【作者】 李晶；

【导师】 袁其朋；

【作者基本信息】 北京化工大学 ， 生物化工， 2009， 硕士

【摘要】 丙烯酸（CH₂=CHCOOH）,是工业生产中重要的不饱和有机酸,也是重要的有机化工原料。它主要用于制备丙烯酸树脂及其它有机合成,另外也用于粘合剂、涂料、化纤、皮革、造纸、纺织工业和光敏树脂板等的生产。今后几年,我国在高吸水树脂、丙烯酸酯、特种丙烯酸酯以及油田领域对丙烯酸的需求都会逐年增加。工业法生产丙烯酸的合成原料基本来自石油提炼所获得的产品,随着石油资源供应日趋紧张,发酵法生产丙烯酸,越来越受到研究人员的重视。本项目通过系统分析微生物在葡萄糖培养基上产丙烯酸的代谢途径,通过代谢调控的手段,分析细胞代谢通量,添加甲萘醌等电子受体和阻断剂发酵生产丙烯酸,降低生产成本,提高丙烯酸产量。主要工作如下:1.本文以埃氏巨球形菌（Megasphaera elsdenii）为出发菌株,对原始菌株进行了理化诱变及抗性突变,获得一株高抗酸性的菌株,其产丙烯酸量达6.46 mmol/L,是诱变前的1.85倍。2.通过研究培养基成分和培养条件对产丙烯酸的影响,并对其进行优化,得到了最佳产丙烯酸培养基为:KH₂PO₄（0.5 g/L）,NH₄Cl（0.5g/L）,MgCl₂·6H₂O（0.3g/L）,可溶性淀粉（20.0 g/L）,葡萄糖（13.0g/L）,酵母提取物（4.0g/L）。3.通过研究培养条件对产丙烯酸的影响,并对其进行优化,得到了最佳培养条件为:接种量10%,装液量20%,37℃,pH 7.5,120 rpm,72 h。4.通过探讨阻断剂和电子受体对菌株产丙烯酸的影响,确定了最佳添加物及条件为:0.015%（v/v）甲萘醌,0.15%的3-丁炔酸,pH 7.5,温度37℃,反应72h,丙烯酸产量为8.96 mmol/L。更多还原

【Abstract】 Acrylic acid（CH₂=CH-COOH） has attracted much attention due to its wide application in the polymerization of flocculants,dispersants,coatings, paints,adhesives as well asbinders for leather,paper and textile industries.Previous studies have showed that during the biological production of acrylic acid from renewable sources,the continuous exhaustion of reduction equivalents leads to the low productivity.Recent researches have revealed that microbial production of acrylic acid could be one of the potential alternatives for its low cost and the utilization of bulk raw materialsfrom biomass.The thesis primarily investigated the producing process of Acrylic acid and tried to find a route in which the stock is conveniently acceptable and the cost is comparably low,theoperation is simple and the quality of product is excellent.This work will be a preceding step for the production of Acrylic acid,which consist of:（1） Treated by ultraviolet ray,DES and structural analogue,a acid resistance strain was obtained whose acrylic acid productivity is 6.46 mmol/L,1.85 fold of that of the comparing one.（2） Based on the Megasphaera elsdenii,the thesis showed the research on the composition and proportion of the medium,the optimization of theenzymatic reaction condition,the affecting factors of electron acceptors on the production of acrylic acid,and the screening of the acid resistance strain.（3） The results show that, 2-hydroxy-p-naphthoquinone,due to its low cell toxicity,good membrane penetrability,easy capture for electrons and stable maintenance in vivo,successfully removed available electrons derived from the metabolic flux,which lead most acrylyl-COA to transform into acrylic acid by acrylyl-COA transferase.（4） By investigating the effects of medium components and the cultivation condition,the optimum culture medium and cultivation condition were obtained.The optimum medium was listed as follows（%）:KH₂PO₄（0.5 g/L）,NH₄Cl（0.5 g/L）, MgCl₂·6H₂O（0.3g/L）,soluble starch（20.0 g/L）,glucose（13.0g/L）, yeast extract（4.0g/L）;the optimum cultivation conditionincluded inoculation 10%,medium volume 100 ml/250ml,37℃,pH 7.5,120 rpm,cultivation time 72 h.In order to improve the acrylic acid production, In shaker flask cultivations,the highest acrylic acid concentration of 8.96 mmol/L,which was 4.5 times higherthanthe reported,was achieved at pH 7.5,with the added 2-hydroxy-p-naphthoquinone additive concentration of 0.015%（v/v）.更多还原