Distinct microbial hydrogen and reductant disposal pathways explain interbreed variations in ruminant methane yield.

Ruminants are essential for global food security, but these are major sources of the greenhouse gas methane. Methane yield is controlled by the cycling of molecular hydrogen (H2), which is produced during carbohydrate fermentation and is consumed by methanogenic, acetogenic, and respiratory microorganisms. However, we lack a holistic understanding of the mediators and pathways of H2 metabolism and how this varies between ruminants with different methane-emitting phenotypes. Here, we used metagenomic, metatranscriptomic, metabolomics, and biochemical approaches to compare H2 cycling and reductant disposal pathways between low-methane-emitting Holstein and high-methane-emitting Jersey dairy cattle. The Holstein rumen microbiota had a greater capacity for reductant disposal via electron transfer for amino acid synthesis and propionate production, catalyzed by enzymes such as glutamate synthase and lactate dehydrogenase, and expressed uptake [NiFe]-hydrogenases to use H2 to support sulfate and nitrate respiration, leading to enhanced coupling of H2 cycling with less expelled methane. The Jersey rumen microbiome had a greater proportion of reductant disposal via H2 production catalyzed by fermentative hydrogenases encoded by Clostridia, with H2 mainly taken up through methanogenesis via methanogenic [NiFe]-hydrogenases and acetogenesis via [FeFe]-hydrogenases, resulting in enhanced methane and acetate production. Such enhancement of electron incorporation for metabolite synthesis with reduced methanogenesis was further supported by two in vitro measurements of microbiome activities, metabolites, and public global microbiome data of low- and high-methane-emitting beef cattle and sheep. Overall, this study highlights the importance of promoting alternative H2 consumption and reductant disposal pathways for synthesizing host-beneficial metabolites and reducing methane production in ruminants.

Effects of type of substrate and dilution rate on fermentation in serial rumen mixed cultures.

Forages and concentrates have consistently distinct patterns of fermentation in the rumen, with forages producing more methane (CH4) per unit of digested organic matter (OM) and higher acetate to propionate ratio than concentrates. A mechanism based on the Monod function of microbial growth has been proposed to explain the distinct fermentation pattern of forages and concentrates, where greater dilution rates and lower pH associated with concentrate feeding increase dihydrogen (H2) concentration through increasing methanogens growth rate and decreasing methanogens theoretically maximal growth rate, respectively. Increased H2 concentration would in turn inhibit H2 production, decreasing methanogenesis, inhibit H2-producing pathways such as acetate production via pyruvate oxidative decarboxylation, and stimulate H2-incorporating pathways such as propionate production. We examined the hypothesis that equalizing dilution rates in serial rumen cultures would result in a similar fermentation profile of a high forage and a high concentrate substrate. Under a 2 × 3 factorial arrangement, a high forage and a high concentrate substrate were incubated at dilution rates of 0.14, 0.28, or 0.56 h-1 in eight transfers of serial rumen cultures. Each treatment was replicated thrice, and the experiment repeated in two different months. The high concentrate substrate accumulated considerably more H2 and formate and produced less CH4 than the high forage substrate. Methanogens were nearly washed-out with high concentrate and increased their initial numbers with high forage. The effect of dilution rate was minor in comparison to the effect of the type of substrate. Accumulation of H2 and formate with high concentrate inhibited acetate and probably H2 and formate production, and stimulated butyrate, rather than propionate, as an electron sink alternative to CH4. All three dilution rates are considered high and selected for rapidly growing bacteria. The archaeal community composition varied widely and inconsistently. Lactate accumulated with both substrates, likely favored by microbial growth kinetics rather than by H2 accumulation thermodynamically stimulating electron disposal from NADH into pyruvate reduction. In this study, the type of substrate had a major effect on rumen fermentation largely independent of dilution rate and pH.

Invited review: Current enteric methane mitigation options.

