Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans

Polysaccharides produced by microorganisms are utilized for a variety of purposes, including the use in cosmetics and as food additives. More recently, polysaccharides have been exploited by the medical and pharmaceutical industries, and those originated from many species of mushrooms have been especially useful in industrial applications; however, the production and synthesis of these compounds is costly and time consuming. In this study, we developed a method for low-cost production of exopolysaccharide (EPS) that effectively screens components and optimizes medium composition using statistical methods (Plackett-Burman and Box-Behnken design). As a result, we obtained the following optimized medium: sucrose 165.73 g/L, sodium nitrate 3.08 g/L, dipotassium phosphate 1.00 g/L, potassium chloride 0.50 g/L, magnesium sulfate 0.50 g/L, ferrous sulfate 0.01 g/L, and 0.71 g/L of Ashbya gossypii extract. The maximum production of about 29 g/L EPS was achieved in the optimized medium during 84 h batch fermentation.     

Statistical optimization of culture media for growth and lipid production of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> LB572

Botryococcus braunii has an outstanding ability to produce lipid; however, it is a slow-growing green microalgae. Statistical optimization of growth media was performed to faster growth and to increase lipid concentration. The effect of media composition on the growth of B. braunii LB572 was examined using fractional factorial design and central composite design. The media components examined include sodium carbonate, potassium phosphate, calcium chloride, magnesium sulfate, ferric citrate, and sodium nitrate. The results indicated that potassium phosphate and magnesium sulfate were major impact factors. The optimum concentrations of potassium phosphate and magnesium sulphate were found to be 0.058 and 0.09 g/L, respectively, for growth and 0.083 and 0.1 g/L, respectively, for lipid production. These values were validated using bubble column photobioreactors. Lipid productivity increased to 0.19 g/L/day in lipid-optimized media, with an average biomass productivity of 0.296 g/L/day and 64.96% w/w. In growth-optimized media, lipid productivity was 0.18 g/L/day, with an average biomass productivity of 0.304 g/L/day and 59.56% w/w.     

响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

Biogeography of sulfate-reducing prokaryotes in river floodplains

In this study, a large-scale field survey was conducted to describe the biogeography of sulfate-reducing prokaryotes (SRPs) in river floodplains. Fingerprints obtained with three methods, i.e. 16S rRNA gene-based oligonucleotide microarray, dsrB-based denaturing gradient gel electrophoresis (DGGE) and polar lipid-derived fatty acid (PLFA) analyses, were used as a proxy to describe the SRPs community diversity. Each set of profiles was subjected to a combined multivariate/correlation analysis in order to compare SRP community profiles and to highlight the environmental variables influencing the SRPs distribution along environmental gradients. Floodplain soils harbored distinct SRP communities displaying biogeographic patterns. Nearly all profiles from the tidal sites consistently separated from the nontidal sites, independently from the screening method and the multivariate statistics used. The distribution of the microarray/DGGE/PLFA-based fingerprints in the principal component plots could be correlated to eight soil variables, i.e. soil organic matter, total nitrogen, total phosphorous and total potassium, and extractable ammonium, nitrate, phosphate and sulfate, as well as seven pore water variables, i.e. phosphate, sulfate, sulfide, chloride, sodium, potassium and magnesium ions. Indication of a salinity- and plant nutrient-dependent distribution of SRPs related to Desulfosarcina, Desulfomonile and Desulfobacter was suggested by microarray, DGGE and PLFA analyses.     

Selection and optimization of an aqueous two‐phase system for the recovery of 1,3‐propandiol from fermentation broth

In this study a suitable alcohol/salt aqueous two‐phase (ATP) system was selected for the recovery of 1,3‐propandiol (1,3‐PD) from fermentation broth. From the different alcohol/salt systems studied the ethanol and dipotassium hydrogen phosphate ATP system appeared to be favorable. To examine the potential of this ATP system the partition coefficient of 1,3‐PD in synthetic solutions was first optimized with the response surface methodology. The parameters studied were concentrations of ethanol (21.99–38.81% w/w), dipotassium hydrogen phosphate (14.99–31.81% w/w) and 1,3‐PD (6.36–73.64 g/L). The optimum conditions were found to be 35.39% w/w for ethanol, 28.40% w/w for dipotassium hydrogen phosphate and 73.6 g/L for 1,3‐PD. Under these conditions the maximum partition coefficient of 1,3‐PD and the extraction yield were determined as 23.14 and 97.82%, respectively. The optimum extraction conditions were then used to guide the recovery of 1,3‐PD from a real fermentation broth. The partition coefficient and extraction yield of 1,3‐PD reached 20.28–97.20% in this case, respectively. A favorable partition of the organic acids lactate, acetate and butyrate in the bottom phase was also achieved. We have also studied the removal of cells and macromolecules from the broth. Removal ratio of cells and proteins were 96.47 and 93.05%, respectively. Thus, the ethanol/dipotassium hydrogen phosphate ATP system appears to be an interesting alternative or can be used as one useful step in the downstream processing of 1,3‐PD from fermentation broth.     

