Effects of coenzyme M and methyl-coenzyme M on the efficacy of inhibitors of methanogenesis in rumen cultures

Effects of coenzyme M and methyl-coenzyme M on the efficacy of inhibitors of methanogenesis in rumen cultures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of coenzyme M and methyl-coenzyme M on the efficacy of inhibitors of methanogenesis in rumen cultures Emilio M. Ungerfeld, Nathaly Cancino, M. Florencia Samoluk, Gustavo Jaurena, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8419205/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Understanding the mechanisms of action of compounds inhibiting methanogenesis can aid understanding the variation in their efficacy as feed additives for mitigating enteric methane (CH 4 ) emissions from ruminants. 2-Bromoethanesulfonate (BES) and 3-nitrooxypropanol (3-NOP) inhibit methanogenesis by acting as structural analogs of methyl-coenzyme M (methyl-CoM), a methyl donor in the last reaction of methanogenesis. We hypothesized that a high concentration of methyl-CoM and its nonmethylated form, coenzyme M (CoM), would block the antimethanogenic effects of BES and 3-NOP in mixed rumen cultures and would not affect bromoform (BMF), which acts through a different mechanism. This hypothesis and the possible underlying mechanisms were examined in one ruminal serial culture and three batch culture experiments. Results Coenzyme M and methyl-CoM added at 1 mM blocked the inhibition of methanogenesis by BES but not by 3-NOP or BMF. 2-Bromoethanesulfonate strongly decreased the relative abundance of Methanobrevibacter ruminantium but did not affect other methanogens. Similar to CH 4 production, the effect of BES on the relative abundance of M. ruminantium was reversed by CoM and methyl-CoM. Because M. ruminantium cannot synthesize CoM and must take up exogenous CoM, the sensitivity of methanogens to BES may depend on them lacking the genetic capacity to synthesize CoM, as BES and CoM compete for transmembrane transport. In contrast, 3-NOP diffuses across cell membranes and does not compete for transmembrane transport with CoM, which is consistent with its effects not being reversed by CoM or methyl-CoM. Bromoform does not act competitively with CoM or methyl-CoM so that the addition of CoM or methyl-CoM did not reverse the effects of BMF. Conclusions These results may explain previous observations of lack of persistence of BES in vivo and in continuous cultures. Long-term BES supplementation is thought to shift the archaeal community towards methanogens that do not require exogenous CoM and are tolerant to BES. The effects of 3-NOP and BMF were unaffected by the addition of CoM and methyl-CoM presumably because they can diffuse across cell membranes, and in the case of BMF, the inhibition of methyl-tetrahydromethanopterin: coenzyme M methyltransferase may not be relieved by CoM or methyl-CoM. methane methanogens coenzyme M methyl-coenzyme M methanogenesis inhibitors 2-bromoethanesulfonate 3-nitrooxypropanol bromoform Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background The abatement of anthropogenic methane (CH 4 ) emissions would slow global warming in the short-term [ 1 , 2 ]. Approximately 30% of anthropogenic CH 4 originates in the rumen of domestic ruminants i.e., enteric CH 4 [ 1 , 3 ]. Thus, there is ongoing research on the mitigation of emissions of enteric methane (CH 4 ) from ruminants [ 4 ]. Feed additives that inhibit rumen methanogenesis, including 3-nitrooxypropanol (3-NOP) and the bromoform-containing red algae Asparagopsis , are the most potent strategies for mitigating the emissions of enteric CH 4 [ 5 , 6 ]. Yet, there is variation in the extent of inhibition of CH 4 production caused by these additives. Empirically, it is known that the efficacy of 3-NOP and Asparagopsis , defined as the decrease in CH 4 per gram of active compound ingested, is affected by dietary fiber and ether extract, and by the type of animal [ 7 – 9 ], but the specific mechanisms explaining those interactions are not understood. Additionally, there is variation among pure cultures of different methanogens in their sensitivity to inhibitors of methanogenesis, and the reasons behind this variation also remain unclear [ 10 – 13 ]. A mechanistic understanding of the variation in the efficacy of inhibitors of rumen methanogenesis may allow maximizing their effects in the rumen. Variation in the efficacy of inhibitors of methanogenesis may be related, at least in part, to their mechanisms of action. 3-Nitrooxypropanol is a structural analog of methyl-coenzyme M (methyl-CoM) [ 12 ], a methyl donor substrate that reacts with electron donor coenzyme B in the last step of methanogenesis, which is catalyzed by methyl-coenzyme M reductase (MCR), to form CH 4 and a heterodisulfide of coenzyme M (CoM) and coenzyme B [ 14 ]. 3-Nitrooxypropanol binds to MCR and oxidizes a Ni + 1 atom in the porphinoid cofactor F 430 to Ni + 2 thus impeding the formation of CH 4 from methyl-CoM [ 12 ]. Another methanogenesis inhibitor, 2-bromoethanesulfonate (BES), also competes with methyl-CoM [ 15 ] for the catalytic site in MCR and likewise oxidizes the Ni + 1 atom of cofactor F 430 to Ni + 2 [ 16 – 18 ]. Methyl-coenzyme M and also its nonmethylated form coenzyme M (CoM) have been shown to reverse the inhibition of CH 4 formation by BES in pure cultures of the ruminal methanogen Methanobrevibacter ruminantium M1 [ 19 , 20 ], non-ruminal methanogens Methanosarcina strain 227, Methanospirillum hungatei , Methanococcus voltae [ 15 , 21 – 23 ], methanogenic marine sediments [ 24 ], and purified MCR [ 17 ]. Bromoform (BMF) and other CH 4 halogenated analogs inhibit methanogenesis by reacting with a corrinoid cofactor of methyltetrahydromethanopterin: coenzyme M methyltransferase (MTR), thus blocking the formation of the methylated form of the corrinoid and the transfer of a methyl group to CoM to form methyl-CoM [ 25 ]. Because BES and 3-NOP inhibit MCR by competing with methyl-CoM, and CoM has also been shown to reverse BES effects, and BMF inhibits a different enzyme, we hypothesized that excess CoM or methyl-CoM added to mixed rumen cultures would reverse methanogenesis inhibition by BES and 3-NOP, but not by BMF. Second, some methanogens that lack the capacity to synthesize CoM, take up exogenous CoM from the medium at greater rates than those methanogens that can synthesize CoM [ 19 ]. Because CoM, methyl-CoM, and BES share a common transmembrane uptake system [ 23 ], and resistance to BES in methanogens is related to low rates of transport of BES into the cell [ 15 , 21 ], it is conceivable that methanogens that cannot synthesize CoM are more sensitive to BES because they take up BES at faster rates than those methanogens that can synthesize CoM. On the other hand, 3-NOP and BMF are understood to freely diffuse across cell membranes independently of transmembrane transporters [ 12 , 26 ]. We thus further hypothesized that supplementing CoM to serially transferred mixed rumen cultures would stimulate the growth of methanogens that cannot synthesize CoM and require exogenous CoM, resulting in mixed rumen cultures with greater sensitivity to BES after CoM supplementation is discontinued than cultures that had not received CoM; in contrast, the sensitivity of rumen cultures to 3-NOP and BMF would not be expected to be affected by previous supplementation with CoM. Methods Four in vitro experiments were conducted: one with serial mixed rumen cultures (Experiment 1; Fig. 1 ) and three with batch mixed rumen cultures (Experiments 2, 3, and 4). Experiment 1 Experiment 1 was conducted as a 2 × 4 × 2 factorial arrangement of treatments, with the following factors: i) Supplementation of CoM at 0 or 1 µM to serial rumen cultures in serial transfers 1 to 4; ii) Inhibition of methanogenesis: Control (distilled water), BES, 3-NOP, or BMF (all 5 µM), and iii) Spiking of CoM at 0 or 1 mM (Fig. 1 ). General rumen contents sampling and processing procedures were conducted as described before [ 27 ]. Rumen contents were sampled at approximately 9 am before feeding from two ruminally cannulated, nonlactating, nonpregnant, Holstein cows fed once daily 1 kg of concentrate (88.0% DM, and 18.3% CP, 29.2% NDF, 3.4% EE, 3.3% total ashes, all DM basis) and ad libitum ryegrass hay (90.7% DM, and 8.6% CP, 66.4% NDF, 39.5% ADF, 1.9% EE, 6.1% total ashes, all DM basis). Proximate compositions of the cannulated cows feed and the incubation substrate were analyzed according to AOAC [ 28 ]. Rumen contents were strained through an approximately 0.5 mm double synthetic cloth (Eurotelas, Osorno, Chile) and both fluid and solids from each cow were separately transported to the laboratory in insulated flasks. Approximately 150 mL of fluid and 150 mL of solids from the same cow were placed in 500-mL Erlenmeyer flasks under CO 2 and discontinuously (3 s bursts followed by 2 s pauses) blended for 1 min with an eggbeater running at low speed to dislodge microbial cells loosely adhered to plant particles. The Erlenmeyer flasks contents were then strained through two layers of synthetic cloth to obtain the rumen inoculum as the filtrate fraction [ 27 ]. Rumen inoculum from each cow (14 mL) was delivered under CO 2 into two 100-mL serum bottles containing 26 mL of culture medium [ 29 ] modified to contain the Raju [ 30 ] trace mineral solution and 400.4 [400.0, 400.6] (mean [min, max]) mg of a substrate composed of 75% forage and 25% concentrate (DM basis; Supplementary Table 1) ground to pass a 1-mm screen. One serum bottle with inoculum from each of the cows received 25 µL of a CoM (Sigma-Aldrich, China) solution to achieve a final concentration of 1 µM. This concentration of CoM was defined on the basis of the concentration of CoM used to maintain methanogens requiring exogenous CoM [ 31 ]. The serum bottle with the inoculum from each cow that did not receive CoM supplementation received 25 µL of distilled water and constituted the corresponding control treatment. All four serum bottles (two inocula from two cows, each with or without 1 µM CoM supplementation) were gassed and sealed under CO 2 and incubated at 39°C and 60 rpm for 24 h, after which gas pressure was measured with a pressure transducer (Sper Scientific 840065, Scottsdale, AZ, United States). Gas samples (0.5 mL) were analyzed for CH 4 and dihydrogen (H 2 ) content in a Clarus 580 Perkin Elmer GC equipped with a 60/80 Carboxen 1000 packed column (Supelco, Bellefonte, PA, United States) operating at an isothermal temperature of 180°C with N 2 at 30 mL/min as carrier gas and a thermal conductivity detector [ 32 ]. Standards of known concentrations were used to calculate the concentrations of CH 4 and H 2 . Gas production was calculated via the ideal gas law and a gas pressure equal to the gas pressure recorded plus 1 atm (101,325 Pa) and 312 K. Bottles were then vortexed at maximum speed for 2 min to dislodge microbial cells loosely attached to plant particles and subsequently left untouched for 1 min to allow plant particles to sink. Bottles were then opened under CO 2 and 14 mL of fluid was transferred to a new bottle corresponding to the same CoM supplementation treatment containing fresh medium and 400.4 [400.2, 400.6] (mean [min, max]) mg of substrate. The pH (Orion Star A214, Thermo Scientific, Chelmsford, MA, United States) and reducing potential (E h ; Oakton pH 700 meter, Vernon Hills, IL, United States, Ag/AgCl electrode in saturated KCl, Schott Instruments, BlueLine 31 Rx, Mainz, Germany) of the bottle donating the inoculum were then measured. Measured E h values were then corrected to those of the Standard Hydrogen Electrode (SHE) by adding 197 mV [ 33 ]. The process of serially transferring inoculum to a new bottle with fresh medium and new substrate was repeated five times (Fig. 1 ). In serial transfer 4, each bottle corresponding to a combination of a 1 µM CoM supplementation treatment and a cow´s inoculum donated 4 mL of inoculum fluid to each of eight bottles containing 36 mL of fresh medium and 400.4 [400.0, 402.0] (mean [min, max]) mg of substrate. The eight bottles of serial transfer 5 that received their inoculum from the same bottle of serial transfer 4 were blocked so to successively receive their 4 mL inoculum in two doses i.e., 2 mL from the first, and 2 mL from the second, half of the total inoculum delivered from the donor bottle of serial transfer 4. This procedure was conducted to avoid introducing a bias in delivering the upper or lower halves of the liquid volume of the donating bottle to particular treatments. The eight bottles in serial transfer 5 that received inoculum from each bottle in serial transfer 4 received a treatment combination of two factors: i) 1 mL of a CoM solution to achieve a 1 mM final concentration (CoM spiking) or 1 mL distilled water (Control); ii) 25 µL of a solution of BES (Aldrich Chemistry, Taiwan), 3-NOP (RR Scientific LLC, Shanghai, China), or BMF (Sigma-Aldrich, US), to achieve a 5 µM final concentration of each inhibitor of methanogenesis, or 25 µL distilled water (Control). Because BES and 3-NOP were dissolved in water, and bromoform was dissolved in ethanol, 25 µL of water or ethanol was added to the corresponding bottles to equalize the amounts of water and ethanol received by all the bottles. Bottles were sealed under CO 2 and incubated at 39°C and 60 rpm for 72 h. The incubation time of the fifth serial transfer was extended from 48 to 72 h to compensate for the lower volume of inoculum in comparison with the previous four serial transfers. At the end of the 72-h incubation period gas pressure and composition, pH, and E h were measured as described before. Bottles were then opened, and two 1-mL fluid aliquots were delivered into 2-mL microcentrifuge tubes containing 0.20 mL of 20% ( V / V ) o -phosphoric acid and 0.20 mL of 35 mM crotonic acid or 1% ( V / V ) sulfuric acid for subsequent analyses of carboxylic acids and alcohols or ammonium (NH 4 + ), respectively. All fluid samples were stored at -20°C until processing. Bottles contents were then centrifuged at 10,956 × g and 4°C for 15 min into 50-mL pre-weighted centrifuged tubes. The supernatants were discarded and the tubes containing the pellets were frozen at -80°C and then lyophilized. Lyophilized tubes were then weighted, and the dry mass of the incubation residue was calculated by difference. Apparent dry matter disappearance (DMD) was calculated by subtracting the residue dry matter from the initial substrate dry matter and expressed as a percentage. Microcentrifuge tubes for analysis of carboxylic acids and alcohols were thawed and centrifuged at 16,100 × g and 4°C for 10 min. The supernatants were then filtered through 13-mm 0.45 µm filters into 2-mL GC vials. Concentration of volatile fatty acids (VFA), succinate, methanol, and ethanol were analyzed in a PerkinElmer Clarus 580 GC equipped with an Elite-FFAP (PerkinElmer, Shelton, CT, US) capillary column and a flame ionization detector operating at 200°C. Split was set at 33: 1. The initial oven temperature of 50°C was maintained for 2.70 min, followed by a 15°C/min ramp to 150°C. The temperature was held at 150°C for 5 min and then increased at 15°C/min to 210°C and held for 1.5 min. The carrier gas was He at 51.4 mL/min. Calibration curves were made with standards of known concentration and crotonic acid was used as an internal standard. The sum of 2- and 3-methylbutyrate is reported as these two VFA co-eluted. Concentration of NH 4 + was analyzed colorimetrically following the method of Kaplan [ 34 ]. Three incubation rounds were conducted in different weeks. Results of fermentation variables of serial transfer 5 were analyzed as a 2 × 4 × 2 arrangement of treatments with the following model: response ijklm = general mean + CoM supplementation i serial transfers 1–4 (1 µM) + inhibitor j serial transfer 5 + CoM spiking k serial transfer 5 (1 mM) + double interactions + cow l (random) + incubation m (random) + error ijklm The triple interaction was initially included in the model, but it was removed from the model because it was not significant for any response variable ( P ≥ 0.30). When the inhibitor effects were significant ( P 0.05), inhibitors treatment means were compared through Tukey´s Honestly Significant Difference test. When interactions between inhibitors and CoM spiking were significant ( P < 0.05), the inhibitors causing the interaction were identified via three Least Square Difference (LSD) contrasts to compare the difference between each inhibitor and the methanogenesis inhibition control with and without CoM spiking: Contrast 1 = (Control, no CoM – BES, no CoM) – (Control, CoM – BES, CoM) Contrast 2 = (Control, no CoM – 3-NOP, no CoM) – (Control, CoM – 3-NOP, CoM) Contrast 3 = (Control, no CoM – BMF, no CoM) – (Control, CoM – BMF, CoM) Significance was declared at P < 0.05 tendencies at 0.05 ≤ P < 0.10. Results of fermentation variables of serial transfers 1 to 4 were analyzed as follows: response ijkl = general mean + serial transfer i + CoM supplementation j + serial transfer × CoM supplementation ij + cow k (random) + incubation l (random) + error ijkl Residuals distribution was examined through normal quantile plots. Homoscedasticity was examined through plots of observed residuals against predicted observations. Outliers were identified as observations falling out of a 99.9% studentized residuals normal distribution and examined for obvious typing or measurement problems. Outliers with no typing or measurement problems and clustering in the same treatment were considered as biological results and retained. Other outliers without an obvious technical cause were tentatively removed and the analysis re-run. As conclusions did not change substantially, all observations were retained. All statistical analyses were conducted with JMP 19.0.1 [ 35 ]. Experiment 2 Under a 4 × 2 factorial arrangement of treatments, Experiment 2 evaluated the interactions between the same three inhibitors of methanogenesis evaluated in Experiment 1 (BES, 3-NOP, and BMF, each at 5 µM, plus a Control treatment without a methanogenesis inhibitor) and methyl-CoM spiking at 0 or 1 mM on CH 4 production and fermentation of 48-h rumen batch cultures. Sampling and processing of rumen contents was conducted as described for Experiment 1. Under CO 2 , 8 mL of rumen inoculum from each cow, obtained as per Experiment 1, was were delivered into 100-mL serum bottles containing 32 mL of incubation medium [ 29 ] and 400.5 [400.2, 400.6] (mean [min, max]) mg of the same substrate used in Experiment 1. The inoculated medium then received 25 µL of a solution of 3-NOP or BES, or 25 µL of distilled water (methanogenesis inhibition Control). Depending on their methanogenesis inhibition treatment, bottles received 25 µL of ethanol or 25 µL of a BMF solution in ethanol. Final concentration of BES, 3-NOP or BMF was 5 µM. Each methanogenesis inhibition treatment in turn received 1 mL of a methyl-CoM (BLD Pharmatech Co., Ltd., Cincinnati, OH, US) solution to achieve a final concentration of 1 mM (methyl-CoM spiking) or 1 mL distilled water (methyl-CoM control). Bottles were gassed and sealed under O 2 -free CO 2 and incubated at 39°C and 60 rpm for 48 h. Gas production and composition, pH, E h , total and individual VFA, ammonium concentration, and DMD were determined as per Experiment 1. Three incubation runs were conducted in different weeks. Results of Experiment 2 were analyzed as a 3 × 2 factorial arrangement of treatments as follows: response ijkl = general mean + inhibitor i + methyl-CoM spiking j (1 mM) + inhibitor × methyl-CoM spiking ij + cow k + incubation l + error ijkl Residuals normality and outlier analyses were conducted as in Experiment 1. Experiment 3 Experiment 3 examined the interaction between BES at 5 µM and CoM or methyl-CoM at 0, 1, 10, 100, or 1000 µM, on CH 4 production, other fermentation variables, and the prokaryote community composition of 48-h rumen batch cultures. Rumen collection and processing were conducted as in Experiments 1 and 2. Rumen inoculum from each cow (8 mL) was delivered to 100-mL incubation bottles containing 32 mL of the Mould et al. [ 29 ] incubation medium and 400.3 [400.2, 400.5] (mean [min, max]) mg of the high forage substrate used in Experiments 1 and 2. Aliquots (5 mL) of each inoculum were kept in 15-mL polypropylene conical tubes at -20°C for subsequent analysis of CoM and methyl-CoM concentration in rumen fluid. Each bottle received 25 µL of a BES solution to achieve a final concentration of 5 µM or 25 µL of distilled water (methanogenesis inhibition Control). In turn, each bottle received 1 mL of one of four different CoM or methyl-CoM solutions to achieve 1, 10, 100, or 1000 µM final concentration. The CoM/methyl-CoM control received 1 mL of distilled water. Bottles were gassed and sealed under CO 2 and incubated for 48 h at 60 rpm. Measurements and analytical procedures were conducted as in Experiments 1 and 2. Bottle contents were centrifuged and subsequently lyophilized as in Experiments 1 and 2. Centrifugation tubes containing lyophilized incubation residues of treatments supplemented 0 or 1000 µM CoM or methyl-CoM, both with and without BES, were stored at -80°C for DNA extraction. The concentration of CoM and methyl-CoM in samples of ruminal inocula was analyzed by FoodTech, Temuco, Chile. General procedures were from Lovley et al. [ 36 ]. Inocula samples for CoM and methyl-CoM analysis were thawed and centrifuged at 13,000 × g and 4°C for 15 min. Supernatants were acidified with 5 mL 0.1 N HCl and filtered through 0.45 µm hydrophilic sterile syringe filters (Millex™ PVDF syringe filter, MilliporeSigma, Burlington, MA, USA) into 2-mL microcentrifuge tubes. Aliquots (1 mL) of the filtrates were frozen at -80°C, lyophilized, 25 µL of Sigma-Sil-A (Sigma-Aldrich, Burlington, MA, US) was added, the mixture was vortexed, and incubated at 70°C for 30 min for thimethylsilyl derivatization [ 37 , 38 ]. Samples were then cooled at room temperature for 30 min and added 1 mL of ethyl acetate (Sigma-Aldrich, Burlington, MA, US). The mix was vortexed and subsequently centrifuged at 16,000 × g at room temperature for 10 min. The ethyl acetate layer (700 µL) containing the derivatized compounds was then transferred to 2-mL GC vials for GC‒MS analysis. Parameters of the GC-MS run were adjusted by injecting standards of CoM and methyl-CoM and identifying retention times and fragments. Samples (1 µL) were injected in splitless mode into a QP2020NX Shimadzu GC-MS (Kyoto, Japan) equipped with a Restek Rtx-5Ms (30 m, 0,25 mm ID, 0.25 µm) capillary column (Bellefonte, PA, US). The initial temperature was 80°C for 3 min, followed by a 10°C/min ramp to 160°C. Temperature was maintained at 160°C for 1 min and then increased at 5°C/min to 230°C, and maintained at 230°C for 5 min. Samples were electron-ionized and fragments were scanned between 40 and 400 m/z at 1250 amu/s. Coenzyme M and methyl-CoM were identified and quantified using chemically pure standards. Limits of detection were 2.84 and 2.78 µM for CoM and methyl-CoM, respectively, and limits of quantification were 8.60 and 8.43 µM in the same order. Retention time was 7 min 31 s for both CoM and methyl-CoM. Both CoM and methyl-CoM were quantified as the sum of intensities of the 73, 103, 117, 147 and 205 m / z fragments. Concentration of CoM and methyl-CoM are reported as their sum as the specific compounds could not be separated. A subset of 24 lyophilized residues from