In vitro ruminal methanogenesis of a hay-rich substrate in response to different combination supplements of nitrocompounds; pyromellitic diimide and 2-bromoethanesulphonate

Sixteen combinations of 5 treatments at 4 levels were designed in a L₁₆(4⁵) orthogonal experimental design to evaluate associative effects of five methanogenesis inhibitors at four dose levels: nitroethane (NE, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitroethanol (NEOH, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitro-1-propanol (NPOH, 0 mM, 5 mM, 10 mM and 15 mM), pyromellitic diimide (PMDI, 0 mM, 0.02 mM, 0.05 mM and 0.07 mM) and 2-bromoethanesulphonate (BES, 0 mM, 0.01 mM, 0.03 mM and 0.05 mM) on in vitro ruminal methane production of the mixed substrate (Chinese wildrye hay:maize meal = 4:1) using a cumulative gas production technique. After 48 h incubation, in vitro dry matter disappearance (IVDMD), total gas production (GP₄₈, ml/g DM) and total volatile fatty acids (VFA) production in various combinations of these inhibitors were decreased by 10.6-56.0, 26.5-44.5 and 20.3-47.6%, respectively (P<0.05). The molar proportion of acetate in the inhibitor combination groups was decreased by 6.6-12.5% while those of propionate and butyrate were increased by 7.0-19.2 and 21.9-56.5% (P<0.01), respectively. Methane proportion (MP) in total gas production was reduced by 79.4-98.5% (P<0.01), and the highest inhibition occurred in the combination of 10 mM NE, 10 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures. The partial correlation coefficients between NE, NEOH, NPOH, PMDI or BES and CH₄ proportion were −0.465 (P<0.01), −0.417 (P<0.01), −0.355 (P<0.05), −0.408 (P<0.01) and −0.345 (P<0.05), respectively, indicating that NE was the most potent inhibitor, followed by NEOH and PMDI, and finally NPOH and BES. In general, VFA production in the inhibitor combinations was substantially shifted to produce much more butyrate and propionate and less acetate. The combination of 15 mM NE, 10 mM NEOH, 5 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures, leading to >95% methane inhibition, may be the optimal application of these inhibitors with less depression of total VFA production. Further feeding trials to validate these combinations is still required on rumen function, methane production, growth performance and milk production.     

Inhibitory effects of 2-bromoethanesulfonate and protection by addition of coenzyme M in hydrogen-oxidizing marine enrichment cultures

Abstract Since bromoethanesulfonate (BES) is an inhibitor of methane production (competitive with methyl-coenzyme M), cells able to accumulate large internal pools of methyl-coenzyme M via uptake of its precursor, HS-CoM, should be protected from BES by addition of HS-CoM to the growth medium. Hydrogen-oxidizing marine methanogen enrichments were prepared from anaerobic sediment samples collected at Sippewisset Salt Marsh and Oyster Bay Inlet near Woods Hole, MA. The three enrichments studied were a mixture of cell types with at least 50% of the culture comprised of methanogens. Methane production was found to be sensitive to BES with half maximal inhibition occurring at 5–20 μM BES depending on the enrichment. For each, half maximal protection against 40 μM BES occurred at a HS-CoM: BES molar ratio of 20: 1 to 40: 1. Since the protected enrichments exhibited normal sensitivity toward BES after removal of HS-CoM, it was concluded that methane production in the presence of both BES and HS-CoM resulted from true protection and not growth of BES-resistant mutants. These results suggest that uptake of HS-CoM may be a general property of methanogens occupying anaerobic marine sediments. It is possible that uptake of this coenzyme is an important nutritional feature of methanogens in their natural habitat.     

Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

Isolation of key methanogens for global methane emission from rice paddy fields: a novel isolate affiliated with the clone cluster rice cluster I

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H(2)) continuously provided by heterotrophic H(2)-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H(2)-producing syntroph, Syntrophobacter fumaroxidans, as the H(2) supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.     

Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.     

Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values.

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.     

Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs

We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.     

Isolation of Key Methanogens for Global Methane Emission from Rice Paddy Fields: a Novel Isolate Affiliated with the Clone Cluster Rice Cluster I

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H₂) continuously provided by heterotrophic H₂-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H₂-producing syntroph, Syntrophobacter fumaroxidans, as the H₂ supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.     

