Electron transport phosphorylation in rumen butyrivibrios: unprecedented ATP yield for glucose fermentation to butyrate.

From a genomic analysis of rumen butyrivibrios (Butyrivibrio and Pseudobutyrivibrio sp.), we have re-evaluated the contribution of electron transport phosphorylation (ETP) to ATP formation in this group. This group is unique in that most (76%) genomes were predicted to possess genes for both Ech and Rnf transmembrane ion pumps. These pumps act in concert with the NifJ and Bcd-Etf to form a electrochemical potential (ΔμH(+) and ΔμNa(+)), which drives ATP synthesis by ETP. Of the 62 total butyrivibrio genomes currently available from the Hungate 1000 project, all 62 were predicted to possess NifJ, which reduces oxidized ferredoxin (Fdox) during pyruvate conversion to acetyl-CoA. All 62 possessed all subunits of Bcd-Etf, which reduces Fdox and oxidizes reduced NAD during crotonyl-CoA reduction. Additionally, 61 genomes possessed all subunits of the Rnf, which generates ΔμH(+) or ΔμNa(+) from oxidation of reduced Fd (Fdred) and reduction of oxidized NAD. Further, 47 genomes possessed all six subunits of the Ech, which generates ΔμH(+) from oxidation of Fdred. For glucose fermentation to butyrate and H2, the electrochemical potential established should drive synthesis of ∼1.5 ATP by the F0F1-ATP synthase (possessed by all 62 genomes). The total yield is ∼4.5 ATP/glucose after accounting for three ATP formed by classic substrate-level phosphorylation, and it is one the highest yields for any glucose fermentation. The yield was the same when unsaturated fatty acid bonds, not H(+), served as the electron acceptor (as during biohydrogenation). Possession of both Ech and Rnf had been previously documented in only a few sulfate-reducers, was rare in other rumen prokaryotic genomes in our analysis, and may confer an energetic advantage to rumen butyrivibrios. This unique energy conservation system might enhance the butyrivibrios' ability to overcome growth inhibition by unsaturated fatty acids, as postulated herein

Relationship between urinary energy and urinary nitrogen or carbon excretion in lactating Jersey cows

Measurement of urinary energy (UE) excretion is essential to determine metabolizable energy (ME) supply. Our objectives were to evaluate the accuracy of using urinary N (UN) or C (UC) to estimate UE and ultimately improve the accuracy of estimating ME. Individual animal data (n = 433) were used from 11 studies with Jersey cows at the University of Nebraska–Lincoln, where samples were analyzed after drying (n = 299) or on an as-is basis (n = 134). Dried samples resulted in greater estimated error variance compared with as-is samples, and thus only as-is samples were used for final models. The as-is data set included a range (min to max) in dry matter intake (11.6–24.6 kg/d), N intake (282–642 g/d), UE excretion (1,390–3,160 kcal/d), UN excretion (85–220 g/d or 20.6–59.5% of N intake), and UC excretion (130–273 g/d). As indicated by a bias in residuals between observed and predicted ME as dietary crude protein (CP; range of 14.9–19.1%) increased, the National Research Council dairy model did not accurately predict ME of diets, as dietary CP varied. The relationship between UE (kcal/d) and UN (g/d) excretion was linear and had an intercept of 880 ± 140 kcal. Because an intercept of 880 is biologically unlikely, the intercept was forced through 0, resulting in linear and quadratic relationships. The regressions of UE (kcal/d) on UN (g/d) excretion were UE = 14.6 ± 0.32 × UN, and UE = 20.9 ± 1.0 × UN − 0.0357 ± 0.0056 × UN2. In the quadratic regression, UE increased, but at a diminishing rate as UN excretion increased. As UC increased, UE linearly and quadratically increased. However, error variance was greater for regression with UC compared with UN as explanatory variables (8.42 vs. 7.42% of mean UE). The use of the quadratic regression between UN and UE excretion to predict ME resulted in a slope bias in ME predictions as dietary CP increased. The linear regression between UE and UN excretion removed slope bias between predicted ME and CP, and thus may be more appropriate for predicting UE across a wider range of dietary CP. Using equations to predict UE from UN should improve our ability to predict diet ME in Jersey cows compared with calculating ME directly from digestible energy

Maximizing efficiency of rumen microbial protein production.

Rumen microbes produce cellular protein inefficiently partly because they do not direct all ATP toward growth. They direct some ATP toward maintenance functions, as long-recognized, but they also direct ATP toward reserve carbohydrate synthesis and energy spilling (futile cycles that dissipate heat). Rumen microbes expend ATP by vacillating between (1) accumulation of reserve carbohydrate after feeding (during carbohydrate excess) and (2) mobilization of that carbohydrate thereafter (during carbohydrate limitation). Protozoa account for most accumulation of reserve carbohydrate, and in competition experiments, protozoa accumulated nearly 35-fold more reserve carbohydrate than bacteria. Some pure cultures of bacteria spill energy, but only recently have mixed rumen communities been recognized as capable of the same. When these communities were dosed glucose in vitro, energy spilling could account for nearly 40% of heat production. We suspect that cycling of glycogen (a major reserve carbohydrate) is a major mechanism of spilling; such cycling has already been observed in single-species cultures of protozoa and bacteria. Interconversions of short-chain fatty acids (SCFA) may also expend ATP and depress efficiency of microbial protein production. These interconversions may involve extensive cycling of intermediates, such as cycling of acetate during butyrate production in certain butyrivibrios. We speculate this cycling may expend ATP directly or indirectly. By further quantifying the impact of reserve carbohydrate accumulation, energy spilling, and SCFA interconversions on growth efficiency, we can improve prediction of microbial protein production and guide efforts to improve efficiency of microbial protein production in the rumen