the CoM 0 and 1000 µM treatments, both with and without BES, were selected for the analysis of the composition of the archaeal and bacterial communities through amplicon sequencing the V3-V4 regions of their 16S rRNA genes. Prokaryote metataxonomic analyses including DNA extraction, library preparation, sequencing, and bioinformatics were conducted by AUSTRAL-omics, Universidad Austral de Chile, Valdivia, Chile [ 39 ]. DNA was extracted from lyophilized incubation residues with a DNeasy PowerSoil Pro Kit (Qiagen) following the manufacturer´s instructions modified according to Yu and Forster [ 40 ], including two negative controls. Lyophilized samples for DNA analysis (250 mg) were placed in 2-mL PowerBead Pro Tube cryotubes containing 800 µL of CD1 solution and a total of 200 µL of 0.1-mm diameter zirconium beads. Microbial cells were mechanically disrupted by applying three 30-s bead beating cycles at 3,400 strokes (Mini-Beadbeater 24, BioSpecproducts, Bartlesville, OK, US). Samples were then incubated with 25 µL of 20 mg/mL proteinase K (New England Biolabs, Ipswich, MA, US) and 7 µL of 50 mg/mL lysozyme solution (Thermo Fisher, Rockford, IL, US) at 37°C for 4 h followed by an incubation at 55°C for 12 h. Preparations obtained were then passed through a DNA-binding column (MB Spin Columns, Quiagen, Germantown, MD, US) with buffer C6 (50 µL) added for DNA elusion. Samples were then incubated at room temperature for 1 h and centrifuged at 15,000 × g at room temperature for 1 min. Extracted DNA was quantified with fluorometry with a Qubit dsDNA HS Assay Kit (Thermo Fischer, Eugene, OR, US). DNA integrity was assessed via 1.5% agarose gel electrophoresis. Concentration of DNA in all samples used for amplification was equalized to 2 ng DNA/µL. Amplicons for sequencing libraries were generated following the Illumina 16S Metagenomic Sequencing Library Preparation protocol [ 41 ]. A total of 28 cycles of DNA amplification were conducted (30 s denaturing at 95°C, 30 s annealing at 55°C, and 30 s extension at 72°C) followed by a 5 min final extension at 72°C. KAPA HiFi DNA polymerase (Roche Sequencing Solutions, Inc., Cape Town, South Africa) was used in the PCR reactions. Archaeal and bacterial 16S rRNA gene V3-V4 regions were amplified using the primers of Mesa et al. [ 42 ] and Walters et al. [ 43 ], respectively. Negative extraction and PCR controls were included in the PCR amplification reactions. Products of PCR were purified from nonspecific products and primer dimers with Mag-Bind® TotalPure NGS (Omega Bio-Tek, Norcross, GA, US) magnetic pearls and quantified using a Qubit 4.0 fluorometer and the Qubit dsDNA HS Assay Kit (Thermo Fischer, Eugene, OR, US). Amplicons at equal amounts (10 ng) were pooled for a second PCR reaction (3 min denaturing at 95°C followed by 8 cycles of 30 s denaturing at 95°C, 30 s annealing at 55°C, and 30 s extension at 72°C, followed by 5 min extension at 72°C, with KAPA HiFi DNA polymerase (Roche Sequencing Solutions, Inc., Cape Town, South Africa) for ligating Illumina P5 and P7 adaptors attached to Illumina Nextera XT Index Kit v2 indices. The obtained library was purified with Mag-Bind® TotalPure NGS (Omega Bio-Tek, Norcross, GA, US) magnetic pearls and eluted with 22 µL 10 mM Tris. Integrity of DNA was assessed on a 1.5% agarose gel and DNA size was examined with capillary electrophoresis Fragment Analyzer (Agilent Technologies, Santa Clara, CA, US). Concentration of DNA was quantified by fluorometry (Qubit 4.0 fluorometer and Qubit dsDNA HS Assay Kit, Thermo Fischer, Eugene, OR, US), adjusted to 7 nM, and denatured with 5 µL of 0.2 mM NaOH for 5 min to a 750 pM final concentration. Libraries were mixed with a PhiX control library at 20% ( V / V ) to add heterogeneity and the final mixture was loaded on the Illumina platform at 2 × 300 Illumina paired forward and reverse cycles run with FastQ. Raw sequences generated by Illumina NextSeq 1000 were demultiplexed with Cutadapt 2.10 [ 44 ]. Demultiplexed sequences were filtered for quality with Trimomatic v. 0.39 [ 45 ] and PRINSEQ v. 0.20.4 [ 46 ], retaining sequences with a Phred ≥ Q30. Amplicon Sequence Variants (ASVs) were inferred with R DADA2 1.36 [ 47 ]. Flow work included the estimation of sequencing error per base, dereplication, ASV inference, concatenation of paired readings, and removal of chimeras [ 47 ]. Taxonomic assignment was performed with RDP Naïve Bayesian Classifier of DADA2 [ 48 ] with a 50% bootstrap threshold at the species level. Two filters of abundance were applied to minimize false positives and negatives. A filter per sample was applied keeping only those sequences with an abundance over 0.1% of total sequences in their sample. Second, a filter per ASV was applied by eliminating reads with occurrences of less than 1% of total reads of their particular ASV [ 49 – 51 ]. Diversity analyses were conducted with rarefied results normalizing samples by sequencing depth based on the sample with the least number of reads. Principal component analyses were conducted based on Bray-Curtis dissimilarity distances with the phyloseq 1.52 and R 4.5.1 packages [ 52 ] of R [ 53 ]. Alfa-diversity was estimated with vegan [ 54 ] in R [ 53 ]. A total of 11,471 and 24,104 archaeal bacterial reads, respectively, were obtained and taxonomically assigned of with SILVA 138 [ 55 ]. Eukaryotic ASVs and ASVs unassigned at the phylum level were eliminated from the analysis, resulting in a total of 5,343 and 68 filtered bacterial and archaeal ASVs, respectively. We searched GenBank [ 56 ] for methanogens carrying the genes comA , comB , comC , and comD and comE , encoding enzymes involved in the synthesis of CoM in in the Methanobacteriales order, as well as the CoM biosynthesis genes CS in Methanomicrobiales and Methanosarcinales and genes XcbB , XcbC , XcbD and XcbE in bacteria [ 57 – 59 ]. We used that information to estimate which percentage of methanogens in each sample with the genetic capacity to synthesize CoM as well as to transport CoM into their cells. Two incubation runs were conducted in different weeks. Fermentation results from Experiment 3 were analyzed as follows: response ijklmn = general mean + methanogenesis inhibition i (Control or BES) + CoM j + methyl-CoM k + methanogenesis inhibition × CoM ij + methanogenesis inhibition × methyl-CoM ik + cow l + incubation m + error ijklmn Concentration of CoM and methyl-CoM was added unity and decimal log-transformed for the statistical analyses as follows: $$\:{\text{log}}_{10}(CoM+1)\:\text{a}\text{n}\text{d}\:{\text{log}}_{10}(methyl-CoM+1)$$ The composition and diversity of the archaeal and bacterial communities were analyzed in the subset of treatments with 0, 1000 µM CoM, or 1000 µM methyl-CoM as a factorial arrangement of treatments with two factors: i) methanogenesis inhibition (Control or BES), and ii) addition of methyl group carriers (Control, CoM, or methyl-CoM), and their interaction: response ijkl = general mean + methanogenesis inhibition i (Control or BES) + methyl group carrier j (Control, CoM, or methyl-CoM) + interaction ij + cow k + incubation l + error ijklm When the addition of CoM or methyl-CoM was significant ( P < 0.05) or tendencies (0.05 ≤ P ≤ 0.10), CoM and methyl-CoM addition were each separately compared with the Control treatment with 0 CoM or methyl-CoM. When interactions between methanogenesis inhibition and CoM or methyl-CoM were significant ( P < 0.05) or tendencies (0.05 ≤ P ≤ 0.10), the effect of BES addition was separately analyzed across the addition of CoM or methyl-CoM as follows: Contrast 1 (CoM) = [(Control, Control) – (BES, Control)] – [(Control, CoM) – (BES, CoM)] Contrast 2 (methyl-CoM) = [(Control, Control) – (BES, Control)] – [(Control, methyl-CoM) – (BES, methyl-CoM)] Residuals normality and outlier analyses were conducted as in Experiments 1 and 2. Experiment 4 Experiment 4 examined the effects of molar ratios of CoM or methyl-CoM to 3-NOP or BMF greater than those examined in Experiments 1 and 2, on CH 4 production and H 2 accumulation in 48-h mixed ruminal cultures. General rumen inocula sampling and preparation procedures were as described for Experiments 1, 2, and 3. Rumen inoculum from each cow (2.4 mL) was delivered under O 2 -free CO 2 into 27-mL 18 × 150 mm anaerobic serum tubes (Bellco Glass, Inc., Vineland, NJ, US) containing 9.6 mL of medium [ 29 ] and 120.6 [120.4, 120.8] (mean [min, max]) mg of the same high forage substrate incubated in the previous experiments. Each tube received 0.5 mL of distilled water, or CoM or methyl-CoM solutions prepared as in the previous experiments to achieve final concentrations of 0 (methyl carrier Control), 2.5 mM CoM, or 2.5 mM methyl-CoM. In turn, each tube received 10 µL distilled water (methanogenesis inhibition Control) or stock solutions of 3-NOP or BMF to achieve different final concentrations. Initially, 3-NOP final concentrations were 0.1, 0.5, or 1.0 µM; however, as 3-NOP at those concentrations did not inhibit methanogenesis, they were increased to 1.0, 2.5, or 5.0 µM for incubations 2 and 3 of Experiment 4. Bromoform final concentrations were 1.0, 0.5, or 0.1 µM in all three incubation runs of Experiment 4. A treatment containing 5.0 µM BES final concentration was included as a positive control. As BMF was dissolved in ethanol, each Control, 3-NOP, and BES tube also received 10 µL of ethanol, and each BMF tube also received 10 µL distilled water. Tubes were sealed under O 2 -free CO 2 and incubated in an oscillating water bath at 39°C and 60 rpm for 48 h. At the end of the incubation, gas pressure and gas contents of CH 4 and H 2 were measured following the procedures of the previous experiments. Tubes were then opened and pH and E h measured as before. Three incubation runs were conducted in different weeks. Results were analyzed as: response = intercept + BES + 3-NOP + BMF + methyl group addition (Control, CoM, or methyl-CoM) + BES × methyl group addition + 3-NOP × methyl group addition + BMF × methyl group addition + cow + incubation + error Residuals normality and outlier analyses were conducted as previously described. Results Experiment 1 Table 1 shows the main effects and interactions of methanogenesis inhibitors and spiking of CoM at 1 mM. To avoid showing 16 treatment means in one table (i.e., all combinations of the 2 × 4 × 2 factorial), the main effect and interactions of CoM supplementation at 1 µM in serial transfers 1 to 4 are shown separately in Supplementary Table S2. Spiking CoM at 1 mM in serial transfer 5 increased total gas production ( P < 0.001; Table 1 ). Bromoform and to a lesser extent 3-NOP decreased total gas production ( P < 0.001). There was an interaction between methanogenesis inhibition and CoM spiking at 1 mM on CH 4 production ( P = 0.018), by which BES, unlike 3-NOP and bromoform, inhibited methanogenesis by 58% without 1 mM CoM spiking but did not inhibit CH 4 production with 1 mM CoM ( P < 0.05; Table 1 , Fig. 2 ). Spiking CoM at 1 mM did not affect CH 4 production in the absence of inhibitors of methanogenesis ( P = 0.88; result not shown). Inhibiting methanogenesis with 3-NOP and BMF increased H 2 accumulation by 3.45- and 5.78-fold, respectively ( P < 0.001). Incubations with 3-NOP and bromoform had a higher final pH than Control incubations ( P < 0.001). Bromoform and to a lesser extent 3-NOP ( P < 0.001), and CoM spiking at 1 mM ( P < 0.001), increased E h . Both CoM supplementation at 1 µM in serial transfers 1 to 4 ( P = 0.040) and CoM spiking at 1 mM in serial transfer 5 ( P = 0.005), and BMF and to a lesser extent 3-NOP ( P < 0.001) decreased apparent DM disappearance. Bromoform and to a lesser extent 3-NOP decreased total VFA concentration, acetate concentration, and the acetate to propionate molar ratio ( P < 0.001). 3-Nitrooxypropanol increased, and BMF decreased, propionate concentration ( P < 0.05), whereas BMF increased butyrate concentration ( P < 0.05). Coenzyme M spiking at 1 mM increased isobutyrate concentration only when methanogenesis was inhibited (interaction P = 0.008). 3-Nitrooxypropanol and BMF decreased the concentrations of valerate, caproate, heptanoate, and NH 4 + , and increased methanol ( P < 0.001). Coenzyme M spiking at 1 mM increased the concentration of NH 4 + ( P < 0.001). All three inhibitors of methanogenesis decreased succinate concentration ( P = 0.002). pH and heptanoate normal quantile plots exhibited S-shaped deviations. The plots of residuals against predicted observations of CH 4 , H 2 , valerate, caproate, heptanoate, succinate, methanol, and ethanol, were funnel-shaped; however, log-transformation of those y variables did not alter the conclusions of the analyses. No outliers unrelated to the rest of the observations of their treatment were found. As the serial incubations progressed in serial transfers 1 to 4, production of total gas ( P < 0.001; Supplementary Figure S1 ) and CH 4 ( P < 0.001; Supplementary Figure S2) decreased, whereas E h increased ( P < 0.001; Supplementary Figure S3). Accumulation of H 2 ( P = 0.85) and pH ( P = 0.32) were unaffected (not shown). None of the response variables in serial transfers 1 to 4 were affected by CoM supplementation at 1 µM ( P ≥ 0.41; not shown). Experiment 2 Spiking 1mM methyl-CoM relieved the effects of BES ( P ≤ 0.003) but not of 3-NOP ( P ≥ 0.62) or BMF ( P ≥ 0.24) on CH 4 production, H 2 accumulation, acetate and isobutyrate concentrations, the acetate to propionate molar ratio, and the concentration of succinate and ethanol (interaction methanogenesis inhibition × methyl-CoM P ≤ 0.029; Table 2 ). In the absence of methyl-CoM spiking, BES decreased CH 4 production (-52%) and acetate concentration, and increased the accumulation of H 2 (5.01-fold), caproate, succinate, and ethanol ( P < 0.05). Both with and without 1 mM methyl-CoM, 3-NOP decreased CH 4 (-29.6%) and the acetate to propionate molar ratio, and increased H 2 accumulation (3.28-fold), caproate, and ethanol ( P < 0.05), and BMF decreased total gas production, CH 4 (-97.1%), total VFA concentration, acetate, isobutyrate, 2- and 3-methylbutyrate, caproate, the acetate to propionate molar ratio, succinate, and NH 4 + ( P < 0.05), and increased the accumulation of H 2 (14.7-fold), and methanol ( P < 0.05). Bromoform tended to increase butyrate concentration ( P = 0.081). Residuals as a function of predicted values were not uniformly distributed for H 2 , the acetate to propionate molar ratio, and succinate; however, log-transformation of the response variables did not alter the shape of the plots or the conclusions of those analyses. Normal quantile plots of pH and 2- and 3-methylbutyrate were S-shaped, but log-transformation of the response variables did not correct them or change the conclusions of the analyses. Noninfluential outliers following the overall responses of their treatments were detected for H 2 and were not eliminated from the analyses. Experiment 3 Both CoM and methyl-CoM relieved the inhibition of total gas and CH 4 production, the accumulation of H 2 and ethanol, the decrease in the acetate to propionate ratio, and the decrease in the concentrations of 2- and 3-methylbutyrate, succinate, and NH 4 + , caused by BES (interactions BES by CoM or methyl-CoM P ≤ 0.044; Figs. 3 – 5 and Supplementary Figures S4-S8). At 100 µM, CoM and methyl-CoM reversed approximately 62 and 68% of methanogenesis inhibition by BES, respectively, and at 1000 µM, CoM and methyl-CoM reversed 93 and 100% methanogenesis inhibition, in the same order. 2-Bromoethanesulfonate decreased ( P ≤ 0.036) total VFA and isobutyrate concentration whereas methyl-CoM tended ( P = 0.066) to increase isobutyrate (Supplementary Figures S9 and S10). Methyl-coenzyme M (interaction BES by methyl-CoM P = 0.010) but not CoM (interaction BES by methyl-CoM P = 0.23) reversed the decrease in acetate concentration caused by BES (Supplementary Figure S11). There was a tendency for CoM to decrease propionate ( P = 0.056), especially with BES (interaction BES × CoM; P = 0.062; Supplementary Figure S12). In the presence of BES, CoM and methyl-CoM decreased or tended to decrease valerate, caproate, and heptanoate (interaction of BES with CoM or methyl-Com P ≤ 0.095; Supplementary Figures S13-S15). There were no effects of BES, CoM, methyl-CoM, or the interactions of BES with CoM or methyl-CoM on DM disappearance and butyrate and methanol concentrations ( P ≥ 0.17; not shown). Coenzyme M and methyl-CoM increased or tended to increase pH when methanogenesis was inhibited by BES ( P ≤ 0.055; Supplementary Figure S16). 2-Bromoethanesulfonate tended to decrease ( P = 0.071), and methyl-CoM increased, E h ( P = 0.017; Supplementary Figure S17). Log-transforming total gas production and butyrate, caproate, heptanoate, and NH 4 + concentrations did not correct S-shaped residual normality plots or funnel-shaped plots of residuals against predicted values, nor changed the conclusions of those analyses, so the analyses with the untransformed variables are reported. An outlier with no H 2 accumulation in the BES treatment with 1 µM CoM was removed from the analysis. Noninfluential outliers with high methanol and ethanol concentrations were left in the analyses. The rumen fluid inocula from both cows in both incubations had 55.3 ± 1.81 (mean ± SD) µM CoM plus methyl-CoM, ranging between 53.2 and 57.4 µM (data not shown). A total of 152 unique archaeal ASVs were assigned at the phylum level. The rarefaction curve revealed adequate coverage (Supplementary Figure S18). At the phylum level, the addition of BES decreased the relative abundance of total Euryarchaeota 16S rRNA gene ( P = 0.011; Supplementary Figure S19), whereas methyl-CoM at 1 mM increased total Euryarchaeota relative abundance ( P = 0.005); the opposite changes were observed in the relative abundance of Thermoplasmatota ( P ≤ 0.015). At the genus level, there was an interaction between methanogenesis inhibition and methyl group carriers on the relative abundance of Methanobrevibacter ruminantium ( P = 0.005), through which CoM ( P = 0.028) and methyl-CoM ( P = 0.002) at 1 mM reversed the decrease in M. ruminantium caused by BES (Fig. 6 ). Other Methanobrevibacter , Methanosphaera spp., and Candidatus Methanomethylophilus were unaffected by any of the treatments or their interactions ( P ≥ 0.16). The relative abundance of unclassified Methanomethylaceae was increased by BES ( P = 0.010) and decreased by methyl-CoM ( P = 0.005); changes in the relative abundance of unclassified Methanomethylaceae reflected changes in M. ruminantium , as absolute reads of unclassified Methanomethylaceae were unaffected ( P ≥ 0.15; Supplementary Fig. 20). A PCA plot revealed that most of the treatments supplemented with BES and no CoM or methyl-CoM separated from their functional methanogenesis controls, whereas the treatments receiving BES plus CoM or methyl-CoM appeared closer to the methanogenesis inhibition controls (Supplementary Figure S21). There were no effects of BES, CoM, or methyl-CoM or their interactions on archaeal diversity indices ( P ≥ 0.24; Supplementary Table S3). A total of 23,929 unique bacterial ASVs were assigned at the phylum level. The rarefaction curve revealed adequate coverage (Supplementary Figure S22). Only those bacterial clades whose relative abundance was equal or greater than 0.5% of the total bacterial 16S rRNA gene in at least one combination of BES and CoM or methyl-CoM treatment are presented. At the phylum level, BES decreased the relative abundance of Actinobacteriota ( P = 0.033; Supplementary Table S4). Coenzyme M tended to increase the relative abundance of Spirochaetota ( P < 0.10). Methyl-CoM relieved ( P = 0.048) and CoM tended to relieve ( P = 0.093) a decrease in the relative abundance of Verrucomicrobiota caused by BES (interaction BES × CoM/methyl-CoM P = 0.10). There were no effects of BES, CoM, methyl-CoM, or their interactions, on the relative abundance of other bacterial phyla ( P ≥ 0.11). At the genus level, BES decreased the relative abundances of Olsenella ( P = 0.029; Supplementary Table S5) and Butyrivibrio ( P = 0.007) and tended to increase those of the Rikenellaceae RC9 group ( P = 0.050). Coenzyme M increased the relative abundance of [Eubacterium] halli group ( P = 0.024). Coenzyme M tended ( P = 0.067) and methyl-CoM decreased ( P = 0.010) the relative abundance of Ruminococcus . Methyl-CoM decreased the relative abundance of Horsej-a03 ( P = 0.036). Coenzyme M and methyl-CoM relieved or partially relieved the decrease in the relative abundance of the Lachnospiraceae UCG-008 group (interaction BES × CoM P = 0.028) and Papillibacter ( P = 0.018), respectively. Most treatments receiving methyl-CoM and BES clustered with Control treatments with no added BES in a PCA plot (Supplementary Fig. 23). Experiment 4 There were no effects of BES (not shown), 3-NOP, BMF, methyl group carriers, or their interactions, on total gas production ( P ≥ 0.19; Supplementary Figures S24A, B). 2-Coenzyme M and methyl-CoM overcame methanogenesis inhibition (BES × methyl group carriers P = 0.006; Supplementary Figure S25) and H 2 accumulation (BES × methyl group carriers P = 0.025; Supplementary Figure S26) by BES. 3-Nitrooxypropanol tended ( P = 0.095) to decrease CH 4 production by a maximum of 9.5% (Fig. 7 ) and BMF decreased ( P < 0.001; Fig. 8 ) CH 4 production by a maximum of 38%, without interactions with the addition of methyl group carriers ( P ≥ 0.56; Figs. 7 and 8 ). Accumulation of H 2 was decimal log-transformed to correct for residual normality and positive outliers. Nitrooxypropanol ( P = 0.012; Fig. 9) and BMF increased ( P < 0.001; Fig. 10) H 2 accumulation. The addition of methyl group carriers did not interact with 3-NOP ( P = 0.55; Fig. 9) or BMF ( P = 0.14; Fig. 10) on H 2 accumulation. Both BES ( P = 0.051; Supplementary Figure S27) and BMF ( P = 0.087; Supplementary Figure S28) tended to increase final pH. Bromoform ( P = 0.028; Supplementary Figure S29), CoM ( P < 0.001; Supplementary Figure S30), and methyl-CoM ( P < 0.001; Supplementary Figure S30) decreased final E h . Discussion As we hypothesized, and in agreement with previous observations in pure cultures of methanogens, methanogen cell extracts, purified MCR, and methanogenic sediments [ 15 , 17 , 21 , 22 , 24 ], the addition of CoM or methyl-CoM to rumen cultures blocked the inhibitory effect of BES on methanogenesis. In Experiment 3, molar ratios of added CoM and methyl-CoM to BES of 20 µM/µM (i.e., at 100 µM CoM or methyl-CoM) reversed most of the inhibition of methanogenesis by BES. Similarly, Konisky [ 24 ] reported that in CH 4 -producing marine sediments, molar ratios of CoM to BES between 20 and 40 µM/µM reversed approximately half of the inhibition of methanogenesis. The question arises whether the reversal of methanogenesis inhibition demonstrated herein with rumen cultures could influence BES efficacy at typical in vivo concentrations of CoM and methyl-CoM in the rumen, as the concentration of CoM plus methyl-CoM in the inoculum of Experiment 3 was comparable to the concentration of CoM or methyl-CoM affecting BES efficacy in Experiment 3. We are not aware of previous reports of the concentration of CoM or methyl-CoM in the rumen, or in mixed rumen cultures. Although the range of concentrations of CoM plus methyl-CoM in the rumen inocula used in Experiment 3 was relatively narrow, the inocula were sampled in two consecutive weeks from the same two rumen fistulated cows penned together and fed the same diet. It is possible that the concentrations of CoM and methyl-CoM vary more amply among different types and species of ruminants eating different diets and in different geographical locations, and that variation in the concentrations of CoM and methyl-CoM in the rumen could thus influence BES efficacy. Elias et al. [ 60 ] reported concentrations of CoM ranging between 0.02 and 0.22 µM for pond and landfill sediments, sewage sludge, and a hydrocarbon-contaminated sediment below 1.2 m deep. We found considerably greater concentrations of CoM plus methyl-CoM (between 50 and 60 µM) in the rumen inocula utilized for Experiment 3. However, the concentration of CoM in the rumen might be higher than in other environments, as the rumen environment contains high concentrations of metabolites derived from microbial activities [ 61 ], and some of the densities of methanogens in the rumen conducted using classical cultivation techniques summarized by Joblin [ 62 ], appear to be greater than the density of methanogens reported by Elias et al. [ 60 ] for other methanogenic environments. Perhaps more importantly, CoM and methyl-CoM may have implications for the persistence of the antimethanogenic effect of BES on rumen fermentation. 