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 pulse-labelled 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. © 2008 International Society for Microbial Ecology.