FISH-NanoSIMS技术在环境微生物生态学上的应用研究

微生物分子生态学技术的不断发展,使得同时分析复杂生态系统中微生物的分布和功能特征成为可能.为了研究荧光原位杂交-纳米二次离子质谱技术(fluorescence in situ hybridization-Nano secondary ion mass spectroscopy,FISH-Nano SIMS)在环境微生物生态学上的应用,本研究采用稳定同位素标记的化合物13C-C6H12O6、15N-NH4Cl作为C源和N源,分别对纯培养锰氧化细菌假单胞菌Pseudomonas sp.QJX-1(培养基加锰及不加锰两种条件下),以及浅层土壤及厌氧污泥两种环境样品进行培养.利用FISH-NanoSIMS技术检测培养后样品中微生物体内12C-、13C-、12C14N-、12C15N-的分布特征及其丰度值,进而探讨纯菌及环境样品中微生物利用同位素碳氮源的情况.结果显示所有样品细菌分布区域对应的同位素碳氮(13C、15N)的含量均显著大于其自然丰度值,这表明Pseudomonas sp.QJX-1及环境样品中的微生物均能代谢13C-C6H12O6和15N-NH4Cl.研究进一步发现,Pseudomonas sp.QJX-1在碳氮源消耗至较低浓度时才进行锰氧化;浅层土壤和厌氧污泥中可能都存在同步硝化反硝化细菌群落.FISH和Nano SIMS技术联用能同时分析环境样品中特定微生物的分布特征及代谢功能,进而能更好地掌握环境样品中微生物群落的生理生态学特征.

Application of FISH-NanoSIMS Technique in Environmental Microbial Ecology Study

With the development of microbial ecology techniques, it is possible to analyze the distribution and function of microorganisms simultaneously in complex ecosystems. To explore the application of FISH-NanoSIMS in environmental microbial ecology study, our study used the stable isotope labeled compounds ¹³C-C₆H₁₂O₆ and ¹⁵N-NH₄Cl as C and N sources for cultivating the pure culture(manganese oxidizing bacteria, Pseudomonas sp. QJX-1) and environmental samples(the shallow soil and anaerobic sludge). FISH-NanoSIMS was used to detect the distribution of microorganisms and relatively quantify secondary ions(¹²-, ¹³C^-, ¹²C¹⁴N^-, ¹² C¹⁵N^-) in cultivated samples, in order to explore the utilization of C and N isotopes sources by the pure culture and microorganisms in environment samples. The results showed that the contents of ¹³C and ¹⁵N in the area of bacteria were significantly greater than the natural abundance in all samples. It indicated that Pseudomonas sp. QJX-1 and some specific bacteria in environmental samples could metabolize ¹³C-C₆H₁₂O₆ and ¹⁵N-NH₄Cl. Furthermore, this study revealed that for Pseudomonas sp. QJX-1, the manganese oxidation only occurred when the carbon and nitrogen were consumed to a low level. For environmental samples, the bacterial nitrification and denitrification were both observed in the shallow soil and anaerobic sludge. In a word, our study demonstrated that the combination of FISH and NanoSIMS could simultaneously examine microbial distribution and microbial metabolic activity in environmental samples, which will help us to obtain the eco-physiology information of microbial community.