Ruminant livestock are an important source of anthropogenic methane (CH4). Decreasing the emissions of enteric CH4 from ruminant production is strategic to limit the global temperature increase to 1.5°C by 2050. Research in the area of enteric CH4 mitigation has grown exponentially in the last 2 decades, with various strategies for enteric CH4 abatement being investigated: production intensification, dietary manipulation (including supplementation and processing of concentrates and lipids, and management of forage and pastures), rumen manipulation (supplementation of ionophores, 3-nitrooxypropanol, macroalgae, alternative electron acceptors, and phytochemicals), and selection of low-CH4-producing animals. Other enteric CH4 mitigation strategies are at earlier stages of research but rapidly developing. Herein, we discuss and analyze the current status of available enteric CH4 mitigation strategies with an emphasis on opportunities and barriers to their implementation in confined and partial grazing production systems, and in extensive and fully grazing production systems. For each enteric CH4 mitigation strategy, we discuss its effectiveness to decrease total CH4 emissions and emissions on a per animal product basis, safety issues, impacts on the emissions of other greenhouse gases, as well as other economic, regulatory, and societal aspects that are key to implementation. Most research has been conducted with confined animals, and considerably more research is needed to develop, adapt, and evaluate antimethanogenic strategies for grazing systems. In general, few options are currently available for extensive production systems without feed supplementation. Continuous research and development are needed to develop enteric CH4 mitigation strategies that are locally applicable. Information is needed to calculate carbon footprints of interventions on a regional basis to evaluate the impact of mitigation strategies on net greenhouse gas emissions. Economically affordable enteric CH4 mitigation solutions are urgently needed. Successful implementation of safe and effective antimethanogenic strategies will also require delivery mechanisms and adequate technical support for producers, as well as consumer involvement and acceptance. The most appropriate metrics should be used in quantifying the overall climate outcomes associated with mitigation of enteric CH4 emissions. A holistic approach is required, and buy-in is needed at all levels of the supply chain.

Dietary selection of metabolically distinct microorganisms drives hydrogen metabolism in ruminants.

Ruminants are important for global food security but emit the greenhouse gas methane. Rumen microorganisms break down complex carbohydrates to produce volatile fatty acids and molecular hydrogen. This hydrogen is mainly converted into methane by archaea, but can also be used by hydrogenotrophic acetogenic and respiratory bacteria to produce useful metabolites. A better mechanistic understanding is needed on how dietary carbohydrates influence hydrogen metabolism and methanogenesis. We profiled the composition, metabolic pathways, and activities of rumen microbiota in 24 beef cattle adapted to either fiber-rich or starch-rich diets. The fiber-rich diet selected for fibrolytic bacteria and methanogens resulting in increased fiber utilization, while the starch-rich diet selected for amylolytic bacteria and lactate utilizers, allowing the maintenance of a healthy rumen and decreasing methane production (p < 0.05). Furthermore, the fiber-rich diet enriched for hydrogenotrophic methanogens and acetogens leading to increased electron-bifurcating [FeFe]-hydrogenases, methanogenic [NiFe]- and [Fe]-hydrogenases and acetyl-CoA synthase, with lower dissolved hydrogen (42%, p < 0.001). In contrast, the starch-rich diet enriched for respiratory hydrogenotrophs with greater hydrogen-producing group B [FeFe]-hydrogenases and respiratory group 1d [NiFe]-hydrogenases. Parallel in vitro experiments showed that the fiber-rich selected microbiome enhanced acetate and butyrate production while decreasing methane production (p < 0.05), suggesting that the enriched hydrogenotrophic acetogens converted some hydrogen that would otherwise be used by methanogenesis. These insights into hydrogen metabolism and methanogenesis improve understanding of energy harvesting strategies, healthy rumen maintenance, and methane mitigation in ruminants.

3-Nitrooxypropanol substantially decreased enteric methane emissions of dairy cows fed true protein- or urea-containing diets.