2-Bromoethanesulfonate is considered a transient inhibitor of methanogenesis [ 63 , 64 ], although in vitro and in vivo experimental results concerning the persistence of BES are scarce and partially conflicting. In 24- or 48-h mixed rumen batch cultures, BES at relatively low concentrations ≤ 30 µM has consistently inhibited methanogenesis [ 65 – 71 ]. One pulse dose of 50 µM BES to continuous cultures also inhibited CH 4 production for approximately 24 h [ 72 ]. Conversely, in vivo inhibition of methanogenesis by BES daily dosed to a sheep was shown to last for only 4 d, after which the rumen microbiota seemed to adapt to BES, and CH 4 production increased again [ 73 ]. In rumen continuous cultures, BES at the relatively high concentration of 250 µM inhibited CH 4 production in the first 2 d of incubation, but was ineffective after a 7-d adaptation period [ 74 ]; in contrast, in a semicontinuous culture study, BES at 36 or 72 µM effectively inhibited CH 4 production and decreased methanogen abundance even after 8 d of adaptation [ 75 ]. Ahring et al. [ 76 ] dosed a long-term (30 d) semicontinuous rumen-inoculated bioreactor with very high 10 mM pulse doses of BES on d 1 and 15 of incubation and reported profound inhibition of methanogenesis. Similarly, Webster et al. [ 77 ] reported severe inhibition of CH 4 production 9 d after dosing with a relatively high dose of 0.5 mM BES a bioreactor inoculated with cow dung and wastewater. The transient effects of BES on methanogenesis may be related to changes in the composition of the rumen archaeal community triggered by BES supplementation, as differences in the sensitivity of different methanogens to BES have been reported [ 11 , 13 , 22 , 30 ]. The uptake of CoM by the ruminal methanogen M. ruminantium strain M1 was inhibited by BES, indicating competition between CoM and BES for transmembrane transport [ 19 ]. There are important differences among methanogens in the uptake of CoM [ 61 ]. The ruminal methanogen M. ruminantium strain M1, which lacks three genes of the CoM biosynthetic pathway [ 78 ], requires exogenous CoM [ 79 ]. On the other hand, the non-ruminal strain M. ruminantium strain PS, and other ruminal and non-ruminal methanogens, do not take up CoM or take it up at much lower rates than M. ruminantium M1 [ 19 ]. The ruminal methanogen Methanomicrobium mobile 1, which is considerably more resistant to BES than M. ruminantium M1 [ 11 ], takes up CoM at a rate less than 10% the CoM uptake rate of M. ruminantium M1 [ 19 ]. In agreement with those findings, resistance to BES in non-ruminal methanogens Methanococcus voltae and Methanosarcina 227 was related to low rates of transport of BES into the cell [ 15 , 23 ]. The existence of a common transmembrane transport system for BES and its structural analogs CoM and methyl-CoM has been proposed [ 23 ]. We searched GenBank for the capacity of the archaeal clades present in Experiment 3 samples to synthesize CoM, based on the presence of genes comA , comB , comC , comD , and comE in their genomes. Within Methanobacteriales , with the exception of M. ruminantium (strain M1, Leahy et al. [ 78 ]), all other Methanobacteriales identified in Experiment 3 seemed to have the genetic capacity to synthesize CoM [ 56 ]. In Experiment 3 the relative abundance of Methanobacteriales other than M. ruminantium was unaffected by BES or by BES interactions with CoM or methyl-CoM. M. ruminantium M1 is highly sensitive to BES [ 11 , 13 , 30 ], and in agreement, the abundance of M. ruminantium was decreased by BES by more than two thirds in Experiment 3, and almost completely recovered when 1 mM CoM or methyl-CoM was supplemented along BES. Our observation that CoM and methyl-CoM negate the effects of BES on M. ruminantium abundance and on CH 4 production agrees with the sensitivity of methanogens to BES being mediated by their capacity to synthesize CoM. Ruminal Methanomassiliicoccales isolates ISO4-G1, ISO4-H5, and Thermoplasmatales archaeon BRNA1 lack CoM biosynthetic genes comADE and thus cannot synthesize CoM [ 61 , 80 , 81 ]. Methanomassiliicoccales ISO4-H5 is highly sensitive to BES [ 30 ], suggesting that, as in M. ruminantium , the requirement to take up exogenous CoM also results in high rates of intracellular transport of BES. In Experiment 3, we did not find effects of BES or BES interactions with CoM or methyl-CoM on the relative abundance of a relatively small population of Candidatus Methanomethylophilus sp., or on other Methanomassiliicoccales ; however, because our metataxonomic analysis did not have enough depth to identify all archaeal ASVs at the species or strain level, it is difficult to speculate on whether the Methanomassiliicoccales in our cultures had the genetic capacity to synthesize CoM. Future studies with ruminal mixed cultures or in vivo could use metagenomics or qPCR to directly quantify the effects of BES on CoM biosynthetic genes, to accurately confirm whether the effects of BES and its interactions with CoM and methyl-CoM on CH 4 production are related to the genetic capacity of the mixed methanogen community to synthesize CoM. Therefore, in addition to oxidizing the Ni + 1 atom in cofactor F 430 in MCR [ 16 , 82 ], BES inhibits methanogenesis in M. ruminantium M1 and other methanogens that cannot synthesize CoM by blocking the uptake of CoM. An implication of this second mechanism of action is that those methanogens that can synthesize their own CoM and are not reliant on exogenous CoM would be more tolerant to BES, as they may not take up external CoM or they would do it at slower rates [ 19 ], thus also taking up BES at slower rates or not taking it up. Thus, it appears possible that long-term supplementation of BES could select for those methanogens that can synthesize CoM and do not transport CoM and BES into their cells, resulting in lower sensitivity to BES and ultimately in transient inhibition of methanogenesis. In addition, MCRs of different rumen methanogens may differ in their sensitivity to BES. For example, MCR isolated from M. ruminantium [ 83 ] was more sensitive to BES than MCR isolated from non-ruminal methanogen Methanothermobacter marburgensis [ 14 , 17 ], although it should be cautioned that those determinations were not direct comparisons conducted in the same study. In Experiment 1, we investigated whether supplementation of CoM at 1 µM in the first four of five serial transfers of rumen serial cultures would favor those methanogens lacking the capacity to synthesize CoM and presumably more sensitive to BES, increasing the efficacy of BES to inhibit methanogenesis. However, supplementing CoM at 1 µM in serial transfers 1 to 4 in Experiment 1, however, had minimal effects on fermentation. This may be related to the presumably relatively small increase in CoM concentration resulting from the 1 µM supplementation dose utilized in serial transfers 1 to 4 in Experiment 1, in comparison with the concentration of CoM that the non-supplemented cultures may have had. Inhibiting methanogenesis affects the rumen microbial community beyond methanogens through the resulting accumulation of H 2 , which profoundly affects electron flows in rumen fermentation through the thermodynamics of H 2 -producing and H 2 -incorporating pathways [ 84 – 86 ]. We observed a decrease in Olsenella when methanogenesis was inhibited with BES. Olsenella isolated from the sheep rumen and pig jejunum produces acetate as a minor product in pure culture [ 87 ] but it is possible that in mixed cultures in the presence of methanogens and other hydrogenotrophs removing H 2 Olsenella produces greater amounts of acetate than in pure culture. If that is the case, the accumulation of H 2 caused by BES could inhibit the release of H 2 associated to acetate production [ 85 ], thus explaining the inhibitory effect of BES on Olsenella ´s abundance in Experiment 3. Similarly, butyrate production involves a release of H 2 [ 85 ] and the H 2 built up resulting from methanogenesis inhibition by BES might have thus decreased butyrate-producing Butyrivibrio ´s relative abundance in Experiment 3. Even though hydrogenotrophic bacterial genera such as Prevotella and Selenomonas are expected to increase as a consequence of methanogenesis inhibition and H 2 accumulation, experimental results in this respect have been variable [ 86 ], which is in agreement with the lack of effects on those genera observed in Experiment 3. The effects of BES, CoM and methyl-CoM on the Rikenellaceae RC9 group , [Eubacterium] halli group , and Horsej-a03 are difficult to interpret because of the lack of known isolates characterized in pure cultures or cocultures. In bacteria, CoM is involved in the oxidation of alkenes [ 58 ]. Searching GenBank, we did not find any of the bacterial genera found in Experiment 3 to possess genes related to the biosynthesis of CoM. There has been speculation that, because 3-NOP inhibits MCR by having a similar molecular shape as methyl-CoM [ 12 ], the concentration of CoM and methyl-CoM in rumen fluid may influence 3-NOP efficacy [ 88 ]. However, spiking rumen cultures with CoM or methyl-CoM at 1 mM in Experiments 1 and 2 did not hinder the inhibition of methanogenesis by 3-NOP. In Experiment 4, we examined molar ratios of CoM and methyl-CoM as high as 25,000 to 1 and could not find hindering effects of CoM or methyl-CoM on the antimethanogenic effect of 3-NOP; however, because of reasons unrelated to CoM or methyl-CoM supplementation, the effects of 3-NOP on CH 4 production were considerably milder than those found in Experiments 1 and 2. Both BES and 3-NOP irreversibly inactivate MCR through oxidation of the Ni 1+ atom in cofactor F 430 bound to MCR, to Ni 2+ [ 12 , 16 , 17 ]. Unlike BES, which is a charged molecule that needs to be actively transported into the cells, 3-NOP is a polar but uncharged molecule thought to freely diffuse across cell membranes [ 12 , 16 , 26 ]. The requirement for a transmembrane transporter for BES and the resulting competition between BES and CoM for intracellular transport [ 23 ], but not for 3-NOP, could thus explain why CoM and methyl-CoM hindered the antimethanogenic effect of BES, but not the antimethanogenic effect of 3-NOP which would not compete for transporters. The active uptake of CoM in M. ruminantium M1 and other methanogens requiring exogenous CoM, might theoretically be expected to competitively displace 3-NOP from the MCR catalytic site, thus affecting 3-NOP efficacy even if it does not compete for transmembrane transporters. However, the active transport of CoM in M. ruminantium displays saturation kinetics, with the rate of uptake of CoM plateauing at approximately 0.63 µM [ 19 ]. Therefore, increasing the concentration of CoM in the medium might not have altered the intracellular CoM concentration to compete with 3-NOP for access to the catalytic site in MCR. In the present study, the addition of CoM or methyl-CoM did not interfere with the inhibition of methanogenesis by BMF, even at molar ratios of CoM or methyl-CoM to BMF as high as 25,000 to 1 in Experiment 4. Bromoform and other CH 4 halogenated analogs inhibit methanogenesis by reacting with the corrinoid unit in MTR, blocking the transfer of a methyl group from MTR to CoM to form methyl-CoM [ 25 ]. Yu and Smith [ 89 ] proposed that CH 4 halogenated analogs can inhibit methanogenesis by binding not only corrinoid-containing MTR but also porphyrin-containing MCR, as well as free intracellular corrinoids and porphinoids. Observations showing that corrinoids and also cofactor F 430 stimulated dechlorination of chlorohydrocarbons by Methanosarcina barkeri [ 90 , 91 ] and that MCR conducted dechlorination of 1, 2-dichloroethane [ 92 ], also support the existence of an interaction of halohydrocarbons with MCR, implying that BMF and other CH 4 halogenated analogs could have two targets, MTR and MCR [ 93 ]. Bromoform has been computationally predicted to bind cofactor F 430 with high affinity [ 26 ]. However, Karuso and Liu [ 94 ] reported that BMF inhibited MCR isolated from Methanothermobacter marburgensis only at considerably high concentrations above 0.50 mM (greater than the methyl-CoM to BMF molar ratio of 20: 1 in that study), which agrees with earlier observations with chloroform [ 95 ]. In contrast to the enzymology study by Karuso and Liu [ 94 ] with BMF and purified MCR, in mixed cultures, BMF could potentially inhibit both MCR and MTR. The fact that we found no alleviation of methanogenesis inhibition by BMF with the addition of methyl-CoM or CoM at much larger molar ratios to BMF than the 20 to 1 molar ratio found to partially relieve inhibition of MCR by Karuso and Liu [ 94 ], considering that BMF rapidly diffuses across cell membranes [ 26 ], suggests that in our study BMF inhibited CH 4 production mainly through its reaction with corrinoids in MTR without affecting MCR. Vitamin B 12 and other corrinoids play a role in various cellular methylation reactions, including the conversion of succinyl-CoA to methyl-malonyl-CoA in propionate randomizing pathway in the rumen [ 96 ]. Therefore, it could be conceivable therefore that BMF interferes with propionate formation via randomizing pathway. However, BMF addition did not cause consistent effects on propionate production and did not induce succinate accumulation in Experiments 1 and 2, suggesting that the conversion of succinyl-CoA to methyl-malonyl-CoA was not impaired by BMF. These results agree with previous rumen batch cultures work in which bromoform and other CH 4 -halogenated analogs did not impaired or improved, propionate production [ 13 , 97 , 98 ], although a decrease in propionate was observed in continuous cultures supplemented bromochloromethane [ 97 ]. Conclusions Both CoM and methyl-CoM hindered the effects of BES to inhibit methanogenesis and decrease the abundance of M. ruminantium . These results suggest that the inhibition of methanogenesis by BES and its reversal by CoM and methyl-CoM may be related to the capacity of methanogens to synthesize CoM, which can have implications regarding the lack of persistence of BES on CH 4 production in vivo. Whether the sensitivity of methanogens in mixed cultures and in vivo to BES is related to their genetic capacity to synthesize CoM affecting BES transmembrane transport needs to be confirmed through metagenomics or qPCR quantification of CoM biosynthetic genes. The efficacy of 3-NOP and BMF was not influenced by CoM or methyl-CoM. Although both BES and 3-NOP affect MCR through similar mechanisms, CoM and methyl-CoM compete for transmembrane transport with BES, but CoM and methyl-CoM do not seem to compete with 3-NOP for transmembrane transport. In our study, BMF inhibition of methanogenesis seemed to be mediated through the inhibition of MTR rather than MCR. Abbreviations ADF, Acid detergent fiber ASV, Amplicon sequence variant BES, 2-Bromoethanesulfonate BMF, Bromoform CoM, Coenzyme M CP, Crude protein DM, Dry matter DMD, Apparent dry matter disappearance EE, Ether extract GC, Gas chromatography GC-MS, Gas chromatography coupled to mass spectrometry HSD, Honestly Significant Difference LSD, Least squares difference Methyl-CoM, Methyl-coenzyme M MCR, Methyl-coenzyme M reductase MTR, Methyltetrahydromethanopterin: coenzyme M methyltransferase NDF, Neutral detergent fiber 3-NOP, 3-Nitrooxypropanol PCA, Principal components analysis VFA, Volatile fatty acids Declarations Ethics approval and consent to participate All animal procedures were approved by Instituto de Investigaciones Agropecuarias (approval number 03/2024). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials The 16S rRNA gene sequence files generated in this study are available at https://www.ncbi.nlm.nih.gov/sra/PRJNA1375672. The datasets supporting the conclusions of this article are available in the Open Science Framework repository, project dxy8e https://osf.io/dxy8e/overview Funding This research was funded by Agencia Nacional de Investigación y Desarrollo, Santiago, Chile, Proyecto Fondecyt 1240264. We are grateful to Universidad de Buenos Aires for a UBAINT Doctoral 2023-2024 fellowship granted to Florencia Samoluk and to the Global Research Alliance on Agricultural Greenhouse Gases (GRA) and the CGIAR Initiative on Low-Emission Food Systems (Mitigate+) for supporting Boma Iriso through their CLIFF-GRADS program. Authors´ contributions EMU conceived, designed, and conducted the experiments and analyzed the samples, analyzed the data, and wrote the manuscript. 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Effect of iodoform in maize and clover grass silages: An in vitro study. Ruminants. 2024, 4 : 418-432. doi: 10.3390/ruminants4030030 Tables Table 1. Effects of spiking coenzyme M (CoM) at 1 mM on the efficacy of methanogenesis inhibitors (Inh) 2-bromoethanesulfonate (BES), 3-nitrooxypropanol (3-NOP) or bromoform (BMF), all at 5 µM, in serial transfer 5 of mixed serial rumen cultures that had received CoM at 0 or 1 µM in serial transfers 1 to 4. Results are presented across the average of 0 and 1 µM CoM supplementation in serial transfers 1 - 4 (Experiment 1). Methanogenesis inhibition Control BES 3-NOP BMF SEM P = Inh CoM Inh × CoM CoM 0 1 mM 0 1 mM 0 1 mM 0 1 mM Total gas (mmol) 4.17 ab 1 4.23 ab 4.12 a 4.23 a 4.11 b 4.17 b 4.03 c 4.07 c 0.082 <0.001 <0.001 0.12 CH 4 (µmol) 129 a 127 a 54.3 j 2 123 i 33.9 m 36.8 m ND x ND x 30.6 <0.001 0.058 0.018 H 2 (µmol) 17.8 c 12.7 c 21.1 c 10.5 c 49.4 b 53.9 b 79.3 a 87.4 a 8.62 <0.001 0.90 0.71 pH 6.33 a 6.27 a 6.31 ab 6.26 ab 6.27 bc 6.24 bc 6.29 c 6.25 c 0.053 <0.001 <0.001 0.36 E h -105 b -108 b -104 -101 -99.3 n -99.0 m -98.9 -94.8 5.39 <0.001 <0.001 0.004 DMD (%) 57.5 ab 56.7 ab 57.7 a 56.9 a 56.5 b 56.5 b 53.9 c 53.2 c 1.10 <0.001 0.005 0.54 Total VFA (mM) 69.6 a 69.3 a 67.3 a 69.3 a 65.8 b 66.5 b 59.4 c 59.5 c 1.62 <0.001 0.25 0.44 Acetate (mM) 36.2 a 36.5 a 34.6 a 36.7 a 33.2 b 33.7 b 28.9 c 28.5 c 1.30 <0.001 0.21 0.36 Propionate (mM) 24.5 b 24.0 b 24.5 b 24.3 b 25.3 a 25.8 a 22.4 c 22.5 c 0.95 <0.001 0.88 0.63 Butyrate (mM) 4.66 b 4.78 b 4.91 b 4.34 b 5.29 b 4.92 b 6.39 a 6.50 a 0.29 <0.001 0.29 0.35 Isobutyrate (mM) 0.34 0.32 0.29 j 0.33 i 0.29 n 0.33 m 0.26 y 0.30 x 0.030 <0.001 <0.001 0.008 2- and 3-methylbutyrate (mM) 0.53 a 0.47 b 0.42 j 0.50 i 0.42 n 0.50 m 0.33 y 0.43 x 0.072 <0.001 <0.001 <0.001 Valerate (mM) 1.93 a 1.81 a 1.55 a 1.81 a 1.00 b 1.03 b 0.97 b 1.04 b 0.46 <0.001 0.56 0.66 Caproate (mM) 0.87 a 0.80 a 0.61 a 0.78 a 0.17 b 0.17 b 0.14 b 0.18 b 0.32 <0.001 0.69 0.79 Heptanoate (mM) 0.63 a 0.58 a 0.38 a 0.54 a 0.046 b 0.057 b 0.014 b 0.024 b 0.24 <0.001 0.69 0.81 Acetate to propionate (mM/mM) 1.51 1.54 1.42 1.52 1.32 1.31 1.29 1.27 0.084 <0.001 0.40 0.54 Succinate (mM) 0.40 a 0.37 a 0.35 b 0.34 b 0.33 b 0.33 b 0.35 b 0.35 b 0.018 0.002 0.56 0.78 Methanol (µM) 12.1 a ND a ND a ND a 412 b 163 b 652 b 411 b 93.2 <0.001 0.011 0.84 Ethanol (mM) 7.85 a 3 8.08 a 9.87 a 8.07 a 12.8 b 13.1 b 14.6 b 14.6 b 2.00 <0.001 0.61 0.58 NH 4 + (mM) 20.1 a 20.4 a 19.3 ab 20.3 ab 19.0 b 20.0 b 19.0 b 20.2 b 1.14 <0.001 <0.001 0.076 1 When the main effect of methanogenesis inhibition was significant ( P 0.05), unlike superscripts between inhibitors indicate significant differences ( P < 0.05) according to Tukey´s HSD; 2 When the interaction between methanogenesis inhibition and CoM spiking was significant ( P < 0.05), the effect of CoM is separately evaluated on the efficacy of each inhibitor and unlike superscripts within an inhibitor of methanogenesis indicate that the effect of the inhibitor was affected by CoM spiking ( P < 0.05): a, b = CoM effect within Control; i, j = CoM effect within BES; m, n = CoM effect within 3-NOP; x, y = CoM effect within BMF; 3 Calculated initial concentration of ethanol was 10.4 mM. Table 2. Effects of spiking methyl-coenzyme M (methyl-CoM) at 1 mM on the efficacy of methanogenesis inhibitors (Inh) 2-bromoethanesulfonate (BES), 3-nitrooxypropanol (3-NOP) or bromoform (BMF), all at 5 µM, in batch rumen cultures (Experiment 2). CH 4 production inhibition Control BES 3-NOP BMF SEM P = Methyl-CoM 0 1 mM 0 1 mM 0 1 mM 0 1 mM Inh Methyl-CoM Inh × Methyl-CoM Total gas (mmol) 4.67 a 1 4.64 a 4.56 a 4.63 a 4.55 a 4.56 a 4.37 b 4.39 b 0.069 <0.001 0.52 0.65 CH 4 (µmol) 456 a 441 a 220 j 435 i 315 m 325 m 12.8 x 12.9 x 59.7 <0.001 0.016 <0.001 H 2 (µmol) 12.7 a 17.3 a 63.4 i 16.4 j 49.8 m 48.3 m 224 x 217 x 14.0 <0.001 0.033 0.013 pH 6.16 6.16 6.19 6.15 6.19 6.17 6.18 6.18 0.053 0.54 0.27 0.62 DMD (%) 26.9 23.9 23.3 23.3 24.9 24.6 22.9 23.1 6.62 0.49 0.53 0.78 Total VFA (mM) 82.2 a 82.4 a 77.6 a 83.6 a 78.6 a 78.1 a 70.0 b 68.3 b 3.06 <0.001 0.43 0.18 Acetate (mM) 52.8 a 52.5 a 45.4 b 53.4 a 48.0 m 47.8 m 37.7 x 37.2 x 2.16 <0.001 0.048 0.002 Propionate (mM) 15.4 15.6 17.4 15.6 16.3 16.0 17.7 17.1 1.83 0.065 0.22 0.52 Butyrate (mM) 10.9 11.1 11.6 11.3 11.2 11.2 11.9 11.5 0.75 0.081 0.54 0.66 Isobutyrate (mM) 0.69 a 0.73 a 0.65 j 0.75 i 0.67 m 0.68 m 0.54 x 0.50 x 0.058 <0.001 0.079 0.029 2- and 3-methylbutyrate (mM) 1.17 a 1.24 a 1.04 a 1.27 a 1.12 a 1.14 a 0.89 b 0.86 b 0.15 <0.001 0.059 0.11 Valerate (mM) 1.09 1.12 1.21 1.13 1.13 1.14 1.09 1.05 0.052 0.10 0.38 0.48 Caproate (mM) 0.15 ab 0.18 ab 0.26 a 0.17 a 0.21 a 0.20 a 0.10 b 0.095 b 0.042 <0.001 0.27 0.15 Acetate to propionate (mM/mM) 3.52 a 3.45 a 2.66 j 3.48 i 3.04 m 3.07 m 2.15 x 2.19 x 0.35 <0.001 0.030 0.004 Succinate (mM) 0.81 a 0.95 a 0.60 j 0.97 i 0.77 m 0.81 m 0.55 x 0.51 x 0.14 <0.001 0.007 0.016 Methanol (µM) ND ND ND ND ND ND 0.50 0.55 0.055 <0.001 0.75 0.79 Ethanol (mM) 4.84 a 3 5.56 a 10.7 i 5.74 j 8.22 m 8.12 m 13.8 x 13.7 x 1.93 <0.001 0.034 0.001 NH 4 + (mM) 27.1 a 27.0 a 25.8 a 27.3 a 26.2 a 26.7 a 23.4 b 23.9 b 1.60 0.05), unlike superscripts between inhibitors indicate significant differences ( P < 0.05) according to Tukey´s HSD; 2 When the interaction between methanogenesis inhibition and methyl-CoM spiking is significant ( P < 0.05), the effect of methyl-CoM is separately evaluated on the efficacy of each inhibitor and unlike superscripts within an inhibitor of methanogenesis indicate a significant effect of methyl-CoM spiking ( P < 0.05): a, b = methyl-CoM effect within Control; i, j = methyl-CoM effect within BES; m, n = methyl-CoM effect within 3-NOP; x, y = methyl-CoM effect within BMF; 3 Initial concentration of ethanol was 10.4 mM. 