Effects of Nitrate Addition on Rumen Fermentation, Bacterial Biodiversity and Abundance

  • Zhao, Liping (State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University) ;
  • Meng, Qingxiang (State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University) ;
  • Ren, Liping (State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University) ;
  • Liu, Wei (Beijing Computing Center) ;
  • Zhang, Xinzhuang (State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University) ;
  • Huo, Yunlong (State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University) ;
  • Zhou, Zhenming (State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University)
  • Received : 2015.01.31
  • Accepted : 2015.03.25
  • Published : 2015.10.01


This study examined changes of rumen fermentation, ruminal bacteria biodiversity and abundance caused by nitrate addition with Ion Torrent sequencing and real-time polymerase chain reaction. Three rumen-fistulated steers were fed diets supplemented with 0%, 1%, and 2% nitrate (dry matter %) in succession. Nitrate supplementation linearly increased total volatile fatty acids and acetate concentration obviously (p = 0.02; p = 0.02; p<0.01), butyrate and isovalerate concentration numerically (p = 0.07). The alpha (p>0.05) and beta biodiversityof ruminal bacteria were not affected by nitrate. Nitrate increased typical efficient cellulolytic bacteria species (Ruminococcus flavefaciens, Ruminococcus ablus, and Fibrobacter succinogenes) (p<0.01; p = 0.06; p = 0.02). Ruminobactr, Sphaerochaeta, CF231, and BF311 genus were increased by 1% nitrate. Campylobacter fetus, Selenomonas ruminantium, and Mannheimia succiniciproducens were core nitrate reducing bacteria in steers and their abundance increased linearly along with nitrate addition level (p<0.01; p = 0.02; p = 0.04). Potential nitrate reducers in the rumen, Campylobacter genus and Cyanobacteria phyla were significantly increased by nitrate (p<0.01; p = 0.01).To the best of our knowledge, this was the first detailed view of changes in ruminal microbiota by nitrate. This finding would provide useful information on nitrate utilization and nitrate reducer exploration in the rumen.


