cocktail for cellulosic ethanol production.     

Enhancing Endo-nitrilase production by a newly isolated Arthrobacter nitroguajacolicus ZJUTB06-99 through optimization of culture medium

The medium components of nitrilase production by Arthrobacter nitroguajacolicus ZJUTB06-99 were optimized in this study. Effects of factors such as carbon sources, nitrogen sources, and inducers on nitrilase production were investigated. Glucose, yeast extract, and ε-caprolactam were chosen as the suitable components. Moreover, experiments were carried out to fix the concentration of three factors for the zero coded level of variables in the subsequent optimization. Response surface methodology (RSM) and central composite design (CCD) were employed for further optimization. A quadratic model was found to fit the nitrilase activity and the variables. The results revealed that the optimized medium contained (%, w/v) 2.80, glucose; 0.57, yeast extract; and 0.42, ε-caprolactam. Validation experiments were carried out under the optimized conditions and nitrilase activity of 107.49 U/L was close to the predicted activity 110.82 U/L. After optimization, the nitrilase activity attained 2.86 fold of activity compared to the unoptimized conditions and the conversion of acrylonitrile was significantly improved. The strain growth curve and nitrilase activity alteration in the course of culture were tested. The cells were suitably harvested after cultured for 72∼78 h.     

Medium components effecting bacteriocin production by two strains of Lactobacillus plantarum ST414BZ and ST664BZ isolated from boza

Bacteriocins ST414BZ and ST664BZ, produced by Lactobacillus plantarum, inhibited the growth of a number of lactic acid bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae. Optimal production of bacteriocin ST664BZ (12 800 AU/mL) was recorded in MRS broth with an initial pH of 6.0 and 6.5. Bacteriocin ST414BZ was produced in MRS broth at lower pH values, ranging from 6.5 to 5.0. Low levels of bacteriocin activity were produced in BHI, M17, 10% (w/v) soy flour and 10% (w/v) molasses, suggesting that specific nutrients are required for optimal production. Bacteriocin ST414BZ production doubled (from 12 800 to 25 600 AU/mL) in MRS broth with tryptone as sole nitrogen source, or when glucose was replaced with maltose. Bacteriocin ST664BZ production, on the other hand, was less influenced by changes in nitrogen content, but increased two-fold (to 25 600 AU/mL) when glucose was replaced with sucrose, maltose or mannose, or when MRS broth was supplemented with 2.0 g/L KH₂ PO₄. Enrichment of MRS broth with vitamins B₁₂, B₁ or C did not stimulate production of the two bacteriocins. Growth in the presence of DL-6,8-thioctic acid increased bacteriocin ST664BZ production to 25 600 AU/mL. Concluded from these results, optimal levels of bacteriocins ST414BZ and ST664BZ will be produced in boza enriched with tryptone and maltose.     

中国冰川1号产适冷蛋白酶耐冷菌的分离鉴定及产酶条件

从中国冰川 1号样品分离获得一株产适冷蛋白酶耐冷菌株SYP- A2 - 3,鉴定为蜡状芽孢杆菌 (Bacilluscereus)。该菌生长温度范围为 0～ 38℃ ,最适生长温度 2 5℃ ,而最适产酶温度为 15℃。所产蛋白酶为中性金属蛋白酶 ,最适催化温度为 4 2℃ ,低温催化活力较高 ,适宜作用pH为 7. 0～ 8 .5 ,SDS PAGE测定的分子量为 34 2kD。SYP A2 3产酶条件的研究结果显示酪蛋白是较好的氮源 ,葡萄糖、淀粉是较好的碳源 ,产酶最佳pH为 6. 5～ 7. 0 ,在优化的条件下 ,15℃摇瓶产酶达到 380 0U mL ,5L发酵罐通气培养产酶达 4 80 0U mL。     

Five-factor-at-a-time (FFAT) approach for optimal production of an extracellular RNase from Bacillus safensis RB-5

Abstract

A Gram-positive, rod-shaped, endospore-forming, and RNA-degrading bacterium RB-5 was isolated from a soil sample. Based on 16-rDNA gene sequence, the bacterium RB-5 was identified as Bacillus safensis (Accession number KX443714.1). The bacterium appeared to be related to Bacillus safensis KL-052, an other-member of genus Bacillus. One-factor-at-a-time (OFAT) and Response Surface Methodology (RSM) statistical approaches were used to optimize the fermentation broth to obtain an improved extracellular RNase production from B. safensis RB-5. These approaches improved RNase activity of B. safensis KL-052 from 4.26 to 7.85?U/mL. The OFAT approach was used to study the effects of supplementation of carbon, nitrogen and physical conditions, which included temperature, pH and agitation rate on extracellular RNase production by B. safensis KL-052. Five variables screened by Central Composite Design (CCD) were employed to evaluate their interactive effects on RNase production by the organism. CCD selected 2⁵ factorial values obtained by the statistical approach were peptone 1.13% (w/v), sodium nitrate 1.13% (w/v), MgSO₄ 0.06% (w/v), pH 8.5, and temperature 35?°C for RNase production by B. safensis. The highest predicted value of RNase was 7.05?U/ml while actual obtained value was 7.85?U/ml that was ～84% and 1.84-fold higher than OFAT approach.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

Production of Penicillin G acylase from Bacillus sp.: Effect of medium components

A Bacillus sp. producing a high level of intracellular penicillin G acylase (PAC) was isolated. The PAC production in this strain was induced by phenylacetic acid. Various carbon and nitrogen sources were evaluated for their effect on growth and PAC production at 28 °C and pH 7.0. Cells grown in medium supplemented with sucrose as carbon source and tryptone as nitrogen source produced maximum activity of 6.45 and 8.92 U mg^–1, respectively. Maximum concentration of PAC (10.1 Umg^–1) was produced by the cells grown in the medium containing sucrose and tryptone, which was twofold higher than the production in basal medium.     

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> using surface response methodology and inulin hydrolysis

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl₂. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.