Identification of Novel Rumen Bacteria Using Starch As a Selective Nutrient in Batch Cultures

The rumen is a complex and diverse microbial ecosystem composed of bacteria, methanogens, protozoa, and fungi. Among these diverse and complex microbial communities, bacteria are a predominant component in terms of cell densities, taxonomic diversity, and metabolic potential that play a vital role in the fermentation of feedstuff. Since the specific role of most rumen bacteria in the digestion of feedstuff remains to be determined, the primary objective of this research was to identify uncharacterized bacteria that are involved in metabolizing starch. Our general approach consisted of culturing rumen fluid under anaerobic conditions and at a constant temperature, using laboratory-scale bioreactors in the presence of starch. For each experiment, three replicate cultures were supplemented with starch (ADM Corn processing Clinton, Iowa) as substrate, while two replicate cultures received no supplementation. Candidate starch utilizing bacteria were identified using amplicon sequencing for the V1-V3 region of the 16S rRNAgene, by comparing OTU profiles of starch-enriched cultures to non-supplemented cultures. For trial 1, a combined 351,356 high quality sequence reads were used to determine the bacterial composition in control and treatment groups, ranging between 14,460 and 84,655 per sample. Our results showed that one species-level Operational Taxonomic Unit (OTU) named OTU-SD1, corresponding to an uncultured species of Prevotella, was enriched on day 7 (15.6±3.22) and 14 (17.7±5.02) in treatment groups when compared to control cultures (0.17±0.08). For trial 2, a combined 309,387 high quality sequence reads were obtained ranging between 4691 and 77,984 per sample. Our results showed that two species-level Operational Taxonomic Unit (OTUs) named OTU-SD2, and OTU-SD3 corresponding to an uncultured species of Prevotella, was enriched on day 7 (36.7±31.7) and Acetobacter, was enriched on day 14 (27.4±26.2) in treatment groups when compared to control cultures (1.90±1.39). Metagenomic sequence analysis for OTU-SD1 revealed the presence of genes for sugar utilization, including an α-amylase enzyme predicted to be involved in hydrolyzing starch. A better understanding of the role of novel bacteria in feed digestion will provide new avenues for developing supplements or feed additives that support animal health and increase production efficiency of the livestock industry.