Methane is a potent but short-lived greenhouse gas targeted for short-term amelioration of climate change, with enteric methane emitted by ruminants being the most important anthropogenic source of methane. Ruminant production also releases nitrogen to the environment, resulting in groundwater pollution and emissions of greenhouse gas nitrous oxide. We hypothesized that inhibiting rumen methanogenesis in dairy cows with chemical inhibitor 3-nitrooxypropanol (3-NOP) would redirect metabolic hydrogen towards synthesis of microbial amino acids. Our objective was to investigate the effects of 3-NOP on methane emissions, rumen fermentation and nitrogen metabolism of dairy cows fed true protein or urea as nitrogen sources. Eight ruminally-cannulated cows were fed a plant protein or a urea-containing diet during a Control experimental period followed by a methanogenesis inhibition period with 3-NOP supplementation. All diets were unintentionally deficient in nitrogen, and diets supplemented with 3-NOP had higher fiber than diets fed in the Control period. Higher dietary fiber content in the 3-NOP period would be expected to cause higher methane emissions; however, methane emissions adjusted by dry matter and digested organic matter intake were 54% lower with 3-NOP supplementation. Also, despite of the more fibrous diet, 3-NOP shifted rumen fermentation from acetate to propionate. The post-feeding rumen ammonium peak was substantially lower in the 3-NOP period, although that did not translate into greater rumen microbial protein production nor lesser nitrogen excretion in urine. Presumably, because all diets resulted in low rumen ammonium, and intake of digestible organic matter was lower in the 3-NOP period compared to the Control period, the synthesis of microbial amino acids was limited by nitrogen and energy, precluding the evaluation of our hypothesis. Supplementation with 3-NOP was highly effective at decreasing methane emissions with a lower quality diet, both with true protein and urea as nitrogen sources.

Effect of Oats and Wheat Genotype on In Vitro Gas Production Kinetics of Straw.

Increases in cereals grain yield in the last decades have increased the accumulation of straw on the soil after harvest. Farmers typically open burn the straw to prepare the soil for the next crop, resulting in pollution, emission of greenhouse gases, erosion, loss of soil organic matter, and wildfires. An alternative is feeding straw to ruminants, but straw nutritive value is limited by its high content of lignocellulose and low content of protein. Cereal breeding programs have focused on improving grain yield and quality and agronomic traits, but little attention has been paid to straw nutritive value. We screened straw from 49 genotypes of oats and 24 genotypes of wheat from three cereal breeding trials conducted in Chile for in vitro gas production kinetics. We found moderate effects of the genotype on gas production at 8, 24, and 40 h of incubation, and on the maximum extent and rate of gas production. Gas production was negatively associated with lignin and cellulose contents and not negatively associated with grain yield and resistance to diseases and lodging. Effects observed in vitro need to be confirmed in animal experiments before gas production kinetics can be adopted to identify cereal genotypes with more digestible straw.

Association of fibre degradation with ruminal dissolved hydrogen in growing beef bulls fed with two types of forages.

The present study investigated the association between fibre degradation and the concentration of dissolved molecular hydrogen (H2) in the rumen. Napier grass (NG) silage and corn stover (CS) silage were compared as forages with contrasting structures and degradation patterns. In the first experiment, CS silage had greater 48-h DM, neutral-detergent fibre (NDF) and acid-detergent fibre degradation, and total gas and methane (CH4) volumes, and lower 48-h H2 volume than NG silage in 48-h in vitro incubations. In the second experiment, twenty-four growing beef bulls were fed diets including 55 % (DM basis) NG or CS silages. Bulls fed the CS diet had greater DM intake (DMI), average daily gain, total-tract digestibility of OM and NDF, ruminal dissolved methane (dCH4) concentration and gene copies of protozoa, methanogens, Ruminococcus albus and R. flavefaciens, and had lower ruminal dH2 concentration, and molar proportions of valerate and isovalerate, in comparison with those fed the NG diet. There was a negative correlation between dH2 concentration and NDF digestibility in bulls fed the CS diet, and a lack of relationship between dH2 concentration and NDF digestibility with the NG diet. In summary, the fibre of CS silage was more easily degraded by rumen microorganisms than that of NG silage. Increased dCH4 concentration with the CS diet presumably led to the decreased ruminal dH2 concentration, which may be helpful for fibre degradation and growth of fibrolytic micro-organisms in the rumen.

Metabolic Hydrogen Flows in Rumen Fermentation: Principles and Possibilities of Interventions.

