Identification of Candidate Cellulose Utilizing Bacteria from the Rumen of Beef Cattle, Using Bacterial Community Profiling and Metagenomics

The ruminal microbiome allows ruminant animals to convert cellulosic biomass into food products. A majority of ruminal microorganisms remain uncharacterized due, in part, to the complexity of ruminal microbial communities. In order to gain further insight, selection-based batch culturing from bovine rumen fluid, in combination with metagenomics, was used to identify and characterize previously uncharacterized rumen bacteria capable of metabolizing cellulose, which was supplemented as a purified substrate. 16S rRNA-based population analysis was used to identify rumen bacteria enriched within 14 days of culturing. As a result of 4 independent experiments, seven different candidate cellulose-utilizing species-level operational taxonomic units (OTUs) were identified. Six of the enriched OTUs showed increased levels ranging between 46 and 445-fold compared to their respective rumen inocula, representing 14.1% to 41.3% of reads in samples supplemented with cellulose. One OTU corresponded to a known species (Ruminococcus flavefaciens), four OTUs were predicted to be uncultured species of known genera (Ethanoligenens sp., two Prevotella sp., and Rummeliibacillus sp.), and two were assigned to the family Ruminococcaceae. One enriched culture consisting of an uncultured Rummeliibacillus and Prevotella was used for metagenome analysis. Analysis revealed genes with predicted cellulolytic capabilities in the Rummeliibacillus-related organism (cellulase, endoglucanase, and beta-glucanase) and in the Prevotella-related organism (cellulase). Additionally, genes predicted to function in cellulose binding, as well as proteases and glutamate synthases needed for amino acid acquisition, were also found in both OTUs. The identification and characterization of novel cellulolytic species of ruminal bacteria will contribute to a better understanding of ruminal cellulose metabolism