Isolation And Identification Of Methanogenic Bacteria From Digester And Its Distribution Analysis

The bacteria that can generate methane could not be cultured in the complex organic compound but in the strict anaerobic environment. They could synthesize methane and complete the energy metabolism by the simple material, such as hydrogen, carbon dioxide, formic acid, methanol, methylamine, acetic acid and so on. One of the essential steps of methane fermentative process is the biosynthesis of methane, so the distribution of the superior methanogen species and its customs are widely studied. It will be benefited to improve the efficiency of methane fermentation.Two strains SH01 and SH02 were isolated from the overwintered feces digester by the modified Hungate anaerobic techniques. SH01 is curved, G^-, motile, light-yellow colony, which can use H₂/CO₂ and formate but not methanol, acetic sodium and trimethylamine as the substrate. The optimal pH is 6.8-7.2, and the optimal temperature is 35-40℃, and Na⁺ concentration is less than 0.1 mol/L. Strain SH01 is Methanospirllum hungatei judged by thephysiology, morphology, structural characteristics and phylogenetic analysis.SH02 is curved, G^-, non-motile, light-yellow colony, which can use H₂/CO₂ and formate but not methanol, acetic sodium and trimethylamine as the substrate. The optimal pH is 7.5-8.0, and the optimal temperature is 37-42℃, and Na⁺ concentration is less than 0.3mol/L. Strain SH02 is one of species of Methanoculleus judged by the physiology, morphology, structural characteristics and phylogenetic analysis.MPN method (The Most Probable Number Method) was applied to study the annual variety of methanogenic bacteria cluster. The relationships between the efficiency of methane fermentation and its species were estimated by the percentage of the methanogenic bacteria cultured by different kinds of substrates. The under-nutrition acetate (acetic acid or acetate as substrate) of methanogenic bacteria are superior species in the methanogen groups which account for 70%. The amount of methanogenic bacteria varied greatly under different seasons, the minimum is in December, approximately 1.0×10⁶, and the maximum is in June, approximately 1.0×10⁸, according to the changes of methane quantity in the digester.