Rumen fermentation affects ruminants productivity and the environmental impact of ruminant production. The release to the atmosphere of methane produced in the rumen is a loss of energy and a cause of climate change, and the profile of volatile fatty acids produced in the rumen affects the post-absorptive metabolism of the host animal. Rumen fermentation is shaped by intracellular and intercellular flows of metabolic hydrogen centered on the production, interspecies transfer, and incorporation of dihydrogen into competing pathways. Factors that affect the growth of methanogens and the rate of feed fermentation impact dihydrogen concentration in the rumen, which in turn controls the balance between pathways that produce and incorporate metabolic hydrogen, determining methane production and the profile of volatile fatty acids. A basic kinetic model of competition for dihydrogen is presented, and possibilities for intervention to redirect metabolic hydrogen from methanogenesis toward alternative useful electron sinks are discussed. The flows of metabolic hydrogen toward nutritionally beneficial sinks could be enhanced by adding to the rumen fermentation electron acceptors or direct fed microbials. It is proposed to screen hydrogenotrophs for dihydrogen thresholds and affinities, as well as identifying and studying microorganisms that produce and utilize intercellular electron carriers other than dihydrogen. These approaches can allow identifying potential microbial additives to compete with methanogens for metabolic hydrogen. The combination of adequate microbial additives or electron acceptors with inhibitors of methanogenesis can be effective approaches to decrease methane production and simultaneously redirect metabolic hydrogen toward end products of fermentation with a nutritional value for the host animal. The design of strategies to redirect metabolic hydrogen from methane to other sinks should be based on knowledge of the physicochemical control of rumen fermentation pathways. The application of new -omics techniques together with classical biochemistry methods and mechanistic modeling can lead to exciting developments in the understanding and manipulation of the flows of metabolic hydrogen in rumen fermentation.

Inhibiting Methanogenesis Stimulated de novo Synthesis of Microbial Amino Acids in Mixed Rumen Batch Cultures Growing on Starch but Not on Cellulose.

Ameliorating methane (CH4) emissions from ruminants would have environmental benefits, but it is necessary to redirect metabolic hydrogen ([H]) toward useful sinks to also benefit animal productivity. We hypothesized that inhibiting rumen methanogenesis would increase de novo synthesis of microbial amino acids (AA) as an alternative [H] sink if sufficient energy and carbon are provided. We examined the effects of inhibiting methanogenesis with 9, 10-anthraquione (AQ) on mixed rumen batch cultures growing on cellulose or starch as sources of energy and carbon contrasting in fermentability, with ammonium (NH4+) or trypticase (Try) as nitrogen (N) sources. Inhibiting methanogenesis with AQ inhibited digestion with cellulose but not with starch, and decreased propionate and increased butyrate molar percentages with both substrates. Inhibiting methanogenesis with 9, 10-anthraquinone increased de novo synthesis of microbial AA with starch but not with cellulose. The decrease in the recovery of [H] caused by the inhibition of methanogenesis was more moderate with starch due to an enhancement of butyrate and AA as [H] sinks. There may be an opportunity to simultaneously decrease the emissions of CH4 and N with some ruminant diets and replace plant protein supplements with less expensive non-protein nitrogen sources such as urea.

Inhibiting Methanogenesis in Rumen Batch Cultures Did Not Increase the Recovery of Metabolic Hydrogen in Microbial Amino Acids.

There is an interest in controlling rumen methanogenesis as an opportunity to both decrease the emissions of greenhouse gases and improve the energy efficiency of rumen fermentation. However, the effects of inhibiting rumen methanogenesis on fermentation are incompletely understood even in in vitro rumen cultures, as the recovery of metabolic hydrogen ([H]) in the main fermentation products consistently decreases with methanogenesis inhibition, evidencing the existence of unaccounted [H] sinks. We hypothesized that inhibiting methanogenesis in rumen batch cultures would redirect [H] towards microbial amino acids (AA) biosynthesis as an alternative [H] sink to methane (CH4). The objective of this experiment was to evaluate the effects of eight inhibitors of methanogenesis on digestion, fermentation and the production of microbial biomass and AA in rumen batch cultures growing on cellulose. Changes in the microbial community composition were also studied using denaturing gradient gel electrophoresis (DGGE). Inhibiting methanogenesis did not cause consistent changes in fermentation or the profile of AA, although the effects caused by the different inhibitors generally associated with the changes in the microbial community that they induced. Under the conditions of this experiment, inhibiting methanogenesis did not increase the importance of microbial AA synthesis as a [H] sink.

