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[Inhibition of methanogenesis in the rumen of sheep. II. Methanogenesis after administration of inhibitors].

Studies were carried out on 5 sheep 2-4 years old of about 40 kg in weight successively in summer and autumn. The experiment conditions and the method were the same as in part one of this paper. The most effective inhibitors of methanogenesis used included also fatty acids such as linoleic and cis-oleic acid, some saturated fatty acids as; behenic and stearic, chloral hydrate, sodium sulphite and chloroform. Various changes in the composition of the rumen gases and in their ratios were caused by the individual inhibitors. Moreover, an increased dose of the inhibitor always resulted in increased inhibition of methane production, which was the highest in sheep fed on a bulky food. The observations also showed that the application route of the inhibitors had no effect on their action. The methane production inhibitors used did not cause any appetite disturbances in the experimental sheep and not show any toxic effect.

Animals

[Inhibition of methanogenesis in the rumen of sheep. I. Methanogenesis before administration of inhibitors].

Studies were carried out on 5 sheep aged 2-4 years of about 40 kg in body weight successively in summer and autumn. They were fed in 6 feeding groups in the morning between 6.30 and 7.30 and in the afternoon between 13.30 and 14.30. Gas samples were taken with an Orsat apparatus, Kiecka methanometer and a gas chromatograph from the rumen through a stable fistula directly before and after feeding and 0.5, 1, 2, 3, 4, 5, 6 hr after eating the morning food. The presence of CH4, CO2, N2, H2, O2, CO, CnHm, H2S and SO2 was found in the rumen gas samples analysed. The ratios of the following gases were also analysed: CO2:CH4, N2:O2, CO2:O2, H2:N2, CO2:N2 and CH4:N2. It was shown that the level of methane production in the sheep rumen depends on the composition of the food fed, the lapse of time from the last feeding and on the years season, whereas the amount of methane and its ratio to that of other gases of the rumen show a distinct dependence on the content of the food volume fed. The particular gas ratios, especially that of CO2 to CH4 may account for regular fermentation processes developing in the rumen of the sheep studied. The obtained results were mathematically analysed.

Animals

Thermophilic methanogenesis in a hot-spring algal-bacterial mat (71 to 30 degrees C).

Algal-bacterial mats which grow in the effluent channels of alkaline hot springs provided an environment suitable for studying natural thermophilic methane producing bacteria. Methane was rapidly produced in cores taken from the meat and appeared to be an end product of decomposition of the algal-bacterial organic matter. Formaldehyde prevented production of methane. Initial methanogenic rate was lower and methanogenesis became exponential when samples were permitted to cool before laboratory incubation. Methanogenesis occurred and methanogenic bacteria were present over a range of 68 to 30 degrees C, with optimum methanogenesis near 45 degrees C. The temperature distribution of methanogenesis in the mat is discussed relative to published results on standing crop, primary production, and decomposition in the thermal gradient. The depth distribution of methanogenesis was similar to that of freshwater sediments, with a zone of intense methanogenesis near the mat surface. Methanogenesis in deeper mat layers was very low or undetectable despite large numbers of viable methanogenic bacteria and could not be stimulated by addition of anoxic source water, sulfide, or a macronutrient solution.

Anaerobiosis

Integrative analysis of rumen microbiota activity and host metabolism following methanogenesis inhibition in dairy cattle.

Enteric methane emission from dairy cattle is an environmental challenge. The most efficient mitigation strategies nowadays include the use of methanogenesis inhibitors that specifically target the rumen methanogens. Specific inhibitors, such as 3-nitrooxypropanol (3-NOP), reduce methane emissions without negative effects on the products of fermentation that serve as energy metabolites for the host. However, the concomitant effects of methanogenesis inhibition on rumen microbiota and host metabolism are poorly characterized. Thus, the objective of this study was to explore the association between rumen microbiota and host metabolism when methanogenesis is inhibited. Thirteen dairy cows were used as controls, and 12 were supplemented with 3-NOP for 6 weeks. Rumen microbiota composition and activity were characterized using metagenomics and metatranscriptomics. The host metabolism was assessed in a previous publication by a metabolomic analysis of the plasma. Microbiota data were used as explanatory variables of the metabolome data in a multiblock sparse partial least squares analysis. Overall, the association between rumen microbiota and host metabolism was moderate. Notwithstanding this, a few downregulated transcripts related to glycolysis, hydrogen transfer, and protein synthesis, together with a decrease in the proportion of taxa of the Oscillospirales order, showed a correlation with host one-carbon metabolites (|r| > 0.6). These associations raised novel hypotheses that remain to be elucidated, especially with regard to the effects of dihydrogen on the accumulation of microbial glycolysis and methanogenesis metabolite intermediates.IMPORTANCEDairy cattle produce a substantial amount of methane, a potent greenhouse gas. Several strategies have been designed to reduce methane production by targeting the rumen microbiota. One such strategy specifically inhibits methanogens with a molecule called 3-nitrooxypropanol. This study uses an integrative data analysis approach, combining rumen microbiota and host metabolome information, to explore the consequences of inhibiting methanogenesis on the holobiont. This provides additional holistic insight into the effect of methane mitigation strategies on dairy cattle.

