Abstract
Methane is the second most important greenhouse gas contributing to about 20% of global warming. Its mitigation is conducted by methane oxidizing bacteria that act as a biofilter using methane as their energy and carbon source. Since their first discovery in 1906, methanotrophs have been studied using a complementary array of methods. One of the most used molecular methods involves PCR amplification of the functional gene marker for the diagnostic of copper and iron containing particulate methane monooxygenase. To investigate the diversity of methanotrophs and to extend their possible molecular detection, we designed a new set of degenerate methane monooxygenase primers to target an 850 nucleotide long sequence stretch from pmoC to pmoA. The primers were based on all available full genomic pmoCAB operons. The newly designed primers were tested on various pure cultures, enrichment cultures and environmental samples using PCR. The results demonstrated that this primer set has the ability to correctly amplify the about 850 nucleotide long pmoCA product from Alphaproteobacteria, Gammaproteobacteria, Verrucomicrobia and the NC10 phyla methanotrophs. The new primer set will thus be a valuable tool to screen ecosystems and can be applied in conjunction with previously used pmoA primers to extend the diversity of currently known methane-oxidizing bacteria.
Highlights
Methane is the second most important greenhouse gas contributing to about 20% of global warming (Intergovernmental Panel on Climate Change 2014)
While anaerobic methaneoxidizing archaea consume more than 75% of the CH4 produced in certain marine sediments (Reeburgh and Whjalen 2007; Beal et al 2009; Egger et al 2014), aerobic methane-oxidizing bacteria (MOB) that live at the interface between anoxic and oxic zones in marine environments (Bender and Conrad 1992; Lüke et al 2016; Padilla et al 2016), freshwater wetlands and rice fields (Lüke et al 2014) have been estimated to consume up to 90%
The primers were made specific to MOB so that they had more than five mismatches with ammonium monooxygenase amo gene sequences of ammonia oxidizing bacteria (AOB)
Summary
Methane is the second most important greenhouse gas contributing to about 20% of global warming (Intergovernmental Panel on Climate Change 2014). Methanogenic archaea are assumed to be the sole producers of methane that reside in these environments (Cicerone and Oremland 1988; Conrad et al 1999; Joulian et al 1997) These archaea are present in waste treatment systems, intestines of ruminants and termites and landfills acting as additional CH4 sources. While anaerobic methaneoxidizing archaea consume more than 75% of the CH4 produced in certain marine sediments (Reeburgh and Whjalen 2007; Beal et al 2009; Egger et al 2014), aerobic methane-oxidizing bacteria (MOB) that live at the interface between anoxic and oxic zones in marine environments (Bender and Conrad 1992; Lüke et al 2016; Padilla et al 2016), freshwater wetlands and rice fields (Lüke et al 2014) have been estimated to consume up to 90%
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