煤层水中一株产甲烷菌的分离与系统发育分析

Isolation and phylogenic analysis of a methanogen from coal bed water

为了解煤层中产甲烷菌的生理生化特性,结合厌氧培养箱和平板划线分离技术从山东兖煤菏泽能化公司赵楼煤矿距离地表936 m处坑道顶板取得40℃煤层水样品中微生物进行富集分离纯化.结果表明:在该条件下存在产甲烷微生物,并分离得到一株产甲烷菌株M-3,该菌株呈短杆状,菌体大小约(1.0-2.0)μm×0.5μm;革兰氏染色显阳性;在平板上生长出圆形黄色菌落,边缘光滑整齐;可以利用乙酸、甲酸、甲醇和H2+CO2(V/V=2:1)作为唯一碳源生长;最适生长温度为45℃;对酸碱具有良好的适应性,中性条件下甲烷产量最多;最适NaCl浓度为0.2-0.6 mol L-1.对菌株M-3的16S rRNA基因序列同源性分析表明该菌株与Methanobacterium bryantii同源性高达99%,G+C含量32.9%.本研究通过形态、生理生化特性以及16S rRNA分析,鉴定菌株M-3为M.brytantii.

For a comprehensive understanding of the characteristics of methanogen in coal bed seams, the species of the strain and its characters are needed to be detected. The microbes were enriched and purified from the coal bed water at the top of tunnel 936 m underground of coal mine at Shandong Province. The enrichment and purification were undergone at environmental temperature of 40 ℃ with combined anaerobic glove box and streak plate method. In addition, according to the production of methane in different conditions, the optimal growth conditions were obtained. The results showed that there existed methanogen in this environment and a strain named M-3 was acquired. This isolated strain was about （1.0-2.0） gm x 0.5 pm, short rod and gram positive. Its colony was pale yellow on the culture solid plate, suborbicular with smooth edge. For physiological characteristics, the catabolic substrates included acetic acid, formate, methanol and H： ＋ CO, （V/V = 2：1）. The optimum methane production condition was 45℃, 7.0 ofpH and 0.2-0.6 mol L t of NaCI concentration. In addition, comparison of 16S rRNA genes showed 99% sequence similar between M-3 and Methanobacterium brvantii, and 32.9% ofG ＋ C content. The results of physiology, morphology and characteristics indicate that the strain M-3 is M. brvantii.