Optimization of Phosphatidylinositol-specific Phospholipase C Production Using Response Surface Methodology

Summary Response surface methodology was employed in optimizing the nutrient levels needed towards the optimal production of phosphatidylinositol-specific phospholipase C enzyme by Bacillus thuringiensis serovar. kurstaki. A 2³ factorial central composite experimental design was used. The multiple regression equation, relating the enzyme activity to the nutrient medium, was used to find the optimum values of glucose, peptone and dipotassium hydrogen phosphate. The optimum values of these variables for maximal enzyme production were found to be: glucose, 6.5 g l⁻¹; peptone, 5.38 g l⁻¹ and dipotassium hydrogen phosphate, 6.36 g l⁻¹ with the predicted enzyme activity of 0.96 U ml⁻¹.     

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.     

北柴胡不定根系生物转化合成皂苷研究

通过在北柴胡不定根系培养体系中添加目的产物柴胡皂苷的前体物质和生物合成促进物质以及进行高糖渗透胁迫试验,探讨了利用不定根系生产柴胡皂苷的条件和方法。结果表明,在1/2 MS培养基中添加硫酸镁、硝酸钾、丙酮酸钠以及高糖渗透胁迫均有利于柴胡皂苷的合成。1/2 MS培养基+硫酸镁0.37 g/L+硝酸钾5.7 g/L+丙酮酸钠2.0 mmol/L+蔗糖50 g/L是最适合柴胡皂苷转化合成的培养基,此条件下使柴胡皂苷的含量提高了9.74倍。     

响应面设计法优化单葡萄糖醛酸基甘草次酸（GAMG）发酵转化培养基

以甘草酸（dycyfrhizin,GL）为底物,利用产紫青霉（Penicillium purpurogenum Li-3）液态发酵转化单葡萄糖醛酸甘草次酸（GAMG）,采用响应面设计法对初始发酵培养基进行优化。用部分因子分析法研究原始发酵培养基各成分对响应值的显著程度,发现甘草酸（GL）、NaNO3和K2HPO4的质量浓度对发酵产生GAMG的影响显著（P〈0．01）。用中心组合设计确立甘草酸、NaNO3和K2HPO4的适宜质量浓度分别为2．8、3．0和0．8g／L。在优化条件下进行发酵时,GAMG的转化率从75．49％提高到89．11％,比优化前提高了13．62％。     

Medium optimization for antifungal active substances production from a newly isolated Paenibacillus sp. using response surface methodology

Statistics based experimental designs were used to optimize the medium for antifungal active substances production from a newly isolated Paenibacillus polymyxa Cp-S316 in shaker flask cultivation. The medium components having significant effect on the production were first identified using a fractional factorial design. Then steepest ascent method was employed to approach the experimental design space, followed by an application of response surface methodology for further optimization. A quadratic model was found to fit the antifungal active substances production. Response surface analysis revealed that the optimum values of the tested variables for the production of active substances were 12.3 (g/l) lactose, 17.5 (g/l) peptone, 0.4 (g/l) sodium nitrate, 4.5 (g/l) magnesium sulfate and 100 (g/l) potato. A production of 4687.71mug/ml, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of basal medium, 3.05-fold increase had been obtained.     

Isolation of Aureobasidium pullulans and the effect of different conditions for pullulanase and pullulan production

Isolation and production of pullulahase by a new Aureobasidium pullulans isolate from the Fayoum Governorate (AUMC 2997) which was identified by the Assiut University Mycological Center was investigated. Another isolate from the Aswan Governorate (AUMC 1695) was kindly provided by the Assiut University Mycological Center. Acetone 2× gave better results for the precipitation of protein than 80% ammonium sulfate in the case of the media containing yeast extract. Very low protein production occurred in media without yeast extract. No enzyme production occurred in the first two days and the production of the enzyme started on the third day. Statistical analysis determined that the optimum conditions for the production of pullulanase were: incubation at 25°C for 5 days, pH 5.5, with sucrose as carbon source at 100 g/L and sodium nitrate as nitrogen source at 2 g/L. Addition of manganese chloride to the medium (1, 2 and 3 g/L) caused inhibition of pullulanase. Also, while the lowest pullulan + pigment concentrations were attained at the fifth day, pH 5.5, at 15°C, 100 g/L sucrose, 2 g/L nitrogen sources, the pullulan + pigment production increased with increasing the concentrations of manganese chloride.     

