Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2013
Publication Date: 9/20/2013
Publication URL: http://handle.nal.usda.gov/10113/58030
Citation: Lin, M., Guo, W.S., Meng, Q.X., Stevenson, D.M., Weimer, P.J., Schaefer, D.M. 2013. Changes in ruminal bacterial community composition in steers in response to dietary nitrate. Applied Microbiology and Biotechnology. 97:8719-8727. Interpretive Summary: Virtually any forage crop fed to dairy cattle can accumulate nitrate if the soil concentration is high and the rate of plant growth is rapid. Nitrate itelf is not a toxic substance, but it can be reduced to nitrite by rumen bacteria and nitrite is poisonous to cattle. When nitrate is consumed in small amounts, rumen bacteria have time to reduce nitrite to ammonia, which is not toxic. Even when nitrate is ingested in large amounts, the rumen bacterial population can adapt over time to detoxify nitrate to ammonia. The ability of rumen bacterial populations to reduce nitrate to ammonia (without an intermediate buildup of toxic nitrite) is a crucial aspect in ruminant health and nutrition. However, we do not know which bacteria are responsible for ruminal nitrate metabolism. We used a “molecular fingerprinting” technique to characterize changes in the rumen bacterial community of 4 Hereford steers when the primary source of dietary nitrogen was switched over a 15-day period from urea to nitrate, or from nitrate to urea. The minimum number of bacterial species identified across all the steers was 210. Substantial shifts in the overall composition of the rumen bacterial community were observed for all 4 steers, but a minimum of only 7 species were significantly affected in relative population size due to changes in the type of nitrogen supplement. The results indicated that the rumen bacterial community stabilizes within 15 days of a dietary shift, and that only a few species are likely to account for most of the changes in the rumen bacterial population. These results lay the groundwork for identifying individual bacterial species that may be responsible for ruminal nitrate metabolism.
Technical Abstract: The aim of this study was to determine the effect of dietary nitrate supplementation on rumen bacterial community composition. Beef steers were fed either a nitrate-nitrogen diet or urea-nitrogen diet. An automated method of ribosomal intergenic spacer analysis was applied to solid and liquid fractions of ruminal contents to allow comparison of bacterial communities. Supplemental nitrogen (N) source affected relative population size of 4 amplicon lengths (ALs) in the liquid fraction and 3 ALs in the solid fraction. Five ALs were more prevalent after adaptation to nitrate. Correspondence analysis indicated that feeding the nitrate-N diet versus urea-N diet changed the bacterial community composition in the liquid, but not in the solid fraction. This led to an investigation of the relative sizes of potential nitrate-reducing populations. Mannheimia succiniciproducens, Veillonella parvula, and Campylobacter fetus were obtained from nitrate enrichment culture and quantified by real-time polymerase chain reaction (PCR), with 16S rRNA sequence. Nitrate supplementation increased the percentage of C. fetus in the liquid and solid phases; in the solid phase, the percentage of M. succiniciproducens increased. No change in species prevalence was observed for V. parvula. However, even after adaptation to dietary nitrate, the relative population sizes for all three putative nitrate-reducing species were very low (<0.06% of 16S rRNA gene copy number). The liquid-associated bacterial community composition changed due to nitrate supplementation. At least part of this change reflects an increase in the species prevalence of C. fetus, a species which is not typically regarded as a ruminal inhabitant.