摘要
甲烷氧化菌能在温和条件下特异的利用甲烷,具有应用于甲烷减排和煤矿瓦斯治理的潜力。本论文在对我国典型煤矿土壤的甲烷氧化菌进行分子生态学分析的基础上,确定甲烷氧化混合菌的富集培养策略,获得高效利用甲烷的稳定混合菌群,对其特性和功能进行研究,实现大规模发酵培养,并在实验室和煤矿现场进行了瓦斯治理实验。
用多种分子生态学方法解析了瓦斯煤矿土壤中的甲烷氧化菌及相关微生物的组成信息。该样品含有大量的甲烷氧化菌及甲基氧化菌,其中甲烷氧化菌以Ⅰ型为主,Ⅰ型、Ⅱ型和Ⅹ型甲烷氧化菌的含量分别为78.6%、7.1%和14.3%。
分别以甲烷气和人工瓦斯气作为唯一碳源对土壤样品进行好氧选择性传代培养。富集过程中甲烷和乙烷的消耗能力得到显著强化,总细菌的多样性逐渐减少,Ⅰ型甲烷氧化菌则得到富集,20代以后菌群结构和甲烷去除能力趋于稳定。
研究了两种气体条件富集的混合菌群的特性,包括菌体形态、生长特性、代谢特性和微生物群落结构。驯化后Ⅱ型甲烷氧化菌数量增加,人工瓦斯气富集的混合菌菌群更丰富。混合菌比纯菌的甲烷消耗能力更强,且无代谢产物积累。菌群间的协同作用能有效避免产物抑制,有利于菌群结构和功能的稳定。
对两种条件得到的混合菌的功能进行了系统分析。其在甲烷浓度为3~50%时都有较好的去除效果,最高可达241 ml CH4 h~(-1) OD660~(-1) (L culture)~(-1)。在20~42oC都能有效的去除甲烷。对H_2S、CO和SO_2等毒性气体有很强的耐受性。混合菌保藏可用4oC充甲烷密封保存法、-80oC冻存菌体法和-80oC添加石蜡油冻存法。
以甲烷气富集的混合菌为对象,研究了甲烷氧化混合菌的大规模发酵培养。成功开展了5L-300L和5L-600L的放大发酵实验。混合菌在放大培养时,生长速度稳定,且随着发酵逐级放大,消耗甲烷能力以及菌群结构都能够保持稳定。
在实验室模拟煤层、生物过滤实验中研究混合菌去除甲烷的能力,并在河南三个矿井进行现场中试,将制备的菌液注入煤层并达饱和,甲烷去除效果显著,说明富集的甲烷氧化混合菌有在煤矿中治理瓦斯的应用潜力。
Methanotrophs have great potential for applications in CH_4 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 CH_4 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%.
CH_4 and artificial mash gas were used to domesticate mixed methanotrophic cultures capable of stably and effectively abating CH_4, respectively. The capability of CH_4 and C_2H_6 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 CH_4 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 CH_4 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 CH_4 removal performance under different CH_4 concentrations (3~50%) and different temperatures (20~42oC). And they were tolerant to the toxic gases consisting of H_2S, CO and SO_2 which always exist in the coal mine gas. The mixed methanotrophic communities were well preserved by three methods: 4oC sealing with CH_4, -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 CH_4 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 CH_4 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 CH_4 was removed effectively.
引文
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