| Methanotrophs have great potential for applications in CH4 removal and gas control of coal mines. But the methanotrophs that can be used in complex environment are limited due to low growth rate and unstable MMO activity. Also, there is no substantial applied research on gas control by methantophs.In this study, the methanotroph community of the soil from a typical coal mine in China was analyzed by multiple molecular ecology techniques to estabilish the strategies of methanotrophic enrichment cultures. Two mixed methanotrophic communities were enriched by different domesticating conditions. The characteristics and capabilities of the two methanotrophic communities were evaluated, and large-scale fementation was studied. Lab and field pilot experiments were designed to explore the feasibility of using the mixed methanotrophic community for CH4 removal and gas control.Several complementary molecular ecology techniques were used to elucidate the methanotrophic community from a coal mine. There were lots of methanotrophs and methylotrophs found in the coal mine soils, in which typeⅠwas the main part. The ratios of the typeⅠ, typeⅡand typeⅩmethanotrophs were 78.6%, 7.1% and 14.3%.CH4 and artificial mash gas were used to domesticate mixed methanotrophic cultures capable of stably and effectively abating CH4, respectively. The capability of CH4 and C2H6 removal was enhanced apparently during the enrichment. The diversity of the total bacteria community decreased, and the amount of the typeⅠmethanotrophs increased obviously. After 20 generations, the microbial community and CH4 removal ability became stable.The characteristics of the two mixed methanotrophic communities enriched above, including morphology, growth, metabolism and microbial community structure, were then studied. The amount of the typeⅡmethanotrophs increased in both cultures, and there were more kinds of bacteria in the community enriched with the artificial mash gas. The CH4 removal abilities of the two mixed methanotrophic communities were stronger than the pure methanotroph. There were no metabolites accumulated during the cultivation. The synergistic effects of the different microorganisms were beneficial for maintaining the stability of the microbial community and the capability of avoiding product inhibition.The mixed methanotrophic communities showed good CH4 removal performance under different CH4 concentrations （350%） and different temperatures （2042oC）. And they were tolerant to the toxic gases consisting of H2S, CO and SO2 which always exist in the coal mine gas. The mixed methanotrophic communities were well preserved by three methods: 4oC sealing with CH4, -80oC freezing collected cells, and -80oC freezing with paraffin.The scale-up fermentation for a mixed methanotrophic community was realized from 5L to 300L and from 5L to 600L systems, respectively. The mixed methanotrophic culture grew quickly in large-scale fermentation. The CH4 removal ability and microbial community were kept stable during the different scale fermentation.In the lab-scale experiments of simulate coal bed and biofitration, the performance of CH4 removal by the mixed methnaotrophic community was investigated. And then, pilot-scale study for gas control in coal beds was carried out in three coal mines in Henan. The prepared culture of the mixed methanotrophic community was injected into the coal bed, and the CH4 was removed effectively. |
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