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09:20:51","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":247354,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/c7eac2f8bdb9f950d6cbb221.png"},{"id":99594613,"identity":"5780986e-01d6-4f9e-84d7-ecdb3b0945ae","added_by":"auto","created_at":"2026-01-06 09:20:51","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":241581,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/3db9d2380288113877351985.png"},{"id":99594620,"identity":"a2c22cb1-0e84-40cb-bf2c-79a515dd8f54","added_by":"auto","created_at":"2026-01-06 09:20:51","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":441917,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/7b2a6f732ecfb7e0df2ea3f5.png"},{"id":99794173,"identity":"1c4d0607-f39e-46b5-80e7-8675c2f8374a","added_by":"auto","created_at":"2026-01-08 13:34:10","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":269639,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/4096ca13fdf023eba3eda5e0.png"},{"id":99792539,"identity":"acffb6f1-18c6-4dcf-84f2-b11cbb11f9ac","added_by":"auto","created_at":"2026-01-08 13:22:00","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":256804,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/12a79128de1625887a50cd9d.png"},{"id":99792342,"identity":"cc871fdc-d15c-4a3e-9f25-297302a1ad40","added_by":"auto","created_at":"2026-01-08 13:18:21","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":238024,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/b709a5b57068081f04bc8ef0.png"},{"id":99793085,"identity":"9cb0bcaf-89d0-4160-b353-8c81faaa0710","added_by":"auto","created_at":"2026-01-08 13:30:59","extension":"xml","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":298919,"visible":true,"origin":"","legend":"","description":"","filename":"0782a4d3a94b4c2da2abd59da7f470f81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/59df5c11592d31f0aa817ff7.xml"},{"id":99594617,"identity":"feccdf99-4018-4d82-a0c0-11048be6be41","added_by":"auto","created_at":"2026-01-06 09:20:51","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":315796,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/aa7fcb0a227735c96f6156bb.html"},{"id":99594580,"identity":"ac737f66-ce9f-4e16-bd13-0fc94ad771d4","added_by":"auto","created_at":"2026-01-06 09:20:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138283,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of serial transfer of rumen cultures (Experiment 1). Coenzyme M was supplemented at 0 or 1 µM in serial transfers 1 to 4 and, in serial transfer 5, spiked at 0 or 1 mM, with 5 µM BES, 3-NOP, BMF, or an equivalent volume of distilled water.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/7cd61a6c2d630313a5a9d77e.png"},{"id":99594582,"identity":"609d69f2-b271-4652-a94d-5d836564c012","added_by":"auto","created_at":"2026-01-06 09:20:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":656995,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of methanogenesis inhibitors 2-bromoethanesulfonate (BES), 3-nitrooxypropanol (3-NOP) and bromoform (BMF), all at 5 µM, and of spiking coenzyme M (CoM) at 1 mM, on methane (CH\u003csub\u003e4\u003c/sub\u003e) production in the last transfer of 5-transfer, 48-h mixed serial rumen batch cultures that had been supplemented 0 or 1 µM CoM in transfers 1 to 4 (Experiment 1). Results are presented as averages of the 0 or 1 µM CoM supplementation treatments in transfers 1 to 4 as CoM supplementation at 1 µM and its interactions with the addition of inhibitors of methanogenesis and CoM were not significant (\u003cem\u003eP\u003c/em\u003e ≥ 0.61). Inhibitors, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; CoM spiking, \u003cem\u003eP\u003c/em\u003e = 0.058; Inhibitors × CoM spiking, \u003cem\u003eP\u003c/em\u003e = 0.016. Both 3-NOP and BMF inhibited CH\u003csub\u003e4\u003c/sub\u003e production with and without CoM spiking (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). BES inhibited CH\u003csub\u003e4\u003c/sub\u003e) production without CoM spiking (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) but not with CoM spiking (\u003cem\u003eP\u003c/em\u003e = 0.83).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/c44b6819ff939dd19878ddf8.png"},{"id":99792630,"identity":"cacba7fa-2ba6-4025-9065-255563e65d4c","added_by":"auto","created_at":"2026-01-08 13:23:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":760731,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 2-bromoethanesulfonate (BES, 0 or 5 µM), coenzyme M (CoM; (A)), and methyl-coenzyme M (methyl-CoM; (B)) on methane (CH\u003csub\u003e4\u003c/sub\u003e) production in 48-h mixed rumen batch cultures (Experiment 3): CH\u003csub\u003e4\u003c/sub\u003e (µmol) = 339 (±41.7; \u003cem\u003eP\u003c/em\u003e = 0.047) + (80.2 if Control, -80.2 if BES) (±6.75; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) + 27.9 (±4.45; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) CoM + 26.7 (±4.45; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) methyl-CoM + (-22.1 if Control, 22.1 if BES) (±4.45; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) CoM + (-28.1 if Control, 28.1 if BES) (±4.45; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) methyl-CoM; cow (random), \u003cem\u003eP\u003c/em\u003e = 0.48; incubation (random), \u003cem\u003eP\u003c/em\u003e = 0.52. Concentration of CoM and methyl-CoM expressed in µM was added unity and the resulting sum log-transformed before the statistical analysis.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/2d0c218c62b7b962c5335b2a.png"},{"id":99594581,"identity":"37638c1e-fc19-4a9b-b44a-5a4a0cbd31c5","added_by":"auto","created_at":"2026-01-06 09:20:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":756310,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 2-bromoethanesulfonate (BES, 0 or 5 µM), coenzyme M (CoM; (A)), and methyl-coenzyme M (methyl-CoM; (B)) on dihydrogen (H\u003csub\u003e2\u003c/sub\u003e) accumulation in 48-h mixed rumen batch cultures (Experiment 3): H\u003csub\u003e2\u003c/sub\u003e (µmol) = 24.1 (±1.47; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) + (-12.8 if Control, 12.8 if BES) (±1.60; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) 4.53 (±1.06; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) CoM – 4.69 (±1.06; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) methyl-CoM + (4.10 if Control, -4.10 if BES) (±1.06; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) CoM + (4.65 if Control, -4.65 if BES) (±1.06; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) methyl-CoM; cow (random), \u003cem\u003eP\u003c/em\u003e = 0.88; incubation (random), \u003cem\u003eP\u003c/em\u003e = 0.10. Concentration of CoM and methyl-CoM expressed in µM was added unity and the resulting sum log-transformed before the statistical analysis.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/a26a2ed5a73ffd320ec3c047.png"},{"id":99792675,"identity":"59b06539-e5f1-452f-a0cd-83a269601c9d","added_by":"auto","created_at":"2026-01-08 13:24:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":705513,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 2-bromoethanesulfonate (BES, 0 or 5 µM), coenzyme M (CoM; (A)), and methyl-coenzyme M (methyl-CoM; (B)) on ethanol accumulation in 48-h mixed rumen batch cultures (Experiment 3): ethanol (mM) = 0.19 (±0.046; \u003cem\u003eP\u003c/em\u003e = 0.10) + (-0.18 if Control, 0.18 if BES) (±0.023; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) - 0.073 (±0.015; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) CoM – 0.073 (±0.15; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) methyl-CoM + (0.073 if Control, -0.073 if BES) (±0.0.015; \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001) CoM + (0.073 if Control, -0.073 if BES) (±0.015; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) methyl-CoM; cow (random), \u003cem\u003eP\u003c/em\u003e = 0.53; incubation (random), \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.99. Concentration of CoM and methyl-CoM expressed in µM was added unity and the resulting sum log-transformed before the statistical analysis.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/14ce1047b3913fb1b8d5d97c.png"},{"id":99594584,"identity":"8a15aa21-0a05-40e0-a8c2-5656d454de51","added_by":"auto","created_at":"2026-01-06 09:20:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1092111,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 2-bromoethanesulfonate (BES, 0 or 5 µM), coenzyme M (CoM, 0 or 1 mM; (A)), and methyl-coenzyme M (methyl-CoM, 0 or 1 mM; (B)) on the archaeal community composition of 48-h mixed rumen batch cultures (Experiment 3).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/535c450f1f63a342fe28a7d6.png"},{"id":99594591,"identity":"36984fd5-8df2-49b2-8ff1-3c7adeaa6ad5","added_by":"auto","created_at":"2026-01-06 09:20:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1581612,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 2-bromoethanesulfonate (BES, 5 µM; refer to Supplementary Figure S25), 3-nitrooxypropanol (3-NOP; (A)) and bromoform (BMF; (B)), combined with 2.5 mM coenzyme M (CoM), methyl-coenzyme M (methyl-CoM), or a control treatment without added methyl group carriers (Control), on CH\u003csub\u003e4\u003c/sub\u003e production of mixed rumen batch cultures: BES, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 3-NOP, \u003cem\u003eP\u003c/em\u003e = 0.095; BMF, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; methyl group carrier, \u003cem\u003eP\u003c/em\u003e = 0.13; BES\u0026nbsp; × methyl group carrier, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 3-NOP × methyl group carrier, \u003cem\u003eP\u003c/em\u003e = 0.78; BMF × methyl group carrier, \u003cem\u003eP\u003c/em\u003e = 0.56; cow (random), \u003cem\u003eP\u003c/em\u003e = 0.50; incubation (random), \u003cem\u003eP\u003c/em\u003e = 0.34 (Experiment 4).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/4339f46bc68dc33009a9ab66.png"},{"id":99792683,"identity":"20722af8-f4ee-48ea-bdbe-55ce13fe69d8","added_by":"auto","created_at":"2026-01-08 13:24:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1500949,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 2-bromoethanesulfonate (BES, 5 µM; refer to Supplementary Figure S26), 3-nitrooxypropanol (3-NOP; (A)) and bromoform (BMF; (B)), combined with 2.5 mM coenzyme M (CoM), methyl-coenzyme M (methyl-CoM), or a control treatment without added methyl group carriers (Control), on H\u003csub\u003e2\u003c/sub\u003e accumulation of mixed rumen batch cultures: BES, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 3-NOP, \u003cem\u003eP\u003c/em\u003e = 0.012; BMF, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; methyl group carrier, \u003cem\u003eP\u003c/em\u003e = 0.001; BES\u0026nbsp; × methyl group carrier, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 3-NOP × methyl group carrier, \u003cem\u003eP\u003c/em\u003e = 0.55; BMF × methyl group carrier, \u003cem\u003eP\u003c/em\u003e = 0.14; cow (random), \u003cem\u003eP\u003c/em\u003e = 0.49; incubation (random), \u003cem\u003eP\u003c/em\u003e = 0.33 (Experiment 4). The response variable H\u003csub\u003e2\u003c/sub\u003e accumulation was decimal log-transformed for the analysis but it is shown untransformed in the figure.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/934c328fe6906bad1cfa2a9f.png"},{"id":102747175,"identity":"3bcd6cb4-87cf-4f2e-bf7a-7319a8a6891e","added_by":"auto","created_at":"2026-02-16 09:04:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6660127,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/66129af2-f6a3-418d-8c71-4311d9c034bf.pdf"},{"id":99594589,"identity":"d8dda11f-6f70-422a-9d62-09d623e48bee","added_by":"auto","created_at":"2026-01-06 09:20:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":882037,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8419205/v1/1f5f137e25131e971dba0378.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of coenzyme M and methyl-coenzyme M on the efficacy of inhibitors of methanogenesis in rumen cultures","fulltext":[{"header":"Background","content":"\u003cp\u003eThe abatement of anthropogenic methane (CH\u003csub\u003e4\u003c/sub\u003e) emissions would slow global warming in the short-term [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Approximately 30% of anthropogenic CH\u003csub\u003e4\u003c/sub\u003e originates in the rumen of domestic ruminants i.e., enteric CH\u003csub\u003e4\u003c/sub\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, there is ongoing research on the mitigation of emissions of enteric methane (CH\u003csub\u003e4\u003c/sub\u003e) from ruminants [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Feed additives that inhibit rumen methanogenesis, including 3-nitrooxypropanol (3-NOP) and the bromoform-containing red algae \u003cem\u003eAsparagopsis\u003c/em\u003e, are the most potent strategies for mitigating the emissions of enteric CH\u003csub\u003e4\u003c/sub\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Yet, there is variation in the extent of inhibition of CH\u003csub\u003e4\u003c/sub\u003e production caused by these additives. Empirically, it is known that the efficacy of 3-NOP and \u003cem\u003eAsparagopsis\u003c/em\u003e, defined as the decrease in CH\u003csub\u003e4\u003c/sub\u003e per gram of active compound ingested, is affected by dietary fiber and ether extract, and by the type of animal [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], but the specific mechanisms explaining those interactions are not understood. Additionally, there is variation among pure cultures of different methanogens in their sensitivity to inhibitors of methanogenesis, and the reasons behind this variation also remain unclear [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A mechanistic understanding of the variation in the efficacy of inhibitors of rumen methanogenesis may allow maximizing their effects in the rumen. Variation in the efficacy of inhibitors of methanogenesis may be related, at least in part, to their mechanisms of action.\u003c/p\u003e \u003cp\u003e3-Nitrooxypropanol is a structural analog of methyl-coenzyme M (methyl-CoM) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], a methyl donor substrate that reacts with electron donor coenzyme B in the last step of methanogenesis, which is catalyzed by methyl-coenzyme M reductase (MCR), to form CH\u003csub\u003e4\u003c/sub\u003e and a heterodisulfide of coenzyme M (CoM) and coenzyme B [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 3-Nitrooxypropanol binds to MCR and oxidizes a Ni\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e atom in the porphinoid cofactor F\u003csub\u003e430\u003c/sub\u003e to Ni\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e thus impeding the formation of CH\u003csub\u003e4\u003c/sub\u003e from methyl-CoM [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Another methanogenesis inhibitor, 2-bromoethanesulfonate (BES), also competes with methyl-CoM [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] for the catalytic site in MCR and likewise oxidizes the Ni\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e atom of cofactor F\u003csub\u003e430\u003c/sub\u003e to Ni\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMethyl-coenzyme M and also its nonmethylated form coenzyme M (CoM) have been shown to reverse the inhibition of CH\u003csub\u003e4\u003c/sub\u003e formation by BES in pure cultures of the ruminal methanogen \u003cem\u003eMethanobrevibacter ruminantium\u003c/em\u003e M1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], non-ruminal methanogens \u003cem\u003eMethanosarcina\u003c/em\u003e strain 227, \u003cem\u003eMethanospirillum hungatei\u003c/em\u003e, \u003cem\u003eMethanococcus voltae\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], methanogenic marine sediments [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and purified MCR [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Bromoform (BMF) and other CH\u003csub\u003e4\u003c/sub\u003e halogenated analogs inhibit methanogenesis by reacting with a corrinoid cofactor of methyltetrahydromethanopterin: coenzyme M methyltransferase (MTR), thus blocking the formation of the methylated form of the corrinoid and the transfer of a methyl group to CoM to form methyl-CoM [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Because BES and 3-NOP inhibit MCR by competing with methyl-CoM, and CoM has also been shown to reverse BES effects, and BMF inhibits a different enzyme, we hypothesized that excess CoM or methyl-CoM added to mixed rumen cultures would reverse methanogenesis inhibition by BES and 3-NOP, but not by BMF.\u003c/p\u003e \u003cp\u003eSecond, some methanogens that lack the capacity to synthesize CoM, take up exogenous CoM from the medium at greater rates than those methanogens that can synthesize CoM [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Because CoM, methyl-CoM, and BES share a common transmembrane uptake system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and resistance to BES in methanogens is related to low rates of transport of BES into the cell [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], it is conceivable that methanogens that cannot synthesize CoM are more sensitive to BES because they take up BES at faster rates than those methanogens that can synthesize CoM. On the other hand, 3-NOP and BMF are understood to freely diffuse across cell membranes independently of transmembrane transporters [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We thus further hypothesized that supplementing CoM to serially transferred mixed rumen cultures would stimulate the growth of methanogens that cannot synthesize CoM and require exogenous CoM, resulting in mixed rumen cultures with greater sensitivity to BES after CoM supplementation is discontinued than cultures that had not received CoM; in contrast, the sensitivity of rumen cultures to 3-NOP and BMF would not be expected to be affected by previous supplementation with CoM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eFour in vitro experiments were conducted: one with serial mixed rumen cultures (Experiment 1; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and three with batch mixed rumen cultures (Experiments 2, 3, and 4).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eExperiment 1\u003c/h2\u003e\n \u003cp\u003eExperiment 1 was conducted as a 2 \u0026times; 4 \u0026times; 2 factorial arrangement of treatments, with the following factors: i) Supplementation of CoM at 0 or 1 \u0026micro;M to serial rumen cultures in serial transfers 1 to 4; ii) Inhibition of methanogenesis: Control (distilled water), BES, 3-NOP, or BMF (all 5 \u0026micro;M), and iii) Spiking of CoM at 0 or 1 mM (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eGeneral rumen contents sampling and processing procedures were conducted as described before [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Rumen contents were sampled at approximately 9 am before feeding from two ruminally cannulated, nonlactating, nonpregnant, Holstein cows fed once daily 1 kg of concentrate (88.0% DM, and 18.3% CP, 29.2% NDF, 3.4% EE, 3.3% total ashes, all DM basis) and ad libitum ryegrass hay (90.7% DM, and 8.6% CP, 66.4% NDF, 39.5% ADF, 1.9% EE, 6.1% total ashes, all DM basis). Proximate compositions of the cannulated cows feed and the incubation substrate were analyzed according to AOAC [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Rumen contents were strained through an approximately 0.5 mm double synthetic cloth (Eurotelas, Osorno, Chile) and both fluid and solids from each cow were separately transported to the laboratory in insulated flasks.\u003c/p\u003e\n \u003cp\u003eApproximately 150 mL of fluid and 150 mL of solids from the same cow were placed in 500-mL Erlenmeyer flasks under CO\u003csub\u003e2\u003c/sub\u003e and discontinuously (3 s bursts followed by 2 s pauses) blended for 1 min with an eggbeater running at low speed to dislodge microbial cells loosely adhered to plant particles. The Erlenmeyer flasks contents were then strained through two layers of synthetic cloth to obtain the rumen inoculum as the filtrate fraction [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Rumen inoculum from each cow (14 mL) was delivered under CO\u003csub\u003e2\u003c/sub\u003e into two 100-mL serum bottles containing 26 mL of culture medium [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] modified to contain the Raju [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] trace mineral solution and 400.4 [400.0, 400.6] (mean [min, max]) mg of a substrate composed of 75% forage and 25% concentrate (DM basis; Supplementary Table\u0026nbsp;1) ground to pass a 1-mm screen. One serum bottle with inoculum from each of the cows received 25 \u0026micro;L of a CoM (Sigma-Aldrich, China) solution to achieve a final concentration of 1 \u0026micro;M. This concentration of CoM was defined on the basis of the concentration of CoM used to maintain methanogens requiring exogenous CoM [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. The serum bottle with the inoculum from each cow that did not receive CoM supplementation received 25 \u0026micro;L of distilled water and constituted the corresponding control treatment.\u003c/p\u003e\n \u003cp\u003eAll four serum bottles (two inocula from two cows, each with or without 1 \u0026micro;M CoM supplementation) were gassed and sealed under CO\u003csub\u003e2\u003c/sub\u003e and incubated at 39\u0026deg;C and 60 rpm for 24 h, after which gas pressure was measured with a pressure transducer (Sper Scientific 840065, Scottsdale, AZ, United States). Gas samples (0.5 mL) were analyzed for CH\u003csub\u003e4\u003c/sub\u003e and dihydrogen (H\u003csub\u003e2\u003c/sub\u003e) content in a Clarus 580 Perkin Elmer GC equipped with a 60/80 Carboxen 1000 packed column (Supelco, Bellefonte, PA, United States) operating at an isothermal temperature of 180\u0026deg;C with N\u003csub\u003e2\u003c/sub\u003e at 30 mL/min as carrier gas and a thermal conductivity detector [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Standards of known concentrations were used to calculate the concentrations of CH\u003csub\u003e4\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003e. Gas production was calculated via the ideal gas law and a gas pressure equal to the gas pressure recorded plus 1 atm (101,325 Pa) and 312 K.\u003c/p\u003e\n \u003cp\u003eBottles were then vortexed at maximum speed for 2 min to dislodge microbial cells loosely attached to plant particles and subsequently left untouched for 1 min to allow plant particles to sink. Bottles were then opened under CO\u003csub\u003e2\u003c/sub\u003e and 14 mL of fluid was transferred to a new bottle corresponding to the same CoM supplementation treatment containing fresh medium and 400.4 [400.2, 400.6] (mean [min, max]) mg of substrate. The pH (Orion Star A214, Thermo Scientific, Chelmsford, MA, United States) and reducing potential (E\u003cem\u003eh\u003c/em\u003e; Oakton pH 700 meter, Vernon Hills, IL, United States, Ag/AgCl electrode in saturated KCl, Schott Instruments, BlueLine 31 Rx, Mainz, Germany) of the bottle donating the inoculum were then measured. Measured E\u003cem\u003eh\u003c/em\u003e values were then corrected to those of the Standard Hydrogen Electrode (SHE) by adding 197 mV [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe process of serially transferring inoculum to a new bottle with fresh medium and new substrate was repeated five times (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In serial transfer 4, each bottle corresponding to a combination of a 1 \u0026micro;M CoM supplementation treatment and a cow\u0026acute;s inoculum donated 4 mL of inoculum fluid to each of eight bottles containing 36 mL of fresh medium and 400.4 [400.0, 402.0] (mean [min, max]) mg of substrate. The eight bottles of serial transfer 5 that received their inoculum from the same bottle of serial transfer 4 were blocked so to successively receive their 4 mL inoculum in two doses i.e., 2 mL from the first, and 2 mL from the second, half of the total inoculum delivered from the donor bottle of serial transfer 4. This procedure was conducted to avoid introducing a bias in delivering the upper or lower halves of the liquid volume of the donating bottle to particular treatments.