Supported by : National Natural Science Fund of China


  1. Andries, J. I., F. X. Buysse, D. L. Debrabander, and B. G.Cottyn. 1987. Isoacids in ruminant nutrition: Their role in ruminal and intermediary metabolism and possible influences on performances -A review. Anim. Feed Sci. Technol. 18:169-180.
  2. Ballard, F. J. 1972. Supply and utilization of acetate in mammals. Am. J. Clin. Nutr. 25:773-779.
  3. Broderick, G. A. and J. H. Kang. 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63:64-75.
  4. Bru, D., A. Sarr,and L. Philippot. 2007. Relative abundances of proteobacterial membrane-bound and periplasmic nitrate reductases in selected environments. Appl. Environ. Microbiol. 73:5971-5974.
  5. Caporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. D. Bushman, E. K. Costello, N. Fierer, A. G. Pena, J. K. Goodrich, and J. I. Gordon et al. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7:335-336.
  6. Dai, J. F., Q. X. Meng, and Z. M. Zhou. 2009. Effect of nitrate addition level on in vitro ruminal fermentation characteristics and microbial efficiency. Scientia AgricSinica. 43:3418-3424.
  7. Denman, S. E. and C. S. McSweeney. 2006. Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol. Ecol. 58:572-582.
  8. Erwin, E. S., G. J. Marco, and E. M. Emery. 1961. Volatile fatty acid analyses of blood and rumen fluid by gas chromatography. J Dairy Sci. 44:1768-1771.
  9. Evans, N. J., J. M. Brown, R. D. Murray, B. Getty, R. J. Birtles, C. A. Hart, and S. D. Carter. 2011. Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison with bovine digital dermatitis treponemes. Appl. Environ. Microbiol. 77:138-177.
  10. Flores, E., J. E. Frias, L. M. Rubio, and A. Herrero. 2005. Photosynthetic nitrate assimilation in cyanobacteria. Photosyn. Res. 83:117-133.
  11. Group Jumpstart Consortium Human Microbiome Project Data Generation Working Group. 2012. Evaluation of 16S rDNA-based community profiling for human microbiome research. PLoS ONE. 7(6):e39315.
  12. Guo, W. S., D. M. Schaefer, X. X. Guo, L. P. Ren, and Q. X.Meng. 2009. Use of nitrate-nitrogen as a sole dietary nitrogen source to inhibit ruminal methanogenesis and to improve microbial nitrogen synthesis in vitro. AsianAustralas. J. Anim. Sci. 22: 542-549.
  13. Hulshof, R., A. Berndt, W. Gerrits, J. Dijkstra, S. M. van Zijderveld, J. R. Newbold, and H. B. Perdok. 2012. Dietary nitrate supplementation reduces methane emission in beef cattle fed sugarcane-based diets. J. Anim. Sci. 90:2317-2323.
  14. Isaacson, R. and H. B. Kim. 2012. The intestinal microbiome of the pig. Anim. Health Res. Rev. 13:100-109.
  15. Lee, H. J., J. Y. Jung, Y. K. Oh, S. Lee, E. L. Madsen, and C. O. Jeon. 2012. Comparative survey of rumen microbial communities and metabolites across one caprine and three bovine groups, using bar-coded pyrosequencing and 1H nuclear magnetic resonance spectroscopy. Appl. Environ. Microbiol. 78: 5983-5993.
  16. Lewis, D. 1951. The metabolism of nitrate and nitrite in the sheep. 1. The reduction of nitrate in the rumen of the sheep. Biochem J. 48:175-180.
  17. Lin, M., D. M. Schaefer, W. S. Guo, L. P. Ren,and Q. X. Meng. 2011. Comparisons of in vitro nitrate reduction, methanogenesis, and fermentation acid profile among rumen bacterial, protozoal and fungal fractions. Asian Australas. J. Anim. Sci. 24:471-478.
  18. Lin, M., W. Guo, Q. Meng, D. M. Stevenson, P. J. Weimer, and D. M. Schaefer. 2013. Changes in rumen bacterial community composition in steers in response to dietary nitrate. Appl. Environ.Biotech. 97:8719-8727.
  19. Marais, J. P., J. J. Therion, R. I. Mackie, A. Kistner, and C. Dennison. 1988. Effect of nitrate and its reduction products on the growth and activity of the rumen microbialpopulation. Br. J. Nutr. 59: 301-313.
  20. Miller, W. G., G. Wang, T. T. Binnewies, and C. T. Parker. 2008. The complete genome sequence and analysis of the human pathogen Campylobacter lari. Foodborne Pathog. Dis. 5:371-386.
  21. Palmer, K. andM. A. Horn. 2012. Actinobacterial nitrate reducers and proteobacterial denitrifiers are abundant in N2O-metabolizing palsa peat. Appl. Environ. Microbiol. 78:5584-5596.
  22. Parkhill, J., B. W. Wren, K. Mungall,J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, and S. Holroyd et al. 2000. The genome sequence of the food-borne pathogen Campylobacter jejunireveals hypervariable sequences. Nature 403(6770):665-668.
  23. Patel, R. K. and M. Jain. 2012. NGS QC Toolkit: A toolkit for quality control of next generation sequencing data. PLoS ONE, 7(2):e30619.
  24. Pitta, D. W., W. E. Pinchak, S. E. Dowd, J. Osterstock, V. Gontcharova, E. Youn, K. Dorton, I. Yoon, B. R. Min, J. D. Fulford, T. A. Wickersham, and D. P. Malinowski. 2010. Rumen bacterial diversity dynamics associated with changing from bermudagrass hay to grazed winter wheat diets. Microb.Ecol. 59:511-522.
  25. Price, M. N., P. S. Dehal, and A. P. Arkin. 2009. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26:1641-1650.
  26. Prasanna, R., V. Kumar, S. Kumar, A. Kumar Yadav, U. Tripathi, A. Kumar Singh, M. C. Jain, P. Gupta, P. K. Singh, and N. Sethunathan. 2002. Methane production in rice soil is inhibited by cyanobacteria. Microbiol.Res. 157:1-6.
  27. Sar, C., B. Mwenya, B. Santoso, K. Takaura, R. Morikawa, N. Isogai, Y. Asakura, Y. Toride, and J. Takahashi. 2005. Effect of Escherichia coli wild type or its derivative with high nitrite reductase activity on in vitro ruminal methanogenesis and nitrate/nitrite reduction. J. Anim. Sci. 83:644-652.
  28. Slyter, L. L. and P. A. Putnam. 1967. In vivo vs in vitro continuous culture of ruminal microbial populations.J. Anim. Sci. 26: 1421-1427.
  29. Stewart, C. S., H. J. Flint, and M. P. Bryant. 1997. The rumen bacteria. In:The Rumen Microbial Ecosystem, 2nd ed.,By (P. N. Hobson, and C. S. Stewart). Blackie Academic and Professional, London, UK. 10-72.
  30. Stevenson, D. M. and P. J. Weimer. 2007. Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl. Environ.Biotech.75:165-174.
  31. Thoetkiattikul, H., W. Mhuantong, T. Laothanachareon, S. Tangphatsornruang, V. Pattarajinda, L. Eurwilaichitr, and V. Champreda. 2013. Comparative analysis of microbial profiles in cow rumen fed with different dietary fiber by tagged 16S rRNA gene pyrosequencing. Curr. Microbiol. 67:130-137.
  32. Van Zijderveld, S. M., W. J. J. Gerrits, J. A. Apajalahti, J. R. Newbold, J. Dijkstra, R. A. Leng, and H. B. Perdok. 2010. Nitrate and sulfate: Effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93:5856-5866.
  33. Weakley, D. G. and F. N. Owens. 1983. Influence of ammonia concentration on microbial protein synthesis in the rumen. Oklahoma Agr. Exp. Sta. MP-114, 39.
  34. Zened, A., S. Combes, L. Cauquil, J. Mariette, C. Klopp, O. Bouchez, M. A. Troegeler, and F. Enjalbert. 2013. Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets. FEMS Microbiol. Ecol. 83:504-514.
  35. Zhou, Z., Q. Meng, and Z. Yu. 2011. Effects of methanogenic inhibitors on methane production and abundances of methanogens and cellulolytic bacteria in in vitro ruminal cultures. Appl. Environ. Microbiol. 77:2634-2639.
  36. Zhou, Z., Z. Yu, andQ. Meng. 2012. Effects of nitrate on methane production, fermentation, and microbial populations in in vitro ruminal cultures. Bioresour. Technol.103:173-179.

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