Applying stable isotope probing of phospholipid fatty acids and rRNA in a Chinese rice field to study activity and composition of the methanotrophic bacterial communities in situ

Abstract

Methanotrophs in the rhizosphere play an important role in global climate change since they attenuate methane emission from rice field ecosystems into the atmosphere. Most of the CH 4 is emitted via transport through the plant gas vascular system. We used this transport for stable isotope probing (SIP) of the methanotrophs in the rhizosphere under field conditions and pulselabelled rice plants in a Chinese rice field with CH 4 (99% 13 C) for 7 days. The rate of 13 CH 4 loss rate during 13 C application was comparable to the CH 4 oxidation rate measured by the difluoromethane inhibition technique. The methanotrophic communities on the roots and in the rhizospheric soil were analyzed by terminal-restriction fragment length polymorphism (T-RFLP), cloning and sequencing of the particulate methane monooxygenase (pmoA) gene. Populations of type I methanotrophs were larger than those of type II. Both methane oxidation rates and composition of methanotrophic communities suggested that there was little difference between urea-fertilized and unfertilized fields. SIP of phospholipid fatty acids (PLFA-SIP) and rRNA (RNA-SIP) were used to analyze the metabolically active methanotrophic community in rhizospheric soil. PLFA of type I compared with type II methanotrophs was labelled more strongly with 13 C, reaching a maximum of 6.8 atom-%. T-RFLP analysis and cloning/sequencing of 16S rRNA genes showed that methanotrophs, especially of type I, were slightly enriched in the 'heavy' fractions. Our results indicate that CH 4 oxidation in the rice rhizosphere under in situ conditions is mainly due to type I methanotrophs.

Distributions of putative aerobic methanotrophs in diverse pelagic marine environments

“…MISA complements existing pmo profiling methods including terminal restriction fragment length polymorphism and microarray analyses (Bodrossy et al, 2003;Horz et al, 2005;Qiu et al, 2008) by targeting an intergenic spacer within the pmo operon. Variations in amplicon length are determined directly without requiring downstream manipulations (for example, digestion and hybridization).…”

Applications and impacts of stable isotope probing for analysis of microbial interactions

“…In this study microcosms supplemented with mineral salts medium in addition to 13 C-methane showed on the one hand rapid incorporation of the label into the biomass but a less diverse population and no label in type II methane-oxidizing bacteria (Cebrón et al 2007). This experiment highlights again the dangers coming from unnaturally high substrate concentrations which, however, are often required for sufficient enrichment of nucleic acids for analyses but carefully selected experimental conditions can avoid this bias (Qiu et al 2008). In an interesting combination of fatty acid and ether lipids isotope ratios and FISH-SIMS Orphan et al used the natural 13 C-depletion of methane to elucidate the fate of methane in consortia of metabolically interdependent bacteria and archaea.…”

Revisiting Methanotrophic Communities in Sewage Treatment Plants

“…2). Generally, type I methanotrophs were found to be active in other highmethane environments (30,33,(36)(37)(38)(39). The proliferation of methanotrophs after methane addition indicates their potential to mitigate methane emission in wastewater sludges.…”