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United States Department of Agriculture

Agricultural Research Service

Research Project: UNDERSTANDING THE ROLE OF COMMENSAL ANAEROBIC BACTERIA IN ODOR, EMISSIONS, AND ANTIBIOTIC RESISTANCE FROM STORED LIVESTOCK MANURE

Location: Bioenergy Research Unit

Title: Analyses of methanogenic archaea populations in swine feces and stored swine manure using 16S rDNA and mcrA PCR and pure culture isolation

Authors
item WHITEHEAD, TERENCE
item Spence, Cheryl -
item COTTA, MICHAEL

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 19, 2012
Publication Date: N/A

Technical Abstract: Background: Storage of swine manure is associated with the microbial production of odorous compounds and gaseous emissions which result from anaerobic microbial digestion of materials present in the manure. 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 feces and stored swine manure, and surprisingly few methanogens have been isolated from these ecosystems. Methods: Total deoxyribonucleic acid (DNA) isolated from stored swine manure from local swine facilities and enrichment cultures were used as templates 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 or methanogen-specific primers amplifying a 1,254 bp region of the 16S rDNA gene. The mcrA gene encodes the alpha subunit of methyl coenzyme-M reductase (MCR), a ubiquitous enzyme in methanogens. The subunits of MCR are phylogenetically conserved and mcrA can be used as a phylogenetic marker for methanogen populations. Pure cultures were isolated from feces and manure after enrichments in anaerobic medium containing 10% rumen fluid. Results: Comparative phylogenetic analyses demonstrated various clusters of methanogenic archaea are present in stored swine manure and feces. Representatives of Methanomicrobiales, Methanobacteriales, and Methanosarcinales orders were identified. Many of the cloned 16S rDNA and mcrA sequences showed little similarity to any known methanogens suggesting these sequences represent novel, as yet unidentified methanogens. Two strains of methanogens have been isolated thus far and the closest relative appears to be Methanobrevibacterium smithii. Conclusions: Phylogenetic analyses of swine feces and stored manure indicate that these ecosystems contain both known and unidentified methanogenic archaea. Isolation of pure cultures would enhance our knowledge of these microorganisms and their role in production of greenhouse gases from agricultural sources.

Last Modified: 9/10/2014
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