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Development of bio-production process from biogas

沼气生物生产工艺的开发

基本信息

批准号：
17360401
负责人：
NISHIO Naomichi
金额：
$ 10.36万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17360401/
关键词：
Ethanol Moorella sp Hydrogen Carbon dioxide Carbon monoxide Biogas Resting cells Acetic acid Clostridium Ijungdahlii 嫌気性菌 Moorella アルコール脱水素酵素アルデヒド脱水素酵素

项目摘要

(1) Breeding of high ethanol producing Moorella sp. HUC22-1:To improve the ethanol productivity of Moorella sp. HUC22-1, development of its genetic transformation system was tried. As the results, an uracil auxotroph that lacks dehydrogenase dehydrogenam gene(pyrD) and orotate phosphonlosyltransferaro gene(pyreE) was obtained by the transformation system using homologous recombination and electroporation. Now, we are carrying on a development of genetic transformation system by using the uracil auxotroph, pyrD and pyrE as a host and selection markers.(2) Analysis of metabolic characterization of Moarella sp. HUC22-1 in various substrates:Moorella sp. HUC22-1 ferments glyoxylate to acetate roughly according to 2 glyoxylate→acetate 2 CO_2. Based on the data obtained in a batch culture, the ATP yield per substrate dissimilated was calculated to be 1.11 mol/mol, which was higher than that obtained from only Wood-Ljungdahl pathway. Crude extracts of glyoxylate-grown cells catalyzed the ADP- … More and NADP-dependent condensation of glyoxylate and acetyl-CoA to pyruvate and CO_2 as well as the conversion of pyruvate to acetyl-CoA and CO_2. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, L-malate, an intermediate in the malyl-CoA pathway, was consumed and acetate was produced in this bacterium. These findings suggest that Moorella sp. HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway. But, ethanol was not produced in all substrates tested including glyoxylate, glycolate, oxalate and malate.(3) Ethanol production in gaseous substrate in batch culture:In this year, we used CO as substrate instead of H_2/CO_2. Although the cell could not grow up to 20% CO in the first batch culture, it can grow 100% CO after repeating the acclimatization in the CO gas. At the 100% CO gas substrate, cell growth could increase 8 times and the product could be sifted to ethanol production by increasing the acetate/ethanol ratio.(4) Conversion of acetate to ethanol in resting cell system:Conversion of acetate to ethanol was carried out in the resting cell system. As the results, ethanol production was increased to 147 g/kg dry cell under mechanical shaking in the presence of methyl-viologen at pH7. Less

(1)高产乙醇Moorella sp. HUC22-1的选育：为提高Moorella sp. HUC22-1的乙醇产量，尝试开发其遗传转化体系。结果表明，通过同源重组和电穿孔转化体系，获得了缺乏脱氢酶脱氢基因（pyrD）和旋转磷酸基转移基因（pyreE）的尿嘧啶类营养不良分子。目前，我们正在以尿嘧啶营养不良分子、pyrD和pyrE为寄主和选择标记，进行遗传转化系统的开发。(2) Moarella sp. HUC22-1在不同底物中的代谢特性分析：Moarella sp. HUC22-1大致按照2- glyoxylate→乙酸2 CO_2的顺序将glyoxylate发酵为乙酸。根据分批培养得到的数据，计算出每个底物的ATP产率为1.11 mol/mol，高于单一Wood-Ljungdahl途径的产率。乙酸乙酯细胞粗提物催化乙酸乙酯和乙酰辅酶a的ADP-…More和nadp依赖性缩合成丙酮酸和CO_2以及丙酮酸转化成乙酰辅酶a和CO_2。在该途径的关键酶反应中也检测到ATP的产生。此外，l-苹果酸（丙二酰辅酶a途径的中间产物）在该细菌中被消耗并产生醋酸盐。这些研究结果表明，Moorella sp. hu22 -1可以通过甲基辅酶a途径在乙醛酸分解代谢过程中通过底物磷酸化产生ATP。但是，并不是所有的底物都能产生乙醇，包括乙醛酸盐、乙醇酸盐、草酸盐和苹果酸盐。(3)分批培养中气态底物生产乙醇：今年我们用CO代替H_2/CO_2作为底物。虽然细胞在第一批培养中不能生长到20%的CO，但在CO气体中重复驯化后可以生长到100%的CO。在100% CO气体底物条件下，细胞生长速度可提高8倍，通过提高乙酸/乙醇比，产物可筛选为乙醇生产。(4)静息细胞系统中醋酸酯向乙醇的转化：醋酸酯向乙醇的转化在静息细胞系统中进行。结果表明，在ph为7的甲基紫胶存在下，在机械振荡下，乙醇产量提高到147 g/kg。少