Nitrate improves ammonia incorporation into rumen microbial protein in lactating dairy cows fed a low-protein diet.

Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control. Eight lactating cows were blocked in 4 pairs according to days in milk, parity, and milk yield and allocated to urea (7.0 g urea/kg of dry matter of basal diet) or nitrate (14.6 g of NO3-/kg of dry matter of basal diet, supplemented as sodium nitrate) treatments, which were formulated on 75% of metabolizable protein requirements. Nitrate supplementation decreased ammonia concentration in the rumen liquids (-33.1%) and plasma (-30.6%) as well as methane emissions (-15.0%) and increased dissolved hydrogen concentration (102%), microbial N (22.8%), propionate molar percentage, milk yield, and 16S rRNA gene copies of Selenomonas ruminantium. Ruminal dissolved hydrogen was positively correlated with the molar proportion of propionate (r = 0.57), and negatively correlated with acetate-to-propionate ratio (r = -0.57) and estimated net metabolic hydrogen production relative to total VFA produced (r = -0.58). Nitrate reduction to ammonia redirected metabolic hydrogen away from methanogenesis, enhanced ammonia incorporation into rumen microbial protein, and shifted fermentation from acetate to propionate, along with increasing S. ruminantium 16S rRNA gene copies, likely leading to the increased milk yield.

Influence of Season and Diet on Fiber Digestion and Bacterial Community Structure in the Rumen of Muskoxen (Ovibos moschatus).

We studied the relationship between fiber digestion and the composition of the bacterial community in the rumen of muskoxen at the start and the end of the annual window of plant growth from spring to fall. Eight ruminally cannulated castrated males were fed brome hay or triticale straw (69.6% vs. 84.6% neutral detergent fiber, respectively) that were similar in fiber content to the sedges consumed by wild muskoxen (64.5 to 71.7% neutral detergent fiber). Muskoxen digested fiber from both forages faster and to a greater extent when straw rather than hay was consumed. Fiber digestion was therefore inducible by diet 4 in each season. We used 16S rRNA sequences from ruminal contents to study how season and diet affected the bacterial community and how the latter related to fiber digestion. We found that Bacteroidetes and Firmicutes accounted for 90% of the sequences at the level of Phylum, which is typical for the mammal gut microbiome. Using partial least square regressions, it was found that between 48% and 72% of the variation in fiber digestion was associated with 36⁻43 genera of bacteria. The main fibrolytic bacteria typical of domestic ruminants were generally not among the most important bacteria associated with fiber digestion in muskoxen. This reveals that muskoxen rely upon on a large suite of bacterial genera that are largely distinct from those used by other ruminants to digest the cell walls of plants that vary widely in both abundance and nutritional quality through the year.

Inhibition of Rumen Methanogenesis and Ruminant Productivity: A Meta-Analysis.

Methane (CH4) formed in the rumen and released to the atmosphere constitutes an energy inefficiency to ruminant production. Redirecting energy in CH4 to fermentation products with a nutritional value to the host animal could increase ruminant productivity and stimulate the adoption of CH4-suppressing strategies. The hypothesis of this research was that inhibiting CH4 formation in the rumen is associated with greater ruminant productivity. The primary objective of this meta-analysis was to evaluate how inhibiting rumen methanogenesis relates with the efficiencies of milk production and growth and fattening. A systematic review of peer-reviewed studies in which rumen methanogenesis was inhibited with chemical compounds was conducted. Experiments were clustered based on research center, year of publication, experimental design, feeding regime, type of animal, production response, inhibitor of CH4 production, and method of CH4 measurement. Response variables were regressed against the random experiment effect nested in its cluster, the random effect of the cluster, the linear and quadratic effects of CH4 production, and the random interaction between CH4 production and the experiment nested in the cluster. When applicable, responses were adjusted by intake of different nutrients included as regressors. Inhibiting rumen methanogenesis tended to associate positively with milk production efficiency, although the relationship was influenced by individual experiments. Likewise, a positive relationship between methanogenesis inhibition and growth and fattening efficiency depended on the inclusion and weighting of individual experiments. Inhibiting rumen methanogenesis negatively associated with dry matter intake. Interpretation of the effects of inhibiting methanogenesis on productivity is limited by the availability of experiments simultaneously reporting energy losses in feces, H2, urine and heat production, as well as net energy partition. It is concluded that inhibiting rumen methanogenesis has not consistently translated into greater animal productivity, and more animal performance experiments are necessary to better characterize the relationships between animal productivity and methanogenesis inhibition in the rumen. A more complete understanding of changes in the flows of nutrients caused by inhibiting rumen methanogenesis and their effect on intake also seems necessary to effectively re-channel energy gained from CH4 suppression toward consistent gains in productivity.

