16S rRNA based T-RFLP analysis of methane oxidising bacteria—Assessment, critical evaluation of methodology performance and application for landfill site cover soils

Abstract

Methanotrophic bacteria have a ubiquitous distribution in the environment and play an important role in global climate warming by lowering methane emission into the atmosphere. Globally, landfill sites produce about 10% of the methane entering the atmosphere, and soils above landfill sites have been shown to contain methanotrophic populations with the highest methane oxidation capacity measured. Landfill site simulating lysimeters were set up in which different vegetation regimes were tested for their effect on methane oxidation. Four different plants ( Miscanthus, poplar, grass, alfalfa–grass mixture alongside with an unplanted control) were grown with and without landfill gas (LFG) supply. One year after planting, rhizosphere samples in the upper layer of the lysimeters were collected and the methanotroph community was analysed using a cultivation-independent approach. Terminal restriction fragment length polymorphism (T-RFLP) and RNA dot blot hybridisations with primers/probes targeting specifically type I and type II methanotrophs were used. In addition, methanotrophic 16S rRNA gene clone libraries were compared and dominant clones were sequenced. Methylobacter and Methylocystis were found to be dominant members of the type I and type II methanotroph communities, respectively. The type of plant cover affected both type I and type II methanotrophs, however, biogas had more impact on type I populations displaying higher diversity and abundance in samples supplied with LFG than in the LFG-free controls.