Consolidated storage of swine manure is associated with the microbial production of a variety of odors and emissions, including ammonia, organic acids, alcohols and hydrogen sulfide. Large quantities of methane are also produced from such facilities. In the United States, methane emissions from lagoons and manure storage pits are estimated to account for approximately 30% of the total methane emissions from agriculture. Methanogenic archaea are a diverse group of anaerobic microorganisms responsible for the production of methane. Little is known about the populations or concentrations of methanogens in stored swine manure and surprisingly few methanogens have been isolated from this environment. This study was carried out to identify the methanogen populations in stored swine manure. An understanding of the diversity of methanogenic populations in this environment will enable the development of assays to quantify these organisms in response to different manure treatment strategies aimed at reducing odor and methane emissions. Methyl coenzyme-M reductase (MCR) catalyzes the final step of methane production and is phylogenetically conserved and ubiquitous in methanogens. Sequencing of both the mcrA functional gene, encoding the alpha subunit of the MCR holoenzyme, as well as archaeal 16S rDNA, was used to identify the methanogen populations of stored swine manure. Total DNA was isolated from stored swine manure from local swine facilities and used as a template for PCR.
Results/Conclusions
Amplification with a degenerate primer set flanking a variable region of the mcrA gene generated a PCR product between 464 and 491 bp. A 16S rDNA archaeal primer set generated a PCR product of approximately 800 bp. 16S rDNA and mcrA clone libraries were created and clones were selected at random for sequencing and comparative phylogenetic analysis. Phylogenetic trees were constructed for both mcrA and 16S rDNA sequencing data and comparatively analyzed. A predominant number of clones from both libraries were found to group within the Methanobacteriales and Methanomicrobiales orders. Some of the clones from the mcrA library, but not from the 16S rDNA library, grouped within the Methanosarcinales order. Many sequenced clones showed little similarity to any identified methanogens, suggesting that these sequences represent novel, as yet unidentified methanogenic archaea and a new source of microbial diversity. This is the first mcrA-targeted study of the methanogenic archaea population of swine manure and results from this study will be used to further develop molecular methods targeting the mcrA functional gene to monitor different populations of methanogens in this environment.