Animals

Temperature limitation of methanogenesis in aquatic sediments.

Microbial methanogenesis was examined in sediments collected from Lake Mendota, Wisconsin, at water depths of 5, 10, and 18 m. The rate of sediment methanogenesis was shown to vary with respect to sediment site and depth, sampling date, in situ temperature, and number of methanogens. Increased numbers of methanogenic bacteria and rates of methanogenesis correlated with increased sediment temperature during seasonal change. The greatest methanogenic activity was observed for 18-m sediments throughout the sampling year. As compared with shallower sediments, 18-m sediment was removed from oxygenation effects and contained higher amounts of ammonia, carbonate, and methanogenic bacteria, and the population density of methanogens fluctuated less during seasonal change. Rates of methanogenesis in 18-m sediment cores decreased with increasing sediment depth. The optimum temperature, 35 to 42 C, for sediment methanogenesis was considerably higher than the maximum observed in situ temperature of 23 C. The conversion of H2 and [14C]carbonate to [14C]methane displayed the same temperature optimum when these substrates were added to sediments. The predominant methanogenic population had simple nutritional requirements and were metabolically active at 4 to 45 C. Hydrogen oxidizers were the major nutritional type of sediment methanogens; formate and methanol fermentors were present, but acetate fermentors were not observed. Methanobacterium species were most abundant in sediments although Methanosarcina, Methanococcus, and Methanospirillum species were observed in enrichment cultures. A chemolithotropic species of Methanosarcina and Methanobacterium was isolated in pure culture that displayed temperature optima above 30 C and had simple nutritional requirements.

Cell Count

Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol.

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H(2). Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H(2)-CO(2) by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H(2)-CO(2) may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H(2)-CO(2).

Acetates

Microbial methanogenesis and acetate metabolism in a meromictic lake.

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 mumol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of (14)CH(4) from (14)C-labeled HCOOH, HCO(3) (-), and CH(3)OH and [2-(14)C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H(14)CO(3) (-) by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH(4) and CO(2) in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 mug/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO(2) production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 mug/liter) completely inhibited methanogenesis and stimulated CO(2) formation.

Acetates

Environmental factors influencing methanogenesis in a shallow anoxic aquifer: a field and laboratory study.

The environmental factors influencing methanogenesis in a shallow anoxic aquifer were probed in a combined field and laboratory study. Field data collected over a year revealed that 'in situ' rates of methane production were depressed in winter and elevated in summer. Over the same period, ground water pH values ranged from 6.0 to 7.8 while temperatures varied from 7-22 degrees C. 'In situ' methanogenesis was severely inhibited at temperatures less than 13 degrees C or by pH values less than 7. The influence of these factors on microbial methane formation from both endogenous and exogenous substrates were tested in aquifer slurries adjusted to pH 5-9 and incubated at temperatures ranging from 5-45 degrees C. Temperature optima for methane production from endogenous substrates varied as a function of pH, but the pH optimum was 8 at all temperatures. Optimal conditions for acetoclastic methanogenesis were found at pH 8 and 35 degrees C. An analysis of variance revealed that pH, temperature, and a pH-temperature interaction are all significant variables influencing aquifer methanogenesis. In addition transient sulfate accumulations were also found to limit methane production in some areas. A comparison of field and laboratory methane production patterns suggest that pH, temperature, and sulfate accumulations are important, but not the only environmental variables influencing the mineralization of organic matter in shallow aquifers.

Analysis of Variance

Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis.

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.

Adenosine Triphosphate

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.

Acetates

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.

Acetates

Hydrogen as a substrate for methanogenesis and sulphate reduction in anaerobic saltmarsh sediment.

Hydrogen gas stimulated sulphate reduction in a saltmarsh sediment and the importance of H2 transferred from organotrophic bacteria to the sulphate-reducers is discussed. beta-fluorolactate inhibited sulphate reduction whether lactate, ethanol or hydrogen was being used as growth substrate. When added to sediment beta-fluorolactate inhibited sulphate reduction with a consequent increase in methane production. Addition of H2 stimulated methanogenesis in sediment and this stimulation was greater if CO2 was also present. Hydrogen availability was the primary limitation of methanogenesis but the low concentration of dissolved CO2 in seawater may limit methane production even if H2 is available. The removal of inhibition of methanogenesis by the use of fluorolactate to suppress sulphate reduction or by the provision of hydrogen indicates competitive inhibition of methanogens by sulphate reducers utilizing transferred hydrogen.