Molecular hydrogen generated by elemental magnesium supplementation alters rumen fermentation and microbiota in goats.

We tested the hypotheses that supplementation of a diet with elemental Mg increases ruminal dissolved H2 (dH2) in rumen fluid, which in turn alters rumen fermentation and microbial community in goats. In a randomised block design, twenty growing goats were allocated to two treatments fed the same basal diet with 1·45 % Mg(OH)2 or 0·6 % elemental Mg. After 28 d of adaptation, we collected total faeces to measure total tract digestibility, rumen contents to analyse fermentation end products and microbial groups, and measured methane (CH4) emission using respiration chambers. Ruminal Mg2+ concentration was similar in both treatments. Elemental Mg supplementation increased dH2 at 2·5 h post morning feeding (+180 %, P<0·001). Elemental Mg supplementation decreased total volatile fatty acid concentration (-8·6 %, P<0·001), the acetate:propionate ratio (-11·8 %, P<0·03) and fungal copy numbers (-63·6 %, P=0·006), and increased propionate molar percentage (+11·6 %, P<0·001), methanogen copy numbers (+47·9 %, P<0·001), dissolved CH4 (+35·6 %, P<0·001) and CH4 emissions (+11·7 %, P=0·03), compared with Mg(OH)2 supplementation. The bacterial community composition in both treatments was overall similar. Ruminal dH2 was negatively correlated with acetate molar percentage and fungal copy numbers (P<0·05), and positively correlated with propionate molar percentage and methanogen copy numbers (P<0·05). In summary, elemental Mg supplementation increased ruminal dH2 concentration, which inhibited rumen fermentation, enhanced methanogenesis and seemed to shift fermentation pathways from acetate to propionate, and altered microbiota by decreasing fungi and increasing methanogens.

Redirection of Metabolic Hydrogen by Inhibiting Methanogenesis in the Rumen Simulation Technique (RUSITEC).

A decrease in methanogenesis is expected to improve ruminant performance by allocating rumen metabolic hydrogen ([2H]) to more energy-rendering fermentation pathways for the animal. However, decreases in methane (CH4) emissions of up to 30% are not always linked with greater performance. Therefore, the aim of this study was to understand the fate of [2H] when CH4 production in the rumen is inhibited by known methanogenesis inhibitors (nitrate, NIT; 3-nitrooxypropanol, NOP; anthraquinone, AQ) in comparison with a control treatment (CON) with the Rumen Simulation Technique (RUSITEC). Measurements started after 1 week adaptation. Substrate disappearance was not modified by methanogenesis inhibitors. Nitrate mostly seemed to decrease [2H] availability by acting as an electron acceptor competing with methanogenesis. As a consequence, NIT decreased CH4 production (-75%), dissolved dihydrogen (H2) concentration (-30%) and the percentages of reduced volatile fatty acids (butyrate, isobutyrate, valerate, isovalerate, caproate and heptanoate) except propionate, but increased acetate molar percentage, ethanol concentration and the efficiency of microbial nitrogen synthesis (+14%) without affecting gaseous H2. Nitrooxypropanol decreased methanogenesis (-75%) while increasing both gaseous and dissolved H2 concentrations (+81% and +24%, respectively). Moreover, NOP decreased acetate and isovalerate molar percentages and increased butyrate, valerate, caproate and heptanoate molar percentages as well as n-propanol and ammonium concentrations. Methanogenesis inhibition with AQ (-26%) was associated with higher gaseous H2 production (+70%) but lower dissolved H2 concentration (-76%), evidencing a lack of relationship between the two H2 forms. Anthraquinone increased ammonium concentration, caproate and heptanoate molar percentages but decreased acetate and isobutyrate molar percentages, total microbial nitrogen production and efficiency of microbial protein synthesis (-16%). Overall, NOP and AQ increased the amount of reduced volatile fatty acids, but part of [2H] spared from methanogenesis was lost as gaseous H2. Finally, [2H] recovery was similar among CON, NOP and AQ but was largely lower than 100%. Consequently, further studies are required to discover other so far unidentified [2H] sinks for a better understanding of the metabolic pathways involved in [2H] production and utilization.

