title: Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.
abstract: Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-(13)C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of (13)C into DNA was detectable at in situ acetate concentrations (~7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the (13)C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.
authors.affiliation: Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Quebec, Canada.
authors.forename: Maialen
authors.initials: M
authors.lastname: Barret
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gagnon
authors.forename: Martin L
authors.initials: ML
authors.lastname: Kalmokoff
authors.forename: Edward
authors.initials: E
authors.lastname: Topp
authors.forename: Yris
authors.initials: Y
authors.lastname: Verastegui
authors.forename: Stephen P J
authors.initials: SP
authors.lastname: Brooks
authors.forename: Fernando
authors.initials: F
authors.lastname: Matias
authors.forename: Josh D
authors.initials: JD
authors.lastname: Neufeld
authors.forename: Guylaine
authors.initials: G
authors.lastname: Talbot
mesh_terms: D000693:Anaerobiosis; D000818:Animals; D016000:Cluster Analysis; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D007553:Isotope Labeling; D008372:Manure / Q000382:microbiology*; D008697:Methane / Q000378:metabolism*; D019638:Methanomicrobiaceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013552:Swine
chemical_list: D019641:DNA, Archaeal; D004275:DNA, Ribosomal; D008372:Manure; D012336:RNA, Ribosomal, 16S; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D008697:Methane