Accumulation of Reserve Carbohydrate by Rumen Protozoa and Bacteria in Competition for Glucose

Abstract

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n ‫؍‬ 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n ‫؍‬ 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P ‫؍‬ 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.A diverse assemblage of protozoa, bacteria, methanogens, and fungi inhabit the rumen of ruminant livestock (1). Although they may account for as little as 5% of the microbial biomass (2), protozoa have an important role in stabilizing fermentation (3). Animals with protozoa absent have higher concentrations of short-chain fatty acids (SCFAs) and lower mean pHs (3-5). Furthermore, SCFAs and pH may fluctuate more when protozoa are absent (4, 6).Protozoa have been proposed to stabilize rumen fermentation in part by consuming sugar and starch, preventing the rapid fermentation of these substrates by bacteria (3). According to this proposed mechanism, protozoa synthesize reserve carbohydrate after consuming sugar, and protozoa ferment this reserve carbohydrate and intracellular starch more slowly than do bacteria. This prevents the buildup of SCFAs, depression of pH, and onset of lactic acid acidosis that are detrimental to animal performance (7,8). Sugar consumption has been attributed to protozoa of the family Isotrichidae, and starch consumption has been attributed to protozoa of the family Orphryoscolecidae (3, 9).Besides its importance for animal performance, this sequestration of carbohydrate in reserve carbohydrate and intracellular starch would give protozoa a competitive advantage over bacteria. It would deprive bacteria of a substrate for growth, and it might explain why protozoa can persist alongside bacteria in the rumen, even though ...

In vitro assessment of the factors that determine the activity of the rumen microbiota for further applications as inoculum

“…This interaction may rely on the different community structure and the lower bacterial and protozoal diversity generally reported in the rumen of animals fed high concentrate diets . Further microbial adaptation processes based on the acceleration of the metabolic rate, energy spilling reactions and accumulation of starch particles have been described in the presence of high levels of available energy . These adaptation strategies could help rumen microbes to better prepare their metabolism for further damaging processes, with the protective effect on the microbes being proportional to the severity of the preservation method.…”

Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques

“…Recently, alternatives to the old methods have been introduced for isolation of microbial fractions from digesta. For instance, a procedure has been developed for isolation of culturable mixed ruminal protozoa (Sylvester et al, 2004(Sylvester et al, , 2005Denton et al, 2015) that does not have the major disadvantages of the differential centrifugation method [i.e., contamination with bacteria and feed particles, or use of formalin by some methods for isolation and storage of samples, which can adversely affect AA hydrolysis (Fessenden et al, 2017)].…”

Ruminal bacteria and protozoa composition, digestibility, and amino acid profile determined by multiple hydrolysis times

“…An example can be found in the isolation of protozoa, where differential centrifugation has historically been used to isolate microbial cells with significant contamination from bacteria and feed particles. A procedure developed for isolation of cultivatable mixed ruminal protozoa for quantitative PCR and competi-tion studies (Sylvester et al, 2004(Sylvester et al, , 2005Denton et al, 2015) can be used to isolate protozoa for more accurate nutrient analysis. In addition, several of the methods employ the use of formalin in the isolation and storage of microbial and specifically protozoal samples (Martin et al, 1994;Rossi et al, 2013); this might be problematic, as formaldehyde is known to react with AA (Barry, 1976).…”