含有甲烷氧化菌的混合菌群特性研究

为获得高效甲烷氧化微生物体系,从农业土壤中采样,以甲烷作为唯一碳源进行好氧选择性传代培养,得到生长性能稳定、生长优于Methylosinus trichosporium OB3b纯培养的具有甲烷单加氧酶(Methane Monooxygenase,MMO)活性甲烷氧化混合菌。利用MMO的共代谢特性,分别以苯酚和环氧丙烷作为目标对象,考察该混合菌对有机污染物的降解及用于生产有用化学物质的催化特性。结果表明,所得混合菌具有高效降解苯酚能力,对初始浓度为600mg/L的苯酚,经过11h培养,苯酚降解率可达99%。另外,以该混合菌为催化剂可以实现丙烯氧化生产环氧丙烷。通过降低磷酸盐浓度可以有效提高环氧丙烷的积累浓度,最大可至5mmol/L。此外,采用纯种分离方法结合PCR扩增、16SrRNA和MMO功能基因分析技术对混合菌群结构进行解析。结果表明,该混合菌群由Ⅱ型甲烷氧化菌及其它至少4种非甲烷氧化菌组成,它们分别属于Methylosinus trichosporium和Acinetobacter junii、Cupriavidusme tallidurans、Comamonas testosteroni和Stenotrophomonas maltophilia。采用PCR方法从混合菌及纯化菌株M.trichosporiums Y9总DNA中都能扩增得到mmoB、mmoX和pmoA基因片段,表明该甲烷氧化菌同时具有sMMO和pMMO两种形式的MMO。通过对从甲烷氧化混合菌中分离纯化得到的甲烷氧化菌进行PCR产物测序,结果发现其与Methylosinus trichosporium的同源性为99.9%。

Study on the structure and function of a stable methane-oxidizing mixed microbial consortium

From an agricultural sample taken in Chongqing, a stable methane-oxidizing mixed microbial consortium was established by enrichment culture with methane as a sole source of carbon and energy. The mixed consortium showed high capability of phenol degradation and 1,2-epoxypropane production from propene. More than 99% of phenol at an initial concentration of 600mg/L could be degraded by the mixed microbial consortium after 11 h of cultivation. The productivity of 1, 2-epoxypropane could be increased with the decrease of phosphate concentration. The concentration of 1, 2-epoxypropane produced could reach to 5.0mmol/L. The bacterial structure of the methane-oxidizing mixed microbial consortium was analyzed by pure culture isolation combining with 16S rRNA and PCR of the related MMO functional genes. The results showed that the methane-oxidizing mixed microbial consortium was composed of a type 1U methanotroph identified as Methylosinus trichosporium and at least 4 kinds of heterotrophs ( Comamonas testosteroni, Cupriavidus metallidurans, Acinetobacter junii and Stenotrophomonas maltophilia ). M. trichosporium Y9, isolated from the mixed consortium, harbored both sMMO and pMMO genes.