Uncultivated Methylocystis Species in Paddy Soil Include Facultative Methanotrophs that Utilize Acetate

Methanotrophs are crucial in regulating methane emission from rice field systems. Type II methanotrophs in particular are often observed in high abundance in paddy soil. Some cultivated species of Methylocystis are able to grow on acetate in the absence of methane. We hypothesize that the dominant type II methanotrophs in paddy soil might facultatively utilize acetate for growth, which we evaluate in the present study. The measurement of methane oxidation rates showed that the methanotrophic activity in paddy soil was inhibited by the addition of acetate compared to the continuous supplementation of methane, but the paddy soil maintained the methane oxidation capacity and recovered following methane supplementation. Terminal restriction fragment length polymorphism analysis (T-RFLP) combined with cloning and sequencing of pmoA genes showed that Methylocystis was enriched after incubation with added acetate, while the type I methanotrophs Methylocaldum/Methylococcus and Methylobacter were enriched by methane supplementation. A comparison of pmoA sequences obtained in this study with those in the public database indicated that they were globally widespread in paddy soils or in associated with rice roots. Furthermore, we performed stable isotope probing (SIP) of pmoA messenger RNA (mRNA) to investigate the assimilation of (13)C-acetate by paddy soil methanotrophs. RNA-SIP revealed that Methylocystis-related methanotrophs which shared the same genotype of the above enriched species were significantly labelled. It indicates that these methanotrophs actively assimilated the labelled acetate in paddy soil. Altogether, these results suggested that uncultivated Methylocystis species are facultative methanotrophs utilizing acetate as a secondary carbon source in paddy soil.