Supersaturation of Dissolved Hydrogen and Methane in Rumen of Tibetan Sheep.

Hydrogen (H2) is an essential substrate for methanogens to produce methane (CH4), and also influences pathways of volatile fatty acids (VFA) production in the rumen. Dissolved H2 (H2 (aq)) is the form of H2 available to microbes, and dissolved CH4 (CH4 (aq)) is important for indicating methanogens activity. Rumen H2 (aq) concentration has been estimated by assuming equilibrium with headspace gaseous H2 (H2 (g)) concentration using Henry's law, and has also been directly measured in the liquid phase in some in vitro and in vivo experiments. In this in vivo study, H2 (aq) and CH4 (aq) concentration measured directly in rumen fluid and their corresponding concentrations estimated from their gaseous phase concentrations, were compared to investigate the existence of equilibrium between the gas and liquid phases. Twenty-four Tibetan sheep were randomly assigned to two mixed diets containing the same concentrate mixed with oat grass (OG diet) or barley straw (BS diet). Rumen gaseous phase and contents were sampled using rumenocentesis and oral stomach tubing, respectively. Rumen H2 (aq) and CH4 (aq) concentration and VFA profile differed between sheep fed OG and BS diets. Measured H2 (aq) and CH4 (aq) concentration were greater than H2 (aq) and CH4 (aq) concentrations estimated using gas concentrations, indicating lack of equilibrium between gas and liquid phase and supersaturation of H2 and CH4 in rumen fluid. As a consequence, Gibbs energy changes (ΔG) estimated for various metabolic pathways were different when calculated using dissolved gases concentrations directly measured and when using dissolved gases concentrations assuming equilibrium with the gaseous phase. Dissolved CH4, but not CH4 (g), was positively correlated with H2 (aq). Both H2 (aq) and H2 (g) concentrations were positively correlated with the molar percentage of butyrate and negatively correlated with the molar percentage of acetate. In summary, rumen fluid was supersaturated with both H2 and CH4, and H2 (aq) was closely associated with the VFA profile and CH4 (aq) concentration. The assumption of equilibrium between dissolved gases and gaseous phase affected ΔG estimation.

Limits to Dihydrogen Incorporation into Electron Sinks Alternative to Methanogenesis in Ruminal Fermentation.

Research is being conducted with the objective of decreasing methane (CH4) production in the rumen, as methane emissions from ruminants are environmentally damaging and a loss of digestible energy to ruminants. Inhibiting ruminal methanogenesis generally results in accumulation of dihydrogen (H2), which is energetically inefficient and can inhibit fermentation. It would be nutritionally beneficial to incorporate accumulated H2 into propionate or butyrate production, or reductive acetogenesis. The objective of this analysis was to examine three possible physicochemical limitations to the incorporation of accumulated H2 into propionate and butyrate production, and reductive acetogenesis, in methanogenesis-inhibited ruminal batch and continuous cultures: (i) Thermodynamics; (ii) Enzyme kinetics; (iii) Substrate kinetics. Batch (N = 109) and continuous (N = 43) culture databases of experiments with at least 50% inhibition in CH4 production were used in this meta-analysis. Incorporation of accumulated H2 into propionate production and reductive acetogenesis seemed to be thermodynamically feasible but quite close to equilibrium, whereas this was less clear for butyrate. With regard to enzyme kinetics, it was speculated that hydrogenases of ruminal microorganisms may have evolved toward high-affinity and low maximal velocity to compete for traces of H2, rather than for high pressure accumulated H2. Responses so far obtained to the addition of propionate production intermediates do not allow distinguishing between thermodynamic and substrate kinetics control.