Aerobic culture of Propionibacterium freudenreichii ET-3 can increase production ratio of 1,4-dihydroxy-2-naphthoic acid to menaquinone

This is the first report on the production of both 1,4-dihydroxy-2-naphthoic acid (DHNA) and menaquinone by Propionibacterium freudenreichii ET-3. DHNA can be a stimulator of bifidogenic growth, and menaquinone has important roles in blood coagulation and bone metabolism. During anaerobic culture, DHNA and menaquinone concentrations reached 0.18 mM and 0.12 mM, respectively. The molar ratio between these products was approximately 3:2, which was not affected by culture pH and temperature over the ranges of 6.0-7.0 and 31-35 degrees C, respectively. As for organic acid, propionate and acetate accumulated at concentrations of 0.3 M and 0.15 M, respectively, and the propionate accumulation particularly inhibited further production of DHNA. To improve DHNA production, we switched from anaerobic condition to aerobic condition during the culture when lactose was depleted. DHNA concentration continued to increase even after lactose exhaustion, reaching 0.24 mM. In contrast to DHNA production, menaquinone production stopped after the switch to aerobic condition. The total molar production of DHNA and menaquinone was 0.3 mM irrespective of aerobic culture and anaerobic-aerobic switching culture. Therefore, the anaerobic-aerobic switching culture could increase the production ratio of DHNA to menaquinone. The DHNA concentration obtained from the anaerobic-aerobic switching culture was 1.3-fold higher than that in the anaerobic culture, because P. freudenreichii ET-3 utilized propionate accumulated in the medium via the reversed methylmalonyl CoA pathway under aerobic condition. The culture method proposed in this study could be applicable to industrial-scale fermentation using 1000 l of media, by which 0.23 mM DHNA was produced.     

响应面法优化产酸丙酸杆菌丙酸发酵条件的研究

采用Box-Behnken设计和响应面分析法(Response surface methodology,RSM),以产酸丙酸杆菌发酵甘油产丙酸的3个关键因素(培养温度、pH和接种量)为自变量,以丙酸产量为响应值,对上述因素的最佳水平范围进行了探讨与优化。实验结果表明,培养温度和pH对丙酸产量有显著性影响,并据此建立了相关的数学模型。得到的工艺参数的优选结果是:培养温度为29.75℃、pH为6.61、接种量为6.15%(v/v),丙酸产量最大预测值为17.96g/L。经过优化,丙酸产量提高了27.9%,实验值与预测值基本相符。     

响应面法优化P.acidipropionici产酸发酵培养基

利用响应面分析法优化产酸丙酸杆菌发酵产酸培养基.在单因素试验的基础上,采用Box-Behnken试验设计,选定甘油、混合氮源(酵母提取物:胰酶大豆肉汤=2∶1)和磷酸氢二钾3个关键因子为响应因子,以丙酸产量为响应值建立多元二次回归方程,在分析各个因素的显著性和交互作用后,确定了产酸丙酸杆菌发酵产酸的最优培养基为:甘油44.4g/L、混合氮源(酵母提取物:胰酶大豆肉汤=2∶1)27.0g/L、K2HPO4 2.0g/L,丙酸产量最大预测值为20.75g/L.经过优化,丙酸产量提高了151％,实验值与预测值基本相符.     

Selection of photosynthetic bacterium Rhodobacter sphaeroides 14F for polyhydroxyalkanoate production with two-stage aerobic dark cultivation

Polyhydroxyalkanoate (PHA) production abilities in a two-stage aerobic dark culture of photosynthetic bacteria were investigated at relatively high temperatures (37-40 degrees C). A 14F strain, identified as Rhodobacter sphaeroides, showed the highest PHA production (3.5 g/l PHA with 60% PHA content). Its productivity was 2-3 times higher than those of other photosynthetic bacteria.     