Physically adjusted neutral detergent fiber system for lactating dairy cow rations. I: Deriving equations that identify factors that influence effectiveness of fiber

Physically effective neutral detergent fiber (peNDF) is the fraction of neutral detergent fiber (NDF) that stimulates chewing activity and contributes to the floating mat of large particles in the rumen. Multiplying dietary NDF by particle size has been used as an estimate of peNDF. In re-evaluating the concept of peNDF, we compared the use of peNDF as dietary NDF × particle size with the use of individual NDF and particle size descriptors (physically adjusted NDF; paNDF) when used with other physical and chemical diet descriptors to predict dry matter (DM) intake (DMI), rumination time, and ruminal pH in lactating dairy cows. The purpose is to ultimately use these equations to estimate diet adequacy to maintain ruminal conditions. Each response variable had 8 models in a 2 (peNDF, paNDF) × 2 (diet, diet and ruminal factors) × 2 (DM, as fed basis) factorial arrangement. Particle size descriptors were those determined with the Penn State Particle Separator. Treatment means (n = 241) from 60 publications were used in backward elimination multiple regression to derive models of response variables. When available, peNDF terms entered equations. Models containing peNDF terms had similar or lower unadjusted concordance correlation coefficients (an indicator of similar or lower accuracy and precision) than did models without peNDF terms. The peNDF models for rumen pH did not differ substantially from paNDF models. This suggests that peNDF can account for some variation in ruminal pH; however, overt advantages of peNDF were not apparent. Significant (P \u3c 0.05) variables that entered the models included estimated mean particle size; as fed or DM proportions retained on 19- and 8-mm sieves of the Penn State Particle Separator; DMI; dietary concentrations of forage; forage NDF; CP; starch; NDF; rumen-degraded starch and rumen-degraded NDF; and the interaction terms of starch × mean particle size, acid detergent fiber/NDF, and rumination time/DMI. Many dietary factors beyond particle size and NDF were identified as influencing the response variables. In conclusion, these results appear to justify the development of a modeling approach to integrate individual physical and chemical factors to predict effects on factors affecting rumen conditions

Inhibition of the Rumen Ciliate Entodinium caudatum by Antibiotics

Axenic cultures of free-living aerobic ciliates, such as Tetrahymena thermophila and Paramecium aurelia, have been established and routinely used in laboratory research, greatly facilitating, or enabling characterization of their metabolism, physiology, and ecology. Ruminal protozoa are anaerobic ciliates, and they play important roles in feed digestion and fermentation. Although, repeatedly attempted, no laboratory-maintainable axenic culture of ruminal ciliates has been established. When axenic ciliate cultures are developed, antibiotics are required to eliminate the accompanying bacteria. Ruminal ciliates gradually lose viability upon antibiotic treatments, and the resultant axenic cultures can only last for short periods of time. The objective of this study was to evaluate eight antibiotics that have been evaluated in developing axenic cultures of ruminal ciliates, for their toxicity to Entodinium caudatum, which is the most predominant ruminal ciliate species. Scanning and transmission electron microscopy (TEM) showed that the antibiotics damaged both the cell surface and nuclei of E. caudatum and increased accumulation of intracellular glycogen. Combinations of the three least toxic antibiotics failed to eliminate the bacteria that are present in the E. caudatum culture. The combination of ampicillin, carbenicillin, streptomycin, and oxytetracycline was able to eliminate all the bacteria, but the resultant axenic E. caudatum culture gradually lost viability. Adding the bacterial fraction (live) separated from an untreated E. caudatum culture reversed the viability decline and recovered the growth of the treated E. caudatum culture, whereas feeding nine strains of live bacteria isolated from E. caudatum cells, either individually or in combination, could not. Nutritional and metabolic dependence on its associated bacteria, accompanied with direct and indirect inhibition by antibiotics, makes it difficult to establish an axenic culture of E. caudatum. Monoxenic or polyxenic cultures of E. caudatum could be developed if the essential symbiotic partner(s) can be identified

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

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

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

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

Annual research review, utilization of recycled fibers.

"April 28, 1993."Utilization of recycled fibers: project 3681 / John Waterhouse ... [et al.] ; The effect of recycling of fibers: project 3681 / John Waterhouse ; The identification of recycled fiber of infrared spectroscopy: project 3681 / Sujit Banerjee ; Removal of contaminations by flotation: project 3681 / Bob Stratton