营养盐初始组合浓度对类波氏真眼点藻生长和油脂积累的影响

为了研究培养基中主要营养元素氮、磷、硫初始组合浓度对类波氏真眼点藻(Eustigmatos cf.polyphem D.J.Hibberd)生长、油脂积累和脂肪酸组成的影响,分别以NaNO_3、K_2HPO_4、MgSO_4为氮、磷、硫源,设置不同的营养盐初始组合浓度对其进行培养,采用干重法、重量法、气相色谱分析法、元素分析仪等依次对生物量、总脂、脂肪酸组成和细胞内元素含量进行测定。结果显示,氮、磷、硫三者的初始组合浓度变化对类波氏真眼点藻的生长和油脂积累具有明显的影响,9 mmol/L NaNO_3、0.92 mmol/L K_2HPO_4、0.3 mmol/L MgSO_4营养条件下其生物量达到最大值,为9.19 g/L;低氮、低磷胁迫或二者共同胁迫均促进其油脂积累,但低硫胁迫对油脂的积累影响不大,最大油脂含量出现在3 mmol/L NaNO_3、0.029 mmol/L K_2HPO_4、0.3 mmol/L MgSO_4条件下,为68.7%(DW)。此外,类波氏真眼点藻富含棕榈油酸,占总脂肪酸含量的最大值达60.88%。选择合理的氮、磷、硫初始组合浓度能够有效促进其生长或油脂及棕榈油酸的积累。     

6 种无机絮凝剂对布朗葡萄藻的絮凝效应

探讨了6种无机絮凝剂对布朗葡萄藻的絮凝效应。采用光密度法测定布朗葡萄藻的生物量,分别研究了硫酸铝、硫酸铝铵、硫酸铝钾、硫酸镁、硫酸铁、氯化铁对布朗葡萄藻的絮凝效应。结果表明,光密度法可应用于布朗葡萄藻生物量的测定,可间接测定絮凝效应,且最佳测定波长为680nm;6种无机絮凝刺的絮凝时间为60min,同时6种无机试剂对布朗葡萄藻均有明显的絮凝效应,硫酸铝、硫酸铝钾、氯化铁对布朗葡萄藻的絮凝效应较硫酸铝铵、硫酸镁、硫酸铁明显（P〈0．05）。硫酸铝浓度控制在1．1g／L,硫酸铝钾浓度控制在0．45g／L以内,氯化铁浓度控制在0．9g／L。     

Maximization of recombinant Helicobacter pylori neutrophil activating protein production in Escherichia coli: improvement of a chemically defined medium using response surface methodology

Medium optimization for the production of constitutive recombinant Helicobacter pylori neutrophil activating protein (NAP) in Escherichia coli was investigated by using response surface methodology. Carbon to nitrogen ratio, concentrations of sodium polyphosphate and magnesium sulfate were considered as independent variables. The optimized medium was a chemically defined medium with a carbon to nitrogen ratio of 14.4 and with concentrations of sodium polyphosphate and magnesium sulfate about 7.1 g l(-1) and 3.04 g l(-1) respectively. The maximum recombinant NAP production level (1184.6 mg l(-1)) was 29.96% higher than that in control medium.     

卵孢小奥德蘑驯化栽培、营养成分及抗氧化活性

卵孢小奥德蘑是一种珍贵的食药用真菌。本研究选用果渣、酒糟与菌糠为部分替代料,采用正交试验优化了野生卵孢小奥德蘑的母种、原种及栽培料配方,并测定了栽培子实体的含水量、蛋白质、总糖、维生素C、多酚含量及醇提物对DPPH、ABTs⁺及羟基自由基的清除能力。结果表明,最佳母种培养基配方为麦麸35 g/L、葡萄糖20 g/L、磷酸氢二钾3.5 g/L、硫酸镁2 g/L和琼脂20 g/L,确定了葡萄糖与磷酸氢二钾是影响菌丝生长的重要因素;最优原种培养基配方为果渣45%、豆秸20%、麦麸15%、石灰3%、石膏1%和刺芹侧耳菌糠16%,在培养基中添加果渣能够显著促进卵孢小奥德蘑原种菌丝的生长;最佳栽培料配方为酒糟35%、棉籽壳30%、麦麸20%、石灰1%、石膏1%和玉米芯13%,酒糟对子实体产量的影响达到了显著水平;卵孢小奥德蘑的营养成分显示其富含蛋白质、糖类、维生素C及多酚,由9种配方栽培出的子实体中最高含量可分别达到42.78 g/100 g、23.54 g/100 g、4.02 mg/100 g及4.19 mg/g,且栽培料的不同组分及用量对其营养物质的含量具有显著差异。此外,卵孢小奥德蘑的醇提物具有较强的抗氧化能力且随着用量的增加抗氧化活性增强,当用量为150、40和250 μL时配方Z1对DPPH和ABTs⁺及配方Z2对羟基自由基的清除能力可分别达到88.64%、99.81%和93.48%,本研究结果为卵孢小奥德蘑的栽培、生理活性、药理研究及进一步的开发利用提供理论依据。     

