Effects of copper on methane emission, methanogens and methanotrophs in the rhizosphere and bulk soil of rice paddy

Abstract

Copper contamination is common in paddy fields due to wastewater irrigation and application of sludge and Cu-containing fungicides. We aimed to study the effects of copper (Cu2+) application on methane emission, methanogens and methanotrophs in both the rhizosphere and bulk soil. The study was conducted in rhizobox in which flooded soil was applied with different Cu2+ concentrations. Methane emission was collected with static chamber method and determined by gas chromatography. The diversity and composition of methanogens and methanotrophs were studied using PCR–DGGE and sequencing analysis of methanogenic 16S rRNA and pmoA genes. The abundance of methanogens and methanotrophs was determined using quantitative real-time PCR of mcrA and pmoA genes, respectively. The results showed that Cu2+ application decreased methane emission along with the diversity and abundance of methanogens and methanotrophs, although the application of 200mgkg−1 Cu2+ did not significantly decrease the diversity of methanogens and methanotrophs in the rhizosphere. In addition, Cu2+ decreased methanotrophs diversity more profoundly than methanogens diversity. Methanogens in both the rhizosphere and bulk soil were closely related to Methanosaeta, Methanosarcina, Methanobacterium and Methanomicrobia archaeon. Methanotrophs in rhizosphere soil were clustered into four groups (type I methanotrophs, Methylobacter, Methylomonas and Methylosarcina) while those in bulk soil were much less diverse. The addition of 200 to 800mgkg−1 Cu2+ did not dramatically change the composition of methanogens; however, for methanotrophs, only one DGGE band belonging to Methylosarcina was present after the addition of 800mgkg−1 Cu2+. We conclude that methanotrophs were more sensitive to Cu2+ addition than methanogens, and that the rhizosphere environment alleviated Cu2+ stress on methanogens and methanotrophs.