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 biodiversity of 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.

An in vitro study was conducted to determine the effect of nitrate-nitrogen used as a sole dietary nitrogen source on ruminal fermentation characteristics and microbial nitrogen (MN) synthesis. Three treatment diets were formulated with different nitrogen sources to contain 13% CP and termed i) nitrate-N diet (NND), ii) urea-N diet (UND), used as negative control, and iii) tryptone-N diet (TND), used as positive control. The results of 24-h incubations showed that nitrate-N disappeared to background concentrations and was not detectable in microbial cells. The NND treatment decreased net CH 4 production, but also decreased net CO 2 production and increased net H 2 production. Total VFA concentration was lower (p<0.05) for NND than TND. Suppression of CO 2 production and total VFA concentration may be linked to increased concentration of H 2 . The MN synthesis was greater (p<0.001) for NND than UND or TND (5.74 vs. 3.31 or 3.34 mg/40 ml, respectively). Nitrate addition diminished methane production as expected, but also increased MN synthesis.

The objectives were to compare the ability of various rumen microbial fractions to reduce nitrate and to assess the effect of nitrate on in vitro fermentation characteristics. Physical and chemical methods were used to differentiate the rumen microbial population into the following fractions: whole rumen fluid (WRF), protozoa (Pr), bacteria (Ba), and fungi (Fu). The three nitrogen substrate treatments were as follows: no supplemental nitrogen source, nitrate or urea, with the latter two being isonitrogenous additions. The results showed that during 24 h incubation, WRF, Pr and Ba fractions had an ability to reduce nitrate, and the rate of nitrate disappearance for the Pr fraction was similar to the WRF fraction, while the Ba fraction needed an adaptation period of 12 h before rapid nitrate disappearance. The WRF fraction had the greatest methane (CH 4 ) production and the Pr fraction had the greatest prevailing H 2 concentration (p<0.05). Compared to the urea treatment, nitrate diminished net gas and CH 4 production during incubation (p<0.05), and ammonia-N (NH 3 -N) concentration (p<0.01). Nitrate also increased acetate, decreased propionate and decreased butyrate molar proportions (p<0.05). The Pr fraction had the highest acetate to propionate ratio (p<0.05). The Pr fraction as well as the Ba fraction appears to have an important role in nitrate reduction. Nitrate did not consistently alter total VFA concentration, but it did shift the VFA profile to higher acetate, lower propionate and lower butyrate molar proportions, consistent with less CH 4 production by all microbial fractions.

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