\u003c/p\u003e\n \u003cp\u003eThe eight bottles in serial transfer 5 that received inoculum from each bottle in serial transfer 4 received a treatment combination of two factors: i) 1 mL of a CoM solution to achieve a 1 mM final concentration (CoM spiking) or 1 mL distilled water (Control); ii) 25 \u0026micro;L of a solution of BES (Aldrich Chemistry, Taiwan), 3-NOP (RR Scientific LLC, Shanghai, China), or BMF (Sigma-Aldrich, US), to achieve a 5 \u0026micro;M final concentration of each inhibitor of methanogenesis, or 25 \u0026micro;L distilled water (Control). Because BES and 3-NOP were dissolved in water, and bromoform was dissolved in ethanol, 25 \u0026micro;L of water or ethanol was added to the corresponding bottles to equalize the amounts of water and ethanol received by all the bottles. Bottles were sealed under CO\u003csub\u003e2\u003c/sub\u003e and incubated at 39\u0026deg;C and 60 rpm for 72 h. The incubation time of the fifth serial transfer was extended from 48 to 72 h to compensate for the lower volume of inoculum in comparison with the previous four serial transfers.\u003c/p\u003e\n \u003cp\u003eAt the end of the 72-h incubation period gas pressure and composition, pH, and E\u003cem\u003eh\u003c/em\u003e were measured as described before. Bottles were then opened, and two 1-mL fluid aliquots were delivered into 2-mL microcentrifuge tubes containing 0.20 mL of 20% (\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e) \u003cem\u003eo\u003c/em\u003e-phosphoric acid and 0.20 mL of 35 mM crotonic acid or 1% (\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e) sulfuric acid for subsequent analyses of carboxylic acids and alcohols or ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e), respectively. All fluid samples were stored at -20\u0026deg;C until processing. Bottles contents were then centrifuged at 10,956 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 15 min into 50-mL pre-weighted centrifuged tubes. The supernatants were discarded and the tubes containing the pellets were frozen at -80\u0026deg;C and then lyophilized. Lyophilized tubes were then weighted, and the dry mass of the incubation residue was calculated by difference. Apparent dry matter disappearance (DMD) was calculated by subtracting the residue dry matter from the initial substrate dry matter and expressed as a percentage.\u003c/p\u003e\n \u003cp\u003eMicrocentrifuge tubes for analysis of carboxylic acids and alcohols were thawed and centrifuged at 16,100 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 10 min. The supernatants were then filtered through 13-mm 0.45 \u0026micro;m filters into 2-mL GC vials. Concentration of volatile fatty acids (VFA), succinate, methanol, and ethanol were analyzed in a PerkinElmer Clarus 580 GC equipped with an Elite-FFAP (PerkinElmer, Shelton, CT, US) capillary column and a flame ionization detector operating at 200\u0026deg;C. Split was set at 33: 1. The initial oven temperature of 50\u0026deg;C was maintained for 2.70 min, followed by a 15\u0026deg;C/min ramp to 150\u0026deg;C. The temperature was held at 150\u0026deg;C for 5 min and then increased at 15\u0026deg;C/min to 210\u0026deg;C and held for 1.5 min. The carrier gas was He at 51.4 mL/min. Calibration curves were made with standards of known concentration and crotonic acid was used as an internal standard. The sum of 2- and 3-methylbutyrate is reported as these two VFA co-eluted. Concentration of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was analyzed colorimetrically following the method of Kaplan [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThree incubation rounds were conducted in different weeks. Results of fermentation variables of serial transfer 5 were analyzed as a 2 \u0026times; 4 \u0026times; 2 arrangement of treatments with the following model:\u003c/p\u003e\n \u003cp\u003eresponse\u003csub\u003eijklm\u003c/sub\u003e = general mean\u0026thinsp;+\u0026thinsp;CoM supplementation\u003csub\u003ei\u003c/sub\u003e serial transfers 1\u0026ndash;4 (1 \u0026micro;M)\u0026thinsp;+\u0026thinsp;inhibitor\u003csub\u003ej\u003c/sub\u003e serial transfer 5\u0026thinsp;+\u0026thinsp;CoM spiking\u003csub\u003ek\u003c/sub\u003e serial transfer 5 (1 mM)\u0026thinsp;+\u0026thinsp;double interactions\u0026thinsp;+\u0026thinsp;cow\u003csub\u003el\u003c/sub\u003e (random)\u0026thinsp;+\u0026thinsp;incubation\u003csub\u003em\u003c/sub\u003e (random)\u0026thinsp;+\u0026thinsp;error\u003csub\u003eijklm\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003eThe triple interaction was initially included in the model, but it was removed from the model because it was not significant for any response variable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.30). When the inhibitor effects were significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and none of the interactions was significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), inhibitors treatment means were compared through Tukey\u0026acute;s Honestly Significant Difference test. When interactions between inhibitors and CoM spiking were significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the inhibitors causing the interaction were identified via three Least Square Difference (LSD) contrasts to compare the difference between each inhibitor and the methanogenesis inhibition control with and without CoM spiking:\u003c/p\u003e\n \u003cp\u003eContrast 1 = (Control, no CoM \u0026ndash; BES, no CoM) \u0026ndash; (Control, CoM \u0026ndash; BES, CoM)\u003c/p\u003e\n \u003cp\u003eContrast 2 = (Control, no CoM \u0026ndash; 3-NOP, no CoM) \u0026ndash; (Control, CoM \u0026ndash; 3-NOP, CoM)\u003c/p\u003e\n \u003cp\u003eContrast 3 = (Control, no CoM \u0026ndash; BMF, no CoM) \u0026ndash; (Control, CoM \u0026ndash; BMF, CoM)\u003c/p\u003e\n \u003cp\u003eSignificance was declared at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 tendencies at 0.05\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.10.\u003c/p\u003e\n \u003cp\u003eResults of fermentation variables of serial transfers 1 to 4 were analyzed as follows:\u003c/p\u003e\n \u003cp\u003eresponse\u003csub\u003eijkl\u003c/sub\u003e = general mean\u0026thinsp;+\u0026thinsp;serial transfer\u003csub\u003ei\u003c/sub\u003e + CoM supplementation\u003csub\u003ej\u003c/sub\u003e + serial transfer \u0026times; CoM supplementation\u003csub\u003eij\u003c/sub\u003e + cow\u003csub\u003ek\u003c/sub\u003e (random)\u0026thinsp;+\u0026thinsp;incubation\u003csub\u003el\u003c/sub\u003e (random)\u0026thinsp;+\u0026thinsp;error\u003csub\u003eijkl\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003eResiduals distribution was examined through normal quantile plots. Homoscedasticity was examined through plots of observed residuals against predicted observations. Outliers were identified as observations falling out of a 99.9% studentized residuals normal distribution and examined for obvious typing or measurement problems. Outliers with no typing or measurement problems and clustering in the same treatment were considered as biological results and retained. Other outliers without an obvious technical cause were tentatively removed and the analysis re-run. As conclusions did not change substantially, all observations were retained.\u003c/p\u003e\n \u003cp\u003eAll statistical analyses were conducted with JMP 19.0.1 [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperiment 2\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eUnder a 4 \u0026times; 2 factorial arrangement of treatments, Experiment 2 evaluated the interactions between the same three inhibitors of methanogenesis evaluated in Experiment 1 (BES, 3-NOP, and BMF, each at 5 \u0026micro;M, plus a Control treatment without a methanogenesis inhibitor) and methyl-CoM spiking at 0 or 1 mM on CH\u003csub\u003e4\u003c/sub\u003e production and fermentation of 48-h rumen batch cultures. Sampling and processing of rumen contents was conducted as described for Experiment 1. Under CO\u003csub\u003e2\u003c/sub\u003e, 8 mL of rumen inoculum from each cow, obtained as per Experiment 1, was were delivered into 100-mL serum bottles containing 32 mL of incubation medium [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] and 400.5 [400.2, 400.6] (mean [min, max]) mg of the same substrate used in Experiment 1. The inoculated medium then received 25 \u0026micro;L of a solution of 3-NOP or BES, or 25 \u0026micro;L of distilled water (methanogenesis inhibition Control). Depending on their methanogenesis inhibition treatment, bottles received 25 \u0026micro;L of ethanol or 25 \u0026micro;L of a BMF solution in ethanol. Final concentration of BES, 3-NOP or BMF was 5 \u0026micro;M. Each methanogenesis inhibition treatment in turn received 1 mL of a methyl-CoM (BLD Pharmatech Co., Ltd., Cincinnati, OH, US) solution to achieve a final concentration of 1 mM (methyl-CoM spiking) or 1 mL distilled water (methyl-CoM control). Bottles were gassed and sealed under O\u003csub\u003e2\u003c/sub\u003e-free CO\u003csub\u003e2\u003c/sub\u003e and incubated at 39\u0026deg;C and 60 rpm for 48 h. Gas production and composition, pH, E\u003cem\u003eh\u003c/em\u003e, total and individual VFA, ammonium concentration, and DMD were determined as per Experiment 1.\u003c/p\u003e\n\u003cp\u003eThree incubation runs were conducted in different weeks. Results of Experiment 2 were analyzed as a 3 \u0026times; 2 factorial arrangement of treatments as follows:\u003c/p\u003e\n\u003cp\u003eresponse\u003csub\u003eijkl\u003c/sub\u003e = general mean\u0026thinsp;+\u0026thinsp;inhibitor\u003csub\u003ei\u003c/sub\u003e + methyl-CoM spiking\u003csub\u003ej\u003c/sub\u003e (1 mM)\u0026thinsp;+\u0026thinsp;inhibitor \u0026times; methyl-CoM spiking\u003csub\u003eij\u003c/sub\u003e + cow\u003csub\u003ek\u003c/sub\u003e + incubation\u003csub\u003el\u003c/sub\u003e + error\u003csub\u003eijkl\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eResiduals normality and outlier analyses were conducted as in Experiment 1.\u003c/p\u003e\n\u003ch3\u003eExperiment 3\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eExperiment 3 examined the interaction between BES at 5 \u0026micro;M and CoM or methyl-CoM at 0, 1, 10, 100, or 1000 \u0026micro;M, on CH\u003csub\u003e4\u003c/sub\u003e production, other fermentation variables, and the prokaryote community composition of 48-h rumen batch cultures. Rumen collection and processing were conducted as in Experiments 1 and 2. Rumen inoculum from each cow (8 mL) was delivered to 100-mL incubation bottles containing 32 mL of the Mould et al. [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] incubation medium and 400.3 [400.2, 400.5] (mean [min, max]) mg of the high forage substrate used in Experiments 1 and 2. Aliquots (5 mL) of each inoculum were kept in 15-mL polypropylene conical tubes at -20\u0026deg;C for subsequent analysis of CoM and methyl-CoM concentration in rumen fluid. Each bottle received 25 \u0026micro;L of a BES solution to achieve a final concentration of 5 \u0026micro;M or 25 \u0026micro;L of distilled water (methanogenesis inhibition Control). In turn, each bottle received 1 mL of one of four different CoM or methyl-CoM solutions to achieve 1, 10, 100, or 1000 \u0026micro;M final concentration. The CoM/methyl-CoM control received 1 mL of distilled water. Bottles were gassed and sealed under CO\u003csub\u003e2\u003c/sub\u003e and incubated for 48 h at 60 rpm. Measurements and analytical procedures were conducted as in Experiments 1 and 2. Bottle contents were centrifuged and subsequently lyophilized as in Experiments 1 and 2. Centrifugation tubes containing lyophilized incubation residues of treatments supplemented 0 or 1000 \u0026micro;M CoM or methyl-CoM, both with and without BES, were stored at -80\u0026deg;C for DNA extraction.\u003c/p\u003e\n\u003cp\u003eThe concentration of CoM and methyl-CoM in samples of ruminal inocula was analyzed by FoodTech, Temuco, Chile. General procedures were from Lovley et al. [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Inocula samples for CoM and methyl-CoM analysis were thawed and centrifuged at 13,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 15 min. Supernatants were acidified with 5 mL 0.1 N HCl and filtered through 0.45 \u0026micro;m hydrophilic sterile syringe filters (Millex\u0026trade; PVDF syringe filter, MilliporeSigma, Burlington, MA, USA) into 2-mL microcentrifuge tubes. Aliquots (1 mL) of the filtrates were frozen at -80\u0026deg;C, lyophilized, 25 \u0026micro;L of Sigma-Sil-A (Sigma-Aldrich, Burlington, MA, US) was added, the mixture was vortexed, and incubated at 70\u0026deg;C for 30 min for thimethylsilyl derivatization [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. Samples were then cooled at room temperature for 30 min and added 1 mL of ethyl acetate (Sigma-Aldrich, Burlington, MA, US). The mix was vortexed and subsequently centrifuged at 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at room temperature for 10 min. The ethyl acetate layer (700 \u0026micro;L) containing the derivatized compounds was then transferred to 2-mL GC vials for GC‒MS analysis.\u003c/p\u003e\n\u003cp\u003eParameters of the GC-MS run were adjusted by injecting standards of CoM and methyl-CoM and identifying retention times and fragments. Samples (1 \u0026micro;L) were injected in splitless mode into a QP2020NX Shimadzu GC-MS (Kyoto, Japan) equipped with a Restek Rtx-5Ms (30 m, 0,25 mm ID, 0.25 \u0026micro;m) capillary column (Bellefonte, PA, US). The initial temperature was 80\u0026deg;C for 3 min, followed by a 10\u0026deg;C/min ramp to 160\u0026deg;C. Temperature was maintained at 160\u0026deg;C for 1 min and then increased at 5\u0026deg;C/min to 230\u0026deg;C, and maintained at 230\u0026deg;C for 5 min. Samples were electron-ionized and fragments were scanned between 40 and 400 \u003cem\u003em/z\u003c/em\u003e at 1250 amu/s. Coenzyme M and methyl-CoM were identified and quantified using chemically pure standards. Limits of detection were 2.84 and 2.78 \u0026micro;M for CoM and methyl-CoM, respectively, and limits of quantification were 8.60 and 8.43 \u0026micro;M in the same order. Retention time was 7 min 31 s for both CoM and methyl-CoM. Both CoM and methyl-CoM were quantified as the sum of intensities of the 73, 103, 117, 147 and 205 \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e fragments. Concentration of CoM and methyl-CoM are reported as their sum as the specific compounds could not be separated.\u003c/p\u003e\n\u003cp\u003eA subset of 24 lyophilized residues from the CoM 0 and 1000 \u0026micro;M treatments, both with and without BES, were selected for the analysis of the composition of the archaeal and bacterial communities through amplicon sequencing the V3-V4 regions of their 16S rRNA genes. Prokaryote metataxonomic analyses including DNA extraction, library preparation, sequencing, and bioinformatics were conducted by AUSTRAL-omics, Universidad Austral de Chile, Valdivia, Chile [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. DNA was extracted from lyophilized incubation residues with a DNeasy PowerSoil Pro Kit (Qiagen) following the manufacturer\u0026acute;s instructions modified according to Yu and Forster [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e], including two negative controls. Lyophilized samples for DNA analysis (250 mg) were placed in 2-mL PowerBead Pro Tube cryotubes containing 800 \u0026micro;L of CD1 solution and a total of 200 \u0026micro;L of 0.1-mm diameter zirconium beads. Microbial cells were mechanically disrupted by applying three 30-s bead beating cycles at 3,400 strokes (Mini-Beadbeater 24, BioSpecproducts, Bartlesville, OK, US). Samples were then incubated with 25 \u0026micro;L of 20 mg/mL proteinase K (New England Biolabs, Ipswich, MA, US) and 7 \u0026micro;L of 50 mg/mL lysozyme solution (Thermo Fisher, Rockford, IL, US) at 37\u0026deg;C for 4 h followed by an incubation at 55\u0026deg;C for 12 h. Preparations obtained were then passed through a DNA-binding column (MB Spin Columns, Quiagen, Germantown, MD, US) with buffer C6 (50 \u0026micro;L) added for DNA elusion. Samples were then incubated at room temperature for 1 h and centrifuged at 15,000 \u0026times; g at room temperature for 1 min. Extracted DNA was quantified with fluorometry with a Qubit dsDNA HS Assay Kit (Thermo Fischer, Eugene, OR, US). DNA integrity was assessed via 1.5% agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003eConcentration of DNA in all samples used for amplification was equalized to 2 ng DNA/\u0026micro;L. Amplicons for sequencing libraries were generated following the Illumina 16S Metagenomic Sequencing Library Preparation protocol [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. A total of 28 cycles of DNA amplification were conducted (30 s denaturing at 95\u0026deg;C, 30 s annealing at 55\u0026deg;C, and 30 s extension at 72\u0026deg;C) followed by a 5 min final extension at 72\u0026deg;C. KAPA HiFi DNA polymerase (Roche Sequencing Solutions, Inc., Cape Town, South Africa) was used in the PCR reactions. Archaeal and bacterial 16S rRNA gene V3-V4 regions were amplified using the primers of Mesa et al. [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e] and Walters et al. [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e], respectively. Negative extraction and PCR controls were included in the PCR amplification reactions. Products of PCR were purified from nonspecific products and primer dimers with Mag-Bind\u0026reg; TotalPure NGS (Omega Bio-Tek, Norcross, GA, US) magnetic pearls and quantified using a Qubit 4.0 fluorometer and the Qubit dsDNA HS Assay Kit (Thermo Fischer, Eugene, OR, US).\u003c/p\u003e\n\u003cp\u003eAmplicons at equal amounts (10 ng) were pooled for a second PCR reaction (3 min denaturing at 95\u0026deg;C followed by 8 cycles of 30 s denaturing at 95\u0026deg;C, 30 s annealing at 55\u0026deg;C, and 30 s extension at 72\u0026deg;C, followed by 5 min extension at 72\u0026deg;C, with KAPA HiFi DNA polymerase (Roche Sequencing Solutions, Inc., Cape Town, South Africa) for ligating Illumina P5 and P7 adaptors attached to Illumina Nextera XT Index Kit v2 indices. The obtained library was purified with Mag-Bind\u0026reg; TotalPure NGS (Omega Bio-Tek, Norcross, GA, US) magnetic pearls and eluted with 22 \u0026micro;L 10 mM Tris. Integrity of DNA was assessed on a 1.5% agarose gel and DNA size was examined with capillary electrophoresis Fragment Analyzer (Agilent Technologies, Santa Clara, CA, US). Concentration of DNA was quantified by fluorometry (Qubit 4.0 fluorometer and Qubit dsDNA HS Assay Kit, Thermo Fischer, Eugene, OR, US), adjusted to 7 nM, and denatured with 5 \u0026micro;L of 0.2 mM NaOH for 5 min to a 750 pM final concentration. Libraries were mixed with a PhiX control library at 20% (\u003cem\u003eV\u003c/em\u003e/\u003cem\u003eV\u003c/em\u003e) to add heterogeneity and the final mixture was loaded on the Illumina platform at 2 \u0026times; 300 Illumina paired forward and reverse cycles run with FastQ.\u003c/p\u003e\n\u003cp\u003eRaw sequences generated by Illumina NextSeq 1000 were demultiplexed with Cutadapt 2.10 [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Demultiplexed sequences were filtered for quality with Trimomatic v. 0.39 [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e] and PRINSEQ v. 0.20.4 [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e], retaining sequences with a Phred\u0026thinsp;\u0026ge;\u0026thinsp;Q30. Amplicon Sequence Variants (ASVs) were inferred with R DADA2 1.36 [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Flow work included the estimation of sequencing error per base, dereplication, ASV inference, concatenation of paired readings, and removal of chimeras [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Taxonomic assignment was performed with RDP Na\u0026iuml;ve Bayesian Classifier of DADA2 [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e] with a 50% bootstrap threshold at the species level. Two filters of abundance were applied to minimize false positives and negatives. A filter per sample was applied keeping only those sequences with an abundance over 0.1% of total sequences in their sample. Second, a filter per ASV was applied by eliminating reads with occurrences of less than 1% of total reads of their particular ASV [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. Diversity analyses were conducted with rarefied results normalizing samples by sequencing depth based on the sample with the least number of reads. Principal component analyses were conducted based on Bray-Curtis dissimilarity distances with the phyloseq 1.52 and R 4.5.1 packages [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e] of R [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Alfa-diversity was estimated with vegan [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e] in R [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eA total of 11,471 and 24,104 archaeal bacterial reads, respectively, were obtained and taxonomically assigned of with SILVA 138 [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. Eukaryotic ASVs and ASVs unassigned at the phylum level were eliminated from the analysis, resulting in a total of 5,343 and 68 filtered bacterial and archaeal ASVs, respectively. We searched GenBank [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e] for methanogens carrying the genes \u003cem\u003ecomA\u003c/em\u003e, \u003cem\u003ecomB\u003c/em\u003e, \u003cem\u003ecomC\u003c/em\u003e, and \u003cem\u003ecomD\u003c/em\u003e and \u003cem\u003ecomE\u003c/em\u003e, encoding enzymes involved in the synthesis of CoM in in the \u003cem\u003eMethanobacteriales\u003c/em\u003e order, as well as the CoM biosynthesis genes \u003cem\u003eCS\u003c/em\u003e in \u003cem\u003eMethanomicrobiales\u003c/em\u003e and \u003cem\u003eMethanosarcinales\u003c/em\u003e and genes \u003cem\u003eXcbB\u003c/em\u003e, \u003cem\u003eXcbC\u003c/em\u003e, \u003cem\u003eXcbD\u003c/em\u003e and \u003cem\u003eXcbE\u003c/em\u003e in bacteria [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. We used that information to estimate which percentage of methanogens in each sample with the genetic capacity to synthesize CoM as well as to transport CoM into their cells.