甲烷氧化混合菌的保藏方法研究

Study on preservation methods of mixed methane-oxidizing bacteria

【目的】甲烷氧化混合菌是自然界中吸收甲烷的关键微生物,在甲烷氧化混合菌的研究和应用中,首先要解决其长期稳定保藏的问题,保藏方法应能有效保持菌群结构和功能的完整性、稳定性。【方法】以从煤矿土壤富集得到的两种结构稳定的甲烷氧化混合菌为实验体系,研究对比了冷藏法、低温冷冻法、石蜡油冷冻法、甘油冷冻法4种保藏方法,考察保藏前后混合菌的生长状况、MMO活性、菌群结构等。【结果】保藏6个月后,除甘油冷冻法以外,经其它3种方法保藏的混合菌,都具有与保藏前相当的细胞密度、甲烷氧化能力、MMO酶活以及传代稳定性,且DGGE图谱显示保藏前后的菌群结构变化不大。【结论】这3种保藏方法都可以有效的保持甲烷氧化混合菌功能和菌群结构的稳定性。

[Objective] Mixed methane-oxidizing bacteria are useful for reduction of methane emission. For research and application of mixed methane-oxidizing bacteria, their long-term stable preservation must be first solved. The preservation methods should be able to maintain the integrity and stability of the community structure and function. [Methods] Two kinds of stable mixed methane-oxidizing cultures enriched from a coal mine soil were used as the bacterial community. Four preservation methods, refrigeration, ultralow freezing, freezing in paraffin oil and freezing in glycerin, were comparatively studied. The growth, MMO activity and community structure before and after the preservation were investigated. [Results] The method with glycerin could not be used for mixed methane-oxidizing bacteria preservation. After preservation using the other three methods, the cell density, methane oxidation capacity, MMO activity and subculture stability reached the same level as before the preservation. DGGE fingerprints of 16S rRNA for the mixed cultures before and after the preservation showed that the change of the community structure during the preservation was not much. [Conclusion] The three preservation methods can effectively maintain the function and community structure of the mixed methane-oxidizing bacteria stable.