Isolation And Identification Of Methanotrophs And Research On Its Ability Of Degradation Of Methane In The Coal Sample

Coal mine gas is one of the important factors affecting coal mining safety operation, CH4explosion limit is4.5-16%, Inflammable and explosive. In the process of mining, coal gas’leak may cause dangerous explosion.Therefore, prevention and control of gas disaster have been an important subject to many researchers. The current gas control technology in our country still has its limitation. Main tunnel ventilation, drainage and other physical means to deal with the coal mine gas. However, due to the complex geological structure, coal seams, seam permeability, coal mine gas disaster is difficult to get a thorough governance. Methanotrophs can catalyze CH4into CO2and H2O. Provides a new direction to solve the problem of coal mine gas. It’a new way worth exploring if the work surface or seam of coal seam methane-oxidizing bacteria to reduce methane content. The activity of Methanotrophs may affected with many factors, such as temperature, air pressure and oxygen condition. And in the coal seam containing gas, CH4is in the form of combined with the coal, released with the reduction of the pressure during mining operation. That are the main question in coal mine gas dealing. Focuses on screening and inducing dominant strains, improving the conversion and the density Methanotrophs culture, simulating eliminating the coal gas, Proved methane oxidizing bacteria absorption and utilization bound seam methane ability to carry out the basic work of the microbial treatment of methane gas hazards. In this study, methane-oxidizing bacteria screening, microbial degradation of methane in coal samples carried out the following research work.Collecting he soil on the bank of Wei River in Henan province Xinxiang city as the experiment material. After enrichment culture isolated three methane-oxidizing bacteria. AEM1204, AEM1235, AEM1296. Methane as sole carbon source to the identification of gas chromatography, AEM1235use the strongest of methane. Optimization of culture conditions of pH, temperature, etc. The most suitable medium and culture conditions of AEM1235:methanotrophs Medium (g/L):NH4Cl0.5, K2HPO40.4, KH2PO4·7H2O0.4, M gSO4·7H2O0.3, CaCl2·2H2O0.02, KNO31.6; NaCl0.3, EDTA0.02, FeSO4·7H2O0.004, CuSO4·5H2O0.0013, M nSO4H2O0.004, ZnSO4·7H2O0.00034, NaMoO4·2H2O0.0024; pH7.0, Culture temperature was30℃, By gas filled O2:CH4=1:1. this bacteria grew will with high oxidation capacity. Methanotroph can also make use of other carbon sources, for example, carbinol. Carbinol can promote the oxidation. So we can add right amount carbinol as the bacteria’s carbon source in culture on large scale before using CH4.Then we studied its form and physiological-biochemical characteristic, Gram-negative Bacillus colonies were round, The diameter size is0.5-1mm, translucent gray and edge tidy.The bacteria can take advantage of methanol, glucose, maltose, sucrose, lactose and starch, the results of catalase test, Citrate utilization test, Hydrogen sulfide test, Starch hydrolysis test and gelatin liquidized test were positive. While the results of indole test and methyl red test were negative. After the16S rDNAsequences analysis and the sequence homology,we found AEM123516S rDNA sequence and addicted to meth silk micro bacteria genera (AB680579.1Hypromicrobium methylovorumg) closest relationship. Measure its growth, it shows that under the condition of methane as the sole carbon source AEM1235strains grew more slowly, cultivate achieve maximum increment after8d, so for subsequent methane oxidation test of coal sample with7-8d culturesTake the cultivation of the7-8d AEM1235plate colony count and adjust the cell concentration of1.0×108cfu/ml, used in the coal sample methane oxidation experiment. Added in the coal sample methane oxidative bacteria bacterium suspension, and filled the CH4respectively after extraction with1:1mixed with pure CH4gas O2isothermal adsorption experiment, then used the gas chromatographic to determine of gas volume and the content of various kinds of ingredients. Results show that the coal sample without Methanotrophs methane adsorption and the adsorption experiment of gas of methane change little. Filling the CH4and O21:1mixed gas, methane content in the gas and the oxygen concentration decreased significantly, showed that Methanotrophs has the ability of absorption and utilization of methane, namely under the condition of high pressure oxygen coal samples of methane oxidation. In inflatable pressure of5MPa inoculation amount under the condition of12.5%, CH4and O2consumption ratio is1:1.933, at the same time there is a small amount of CO2generated, showed that after the absorption of methane oxidation mainly exists in the form of oxidation intermediate products.Filled pure CH4gas into coal samples for adsorptiontest, methane oxygen concentration in the gas emited also had significantly lower, and CO2generated, but methane and oxygen consumption were significantly lower than the consumption with the oxygen mixed in the filled gas. In12.5%inoculation quantity and air pressure under the condition of5.0MPa, absorption of methane from oxygen filling under the condition of58%, but the generation of CO2was obviously higher than that of aerobic conditions, But the generation of CO2was obviously higher than that of aerobic condition, which indicates that Methanotrophs AEM1235under the condition of high pressure oxygen still has the ability of absorption and utilization of methane. Result above prompt us even in high pressure and anaerobic condition underground can Methanotroph keep its capacity for assimilating and oxidizing CH4.