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【目的】分离高效降解木糖的嗜热厌氧杆菌菌株,用于发酵生产生物燃料乙醇,为后继的构建基因工程菌株及联合生物工艺提供材料。【方法】运用亨盖特厌氧操作技术从胜利油田油层采出液两年的富集样中分离到一株嗜热厌氧杆菌xyl-d。采用形态学观察、生理生化指标鉴定及基于16S rRNA的系统发育学分析确定其分类地位。【结果】菌株xyl-d为革兰氏阴性厌氧杆菌,菌体大小为(1.35-5.08)μm×(0.27-0.40)μm,单生、成对或成簇生长,芽胞圆形,端生。温度生长范围30-85℃(最适温度65℃);pH范围3.0-10.0(最适pH 7.5);NaCl浓度范围0%-4%(最适NaCl浓度2.0%)。发酵D-木糖的产物是乙醇、乙酸、CO2及少量的异丁醇、丙酸。菌株xyl-d的(G+C)mol%含量为45.6%,与热厌氧杆菌属模式菌株威吉利热厌氧杆菌(Thermoanaerobacter wiegelii)DSM10319T及嗜热乙醇杆菌(Thermoanaerobacter ethanolicus)DSM2246T的16S rRNA序列相似性均为99.3%。菌株利用D-木糖产乙醇的最佳初始pH为8.5;少量酵母粉能刺激生长并显著提高发酵D-木糖的产醇率,使乙醇成为主要的发酵产物;培养基中乙醇浓度达到7%(V/V)时菌体生长受到抑制,最佳生长条件下D-木糖的降解率可达91.37%,最佳产醇条件下发酵1摩尔D-木糖可产生1.29摩尔的乙醇。【结论】菌株xyl-d是从特殊生境(油藏)中分离到的一株高效降解D-木糖的耐酸、嗜热的厌氧杆菌,其为半纤维素降解产乙醇的联合生物工艺提供了菌源。
【Objective】 Isolation of xylose-producing strains of thermophilic anaerobic bacilli was used to produce biofuel ethanol by fermentation, which provided material for subsequent construction of genetic engineering strains and biotechnology. 【Method】 A strain of thermophilic anaerobic bacterium xyl-d was isolated from the enriched sample of Shengli oilfield production fluid for two years by using Hengte anaerobic technology. Morphological observation, identification of physiological and biochemical indicators and phylogenetic analysis based on 16S rRNA were used to confirm their taxonomic status. 【Result】 The strain xyl-d was Gram-negative anaerobic bacilli with the size of (1.35-5.08) μm × (0.27-0.40) μm, single, paired or clustered growth, . Temperature range of growth 30-85 ℃ (optimum temperature 65 ℃); pH range 3.0-10.0 (optimum pH 7.5); NaCl concentration range 0% -4% (optimum NaCl concentration 2.0%). Fermentation D-xylose products are ethanol, acetic acid, CO2 and a small amount of isobutanol, propionic acid. The (G + C) mol% content of strain xyl-d was 45.6%, and the 16S rRNA sequence of Thermoanaerobacter wiegelii DSM10319T and Thermoanaerobacter ethanolicus DSM2246T Similarities were 99.3%. The optimal initial pH of the strain using D-xylose ethanol was 8.5; a small amount of yeast extract could stimulate growth and significantly increase the alcohol production rate of D-xylose fermentation, making ethanol the main fermentation product; the ethanol concentration in the medium reached 7 (V / V). The degradation rate of D-xylose was 91.37% under optimal growth conditions, and 1.29 mol of ethanol was produced when 1 mol of D-xylose was fermented under the optimal conditions of alcohol production. 【Conclusion】 The strain xyl-d is an acid-tolerant and thermophilic anaerobic bacterium isolated from a special habitat (reservoir) and highly efficient in degrading D-xylose, which is provided by the combined biotechnology of hemicellulose degradation of ethanol production The bacteria source.