This study determined environmental controls on the diversity of Archaea and Bacteria as well as the distribution, diversity and physiology of methanotrophs in geothermal environments.
It is known that animal and plant diversity peaks at the tropics and decreases towards the poles, a trend often attributed to temperature differences with latitude. To test the impacts of temperature and pH on prokaryotic diversity, 16S rRNA gene pyrotag sequencing was used to assess the diversity of soil, sediment and biomat samples from geothermal areas in Canada and New Zealand, covering a temperature range of 7.5-99°C and a pH range of 1.8-9.0. Temperature was found to play an important role in determining microbial community diversity when considered over most of the temperature range within which life is possible.
To investigate the presence and types of Verrucomicrobia in geothermal and acidic environments, the above geothermal data set and community analyses of additional acidic peats were investigated for putative methanotrophic 16S rRNA gene sequences. Methanotrophic Verrucomicrobia were found in geothermal environments below pH 5, but not in acidic peats. Based on soil depth profiles with varying temperatures, the analysis of different geothermal sites and a comparison of isolates there appear a thermophilic and a mesophilic group of methanotrophic Verrucomicrobia.
A stable isotope probing (SIP) approach was used to identify methanotrophs in Canadian geothermal springs (pH range 6.34-8.68, temperature range 19.2-50°C). 16S rRNA gene sequences were analyzed following incubation with 13CH4 and separation of 13C-labelled DNA from unlabelled nucleic acids. 16S rRNA gene sequences from the 13C-labelled fractions from high temperature incubations showed a dominance of methanotrophic Alphaproteobacteria genera.
Methanotrophic Verrucomicrobia have been shown to fix carbon autotrophically. As a result 13CH4-SIP will not detect verrucomicrobial methanotrophs. A modified SIP technique was developed based on labelling with 13CH4 and 13CO2. This protocol was validated using M. infernorum strain V4, which assimilated 13CO2 but not 13CH4. To specifically detect Verrucomicrobia methanotrophs a qPCR assay specific to the verrucomicrobial-pmoA genes was developed and used in combination with SIP. Testing this protocol on acidic geothermal soils demonstrated that the primary active methanotrophs were autotrophs and belonged to the Verrucomicrobia.