Enhanced uridine diphosphate <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine production using whole-cell catalysis

Uridine diphosphate N-acetylglucosamine (UDPAG) can be produced by chemical, enzymatic, chemoenzymatic, and fermentative methods. In this study, we used whole-cell catalysis method to produce UDPAG for the first time by Saccharomyces cerevisiae. In order to increase the ATP utilization efficiency and UDPAG conversion yield, the response surface methodology was applied to optimize the whole-cell catalytic conditions for UDPAG production. Firstly, effects of uridine 5′-monophosphate (5′-UMP), glucosamine, vitamin B1, glycerol, magnesium chloride, potassium chloride, temperature, sodium dihydrogen phosphate, sodium acetate, fructose, and pH on UDPAG production were evaluated by a fractional factorial design. Results showed that UDPAG production was mainly affected by sodium dihydrogen phosphate, temperature, and vitamin B1. Then, the concentrations of sodium dihydrogen phosphate and vitamin B1 and temperature were further investigated with a central composite design and response surface analysis. The cultivation conditions to obtain the optimal UDPAG production were determined: sodium dihydrogen phosphate, 31.2 g/L; temperature, 29°C, and vitamin B1, 0.026 g/L. This optimization strategy led to an enhancement of UDPAG production from 2.51 to 4.25 g/L, yield from 44.6% to 75.6% based on the initial 5′-UMP concentration, and ATP utilization efficiency from 7.43% to 12.6%.     

EXPLORING THE OPTIMUM CONDITIONS FOR MAXIMIZING THE MICROBIAL GROWTH OF Candida intermedia BY RESPONSE SURFACE METHODOLOGY

Exploring optimum and cost-efficient medium composition for microbial growth of Candida intermedia Y–1981 yeast culture growing on whey was studied by applying a multistep response surface methodology. In the first step, Plackett–Burman (PB) design was utilized to determine the most significant fermentation medium factors on microbial growth. The medium temperature, sodium chloride and lactose concentrations were determined as the most important factors. Subsequently, the optimum combinations of the selected factors were explored by steepest ascent (SA) and central composite design (CCD). The optimum values for lactose and sodium chloride concentrations and medium temperature were found to be 18.4 g/L, 0.161 g/L, and 32.4°C, respectively. Experiments carried out at the optimum conditions revealed a maximum specific growth rate of 0.090 1/hr; 42% of total lactose removal was achieved in 24 h of fermentation time. The obtained results were finally verified with batch reactor experiments carried out under the optimum conditions evaluated.     

S-腺苷-L-蛋氨酸高密度发酵工艺优化

利用酿酒酵母在5L发酵罐高密度发酵生产S-腺苷-L-蛋氨酸(SAM)后期存在稳定性差的问题,本研究考察了在补糖中添加磷酸氢二铵、谷氨酸钠、三磷酸腺苷二钠来提高酿酒酵母发酵后期的稳定性。通过4批5L发酵罐高密度流加发酵实验研究发现:在发酵34h左右,菌体干重超过100g/L后,开始添加50克L-蛋氨酸,并在补糖中加入10g/L三磷酸腺苷二钠,发酵65.7h,最高生物量干重达到180g/L,SAM产量达到17.1g/L。     

Spirulina platensis growth in open raceway ponds using fresh water supplemented with carbon,nitrogen and metal ions

To investigate the feasibility of using fresh water from Mangueira Lagoon (Rio Grande do Sul, Brazil) for biomass production in open raceway ponds (0.7 m long, 0.18 m wide, 0.075 m deep) we studied the influence of nutrient addition (carbon as sodium bicarbonate, nitrogen as urea, phosphate, sulfate, ferric iron, magnesium and potassium) on the growth rate of the cyanobacteria Spirulina platensis using a 22 factorial design. In unsupplemented lagoon water production of S platensis was 0.78 +/- 0.01 g/l (dry weight basis) while the addition of 2.88 g/l of sodium bicarbonate (without added urea, phosphate, sulfate or metal ions) resulted in 0.82 +/- 0.01 g/l after 400 hours of culture. The further addition of phosphate and metal ions resulted in growth for up to 750 h and a final S. platensis biomass of 1.23 +/- 0.04 to 1.34 +/- 0.03 g/l.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.