An efficient method for production of kynurenic acid by Yarrowia lipolytica

Kynurenic acid (KYNA) is a compound derived from the tryptophan catabolic pathway. Antioxidant and neuroprotective properties have been confirmed for KYNA, which makes it an interesting and important metabolite of biomedical significance. In the present study, the yeast Yarrowia lipolytica was tested for KYNA biosynthesis. The results showed that Y. lipolytica strain S12 is able to produce KYNA in high concentrations (up to 21.38 μg/ml in culture broth and 494.16 μg/g cell dry weight in biomass) in optimized conditions in a medium supplemented with tryptophan. Different conditions of culture growth, including the source of carbon, its concentration and pH value of the medium, as well as the influence of an inhibitor or precursor of KYNA synthesis, were analysed. The obtained data confirmed the presence of KYNA metabolic pathway in the investigated yeast. To our best knowledge, this is the first study that reports KYNA production in the yeast Y. lipolytica in submerged fermentation.     

Process optimization for enhanced production of diphtheria toxin by submerged cultivation

When stationary culture was replaced by submerged cultivation in a fermentor, a significant increase in the yield of diphtheria toxin in a short cultivation time (less than 48 h as against 7-8 d) was noted. It was found that under optimal conditions of temperature, vortex mixing and surface aeration, an alkaline pH favoured toxin release. Furthermore, to enhance the production volume, two-and three-step semicontinuous batch cultivations were carried out. The toxin produced was of good titre with an adequate antigenic purity. Under optimal conditions, the variation in the Limes of flocculation (Lf titre) was likely due to the quality of the production medium, which in turn depended on the quality of the raw materials used. The process was also optimized in a pilot-scale fermentor.     

利用响应面方法优化产L-乳酸的合成培养基

采用响应面方法对拟干酪乳杆菌Lactobacillus paracasei (L.paracasei)的一种产乳酸的合成培养基进行了优化.该文将培养基中的混合氨基酸、核苷类物质和混合维生素看作3大类营养成分.首先利用Plackett-Burman实验设计考察了培养基中包括该3类营养物质在内的8种营养成分对菌体生长和乳酸合成的影响,筛选出了影响菌体生长和乳酸合成的3个主要因素,即混合氨基酸、KH2PO4和CaCO3;在此基础上利用最陡爬坡实验逼近最大响应区域;然后利用Box-Behnken实验设计及响应面分析法确定了最佳培养条件.经过3次实验验证,乳酸实际浓度与预测浓度十分接近,已经超过了在复合培养基中培养的水平.这些结果为L.paracasei的代谢流定量研究奠定了基础.     

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) – PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth.     

2-酮基-L-古龙酸一步发酵生产菌株发酵过程优化

对1株可以一步发酵生产维生素C直接前体2-酮基-L-古龙酸(2-keto-L-gulonic acid,2-KLG)的氧化葡萄糖酸杆菌工程菌株(Gluconobacter oxydans–ss)的发酵条件进行优化,以提高其发酵效率。结合Box-Behnken实验和响应面法对其初始发酵培养基进行优化,得到优化的发酵培养基组成:山梨醇158.0 g/L,酵母膏18.0 g/L,初始p H5.0。采用该优化培养基在3 L全自动发酵罐上进行发酵过程控制。在考察不同转速对2-KLG积累过程影响的基础上,进一步提出分阶段转速控制策略:即发酵前期(0~48 h)转速控制在600 r/min,48 h至发酵结束转速控制在500 r/min。应用该策略,2-KLG产量达到34.86 g/L,生产强度为0.36 g/(L·h),其分别比恒定转速发酵时2-KLG的最大产量提高24.01%和24.14%。     

3-苯氧基苯甲酸降解酶产生条件的优化

采用Plackett-Burman法考察了K2HPO4、KH2PO4、MgSO4.7H2O、（NH4）2SO4、3-苯氧基苯甲酸浓度和初始pH、装液量及接种量8个因素对产酸克雷伯氏菌生成3-苯氧基苯甲酸降解酶的影响,并利用Box-Behnken实验设计及响应面分析对其产酶条件进行了优化。结果表明,培养基中（NH4）2SO4浓度、培养基装液量和接种量对菌体产生3-苯氧基苯甲酸降解酶的影响具有显著性;当培养基中K2HPO4浓度为2.0g/L、KH2PO4浓度为0.5g/L、MgSO4.7H2O浓度为0.2g/L、（NH4）2SO4浓度为0.9g/L、3-苯氧基苯甲酸浓度为200mg/L、初始pH为7.2、250mL锥形瓶装液量为56.6mL、接种量（种子液OD600为1.000）为5.9%（v/v）时,3-苯氧基苯甲酸降解酶的活力可达25.72U/mL。      