Desulfovibrio

Electron-transport-driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri.

Methanogenesis from formaldehyde or formaldehyde + H2, as carried out by Methanosarcina barkeri, was strictly dependent on sodium ions whereas methane formation from methanol + H2 or methanol + formaldehyde was Na+-independent. This indicates that the reduction of formaldehyde to the formal redox level of methanol exhibits a Na+ requirement. During methanogenesis from formaldehyde, a delta pNa in the range of -62 mV to -80 mV was generated by means of a primary, electron-transport-driven sodium pump. This could be concluded from the following results obtained on cell suspensions of M. barkeri. 1. The addition of proton conductors or inhibitors of the Na+/H+ antiporter had no effect on sodium extrusion. 2. During methanogenesis from formaldehyde + H2 a delta psi of -60 mV to -70 mV was generated even in the presence of proton conductors. 3. ATPase inhibitors, applied in the presence of proton conductors, had no effect on primary sodium extrusion or generation of a delta psi. Evidence for a Na+-translocating ATPase could not be obtained.

Adenosine Triphosphate

Association of hydrogen metabolism with methanogenesis in Lake Mendota sediments.

Lake Mendota sediments were studied to determine the role of H2 in sediment methanogenesis. H2 was generally not detectable in sediment. The addition of H2 to sediment significantly increased methanogenensis. The amount of methane produced was proportional to the concentration of hydrogen added. H2 addition stimulated the reduction of CO2 to methane, but did not significantly stimulate the conversion of methanol or the methyl position of acetate to methane. Various organic compounds also stimulated sediment methanogenesis. Formate, ethanol, and glucose were shown to serve as electron donors for CO2 reduction to methane. The addition of formate to sediment resulted in H2 evolution. H2 was not deith the phenomenon of interspecies hydrogen transfer. The results indicate that hydrogen is an important intermediate and a rate-limiting factor in sediment methanogenesis.

Acetates

Factors affecting hydrogen production and consumption by human fecal flora. The critical roles of hydrogen tension and methanogenesis.

We studied the influence of hydrogen tension (PH2) and methanogenesis on H2 production and consumption by human fecal bacteria. Hydrogen consumption varied directly with PH2, and methanogenic feces consumed H2 far more rapidly than did nonmethanogenic feces. At low PH2, H2 production greatly exceeded consumption and there was negligible accumulation of the products of H2 catabolism, methane and sulfide. Thus, incubation at low PH2 allowed the first reported measurements of absolute as opposed to net H2 production. Feces incubated at high and intermediate PH2 had a net H2 production of only 1/900 and 1/64 of absolute production. Glucose fermentation by fecal bacteria yielded an absolute H2 production of 80 ml/g, a value far in excess of that excreted by volunteers ingesting lactulose. We conclude that most H2 produced by colonic bacteria is consumed and methanogenesis and fecal stirring (via its influence on fecal PH2) are critical determinants of H2 consumption and, hence, net H2 production. Study of fecal samples from four subjects with low breath H2 excretion after lactulose showed that absolute H2 production was normal, and the low H2 excretion apparently reflected increased consumption due to rapid methanogenesis (two subjects) and decreased luminal stirring (two subjects).

Bacteria

Energetics of methanogenesis studied in vesicular systems.

Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats where they play an indispensable role in the anaerobic food chain. Methanogenic bacteria possess a number of unique cofactors and coenzymes that play an important role in their specialized metabolism. Methanogenesis from a number of simple substrates such as H2 + CO2, formate, methanol, methylamines, and acetate is associated with the generation of transmembrane electrochemical gradients of protons and sodium ions which serve as driving force for a number of processes such as the synthesis of ATP via an ATP synthase, reverse electron transfer, and solute uptake. Several unique reactions of the methanogenic pathways have been identified that are involved in energy transduction. Their role and importance for the methanogenic metabolism are described.

Adenosine Triphosphate

Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut.

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H2/CO2, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.

Breath Tests

Methanogenesis from acetate: enrichment studies.

An acetate enrichment culture was initiated by inoculating anaerobic sludge from a mesophilic methane digestor into a mineral salts medium with calcium acetate as the sole carbon and energy source. This enrichment was maintained indefinitely by weekly transfer into medium of the same composition. A study of this enrichment disclosed an unexpected age-dependent inhibition of methanogenesis by H2 and formate which apparently differed from the inhibition by chloroform and benzyl viologen. This age-dependent inhibition indicated that microbial interactions of the mixed enrichment population may play a regulatory role in methane formation. Futhermore, stimulation of methanogenesis in the acetate enrichment by addition of yeast extract showed a nutrient limitation which indicated that syntrophic interactions leading to formation of growth factors may also occur. A model is presented to illustrate the possible interrelationships between methanogenic and nonmethanogenic bacteria in their growth and formation of methane and carbon dioxide from acetate.

Acetates