1 Department of Animal Science - Immunology and microbiology, Department of Animal Science, Science and Technology, Aarhus University2 Department of Animal Science - Immunology and microbiology, Department of Animal Science, Science and Technology, Aarhus University
Introduction Enteric methane (CH4) production by ruminants is a major source of anthropogenic CH4 emissions. The CH4 is derived from complex anaerobic degradation of plant biomass by the rumen microbiota, the terminal group being methanogenic archaea. The methanogens have been targets of a plethora of methane mitigation strategies, often aiming at reducing concentrations of H2; the major energy source of most rumen methanogens known to date. However, not all rumen archaea are yet physiologically characterized. Using a metatranscriptomic approach the present study investigated the effect of dietary manipulation, aiming at reducing enteric CH2 production, on the active rumen microbiota. Materials and methods Four rumen-fistulated Holstein dairy cows were fed a control diet (total fat: 3.5% DM) and a rapeseed oil (RSO)-supplemented diet (total fat: 6.5% DM), in a conventional 4×2 cross-over design (three weeks per feeding period). One cow was excluded from the full experiment due to health considerations not related to the diets. Methane emission from the cows was quantified at the end of each feeding period in transparent polycarbonate chambers (Hellwing et al., 2012). Concomitantly, rumen fluid was sampled from the top and bottom part of the frontal, mid and distal section of the rumen (25 ml per section), pooled, physically disrupted to release microorganisms from organic matter and filtered (pore size: 0.5 mm). Total nucleic acids were extracted using a standard phenol-chloroform bead-beating procedure. DNA was removed and double-stranded complementary DNA (ds-cDNA) was synthetized from purified RNA. The ds-cDNA was sequenced using the Illumina HiSeq2000 system (≈160bp; paired-end sequenced from 2×100bp read lengths). Concatenated small subunit (SSU) rRNA reads were analyzed according to Urich et al. (2008) and putative messenger RNA (mRNA)-related reads were analyzed by Meta-Genome Rapid Annotation using Subsystems Technology, v. 3.1.2. (MG-RAST) with subsystem-based annotation based on the SEED database and with MEGAN4 analysis of BlastX searches against the Genbank Refseq protein database (e-value cut-off 1e-5). Results RSO significantly reduced CH4 emission from the cows, resulting in a 6.2% lower CH4-to-CO2 emission ratio (P<0.05). Illumina deep sequencing resulted in ~10-12 Million high-quality reads per sample, of which putative mRNAs comprised between 380,000 and 485,000 reads. Analysis of SSU rRNA reads revealed that archaea were significantly decreased upon RSO supplementation, whereas bacterial and eukaryotic taxa were basically unaffected. The decrease of archaea could be assigned to a novel group of methanogens distantly related to Thermoplasmata (“Rumen Cluster C”, RCC) (Fig. 1A). In contrast to well-known hydrogenotrophic methanogens (Fig. 1B and D), the novel RCC methanogens decreased in abundance and activity upon RSO amendment, as illustrated by the 16S rRNA and methyl-coenzyme M reductase subunit α (mcrA) transcript levels (Fig. 1A and C). mRNAs of enzymes involved in methanogenesis from methylamines (MA), only distantly related to known variants from methanogens, were among the most abundant archaeal transcripts. These transcripts, decreased in abundance upon RSO amendment (Fig. 1E), concurrently with 16S rRNA and mcrA of RCC, making the RCC methanogens the likely origin of these mRNAs. Conclusions Metatranscriptomic analysis enabled in situ characterization of a novel, yet uncultured group of rumen methanogens. These RCC methanogens are putative methylotrophic archaea, and the second order of methanogens able to utilize methylamines. The RCC methanogens may potentially be a key target for strategies to mitigate CH4 emissions from ruminant livestock. Acknowledgements This project was funded by the Ministry for Food, Agriculture and Fisheries, the Faculty of Science and Technology, Aarhus University, Denmark and the Austrian Federal Ministry of Science and Research (GEN-AU III InflammoBiota). References Hellwing, A.L.F., Lund, P., Weisbjerg, M.R., Brask, M., and Hvelplund, T. 2012. Journal of Dairy Science. 95, 6077-6085. Poulsen, M., Schwab, C., Jensen, B.B., Engberg, R.M., Spang, A., Canibe, N., Højberg, O., Milinovich, G., Fragner, L., Schleper, C., Weckwerth, W., Lund, P., Schramm, A., and Urich, T. 2013. Nature Communications. 4. Urich, T., Lanzén, A., Qi, J., Huson, D.H., Schleper, C., and Schuster, S.C. 2008. PLoS ONE. 3.
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Greenhouse Gases and Animal Agriculture Conference 2013