Next generation sequencing and stable isotope probing of active microorganisms responsible for aerobic methane oxidation in red paddy soils

Abstract:

Abstract:［Objective］This study is aimed to establish an unbiased profiling strategy for investigating the microorganisms responsible for aerobic methane oxidation by pyrosequencing the total soil microbial communities at DNA and RNA levels，and to link aerobic methane oxidation activity with taxonomic identity of active microorganisms by DNA /RNA SIP in red paddy soils.［Methods］Three red paddy soils derived from quaternary red clay were collected from Gushi and Taoyuan cities of Hunan province and Leizhou city of Guangdong province，were incubated with the labeled 13 CH4 or 12 CH4 for determination of aerobic methane oxidation kinetics． Pyrosequencing of the 16S rRNA and16S rRNA gene at the whole microbial community levels were performed over the course of aerobic methane oxidation in soil microcosms． 13 C-DNA and 13 C-RNA were obtained through ultracentrifugation of the total soil DNA and RNA extracts，respectively． Clone library of pmoA genes in 13 C-DNA and 16S rRNA genes in 13 C-RNA were constructed．［Results］ Pyrosequencing of the total microbial communities revealed significant increase in the relative abundance of aerobic methanotrophs in soil microcosms upon the completion of aerobic methane consumption． The proportional increase of aerobic methanotrophs was significantly higher at RNA than DNA levels． Type I and II aerobic methanotrophs significantly increased in Gushi soil，while the significant increase of type II aerobic methanotrophs was observed in Taoyuan soil． In the meantime，type I aerobic methanotrophs appeared to be stimulated exclusively in Leizhou soil． Sequencing analysis of the 13 C-labeled pmoA genes and 16S rRNA further demonstrate that phylogenetically distinct methanotrophs dominated aerobic methane oxidation activity in paddy soils of Gushi (Type I and II)，Taoyuan (Type II) and Leizhou (Type I)．［Conclusion］ Highthroughput pyrosequencing at the whole community level of 16S rRNA genes provides an almost unbiased profiling stragety for measuring characteristic changes in relative proportions of aerobic methanotrophs responsible for aerobic methane oxidation activity in red paddy soils，and higher sensitivity was observed at RNA than DNA levels．DNA/RNA-SIP can accurately reveal the active microorganisms responsible for aerobic methane oxidation in read soil，being largely consistent to pyrosequencing-based fingerprinting analysis of the total microbial communities.

Key words:Keywords: next-generation sequencing，stable-isotope probing，red paddy soil，aerobic methane oxidation