代谢工程改造Escherichia coli生产3-羟基丙酸

以提高3-羟基丙酸的产量为目标,对实验室构建的基因工程大肠杆菌进行改造,敲除形成副产物1,3-丙二醇的主要酶基因——乙醛脱氢酶基因yqh D,得到E.coli W3110Δyqh D(p CDFDuet-tac-gpd1-TUkgsadh/p ACYCDuet-tac-dha B1-4),该工程菌摇瓶发酵产量达到2.53 g/L,相比未敲除yqh D基因的菌株,产量提高了5.8倍。另外,敲除了甘油代谢途径中的抑制因子glpR基因,得到E.coli W3110ΔglpR(p CDFDuet-tac-gpd1-TUkgsadh/p ACYCDuet-tac-dha B1-4),该工程菌摇瓶发酵产量达到2.86 g/L,相比未敲除glpR基因的菌株,产量提高了6.7倍。后经5 L罐发酵培养后,3-羟基丙酸的产量提升到15.4 g/L。该实验为进一步利用大肠杆菌工程菌发酵生产3-羟基丙酸提供了研究基础。     

香菇产抑菌活性物质液态发酵条件优化

以抑菌活性为评价指标,以接种量、初始pH值、摇床转速、发酵时间、发酵温度为考察因素,采用单因素及正交试验优选液态发酵条件.结果表明,产抑菌活性物质的最佳发酵条件是接种量7％,发酵液初始pH值为5,培养时间8d、培养温度为28℃.在此条件下,发酵液对黄曲霉的抑菌圈直径为8.9 cm.发酵液初始pH值和发酵时间对试验结果的影响显著.     

Optimization of L-lactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture

We investigated optimization of the feeding of L-lactic acid for the production of poly-D-3-hydroxybutyric acid [P(3HB)] by Alcaligenes eutrophus in a fed-batch culture system. An acidic substrate solution was fed automatically so as to maintain the pH of the culture liquid at 7.0. Feeding of a substrate solution containing 45% (w/v) L-lactic acid, 6.2% (w/v) sodium L-lactate, 5.8% (w/v) ammonia water and 1.8% (w/v) potassium phosphate [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10], allowed the L-lactate concentration in the culture liquid to be maintained at approximately 2 g/l and the cell concentration reached 27.4 g/l after 15 h of cultivation. To promote P(3HB) production, a two-stage fed-batch culture consisting of a culture for cell growth and one for P(3HB) accumulation was carried out. When the substrate solution, whose C N molar ratio was 23, was fed during the P(3HB) accumulation phase, the cell concentration and the P(3HB) content in the cells reached 103 g/l and 57.6% (w/w), respectively, in 51.5 h.     

益生菌乳酸发酵提高低聚果糖含量生产的新方法

研究报告了一种新的提高低聚果糖(FOS)含量的生产方法。以市售低聚果糖为原料,通过添加甜菜碱进行益生菌乳酸发酵,将原料中可发酵性糖葡萄糖、果糖转化为乳酸,蔗糖部分转化为乳酸,使原料中低聚果糖的含量由51%提高到84%,而发酵液无需进行菌体分离即可用于食品加工,为低聚果糖的生产提供了一种低成本、高效率的纯化方法。     

Development of a novel method for feeding a mixture of L-lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly-D-3-hydroxybutyrate production

Fermentative production of poly-D-3-hydroxybutyrate [P(3HB)] from a mixture of L-lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l.h.     

干法预处理及分酸二步法生产生物柴油新工艺

用化学法生产生物柴油时常规工艺会产生大量的废水,随着国家对环境保护工作的重视,废水处理量大是制约企业生产与发展的一大技术瓶颈。介绍了干法预处理及分酸二步法生产生物柴油的新工艺,主要为原料油经过干法脱杂、油酸分离、分别进行酯化与酯交换、酸碱平衡等工艺过程生产出合格生物柴油。新工艺节能、环保,大大减少了废水排放量。     

