Rumen microbial responses to supplemental nitrate. I. Yeast growth and protozoal chemotaxis in vitro as affected by nitrate and nitrite concentrations

Abstract

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH 4 emissions. In the NO 3 − reduction process, NO 2 − can accumulate, which could directly inhibit methanogens and some bacteria. However, little information is available on eukaryotic microbes in the rumen. Protozoa were hypothesized to enhance nitrate reductase but also have more circling swimming behavior, and the yeast Saccharomyces cerevisiae was hypothesized to lessen NO 2 − accumulation. In the first experiment, a culture of S. cerevisiae strain 1026 was evaluated under 3 growth phases: aerobic, anoxic, or transition to anoxic culture. Each phase was evaluated with a control or 1 of 3 isonitrogenous doses, including NO 3 − , NO 2 − , or NH 4 + replacing peptone in the medium. Gas head phase, NO 3 − , or NH 4 + did not influence culture growth, but increasing NO 2 − concentration increasingly inhibited yeast growth. In experiment 2, rumen fluid was harvested and incubated for 3 h in 2 concentrations of NO 3 − , NO 2 − , or sodium nitroprusside before assessing chemotaxis of protozoa toward glucose or peptides. Increasing NO 2 − concentration decreased chemotaxis by isotrichids toward glucose or peptides and decreased chemotaxis by entodiniomorphids but only toward peptides. Live yeast culture was inhibited dose-responsively by NO 2 − and does not seem to be a viable mechanism to prevent NO 2 − accumulation in the rumen, whereas a role for protozoal nitrate reductase and NO 2 − influencing signal transduction requires further research.

Rumen microbial responses to supplemental nitrate. II. Potential interactions with live yeast culture on the prokaryotic community and methanogenesis in continuous culture

“…Preliminary searches of the genome of Entodinium caudatum returned annotated nitrate reductase (personal communication, Tansol Park, The Ohio State University, Columbus), which should be expected based on previous studies comparing protozoal enriched samples versus bacterial samples (Yang et al, 2016). Increasing concentration of NO 2 − profoundly inhibited motility of entodiniomorphids (of which E. caudatum is a predominant member) and entodiniomorphid chemotaxis in our companion study (Roman-Garcia et al, 2019), so NO 2 − reduction by protozoa awaits further inquiry. There was no difference in generation time between NO 3 − , LYC, or their interaction.…”

“…In addition to consuming O 2 , LYC could also stabilize pH by stimulating lactilytic strains of Selenomonas ruminantium (Pinloche et al, 2013), which also are important in NO 3 − and NO 2 − reduction (Yang et al, 2016). In our companion study (Roman-Garcia et al, 2019), we questioned if LYC would reduce NO 2 − in microaerophilic conditions and thereby help prevent its accumulation after NO 3 − is fed, but the hypotheses were not supported, perhaps because of a lack of microbial adaptation to NO 3 − . Our objectives were to evaluate the effect of dietary NO 3 − and its potential interaction with LYC on fermentation characteristics, microbial protein synthesis, and prokaryotic community composition in continuous cultures that are adapted to NO 3 − for 7 d. We hypothesized that NO 3 − would suppress methanogenesis and shift bacterial populations toward those reducing NO 3 − and NO 2 − , and LYC would suppress methanogenesis additively with feeding NO 3 − while decreasing NO 2 − accumulation to result in increased NDF digestibility and efficiency of bacterial growth.…”

Extending Burk Dehority’s Perspectives on the Role of Ciliate Protozoa in the Rumen

“…Isotrichids are highly chemotactic toward glucose and xylose (Diaz et al, 2014) and almost certainly other sugars. Peptides can be both chemoattractive and chemorepellent to isotrichids (Diaz et al, 2014;Roman-Garcia et al, 2019). Hence, uncontrolled glycogenesis in vitro (Jouany and Ushida, 1999;Hall, 2011) probably is lessened in vivo because of much less abrupt increase in sugar availability.…”

Dietary nitrate and presence of protozoa increase nitrate and nitrite reduction in the rumen of sheep

“…The reduction of disappearance rate after adding to the SOL fraction of FAU and DEF rumen digesta could be due to a toxic effect of on rumen micro‐organisms, although final concentration in the buffer added to the rumen digesta was 0.5 mM which is below the 2 mM , value reported to inhibit microbial growth (Iwamoto, Asanuma, & Hino, 2002). More recently, Roman‐Garcia et al (2019) found that in vitro motility and chemotaxis within protozoa were affected differently by but not for isotrichids versus entodiniomorphids. However, the addition of increased NH 3 accumulation rate in both fractions of rumen digesta, suggesting no adverse effects of on rumen micro‐organisms in this current experiment.…”