ARS | Publication request: Group specific quantitative real-time polymerase chain reaction (qRT-PCR) analysis of methanogenic archaea in stored swine manure

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 22, 2009
Publication Date: N/A

Technical Abstract: Consolidated storage of swine manure is associated with the production of a variety of odors and emissions which result from anaerobic digestion of materials present in the manure. Methanogenic archaea are a diverse group of anaerobic microorganisms responsible for the production of methane. In the United States, methane emissions from lagoons and manure storage pits are estimated to be over 40 Tg/year, accounting for approximately 30% of the total methane emissions from agriculture. Little is known about the populations or concentrations of methanogens in stored swine manure and surprisingly, few methanogens have been isolated from this environment. The final reaction of methanogenesis involves reduction of the methyl group bound to coenzyme–M and results in the release of methane gas. In all methanogens, this step is ubiquitously catalyzed by methyl coenzyme-M reductase (MCR). The mcrA gene encodes the alpha subunit of the MCR holoenzyme. The subunits of MCR are phylogenetically conserved, and mcrA can be used as a phylogenetic marker for methanogen populations. In this study, total deoxyribonucleic acid (DNA) was isolated from stored swine manure from local swine facilities and used as a template for direct polymerase chain reaction (PCR). Clone libraries were created using degenerate primer sets that amplified a 464-491 bp variable region of the mcrA gene. Comparative phylogenetic analysis identified three major clusters of methanogenic archaea in stored swine manure representative of Methanomicrobiales, Methanobacteriales, and Methanosarcinales orders. Many cloned mcrA sequences showed little similarity to any known methanogens, suggesting these sequences represent novel, as yet unidentified methanogens. Quantitative real-time polymerase chain reaction (qRT-PCR) primer sets were developed to specifically target approximately 150 bp of the mcrA gene of the major groups of methanogenic archaea in stored swine manure. Standard curves were generated from different concentrations of cloned target mcrA genes representative of each group, and the linear range of detection was at least six orders of magnitude from 1 x 10**2 to 1 x 10**8 copies per PCR. This newly developed qRT-PCR assay will provide a sensitive and specific new method for detection and enumeration of methanogenic archaea in environmental samples.