甘油发酵生产3-羟基丙酸的代谢改造工程菌研究进展

3-羟基丙酸是一种重要的平台化合物,可以用来合成许多重要的化工中间体及工业化工产品,被美国能源部列为当前最具潜力的12种化工产品之一。文中就近年来以大肠杆菌(Escherichia coli)和肺炎克雷伯氏菌(Klebsiella pneumoniae)作为宿主进行代谢改造合成3-羟基丙酸的相关研究进行了总结,介绍了当前以甘油为底物发酵合成3-羟基丙酸的主要研究成果,并针对目前存在的问题加以展望,旨在为3-羟基丙酸的工业化生产提供参考。     

Long-term outdoor cultivation by perfusing spent medium for biodiesel production from Chlorella minutissima

A unique perfusion process was developed to maintain high concentrations of marine alga, Chlorella minutissima. This method is based on recycling cells by continuous feeding with warm spent sea water from nuclear power plants, which has very similar properties as sea water. A temperature of at least 30 °C in a 200 L photo-bioreactor was maintained in this system by perfusion of the thermal plume for 80 days in the coldest season. The maximum cell concentration and total lipid content was 8.3 g-dry wt./L and 23.2 %, w/w, respectively, under mixotrophic conditions. Lipid production was found to be due to a partially or non-growth related process, which implies that large amounts of biomass are needed for a high accumulation of lipids within the cells. At perfusion rates greater than 1.5 L/h, the temperature of the medium inside the reactor was around 30 °C, which was optimal for cell growth. For this system, a perfusion rate of 2.8 L/h was determined to be optimal for maintaining rapid cell growth and lipid production during outdoor cultivation. It was absolutely necessary to maintain the appropriate perfusion rate so that the medium temperature was optimal for cell growth. In addition, the lipids produced using this process were shown to be feasible for biodiesel production since the lipid composition of C. minutissima grown under these conditions consisted of 17 % (w/w) of C₁₆ and 47% (w/w) of C₁₈. The combined results of this study clearly demonstrated that the discharged energy of the thermal plume could be reused to cultivate marine alga by maintaining a relatively constant temperature in an outdoor photo-bioreactor without the need for supplying any extra energy, which could allow for cheap production of biodiesel from waste energy.     

酿酒酵母工程菌产青蒿酸的发酵动力学研究

以青蒿酸产量为考察指标,在50 L发酵罐中对酿酒酵母（Saccharomyces cerevisiae）工程菌1211发酵产青蒿酸的溶氧参数进行优化。在此基础上,根据Logistic方程及Luedeking-Piret方程构建酿酒酵母工程菌1211分批发酵产青蒿酸的动力学模型。结果表明,当溶氧参数为25%～30%时,青蒿酸产量最高,为（6 269.6±100.3）mg/L。青蒿酸合成与菌体生长呈现部分生长偶联型。通过Origin 9.0软件对动力学模型进行非线性拟合,发现S. cerevisiae工程菌1211的菌体繁殖生长、青蒿酸合成以及基质消耗动力学模型的拟合度R2分别达到了0.995 85、0.979 04和0.995 48,该动力学模型能够很好的描述S. cerevisiae工程菌1211分批发酵过程。该研究为青蒿酸的低成本发酵及工业化大规模发酵生产提供了理论基础。     

微生物发酵法生产去氢表雄酮检测方法的建立

建立了一个微生物发酵生产去氢表雄酮(DHEA)的检测方法,采用气相色谱法同时测定发酵液中雄甾-4-烯-3,17-二酮(4-AD)、去氢表雄酮甲氧基甲基醚和DHEA含量。发酵液经乙酸乙酯萃取,可直接进样分析。气相色谱条件为:FID检测器,DB-1型(30 m×0.53 mm×0.25 m)毛细管柱;柱温280℃,进样口280℃,检测器310℃;载气为高纯N2,流速1 m L/min。在实验室条件下,发酵液中各主要成分分离情况良好。结果表明:4-AD、去氢表雄酮甲氧基甲基醚和DHEA的浓度线性范围分别为:4-AD为0.2~1.0 g/L(R2=0.996);去氢表雄酮甲氧基甲基醚为0.1~0.9 g/L(R2=0.992);DHEA为0.2~1.0 g/L(R2=0.998),平均回收率依次为(97.88±0.74)%,(99.39±1.07)%和(98.91±1.09)%。该方法具有步骤简单,重现性好,准确度和灵敏度高的特点。