|Fouts, Derrick -|
|Szpakowski, Sabastian -|
|Purushe, Janaki -|
|Torralba, Manolito -|
|Nelson, Karen -|
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 23, 2012
Publication Date: November 21, 2012
Repository URL: http://handle.nal.usda.gov/10113/57855
Citation: Fouts, D.E., Szpakowski, S., Purushe, J., Torralba, M., Waterman, R.C., Macneil, M.D., Alexander, L.J., Nelson, K.E. 2012. Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen. PLoS One. 7(11):e48289. Interpretive Summary: The bovine rumen harbors a diverse population of microorganisms that converts ingested plan biomass to microbial proteins, short chain volatile fatty acids, and gases (CO**2, NH**3, and CH**4) via fermentation. End-products of rumen microbial fermentation not only provide the host with essential nutrients for metabolism but also produce waste products that are released into the environment. Studying the microbial populations associated with the bovine gastrointestinal tract holds vast potential for answering questions associated with improving animal production, increasing efficiency of animal feed use, assessing their symbiosis with the host, identifying potential opportunities for renewable fuel production including conversion of cellulosic waste to biogas, and reducing greenhouse gas production and emissions. Here we surveyed the extent of diversity of microbial populations in the rumen of cows fed forage diets and established protocols for use in future investigation. We compared multiple filtration and centrifugation techniques using a combination of Sanger and pyrosequencing of rRNA and mcrA amplicons. The observed bacterial diversity, based on the VI-V3 region of the 16S rRNA gene, was between 3181 to 7483 species-level OTUs, the largest ever observed for a ruminant species. This diversity was dominated by Bacteroidales, unknown bacteria, Clostridiales and Bacteroidetes taxa. Though there was little difference between fractions of the major rumen biosphere, there were differences in the rare biosphere, which may be attributable to depth of coverage. There was a considerable difference in microbial diversity between the two cows from which the samples came. The fungal community was sequenced to saturation and resulted in the identification of a novel unknown fungal group represented by over 600 sequences. Raw bolus should be suitable for future studies on rumen microbial diversity.
Technical Abstract: A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate the microbial diversity in the bovine rumen of 14 pasture-fed animals. The observed bacterial species richness, based on the V1-V3 region of the 15S rRNA gene, was between 1902 to 2596 species-level operational taxonomic units (OTUs). Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. The abundance of Prevotella species varied widely among the 14 animals. An increase in dept of sequencing coverage of one animal to greater than 130,000 reads significantly increased the OTU coverage and identified 14382 non-singleton OTUs, the highest number reported to date. The archaeal and fungal communities were sequenced to saturation. Examination of the fungal community resulted in identification of numerous novel fungal groups. The survey of microbial communities and analysis of cross-kingdom correlations suggested that there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and reduced environmental impact.