温度对甲烷产生和氧化的影响

综述了温度对土壤产甲烷和氧化甲烷的影响及其机制．温度主要通过土壤中产甲烷菌的优势菌发生更替来改变土壤的产甲烷能力．较高温条件下产甲烷菌以乙酸和H2／CO2都能利用的甲烷八叠球菌(Methanosarcinaceae)为主,使得土壤处于较高的产甲烷状态．较低温条件下产甲烷菌以只能利用乙酸的甲烷毛菌(Methanosaetaceae)为主,土壤形成甲烷的能力相对较弱．温度提高可以显著地增加甲烷的产生,Q10为1．5-28,平均4．1,但是温度效应明显受控于底物浓度,提高底物浓度降低了产甲烷菌对底物的亲和力,相应地增加了度效应,因此在较低温条件下提高底物浓度可以促进甲烷的产生．温度对大气甲烷氧化的影响弱于产甲烷,甲烷氧化菌较少受温度变化的影响,即便在较低温条件下,土壤也具有一定的氧化大气甲烷能力,原因尚不清楚,可能与甲烷氧化菌对大气甲烷具有较高的亲和力有关,有待进一步研究．     

Inhibition of methanogens increases photo-dependent nitrogenase activities in anoxic paddy soil amended with rice straw

The interaction between phototrophic dinitrogen fixers and methanogens was examined in soil slurries amended with rice straw using 2-bromoethanesulfonic acid (BES), a specific methanogenic inhibitor. Slurries incubated in light increased phototrophic nitrogenase activity (acetylene reducing activity), and showed growth of phototrophic purple bacteria and reduction of CH(4) emission, indicating outcompetition of purple bacteria with methanogens in photic zones. Adding BES effectively inhibited methane production and markedly increased phototrophic acetylene reducing activity accompanied with acetate accumulation, but did not affect populations of purple bacteria in the slurries. More acetate accumulated in the inhibited slurries incubated in dark. We suggest that increased availability of organic substrates for purple bacteria after stopping methanogenic consumption by BES caused the increased phototrophic acetylene reducing activity. These results indicate that, after purple bacteria grow enough, performance of their N(2) fixation may be limited by substrate availability, which methanogenesis may profoundly influence.     

Kinetic Analysis of Competition Between Sulfate Reducers and Methanogens for Hydrogen in Sediments

The competition between sulfate-reducing and methanogenic bacteria for hydrogen was investigated in eutrophic lake sediments that contained low in situ sulfate concentrations and in sulfate-amended sediments. Sulfate reduction and methane production coexisted in situ in lake surface sediments (0 to 2 cm), but methane production was the dominant terminal process. Addition of 10 to 20 mM sulfate to sediments resulted in a decrease in the hydrogen partial pressure and a concomitant inhibition of methane production over time. Molybdate inhibition of sulfate reduction in sulfate-amended sediments was followed by an increase in the hydrogen partial pressure and the methane production rate to values comparable to those in sediments not amended with sulfate. The sulfate reducer population had a half-saturation constant for hydrogen uptake of 141 pascals versus 597 pascals for the methanogen population. Thus, when sulfate was not limiting, the lower half-saturation constant of sulfate reducers enabled them to inhibit methane production by lowering the hydrogen partial pressure below levels that methanogens could effectively utilize. However, methanogens coexisted with sulfate reducers in the presence of sulfate, and the outcome of competition at any time was a function of the rate of hydrogen production, the relative population sizes, and sulfate availability.     

Response surface methodology analysis of anaerobic syntrophic degradation of volatile fatty acids in an upflow anaerobic sludge bed reactor inoculated with enriched cultures

Anaerobic oxidation of volatile fatty acids (VFAs) as the key intermediates is restricted thermodynamically. Presently, enriched acetogenic and methanogenic cultures were used for syntrophic anaerobic digestion of VFAs in an upflow anaerobic sludge bed reactor fed with acetic, propionic, and butyric acids at maximum concentrations of 5.0, 3.0, and 4.0 g/L, respectively. Interactive effects of propionate, butyrate and acetate were analyzed. Hydraulic retention time (HRT) and acetate oxidizing syntrophs and methanogen (hydrogenotrophs) to syntrophic bacteria (propionate- and butyrate-oxidizing bacteria) population ratio (M/A) were investigated as key microbiological and operating variables of VFA anaerobic degradations. M/A did not affect the size distribution and had little effect on extracellular polymer contents of the granules. Granular sludge with close spatial microbial proximity enhanced syntrophic degradation of VFAs compared to other cultures, such as suspended cultures. Optimum conditions were found to be propionate = 1.93 g/L, butyrate = 2.15 g/L, acetate = 2.50 g/L, HRT = 22 h, and M/A = 2.5 corresponding to maximum VFA removal and biogas production rate. Results of verification experiments and predicted values from fitted correlations were in close agreement at the 95% confidence interval. Granules seemed to be smaller particles and less stable in construction with an irregular fractured surface compared to the original granules.     