\u003c/p\u003e\n\u003cp\u003eTwo incubation runs were conducted in different weeks. Fermentation results from Experiment 3 were analyzed as follows:\u003c/p\u003e\n\u003cp\u003eresponse\u003csub\u003eijklmn\u003c/sub\u003e = general mean\u0026thinsp;+\u0026thinsp;methanogenesis inhibition\u003csub\u003ei\u003c/sub\u003e (Control or BES)\u0026thinsp;+\u0026thinsp;CoM\u003csub\u003ej\u003c/sub\u003e + methyl-CoM\u003csub\u003ek\u003c/sub\u003e + methanogenesis inhibition \u0026times; CoM\u003csub\u003eij\u003c/sub\u003e + methanogenesis inhibition \u0026times; methyl-CoM\u003csub\u003eik\u003c/sub\u003e + cow\u003csub\u003el\u003c/sub\u003e + incubation\u003csub\u003em\u003c/sub\u003e + error\u003csub\u003eijklmn\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eConcentration of CoM and methyl-CoM was added unity and decimal log-transformed for the statistical analyses as follows:\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:{\\text{log}}_{10}(CoM+1)\\:\\text{a}\\text{n}\\text{d}\\:{\\text{log}}_{10}(methyl-CoM+1)$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe composition and diversity of the archaeal and bacterial communities were analyzed in the subset of treatments with 0, 1000 \u0026micro;M CoM, or 1000 \u0026micro;M methyl-CoM as a factorial arrangement of treatments with two factors: i) methanogenesis inhibition (Control or BES), and ii) addition of methyl group carriers (Control, CoM, or methyl-CoM), and their interaction:\u003c/p\u003e\n\u003cp\u003eresponse\u003csub\u003eijkl\u003c/sub\u003e = general mean\u0026thinsp;+\u0026thinsp;methanogenesis inhibition\u003csub\u003ei\u003c/sub\u003e (Control or BES)\u0026thinsp;+\u0026thinsp;methyl group carrier\u003csub\u003ej\u003c/sub\u003e (Control, CoM, or methyl-CoM)\u0026thinsp;+\u0026thinsp;interaction\u003csub\u003eij\u003c/sub\u003e + cow\u003csub\u003ek\u003c/sub\u003e + incubation\u003csub\u003el\u003c/sub\u003e + error\u003csub\u003eijklm\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eWhen the addition of CoM or methyl-CoM was significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or tendencies (0.05\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.10), CoM and methyl-CoM addition were each separately compared with the Control treatment with 0 CoM or methyl-CoM. When interactions between methanogenesis inhibition and CoM or methyl-CoM were significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or tendencies (0.05\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.10), the effect of BES addition was separately analyzed across the addition of CoM or methyl-CoM as follows:\u003c/p\u003e\n\u003cp\u003eContrast 1 (CoM) = [(Control, Control) \u0026ndash; (BES, Control)] \u0026ndash; [(Control, CoM) \u0026ndash; (BES, CoM)]\u003c/p\u003e\n\u003cp\u003eContrast 2 (methyl-CoM) = [(Control, Control) \u0026ndash; (BES, Control)] \u0026ndash; [(Control, methyl-CoM) \u0026ndash; (BES, methyl-CoM)]\u003c/p\u003e\n\u003cp\u003eResiduals normality and outlier analyses were conducted as in Experiments 1 and 2.\u003c/p\u003e\n\u003ch3\u003eExperiment 4\u003c/h3\u003e\n\u003cp\u003eExperiment 4 examined the effects of molar ratios of CoM or methyl-CoM to 3-NOP or BMF greater than those examined in Experiments 1 and 2, on CH\u003csub\u003e4\u003c/sub\u003e production and H\u003csub\u003e2\u003c/sub\u003e accumulation in 48-h mixed ruminal cultures. General rumen inocula sampling and preparation procedures were as described for Experiments 1, 2, and 3. Rumen inoculum from each cow (2.4 mL) was delivered under O\u003csub\u003e2\u003c/sub\u003e-free CO\u003csub\u003e2\u003c/sub\u003e into 27-mL 18 \u0026times; 150 mm anaerobic serum tubes (Bellco Glass, Inc., Vineland, NJ, US) containing 9.6 mL of medium [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] and 120.6 [120.4, 120.8] (mean [min, max]) mg of the same high forage substrate incubated in the previous experiments. Each tube received 0.5 mL of distilled water, or CoM or methyl-CoM solutions prepared as in the previous experiments to achieve final concentrations of 0 (methyl carrier Control), 2.5 mM CoM, or 2.5 mM methyl-CoM. In turn, each tube received 10 \u0026micro;L distilled water (methanogenesis inhibition Control) or stock solutions of 3-NOP or BMF to achieve different final concentrations. Initially, 3-NOP final concentrations were 0.1, 0.5, or 1.0 \u0026micro;M; however, as 3-NOP at those concentrations did not inhibit methanogenesis, they were increased to 1.0, 2.5, or 5.0 \u0026micro;M for incubations 2 and 3 of Experiment 4. Bromoform final concentrations were 1.0, 0.5, or 0.1 \u0026micro;M in all three incubation runs of Experiment 4. A treatment containing 5.0 \u0026micro;M BES final concentration was included as a positive control. As BMF was dissolved in ethanol, each Control, 3-NOP, and BES tube also received 10 \u0026micro;L of ethanol, and each BMF tube also received 10 \u0026micro;L distilled water. Tubes were sealed under O\u003csub\u003e2\u003c/sub\u003e-free CO\u003csub\u003e2\u003c/sub\u003e and incubated in an oscillating water bath at 39\u0026deg;C and 60 rpm for 48 h. At the end of the incubation, gas pressure and gas contents of CH\u003csub\u003e4\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003e were measured following the procedures of the previous experiments. Tubes were then opened and pH and E\u003cem\u003eh\u003c/em\u003e measured as before.\u003c/p\u003e\n\u003cp\u003eThree incubation runs were conducted in different weeks. Results were analyzed as:\u003c/p\u003e\n\u003cp\u003eresponse\u0026thinsp;=\u0026thinsp;intercept\u0026thinsp;+\u0026thinsp;BES\u0026thinsp;+\u0026thinsp;3-NOP\u0026thinsp;+\u0026thinsp;BMF\u0026thinsp;+\u0026thinsp;methyl group addition (Control, CoM, or methyl-CoM)\u0026thinsp;+\u0026thinsp;BES \u0026times; methyl group addition\u0026thinsp;+\u0026thinsp;3-NOP \u0026times; methyl group addition\u0026thinsp;+\u0026thinsp;BMF \u0026times; methyl group addition\u0026thinsp;+\u0026thinsp;cow\u0026thinsp;+\u0026thinsp;incubation\u0026thinsp;+\u0026thinsp;error\u003c/p\u003e\n\u003cp\u003eResiduals normality and outlier analyses were conducted as previously described.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eExperiment 1\u003c/h2\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the main effects and interactions of methanogenesis inhibitors and spiking of CoM at 1 mM. To avoid showing 16 treatment means in one table (i.e., all combinations of the 2 \u0026times; 4 \u0026times; 2 factorial), the main effect and interactions of CoM supplementation at 1 \u0026micro;M in serial transfers 1 to 4 are shown separately in Supplementary Table S2. Spiking CoM at 1 mM in serial transfer 5 increased total gas production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Bromoform and to a lesser extent 3-NOP decreased total gas production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was an interaction between methanogenesis inhibition and CoM spiking at 1 mM on CH\u003csub\u003e4\u003c/sub\u003e production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018), by which BES, unlike 3-NOP and bromoform, inhibited methanogenesis by 58% without 1 mM CoM spiking but did not inhibit CH\u003csub\u003e4\u003c/sub\u003e production with 1 mM CoM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Spiking CoM at 1 mM did not affect CH\u003csub\u003e4\u003c/sub\u003e production in the absence of inhibitors of methanogenesis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.88; result not shown). Inhibiting methanogenesis with 3-NOP and BMF increased H\u003csub\u003e2\u003c/sub\u003e accumulation by 3.45- and 5.78-fold, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Incubations with 3-NOP and bromoform had a higher final pH than Control incubations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Bromoform and to a lesser extent 3-NOP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and CoM spiking at 1 mM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), increased E\u003cem\u003eh\u003c/em\u003e. Both CoM supplementation at 1 \u0026micro;M in serial transfers 1 to 4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040) and CoM spiking at 1 mM in serial transfer 5 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), and BMF and to a lesser extent 3-NOP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decreased apparent DM disappearance. Bromoform and to a lesser extent 3-NOP decreased total VFA concentration, acetate concentration, and the acetate to propionate molar ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). 3-Nitrooxypropanol increased, and BMF decreased, propionate concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas BMF increased butyrate concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Coenzyme M spiking at 1 mM increased isobutyrate concentration only when methanogenesis was inhibited (interaction \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008). 3-Nitrooxypropanol and BMF decreased the concentrations of valerate, caproate, heptanoate, and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, and increased methanol (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Coenzyme M spiking at 1 mM increased the concentration of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). All three inhibitors of methanogenesis decreased succinate concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e\n \u003cp\u003epH and heptanoate normal quantile plots exhibited S-shaped deviations. The plots of residuals against predicted observations of CH\u003csub\u003e4\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003e, valerate, caproate, heptanoate, succinate, methanol, and ethanol, were funnel-shaped; however, log-transformation of those \u003cem\u003ey\u003c/em\u003e variables did not alter the conclusions of the analyses. No outliers unrelated to the rest of the observations of their treatment were found.\u003c/p\u003e\n \u003cp\u003eAs the serial incubations progressed in serial transfers 1 to 4, production of total gas (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) and CH\u003csub\u003e4\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Figure S2) decreased, whereas E\u003cem\u003eh\u003c/em\u003e increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Figure S3). Accumulation of H\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.85) and pH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32) were unaffected (not shown). None of the response variables in serial transfers 1 to 4 were affected by CoM supplementation at 1 \u0026micro;M (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.41; not shown).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperiment 2\u003c/h3\u003e\n\u003cp\u003eSpiking 1mM methyl-CoM relieved the effects of BES (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.003) but not of 3-NOP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.62) or BMF (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.24) on CH\u003csub\u003e4\u003c/sub\u003e production, H\u003csub\u003e2\u003c/sub\u003e accumulation, acetate and isobutyrate concentrations, the acetate to propionate molar ratio, and the concentration of succinate and ethanol (interaction methanogenesis inhibition \u0026times; methyl-CoM \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.029; Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In the absence of methyl-CoM spiking, BES decreased CH\u003csub\u003e4\u003c/sub\u003e production (-52%) and acetate concentration, and increased the accumulation of H\u003csub\u003e2\u003c/sub\u003e (5.01-fold), caproate, succinate, and ethanol (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both with and without 1 mM methyl-CoM, 3-NOP decreased CH\u003csub\u003e4\u003c/sub\u003e (-29.6%) and the acetate to propionate molar ratio, and increased H\u003csub\u003e2\u003c/sub\u003e accumulation (3.28-fold), caproate, and ethanol (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and BMF decreased total gas production, CH\u003csub\u003e4\u003c/sub\u003e (-97.1%), total VFA concentration, acetate, isobutyrate, 2- and 3-methylbutyrate, caproate, the acetate to propionate molar ratio, succinate, and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and increased the accumulation of H\u003csub\u003e2\u003c/sub\u003e (14.7-fold), and methanol (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Bromoform tended to increase butyrate concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.081).\u003c/p\u003e\n\u003cp\u003eResiduals as a function of predicted values were not uniformly distributed for H\u003csub\u003e2\u003c/sub\u003e, the acetate to propionate molar ratio, and succinate; however, log-transformation of the response variables did not alter the shape of the plots or the conclusions of those analyses. Normal quantile plots of pH and 2- and 3-methylbutyrate were S-shaped, but log-transformation of the response variables did not correct them or change the conclusions of the analyses. Noninfluential outliers following the overall responses of their treatments were detected for H\u003csub\u003e2\u003c/sub\u003e and were not eliminated from the analyses.\u003c/p\u003e\n\u003ch3\u003eExperiment 3\u003c/h3\u003e\n\u003cp\u003eBoth CoM and methyl-CoM relieved the inhibition of total gas and CH\u003csub\u003e4\u003c/sub\u003e production, the accumulation of H\u003csub\u003e2\u003c/sub\u003e and ethanol, the decrease in the acetate to propionate ratio, and the decrease in the concentrations of 2- and 3-methylbutyrate, succinate, and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, caused by BES (interactions BES by CoM or methyl-CoM \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.044; Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and Supplementary Figures S4-S8). At 100 \u0026micro;M, CoM and methyl-CoM reversed approximately 62 and 68% of methanogenesis inhibition by BES, respectively, and at 1000 \u0026micro;M, CoM and methyl-CoM reversed 93 and 100% methanogenesis inhibition, in the same order. 2-Bromoethanesulfonate decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.036) total VFA and isobutyrate concentration whereas methyl-CoM tended (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.066) to increase isobutyrate (Supplementary Figures S9 and S10). Methyl-coenzyme M (interaction BES by methyl-CoM \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) but not CoM (interaction BES by methyl-CoM \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.23) reversed the decrease in acetate concentration caused by BES (Supplementary Figure S11). There was a tendency for CoM to decrease propionate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056), especially with BES (interaction BES \u0026times; CoM; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.062; Supplementary Figure S12). In the presence of BES, CoM and methyl-CoM decreased or tended to decrease valerate, caproate, and heptanoate (interaction of BES with CoM or methyl-Com \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.095; Supplementary Figures S13-S15). There were no effects of BES, CoM, methyl-CoM, or the interactions of BES with CoM or methyl-CoM on DM disappearance and butyrate and methanol concentrations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.17; not shown). Coenzyme M and methyl-CoM increased or tended to increase pH when methanogenesis was inhibited by BES (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.055; Supplementary Figure S16). 2-Bromoethanesulfonate tended to decrease (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.071), and methyl-CoM increased, E\u003cem\u003eh\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017; Supplementary Figure S17).\u003c/p\u003e\n\u003cp\u003eLog-transforming total gas production and butyrate, caproate, heptanoate, and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentrations did not correct S-shaped residual normality plots or funnel-shaped plots of residuals against predicted values, nor changed the conclusions of those analyses, so the analyses with the untransformed variables are reported. An outlier with no H\u003csub\u003e2\u003c/sub\u003e accumulation in the BES treatment with 1 \u0026micro;M CoM was removed from the analysis. Noninfluential outliers with high methanol and ethanol concentrations were left in the analyses.\u003c/p\u003e\n\u003cp\u003eThe rumen fluid inocula from both cows in both incubations had 55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81 (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) \u0026micro;M CoM plus methyl-CoM, ranging between 53.2 and 57.4 \u0026micro;M (data not shown).\u003c/p\u003e\n\u003cp\u003eA total of 152 unique archaeal ASVs were assigned at the phylum level. The rarefaction curve revealed adequate coverage (Supplementary Figure S18). At the phylum level, the addition of BES decreased the relative abundance of total \u003cem\u003eEuryarchaeota\u003c/em\u003e 16S rRNA gene (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011; Supplementary Figure S19), whereas methyl-CoM at 1 mM increased total \u003cem\u003eEuryarchaeota\u003c/em\u003e relative abundance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005); the opposite changes were observed in the relative abundance of \u003cem\u003eThermoplasmatota\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.015). At the genus level, there was an interaction between methanogenesis inhibition and methyl group carriers on the relative abundance of \u003cem\u003eMethanobrevibacter ruminantium\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), through which CoM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) and methyl-CoM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) at 1 mM reversed the decrease in \u003cem\u003eM. ruminantium\u003c/em\u003e caused by BES (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Other \u003cem\u003eMethanobrevibacter\u003c/em\u003e, \u003cem\u003eMethanosphaera\u003c/em\u003e spp., and \u003cem\u003eCandidatus Methanomethylophilus\u003c/em\u003e were unaffected by any of the treatments or their interactions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.16). The relative abundance of unclassified \u003cem\u003eMethanomethylaceae\u003c/em\u003e was increased by BES (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) and decreased by methyl-CoM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005); changes in the relative abundance of unclassified \u003cem\u003eMethanomethylaceae\u003c/em\u003e reflected changes in \u003cem\u003eM. ruminantium\u003c/em\u003e, as absolute reads of unclassified \u003cem\u003eMethanomethylaceae\u003c/em\u003e were unaffected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.15; Supplementary Fig.\u0026nbsp;20). A PCA plot revealed that most of the treatments supplemented with BES and no CoM or methyl-CoM separated from their functional methanogenesis controls, whereas the treatments receiving BES plus CoM or methyl-CoM appeared closer to the methanogenesis inhibition controls (Supplementary Figure S21). There were no effects of BES, CoM, or methyl-CoM or their interactions on archaeal diversity indices (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.24; Supplementary Table S3).\u003c/p\u003e\n\u003cp\u003eA total of 23,929 unique bacterial ASVs were assigned at the phylum level. The rarefaction curve revealed adequate coverage (Supplementary Figure S22). Only those bacterial clades whose relative abundance was equal or greater than 0.5% of the total bacterial 16S rRNA gene in at least one combination of BES and CoM or methyl-CoM treatment are presented. At the phylum level, BES decreased the relative abundance of \u003cem\u003eActinobacteriota\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033; Supplementary Table S4). Coenzyme M tended to increase the relative abundance of \u003cem\u003eSpirochaetota\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.10). Methyl-CoM relieved (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048) and CoM tended to relieve (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.093) a decrease in the relative abundance of \u003cem\u003eVerrucomicrobiota\u003c/em\u003e caused by BES (interaction BES \u0026times; CoM/methyl-CoM \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.10). There were no effects of BES, CoM, methyl-CoM, or their interactions, on the relative abundance of other bacterial phyla (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.11). At the genus level, BES decreased the relative abundances of \u003cem\u003eOlsenella\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029; Supplementary Table S5) and \u003cem\u003eButyrivibrio\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007) and tended to increase those of the \u003cem\u003eRikenellaceae RC9 group\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.050). Coenzyme M increased the relative abundance of \u003cem\u003e[Eubacterium] halli group\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024). Coenzyme M tended (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.067) and methyl-CoM decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) the relative abundance of \u003cem\u003eRuminococcus\u003c/em\u003e. Methyl-CoM decreased the relative abundance of \u003cem\u003eHorsej-a03\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036). Coenzyme M and methyl-CoM relieved or partially relieved the decrease in the relative abundance of the \u003cem\u003eLachnospiraceae UCG-008 group\u003c/em\u003e (interaction BES \u0026times; CoM \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) and \u003cem\u003ePapillibacter\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018), respectively. Most treatments receiving methyl-CoM and BES clustered with Control treatments with no added BES in a PCA plot (Supplementary Fig.\u0026nbsp;23).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eExperiment 4\u003c/h2\u003e\n \u003cp\u003eThere were no effects of BES (not shown), 3-NOP, BMF, methyl group carriers, or their interactions, on total gas production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.19; Supplementary Figures S24A, B). 2-Coenzyme M and methyl-CoM overcame methanogenesis inhibition (BES \u0026times; methyl group carriers \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006; Supplementary Figure S25) and H\u003csub\u003e2\u003c/sub\u003e accumulation (BES \u0026times; methyl group carriers \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025; Supplementary Figure S26) by BES. 3-Nitrooxypropanol tended (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.095) to decrease CH\u003csub\u003e4\u003c/sub\u003e production by a maximum of 9.5% (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) and BMF decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) CH\u003csub\u003e4\u003c/sub\u003e production by a maximum of 38%, without interactions with the addition of methyl group carriers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.56; Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). Accumulation of H\u003csub\u003e2\u003c/sub\u003e was decimal log-transformed to correct for residual normality and positive outliers. Nitrooxypropanol (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012; Fig.\u0026nbsp;9) and BMF increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;10) H\u003csub\u003e2\u003c/sub\u003e accumulation. The addition of methyl group carriers did not interact with 3-NOP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.55; Fig.\u0026nbsp;9) or BMF (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.14; Fig.