Studies On The Selection And Molecular Identification Of Bio-oxidizing Methane And The Optimization Test

Coal mine gas is the general name of toxic and harmful gases released from coal and rock. Coal mine gas control and disaster prevention has been the worldwide problem which plagued coal mine production safety for a long time; there is still no good fundamental solution to this problem. Besides, it also brought great store security risks because the gas adsorption and storage properties of coal. The use of microorganisms is a very interesting new way for control gas disaster.In this paper, methane-oxidizing bacteria are isolated in the absence of any other form of carbon without methane by the use of methane-oxidizing bacteria selective medium from coal samples and soil samples. A strain with capable of degrading methane named methane-oxidizing bacteria M-1is isolated after the proliferation of cultured strains, screening, rescreening, crossed the plate. The colony morphology of M-1strains was observed smooth and dense, cells were smaller and cell morphology short rod-shaped.The result of physiological and biochemical identification is G-. The maximum OD600is0.246after48h and the rate of methane oxidation reached68.26%after96h in the Initial culture. It will be sure that M-1belongs to Pseudomonas by16S rDNA sequencing and constructing phylogenetic tree of the genus.The optimal growth of methane-oxidizing bacteria M-1is pH=6.5, optimum growth temperature is35℃, optimum nitrogen source was KNO3by univariate tests. M-1is able to use not only methane as carbon source, but also glucose, starch, methanol, and peptone. Optimized to get the best medium formula of M-1through Response Surface Methodology is Na2HPO40.30g/L, KH2PO40.28g/L, MgSO4-7H2O1g/L, KNO31g/L, CaCl20.08g/L, trace element solution1is587.18μL, trace element solution2is3080.30μL. Predicted maximum response value OD6oo=0.296by Design Expert. The OD600is0.293by verification experiment. The rate average of Methane oxidation is94.68%with the culture temperature35℃, initial pH=7, Inoculation amount3%, injected12ml CH4by the result of orthogonal test. The rate of methane oxidation reached92.60%by verification experiment. The degradation rate of M-1is64.26%under the conditions of the mixed gas. The content of methane was degraded from6.8%to2.43%.