Single-cell Genomic Analysis of Fiber-adhering Rumen Bacteria during an in situ Incubation of Timothy Hay.

Rumen microorganisms adhere to the surface of ingested feed and degrade fibrous compounds into short-chain fatty acids, which are the primary source of energy for milk and meat production. A more detailed understanding of uncultured ruminal bacteria involved in polysaccharide degradation may help improve the feed digestion efficiency of ruminants. The aim of the present study was to identify the ability of uncultured ruminal bacteria to degrade polysaccharides by constructing bacterial genomes and linking their 16S rRNA genes with functional genes using single-cell genome sequencing. The ruminal fibrolytic community was enriched on the surface of Timothy hay via a time-limited in situ incubation. A single-cell genomic anal-ysis was used to construct 83 single-cell-assembled genomes from the ruminal fibrolytic community, and 16S rRNA genes were recovered from 80 genomes. The uncultured Ruminococcus, Ruminococcus_D, and Fibrobacter groups had a high percentage of carbohydrate-binding module genes involved in attachment to fibrous compounds. Uncultured Prevotella showed different profiles of genes encoding major carbohydrate-active enzymes from those of known P. ruminicola spp., and the amino acid sequence of glycoside hydrolase family 43 had various deletions and substitutions. The single-cell genomic anal-ysis enabled the construction of genomes with 16S rRNA genes from fiber-adherent rumen bacteria and linked their 16S rRNA genes with the DNA and amino acid sequences of functional genes involved in carbohydrate utilization. Several uncultured rumen bacteria possess functional genes that contribute to feed digestion and biofilm formation.