\u0026nbsp;10) on H\u003csub\u003e2\u003c/sub\u003e accumulation. Both BES (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.051; Supplementary Figure S27) and BMF (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.087; Supplementary Figure S28) tended to increase final pH. Bromoform (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028; Supplementary Figure S29), CoM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Figure S30), and methyl-CoM (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Figure S30) decreased final E\u003cem\u003eh\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs we hypothesized, and in agreement with previous observations in pure cultures of methanogens, methanogen cell extracts, purified MCR, and methanogenic sediments [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the addition of CoM or methyl-CoM to rumen cultures blocked the inhibitory effect of BES on methanogenesis. In Experiment 3, molar ratios of added CoM and methyl-CoM to BES of 20 \u0026micro;M/\u0026micro;M (i.e., at 100 \u0026micro;M CoM or methyl-CoM) reversed most of the inhibition of methanogenesis by BES. Similarly, Konisky [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] reported that in CH\u003csub\u003e4\u003c/sub\u003e-producing marine sediments, molar ratios of CoM to BES between 20 and 40 \u0026micro;M/\u0026micro;M reversed approximately half of the inhibition of methanogenesis.\u003c/p\u003e \u003cp\u003eThe question arises whether the reversal of methanogenesis inhibition demonstrated herein with rumen cultures could influence BES efficacy at typical in vivo concentrations of CoM and methyl-CoM in the rumen, as the concentration of CoM plus methyl-CoM in the inoculum of Experiment 3 was comparable to the concentration of CoM or methyl-CoM affecting BES efficacy in Experiment 3. We are not aware of previous reports of the concentration of CoM or methyl-CoM in the rumen, or in mixed rumen cultures. Although the range of concentrations of CoM plus methyl-CoM in the rumen inocula used in Experiment 3 was relatively narrow, the inocula were sampled in two consecutive weeks from the same two rumen fistulated cows penned together and fed the same diet. It is possible that the concentrations of CoM and methyl-CoM vary more amply among different types and species of ruminants eating different diets and in different geographical locations, and that variation in the concentrations of CoM and methyl-CoM in the rumen could thus influence BES efficacy.\u003c/p\u003e \u003cp\u003eElias et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] reported concentrations of CoM ranging between 0.02 and 0.22 \u0026micro;M for pond and landfill sediments, sewage sludge, and a hydrocarbon-contaminated sediment below 1.2 m deep. We found considerably greater concentrations of CoM plus methyl-CoM (between 50 and 60 \u0026micro;M) in the rumen inocula utilized for Experiment 3. However, the concentration of CoM in the rumen might be higher than in other environments, as the rumen environment contains high concentrations of metabolites derived from microbial activities [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and some of the densities of methanogens in the rumen conducted using classical cultivation techniques summarized by Joblin [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], appear to be greater than the density of methanogens reported by Elias et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] for other methanogenic environments.\u003c/p\u003e \u003cp\u003ePerhaps more importantly, CoM and methyl-CoM may have implications for the persistence of the antimethanogenic effect of BES on rumen fermentation. 2-Bromoethanesulfonate is considered a transient inhibitor of methanogenesis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], although in vitro and in vivo experimental results concerning the persistence of BES are scarce and partially conflicting. In 24- or 48-h mixed rumen batch cultures, BES at relatively low concentrations\u0026thinsp;\u0026le;\u0026thinsp;30 \u0026micro;M has consistently inhibited methanogenesis [\u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69 CR70\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. One pulse dose of 50 \u0026micro;M BES to continuous cultures also inhibited CH\u003csub\u003e4\u003c/sub\u003e production for approximately 24 h [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Conversely, in vivo inhibition of methanogenesis by BES daily dosed to a sheep was shown to last for only 4 d, after which the rumen microbiota seemed to adapt to BES, and CH\u003csub\u003e4\u003c/sub\u003e production increased again [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. In rumen continuous cultures, BES at the relatively high concentration of 250 \u0026micro;M inhibited CH\u003csub\u003e4\u003c/sub\u003e production in the first 2 d of incubation, but was ineffective after a 7-d adaptation period [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]; in contrast, in a semicontinuous culture study, BES at 36 or 72 \u0026micro;M effectively inhibited CH\u003csub\u003e4\u003c/sub\u003e production and decreased methanogen abundance even after 8 d of adaptation [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Ahring et al. [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] dosed a long-term (30 d) semicontinuous rumen-inoculated bioreactor with very high 10 mM pulse doses of BES on d 1 and 15 of incubation and reported profound inhibition of methanogenesis. Similarly, Webster et al. [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] reported severe inhibition of CH\u003csub\u003e4\u003c/sub\u003e production 9 d after dosing with a relatively high dose of 0.5 mM BES a bioreactor inoculated with cow dung and wastewater.\u003c/p\u003e \u003cp\u003eThe transient effects of BES on methanogenesis may be related to changes in the composition of the rumen archaeal community triggered by BES supplementation, as differences in the sensitivity of different methanogens to BES have been reported [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The uptake of CoM by the ruminal methanogen \u003cem\u003eM. ruminantium\u003c/em\u003e strain M1 was inhibited by BES, indicating competition between CoM and BES for transmembrane transport [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. There are important differences among methanogens in the uptake of CoM [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The ruminal methanogen \u003cem\u003eM. ruminantium\u003c/em\u003e strain M1, which lacks three genes of the CoM biosynthetic pathway [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], requires exogenous CoM [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. On the other hand, the non-ruminal strain \u003cem\u003eM. ruminantium\u003c/em\u003e strain PS, and other ruminal and non-ruminal methanogens, do not take up CoM or take it up at much lower rates than \u003cem\u003eM. ruminantium\u003c/em\u003e M1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The ruminal methanogen \u003cem\u003eMethanomicrobium mobile\u003c/em\u003e 1, which is considerably more resistant to BES than \u003cem\u003eM. ruminantium\u003c/em\u003e M1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], takes up CoM at a rate less than 10% the CoM uptake rate of \u003cem\u003eM. ruminantium\u003c/em\u003e M1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In agreement with those findings, resistance to BES in non-ruminal methanogens \u003cem\u003eMethanococcus voltae\u003c/em\u003e and \u003cem\u003eMethanosarcina\u003c/em\u003e 227 was related to low rates of transport of BES into the cell [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The existence of a common transmembrane transport system for BES and its structural analogs CoM and methyl-CoM has been proposed [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe searched GenBank for the capacity of the archaeal clades present in Experiment 3 samples to synthesize CoM, based on the presence of genes \u003cem\u003ecomA\u003c/em\u003e, \u003cem\u003ecomB\u003c/em\u003e, \u003cem\u003ecomC\u003c/em\u003e, \u003cem\u003ecomD\u003c/em\u003e, and \u003cem\u003ecomE\u003c/em\u003e in their genomes. Within \u003cem\u003eMethanobacteriales\u003c/em\u003e, with the exception of \u003cem\u003eM. ruminantium\u003c/em\u003e (strain M1, Leahy et al. [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]), all other \u003cem\u003eMethanobacteriales\u003c/em\u003e identified in Experiment 3 seemed to have the genetic capacity to synthesize CoM [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In Experiment 3 the relative abundance of \u003cem\u003eMethanobacteriales\u003c/em\u003e other than \u003cem\u003eM. ruminantium\u003c/em\u003e was unaffected by BES or by BES interactions with CoM or methyl-CoM. \u003cem\u003eM. ruminantium\u003c/em\u003e M1 is highly sensitive to BES [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and in agreement, the abundance of \u003cem\u003eM. ruminantium\u003c/em\u003e was decreased by BES by more than two thirds in Experiment 3, and almost completely recovered when 1 mM CoM or methyl-CoM was supplemented along BES. Our observation that CoM and methyl-CoM negate the effects of BES on \u003cem\u003eM. ruminantium\u003c/em\u003e abundance and on CH\u003csub\u003e4\u003c/sub\u003e production agrees with the sensitivity of methanogens to BES being mediated by their capacity to synthesize CoM.\u003c/p\u003e \u003cp\u003eRuminal \u003cem\u003eMethanomassiliicoccales\u003c/em\u003e isolates ISO4-G1, ISO4-H5, and \u003cem\u003eThermoplasmatales\u003c/em\u003e archaeon BRNA1 lack CoM biosynthetic genes \u003cem\u003ecomADE\u003c/em\u003e and thus cannot synthesize CoM [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. \u003cem\u003eMethanomassiliicoccales\u003c/em\u003e ISO4-H5 is highly sensitive to BES [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], suggesting that, as in \u003cem\u003eM. ruminantium\u003c/em\u003e, the requirement to take up exogenous CoM also results in high rates of intracellular transport of BES. In Experiment 3, we did not find effects of BES or BES interactions with CoM or methyl-CoM on the relative abundance of a relatively small population of \u003cem\u003eCandidatus Methanomethylophilus\u003c/em\u003e sp., or on other \u003cem\u003eMethanomassiliicoccales\u003c/em\u003e; however, because our metataxonomic analysis did not have enough depth to identify all archaeal ASVs at the species or strain level, it is difficult to speculate on whether the \u003cem\u003eMethanomassiliicoccales\u003c/em\u003e in our cultures had the genetic capacity to synthesize CoM. Future studies with ruminal mixed cultures or in vivo could use metagenomics or qPCR to directly quantify the effects of BES on CoM biosynthetic genes, to accurately confirm whether the effects of BES and its interactions with CoM and methyl-CoM on CH\u003csub\u003e4\u003c/sub\u003e production are related to the genetic capacity of the mixed methanogen community to synthesize CoM.\u003c/p\u003e \u003cp\u003eTherefore, in addition to oxidizing the Ni\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e atom in cofactor F\u003csub\u003e430\u003c/sub\u003e in MCR [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], BES inhibits methanogenesis in \u003cem\u003eM. ruminantium\u003c/em\u003e M1 and other methanogens that cannot synthesize CoM by blocking the uptake of CoM. An implication of this second mechanism of action is that those methanogens that can synthesize their own CoM and are not reliant on exogenous CoM would be more tolerant to BES, as they may not take up external CoM or they would do it at slower rates [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], thus also taking up BES at slower rates or not taking it up. Thus, it appears possible that long-term supplementation of BES could select for those methanogens that can synthesize CoM and do not transport CoM and BES into their cells, resulting in lower sensitivity to BES and ultimately in transient inhibition of methanogenesis. In addition, MCRs of different rumen methanogens may differ in their sensitivity to BES. For example, MCR isolated from \u003cem\u003eM. ruminantium\u003c/em\u003e [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] was more sensitive to BES than MCR isolated from non-ruminal methanogen \u003cem\u003eMethanothermobacter marburgensis\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], although it should be cautioned that those determinations were not direct comparisons conducted in the same study.\u003c/p\u003e \u003cp\u003eIn Experiment 1, we investigated whether supplementation of CoM at 1 \u0026micro;M in the first four of five serial transfers of rumen serial cultures would favor those methanogens lacking the capacity to synthesize CoM and presumably more sensitive to BES, increasing the efficacy of BES to inhibit methanogenesis. However, supplementing CoM at 1 \u0026micro;M in serial transfers 1 to 4 in Experiment 1, however, had minimal effects on fermentation. This may be related to the presumably relatively small increase in CoM concentration resulting from the 1 \u0026micro;M supplementation dose utilized in serial transfers 1 to 4 in Experiment 1, in comparison with the concentration of CoM that the non-supplemented cultures may have had.\u003c/p\u003e \u003cp\u003eInhibiting methanogenesis affects the rumen microbial community beyond methanogens through the resulting accumulation of H\u003csub\u003e2\u003c/sub\u003e, which profoundly affects electron flows in rumen fermentation through the thermodynamics of H\u003csub\u003e2\u003c/sub\u003e-producing and H\u003csub\u003e2\u003c/sub\u003e-incorporating pathways [\u003cspan additionalcitationids=\"CR85\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. We observed a decrease in \u003cem\u003eOlsenella\u003c/em\u003e when methanogenesis was inhibited with BES. \u003cem\u003eOlsenella\u003c/em\u003e isolated from the sheep rumen and pig jejunum produces acetate as a minor product in pure culture [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e] but it is possible that in mixed cultures in the presence of methanogens and other hydrogenotrophs removing H\u003csub\u003e2\u003c/sub\u003e \u003cem\u003eOlsenella\u003c/em\u003e produces greater amounts of acetate than in pure culture. If that is the case, the accumulation of H\u003csub\u003e2\u003c/sub\u003e caused by BES could inhibit the release of H\u003csub\u003e2\u003c/sub\u003e associated to acetate production [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e], thus explaining the inhibitory effect of BES on \u003cem\u003eOlsenella\u003c/em\u003e\u0026acute;s abundance in Experiment 3. Similarly, butyrate production involves a release of H\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] and the H\u003csub\u003e2\u003c/sub\u003e built up resulting from methanogenesis inhibition by BES might have thus decreased butyrate-producing \u003cem\u003eButyrivibrio\u003c/em\u003e\u0026acute;s relative abundance in Experiment 3. Even though hydrogenotrophic bacterial genera such as \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eSelenomonas\u003c/em\u003e are expected to increase as a consequence of methanogenesis inhibition and H\u003csub\u003e2\u003c/sub\u003e accumulation, experimental results in this respect have been variable [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e], which is in agreement with the lack of effects on those genera observed in Experiment 3. The effects of BES, CoM and methyl-CoM on the \u003cem\u003eRikenellaceae RC9 group\u003c/em\u003e, \u003cem\u003e[Eubacterium] halli group\u003c/em\u003e, and \u003cem\u003eHorsej-a03\u003c/em\u003e are difficult to interpret because of the lack of known isolates characterized in pure cultures or cocultures. In bacteria, CoM is involved in the oxidation of alkenes [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Searching GenBank, we did not find any of the bacterial genera found in Experiment 3 to possess genes related to the biosynthesis of CoM.\u003c/p\u003e \u003cp\u003eThere has been speculation that, because 3-NOP inhibits MCR by having a similar molecular shape as methyl-CoM [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the concentration of CoM and methyl-CoM in rumen fluid may influence 3-NOP efficacy [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. However, spiking rumen cultures with CoM or methyl-CoM at 1 mM in Experiments 1 and 2 did not hinder the inhibition of methanogenesis by 3-NOP. In Experiment 4, we examined molar ratios of CoM and methyl-CoM as high as 25,000 to 1 and could not find hindering effects of CoM or methyl-CoM on the antimethanogenic effect of 3-NOP; however, because of reasons unrelated to CoM or methyl-CoM supplementation, the effects of 3-NOP on CH\u003csub\u003e4\u003c/sub\u003e production were considerably milder than those found in Experiments 1 and 2. Both BES and 3-NOP irreversibly inactivate MCR through oxidation of the Ni\u003csup\u003e1+\u003c/sup\u003e atom in cofactor F\u003csub\u003e430\u003c/sub\u003e bound to MCR, to Ni\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Unlike BES, which is a charged molecule that needs to be actively transported into the cells, 3-NOP is a polar but uncharged molecule thought to freely diffuse across cell membranes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The requirement for a transmembrane transporter for BES and the resulting competition between BES and CoM for intracellular transport [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], but not for 3-NOP, could thus explain why CoM and methyl-CoM hindered the antimethanogenic effect of BES, but not the antimethanogenic effect of 3-NOP which would not compete for transporters.\u003c/p\u003e \u003cp\u003eThe active uptake of CoM in \u003cem\u003eM. ruminantium\u003c/em\u003e M1 and other methanogens requiring exogenous CoM, might theoretically be expected to competitively displace 3-NOP from the MCR catalytic site, thus affecting 3-NOP efficacy even if it does not compete for transmembrane transporters. However, the active transport of CoM in \u003cem\u003eM. ruminantium\u003c/em\u003e displays saturation kinetics, with the rate of uptake of CoM plateauing at approximately 0.63 \u0026micro;M [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, increasing the concentration of CoM in the medium might not have altered the intracellular CoM concentration to compete with 3-NOP for access to the catalytic site in MCR.\u003c/p\u003e \u003cp\u003eIn the present study, the addition of CoM or methyl-CoM did not interfere with the inhibition of methanogenesis by BMF, even at molar ratios of CoM or methyl-CoM to BMF as high as 25,000 to 1 in Experiment 4. Bromoform and other CH\u003csub\u003e4\u003c/sub\u003e halogenated analogs inhibit methanogenesis by reacting with the corrinoid unit in MTR, blocking the transfer of a methyl group from MTR to CoM to form methyl-CoM [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Yu and Smith [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] proposed that CH\u003csub\u003e4\u003c/sub\u003e halogenated analogs can inhibit methanogenesis by binding not only corrinoid-containing MTR but also porphyrin-containing MCR, as well as free intracellular corrinoids and porphinoids. Observations showing that corrinoids and also cofactor F\u003csub\u003e430\u003c/sub\u003e stimulated dechlorination of chlorohydrocarbons by \u003cem\u003eMethanosarcina barkeri\u003c/em\u003e [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e] and that MCR conducted dechlorination of 1, 2-dichloroethane [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e], also support the existence of an interaction of halohydrocarbons with MCR, implying that BMF and other CH\u003csub\u003e4\u003c/sub\u003e halogenated analogs could have two targets, MTR and MCR [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Bromoform has been computationally predicted to bind cofactor F\u003csub\u003e430\u003c/sub\u003e with high affinity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, Karuso and Liu [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e] reported that BMF inhibited MCR isolated from \u003cem\u003eMethanothermobacter marburgensis\u003c/em\u003e only at considerably high concentrations above 0.50 mM (greater than the methyl-CoM to BMF molar ratio of 20: 1 in that study), which agrees with earlier observations with chloroform [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. In contrast to the enzymology study by Karuso and Liu [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e] with BMF and purified MCR, in mixed cultures, BMF could potentially inhibit both MCR and MTR. The fact that we found no alleviation of methanogenesis inhibition by BMF with the addition of methyl-CoM or CoM at much larger molar ratios to BMF than the 20 to 1 molar ratio found to partially relieve inhibition of MCR by Karuso and Liu [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e], considering that BMF rapidly diffuses across cell membranes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], suggests that in our study BMF inhibited CH\u003csub\u003e4\u003c/sub\u003e production mainly through its reaction with corrinoids in MTR without affecting MCR.\u003c/p\u003e \u003cp\u003eVitamin B\u003csub\u003e12\u003c/sub\u003e and other corrinoids play a role in various cellular methylation reactions, including the conversion of succinyl-CoA to methyl-malonyl-CoA in propionate randomizing pathway in the rumen [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. Therefore, it could be conceivable therefore that BMF interferes with propionate formation via randomizing pathway. However, BMF addition did not cause consistent effects on propionate production and did not induce succinate accumulation in Experiments 1 and 2, suggesting that the conversion of succinyl-CoA to methyl-malonyl-CoA was not impaired by BMF. These results agree with previous rumen batch cultures work in which bromoform and other CH\u003csub\u003e4\u003c/sub\u003e-halogenated analogs did not impaired or improved, propionate production [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e], although a decrease in propionate was observed in continuous cultures supplemented bromochloromethane [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBoth CoM and methyl-CoM hindered the effects of BES to inhibit methanogenesis and decrease the abundance of \u003cem\u003eM. ruminantium\u003c/em\u003e. These results suggest that the inhibition of methanogenesis by BES and its reversal by CoM and methyl-CoM may be related to the capacity of methanogens to synthesize CoM, which can have implications regarding the lack of persistence of BES on CH\u003csub\u003e4\u003c/sub\u003e production in vivo. Whether the sensitivity of methanogens in mixed cultures and in vivo to BES is related to their genetic capacity to synthesize CoM affecting BES transmembrane transport needs to be confirmed through metagenomics or qPCR quantification of CoM biosynthetic genes. The efficacy of 3-NOP and BMF was not influenced by CoM or methyl-CoM. Although both BES and 3-NOP affect MCR through similar mechanisms, CoM and methyl-CoM compete for transmembrane transport with BES, but CoM and methyl-CoM do not seem to compete with 3-NOP for transmembrane transport. In our study, BMF inhibition of methanogenesis seemed to be mediated through the inhibition of MTR rather than MCR.