170. A Microbial Enzymatic Comparison Between Liquid and Solid Microenvironments in the Rumen of Beef Steers

Abstract It is well established that liquid and solid fractions within the rumen have distinct microbial communities. Previous studies have reported limited taxonomic and functional results when conducting analysis using the 16S rRNA gene. However, whole genome sequencing (WGS) allows for greater depth of sequencing and identification of potential enzymes produced by different microbial species. The objective of this study was to utilize WGS to characterize the enzymatic capabilities of ruminal microorganisms differentially associated with either the solid or liquid microenvironments. Four ruminally cannulated steers were utilized in a 4 × 4 Latin Square experiment and provided ad libitum access to hay with supplemental alfalfa cubes at 200 g/kg intake per day. Periods lasted 28-d; on d-14, rumen contents were sampled and separated into solid (n = 16) and liquid (n = 16) samples, DNA was extracted from samples and sequenced using WGS. After sequencing, quality sequences were analyzed for functional annotation specifically aligning genes to the Clusters of Orthologous Groups (COG) and Carbohydrate-Active Enzymes (CAZyme) databases. Custom Python scripts were used to identify key methanogenic-related enzymes. Relative abundances of COG, CAZyme categories and methanogenic related enzymes were analyzed using a mixed-effects model, with microenvironment (solid or liquid) and period as fixed effects and steer as a random effect. Differences between microenvironments were assessed using Type III ANOVA. Statistical differences (P ≤ 0.05) across rumen fractions were detected in all COG categories with the exception of the COG associated with nuclear structure. The liquid microenvironment contained a significantly higher (P ≤ 0.01) amount of enzymes associated with the “unknown” COG category as compared to the solid (16.2 versus 15.0%). For CAZyme classes, the solid microenvironment contained more glycoside hydrolase enzymes, which have roles in cellulose and hemicellulose degradation, than the liquid (P ≤ 0.01). For methane-related genes, the liquid microenvironment had higher abundances of three of the five subunits of the MCR complex and higher abundance of the pmoA gene, a biomarker for methanotrophic bacteria that consume methane, than the solid (P ≤ 0.01). These findings confirm the solid rumen fraction is specialized for fiber degradation, evident by the enrichment in glycoside hydrolase enzymes, and the liquid fraction contained more enzymes related to methanogenesis and methane cycling, as indicated by enrichment of genes for both the methane-producing MCR complex and the methane-consuming gene. Further, the higher prevalence of the unknown COG category in the liquid phase suggests a substantial portion of the functional potential of free-floating ruminal microbes remains unclassified, highlighting a gap in our understanding of metabolic processes occurring within the liquid fraction of the rumen. These findings deepen our understanding of the functional potential of microbes associated with different rumen fractions.