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 (ϳ7 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.

Phylogenetic identification of methanogens assimilating acetate-derived carbon in dairy and swine manures

“…The swine manure sample S1 was collected at a different farm than the one used in our preliminary work [3]. Details of the sampling procedure for manure samples have been described in a previous study [5].…”

“…The starved manure samples were used for SIP incubations, as previously detailed [3]. Briefly, [ 12 C]-or [ 13 C]-acetate were added to each 45 mL subsample of S1, D1 and D2 manures to a final concentration of 6 g L −1 .…”

“…DNA extracts were purified using the DNeasy ® Blood and Tissue Kit (Qiagen, Canada) prior to undertaking qPCR. Density gradient fractionation was performed following ultracentrifugation of DNA in a cesium chloride gradient, as previously described [3].…”

“…Then, DNA extraction and density fractionation followed by LH-mcrA fingerprinting and clone library analyses were carried out. Carbon flow associated with acetate metabolism and biogas production during SIP incubations were assessed as previously described [3].…”

“…Recently, we conducted a DNA-SIP proof-of-principle experiment, with a single sample from a swine manure storage tank [3], to demonstrate that this experimental approach could be applied to identify active methanogens in such a carbon-rich ecosystem. Methanoculleus spp., although not representing the dominant genus in the initial sample, assimilated acetate-C and contributed to methanogenesis.…”