一株促甲烷氧化假单胞菌Pseudomonas putida P7的分离及电活性特征

Isolation and electroactive characteristics of a methane-oxidation promoting Pseudomonas putida P7

【目的】解析一株从黄河三角洲湿地甲烷氧化富集物中分离获得的甲烷氧化菌伴生菌的生理学及电化学特性,并探究该菌株对甲烷氧化过程的影响。【方法】使用高通量测序技术解析甲烷氧化富集物的菌群结构,采用稀释涂布法、平板划线法分离甲烷氧化菌的伴生菌,通过16S rRNA基因测序技术进行菌株初步鉴定。利用扫描电子显微镜观察菌株形态,并通过气相色谱(gas chromatography,GC)检测伴生菌利用甲烷情况及对甲烷氧化菌氧化甲烷效率的影响。采用双室微生物燃料电池(microbial fuel cells,MFCs)及差分脉冲伏安法(differential pulse voltammetry,DPV)检测菌株的电化学活性。【结果】黄河三角洲湿地土壤甲烷氧化富集物主要的好氧甲烷氧化菌为甲基杆菌属Methylobacter,同时还发现一些伴生菌。分离得到一株甲醇利用菌P7,其16S rRNA基因序列与恶臭假单胞菌Pseudomonas putida的相似性达99.79%。扫描电镜结果显示该菌株为杆状,长约1.5–2.5μm,宽度约为0.5μm。GC检测结果显示,该菌株不能利用甲烷,但与甲烷氧化菌共培养时,可以促进甲烷氧化(P<0.05)。双室MFCs检测结果显示该菌株具有电活性,最大电流输出密度为28 mA/m2,DPV检测结果显示该菌株主要的氧化峰和还原峰分别位于–0.17 V和–0.25 V。【结论】本研究从黄河三角洲湿地甲烷氧化富集物中获得一株具有电活性的甲烷氧化菌的伴生菌恶臭假单胞菌Pseudomonas putida P7,该菌株可以促进甲烷氧化。本研究加深了对甲烷氧化过程中伴生菌的生理学特性及功能的认识。

[Objective]To analyze the physiological and electrochemical characteristics of a methanotrophs-associated bacteria isolated from the methanotrophic enrichments in the Yellow River Delta wetland soil,and to explore their effect on the methane oxidation process.[Methods]High-throughput sequencing was used to analyze microbial community of methanotrophic enrichments.The methanotrophs-associated strain was isolated using plate smearing and scribing method and initially identified by 16S rRNA gene sequencing.The morphology of the isolate was depicted by scanning electron microscopy.Gas chromatography was used to analyze the methane concentration,which showed the ability of utilizing methane and influence of improving methane oxidation by methanotroph.Two-chamber microbial fuel cells and Differential Pulse Voltammetry were used to test the electrochemical activity.[Results]The microbial community of methanotrophic enrichments from incubation of Yellow River Delta wetland soil included methanotrophs Methylobacter and other accompanying genera.We isolated a methanol-metabolizing bacterium,Pseudomonas putida P7(with the similarity of 99.79%)which was a rod bacterium with the length between 1.5μm and 2.5μm and width about 0.5μm.The GC analysis showed that this strain could not use methane but improved methane oxidation(P<0.05).The maximum current density was 28 mA/m2,and the results of DPV revealed that the oxidation peak and reduction peak occurred at–0.17 V and–0.25 V,respectively.[Conclusion]We successfully isolated an electrochemical activity microbe,Pseudomonas putida P7,with ability of improving methane oxidation.This study deepened the understanding of the physiological characteristics and functions of the methanotroph-associated bacteria in the process of methane oxidation.