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADF, Acid detergent fiber\u003c/p\u003e\n\u003cp\u003eASV, Amplicon sequence variant\u003c/p\u003e\n\u003cp\u003eBES, 2-Bromoethanesulfonate\u003c/p\u003e\n\u003cp\u003eBMF, Bromoform\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCoM, Coenzyme M\u003c/p\u003e\n\u003cp\u003eCP, Crude protein\u003c/p\u003e\n\u003cp\u003eDM, Dry matter\u003c/p\u003e\n\u003cp\u003eDMD, Apparent dry matter disappearance\u003c/p\u003e\n\u003cp\u003eEE, Ether extract\u003c/p\u003e\n\u003cp\u003eGC, Gas chromatography\u003c/p\u003e\n\u003cp\u003eGC-MS, Gas chromatography coupled to mass spectrometry\u003c/p\u003e\n\u003cp\u003eHSD, Honestly Significant Difference\u003c/p\u003e\n\u003cp\u003eLSD, Least squares difference\u003c/p\u003e\n\u003cp\u003eMethyl-CoM, Methyl-coenzyme M\u003c/p\u003e\n\u003cp\u003eMCR, Methyl-coenzyme M reductase\u003c/p\u003e\n\u003cp\u003eMTR, Methyltetrahydromethanopterin: coenzyme M methyltransferase\u003c/p\u003e\n\u003cp\u003eNDF, Neutral detergent fiber\u003c/p\u003e\n\u003cp\u003e3-NOP, 3-Nitrooxypropanol\u003c/p\u003e\n\u003cp\u003ePCA, Principal components analysis\u003c/p\u003e\n\u003cp\u003eVFA, Volatile fatty acids\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by Instituto de Investigaciones Agropecuarias (approval number 03/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene sequence files generated in this study are available at https://www.ncbi.nlm.nih.gov/sra/PRJNA1375672. The datasets supporting the conclusions of this article are available in the Open Science Framework repository, project dxy8e https://osf.io/dxy8e/overview\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Agencia Nacional de Investigaci\u0026oacute;n y Desarrollo, Santiago, Chile, Proyecto Fondecyt 1240264. We are grateful to Universidad de Buenos Aires for a UBAINT Doctoral 2023-2024 fellowship granted to Florencia Samoluk and to the Global Research Alliance on Agricultural Greenhouse Gases (GRA) and the CGIAR Initiative on Low-Emission Food Systems (Mitigate+) for supporting Boma Iriso through their CLIFF-GRADS program.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026acute; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEMU conceived, designed, and conducted the experiments and analyzed the samples, analyzed the data, and wrote the manuscript. NC and MFS conducted the experiments and analyzed the samples and reviewed the manuscript. GJ, BI and NP reviewed the manuscript. All the authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Rudolph Thauer for his valuable comments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSzopa S, Naik V, Adhikary B, Artaxo P, Berntsen T, Collins WD, et al. Short-lived climate forcers. In: Masson-Delmotte V, Zhai P, Pirani A, Connors SL, P\u0026eacute;an C, Berger S, Caud N, Chen Y, Goldfarb L, Gomis MI\u003cem\u003e, et al\u003c/em\u003e, editor. 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Results are presented across the average of 0 and 1 \u0026micro;M CoM supplementation in serial transfers 1 - 4 (Experiment 1).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"860\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 151px;\"\u003e\n \u003cp\u003eMethanogenesis inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3-NOP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 57px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e =\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"38\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003eInh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCoM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003eInh \u0026times; CoM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"55\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eCoM\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eTotal gas (mmol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.17\u003csup\u003eab 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.23\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e (\u0026micro;mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e129\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e127\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e54.3\u003csup\u003ej 2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e123\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e33.9\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e36.8\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e30.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e (\u0026micro;mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e17.8\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e12.7\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e21.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e10.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e49.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e53.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e79.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e87.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.27\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.24\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.25\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eE\u003cem\u003eh\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-105\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-108\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-99.3\u003csup\u003en\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-99.0\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-94.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e5.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eDMD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e57.5\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e56.7\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e57.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e56.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e56.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e56.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e53.9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e53.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eTotal VFA (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e69.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e69.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e67.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e69.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e65.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e66.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e59.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e59.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eAcetate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e36.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e36.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e34.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e36.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e33.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e33.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e28.9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e28.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePropionate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e24.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e24.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e24.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e24.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e25.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e25.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e22.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e22.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eButyrate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.78\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e5.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eIsobutyrate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.29\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.33\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.29\u003csup\u003en\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.33\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.26\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.30\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2- and 3-methylbutyrate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.42\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.50\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.42\u003csup\u003en\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.50\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.33\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.43\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eValerate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eCaproate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.87\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eHeptanoate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.046\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.057\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.014\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.024\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eAcetate to propionate (mM/mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eSuccinate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eMethanol (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e12.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e412\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e163\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e652\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e411\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e93.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eEthanol (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e7.85\u003csup\u003ea 3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e9.87\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e12.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e13.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e14.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e14.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e20.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e20.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e19.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e20.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e19.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e20.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e19.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e20.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eWhen the main effect of methanogenesis inhibition was significant (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and the interaction between methanogenesis inhibition and CoM spiking at 1 mM was not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), unlike superscripts between inhibitors indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) according to Tukey\u0026acute;s HSD; \u003csup\u003e2\u003c/sup\u003eWhen the interaction between methanogenesis inhibition and CoM spiking was significant (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), the effect of CoM is separately evaluated on the efficacy of each inhibitor and unlike superscripts within an inhibitor of methanogenesis indicate that the effect of the inhibitor was affected by CoM spiking (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05): a, b = CoM effect within Control; i, j = CoM effect within BES; m, n = CoM effect within 3-NOP; x, y = CoM effect within BMF; \u003csup\u003e3\u003c/sup\u003eCalculated initial concentration of ethanol was 10.4 mM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable \u003c/strong\u003e2. Effects of spiking methyl-coenzyme M (methyl-CoM) at 1 mM on the efficacy of methanogenesis inhibitors (Inh) 2-bromoethanesulfonate (BES), 3-nitrooxypropanol (3-NOP) or bromoform (BMF), all at 5 \u0026micro;M, in batch rumen cultures (Experiment 2).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"832\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e production inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003eBES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3-NOP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 57px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"55\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e =\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"30\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eMethyl-CoM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eInh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eMethyl-CoM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eInh \u0026times; Methyl-CoM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eTotal gas (mmol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.67\u003csup\u003ea 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e4.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e4.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e4.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e4.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.39\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e (\u0026micro;mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e456\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e441\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e220\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e435\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e315\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e325\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e12.8\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e12.9\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e59.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e (\u0026micro;mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e12.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e17.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e63.4\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e16.4\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e49.8\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e48.3\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e224\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e217\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e6.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eDMD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e26.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e23.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e24.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e24.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e22.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e23.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e6.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eTotal VFA (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e82.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e82.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e77.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e83.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e78.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e78.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e70.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e68.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAcetate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e52.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e52.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e45.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e53.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e48.0\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e47.8\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e37.7\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e37.2\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePropionate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e15.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e15.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e16.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e16.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e17.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eButyrate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e11.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eIsobutyrate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.65\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.75\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.67\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.68\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.54\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.50\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2- and 3-methylbutyrate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eValerate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eCaproate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.18\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.095\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAcetate to propionate (mM/mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e3.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.66\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.48\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.04\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.07\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.15\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2.19\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eSuccinate (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.95\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.60\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.97\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.77\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.81\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.55\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.51\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eMethanol (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eEthanol (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.84\u003csup\u003ea 3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e5.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e10.7\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e5.74\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e8.22\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e8.12\u003csup\u003em\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e13.8\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e13.7\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e27.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e27.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e25.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e27.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e26.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e26.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e23.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e23.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eWhen the interaction between methanogenesis inhibition and methyl-CoM spiking is not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), unlike superscripts between inhibitors indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) according to Tukey\u0026acute;s HSD; \u003csup\u003e2\u003c/sup\u003eWhen the interaction between methanogenesis inhibition and methyl-CoM spiking is significant (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), the effect of methyl-CoM is separately evaluated on the efficacy of each inhibitor and unlike superscripts within an inhibitor of methanogenesis indicate a significant effect of methyl-CoM spiking (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05): a, b = methyl-CoM effect within Control; i, j = methyl-CoM effect within BES; m, n = methyl-CoM effect within 3-NOP; x, y = methyl-CoM effect within BMF; \u003csup\u003e3\u003c/sup\u003eInitial concentration of ethanol was 10.4 mM.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"methane, methanogens, coenzyme M, methyl-coenzyme M, methanogenesis inhibitors, 2-bromoethanesulfonate, 3-nitrooxypropanol, bromoform","lastPublishedDoi":"10.21203/rs.3.rs-8419205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8419205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUnderstanding the mechanisms of action of compounds inhibiting methanogenesis can aid understanding the variation in their efficacy as feed additives for mitigating enteric methane (CH\u003csub\u003e4\u003c/sub\u003e) emissions from ruminants. 2-Bromoethanesulfonate (BES) and 3-nitrooxypropanol (3-NOP) inhibit methanogenesis by acting as structural analogs of methyl-coenzyme M (methyl-CoM), a methyl donor in the last reaction of methanogenesis. We hypothesized that a high concentration of methyl-CoM and its nonmethylated form, coenzyme M (CoM), would block the antimethanogenic effects of BES and 3-NOP in mixed rumen cultures and would not affect bromoform (BMF), which acts through a different mechanism. This hypothesis and the possible underlying mechanisms were examined in one ruminal serial culture and three batch culture experiments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCoenzyme M and methyl-CoM added at 1 mM blocked the inhibition of methanogenesis by BES but not by 3-NOP or BMF. 2-Bromoethanesulfonate strongly decreased the relative abundance of \u003cem\u003eMethanobrevibacter ruminantium\u003c/em\u003e but did not affect other methanogens. Similar to CH\u003csub\u003e4\u003c/sub\u003e production, the effect of BES on the relative abundance of \u003cem\u003eM. ruminantium\u003c/em\u003e was reversed by CoM and methyl-CoM. Because \u003cem\u003eM. ruminantium\u003c/em\u003e cannot synthesize CoM and must take up exogenous CoM, the sensitivity of methanogens to BES may depend on them lacking the genetic capacity to synthesize CoM, as BES and CoM compete for transmembrane transport. In contrast, 3-NOP diffuses across cell membranes and does not compete for transmembrane transport with CoM, which is consistent with its effects not being reversed by CoM or methyl-CoM. Bromoform does not act competitively with CoM or methyl-CoM so that the addition of CoM or methyl-CoM did not reverse the effects of BMF.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results may explain previous observations of lack of persistence of BES in vivo and in continuous cultures. Long-term BES supplementation is thought to shift the archaeal community towards methanogens that do not require exogenous CoM and are tolerant to BES. The effects of 3-NOP and BMF were unaffected by the addition of CoM and methyl-CoM presumably because they can diffuse across cell membranes, and in the case of BMF, the inhibition of methyl-tetrahydromethanopterin: coenzyme M methyltransferase may not be relieved by CoM or methyl-CoM.\u003c/p\u003e","manuscriptTitle":"Effects of coenzyme M and methyl-coenzyme M on the efficacy of inhibitors of methanogenesis in rumen cultures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-06 09:20:45","doi":"10.21203/rs.3.rs-8419205/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9604a4bf-472a-4032-8e0a-7c93fde2b8f2","owner":[],"postedDate":"January 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-05T16:05:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-06 09:20:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8419205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8419205","identity":"rs-8419205","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}