Syntrophic interactions among anode respiring bacteria (ARB) and Non‐ARB in a biofilm anode: electron balances

We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro‐organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real‐time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation (～30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate‐oxidizing ARB out‐competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate‐consuming ARB. When we inhibited methanogenesis with 50 mM 2‐bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo‐acetogenesis. This illustrates the key role of competition among the various H₂ scavengers and that, when the hydrogen‐consuming methanogens were present, they out‐competed the other groups. These findings also demonstrate the importance of a three‐way syntrophic relationship among fermenters, acetate‐consuming ARB, and a H₂ consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current. Biotechnol. Bioeng. 2009;103: 513–523. © 2009 Wiley Periodicals, Inc.     

Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

Effects of hydrogen and formate on the degradation of propionate and butyrate in thermophilic granules from an upflow anaerobic sludge blanket reactor.

Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined. The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules. The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested. Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp. strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate. Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp. isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens. By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure. No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate. This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation.     

Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO(4). 2H(2)O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 microM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels ( approximately 0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 microM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

Effects of disodium fumarate on ruminal fermentation and microbial communities in sheep fed on high-forage diets

This study was conducted to investigate effects of disodium fumarate (DF) on fermentation characteristics and microbial populations in the rumen of Hu sheep fed on high-forage diets. Two complementary feeding trials were conducted. In Trial 1, six Hu sheep fitted with ruminal cannulae were randomly allocated to a 2 × 2 cross-over design involving dietary treatments of either 0 or 20 g DF daily. Total DNA was extracted from the fluid- and solid-associated rumen microbes, respectively. Numbers of 16S rDNA gene copies associated with rumen methanogens and bacteria, and 18S rDNA gene copies associated with rumen protozoa and fungi were measured using real-time PCR, and expressed as proportion of total rumen bacteria 16S rDNA. Ruminal pH decreased in the DF group compared with the control (P < 0.05). Total volatile fatty acids increased (P < 0.001), but butyrate decreased (P < 0.01). Addition of DF inhibited the growth of methanogens, protozoa, fungi and Ruminococcus flavefaciens in fluid samples. Both Ruminococcus albus and Butyrivibrio fibrisolvens populations increased (P < 0.001) in particle-associated samples. Trial 2 was conducted to investigate the adaptive response of rumen microbes to DF. Three cannulated sheep were fed on basal diet for 2 weeks and continuously for 4 weeks with supplementation of DF at a level of 20 g/day. Ruminal samples were collected every week to analyze fermentation parameters and microbial populations. No effects of DF were observed on pH, acetate and butyrate (P > 0.05). Populations of methanogens and R. flavefaciens decreased in the fluid samples (P < 0.001), whereas addition of DF stimulated the population of solid-associated Fibrobacter succinogenes. Population of R. albus increased in the 2nd to 4th week in fluid-associated samples and was threefold higher in the 4th week than control week in solid samples. Analysis of denaturing gradient gel electrophoresis fingerprints revealed that there were significant changes in rumen microbiota after adding DF. Ten of 15 clone sequences from cut-out bands appearing in both the 2nd and the 4th week were 94% to 100% similar to Prevotella-like bacteria, and four sequences showed 95% to 98% similarity to Selenomonas dianae. Another 15 sequences were obtained from bands, which appeared in the 4th week only. Thirteen of these 15 sequences showed 95% to 99% similarity to Clostridium sp., and the other two showed 95% and 100% similarity to Ruminococcus sp. In summary, the microorganisms positively responding to DF addition were the cellulolytic bacteria, R. albus, F. succinogenes and B. fibrisolvens as well as proteolytic bacteria, B. fibrisolvens, P. ruminicola and Clostridium sp.     

Increase in colonic methanogens and total anaerobes in aging rats

Methanogens are present in the colons of our local Wistar rat colony. We studied the changes in concentrations of their fecal methanogenic and nonmethanogenic bacteria with age as a model of the development of these communities in humans. We found that the predominant methanogen in the rats is a Methanobrevibacter species. The log of the concentration of total anaerobes increased from 9.8/g (dry weight) at 3.0 weeks of age (shortly after weaning) to 10.7/g (dry weight) at 96 weeks (shortly before the end of the life span). In contrast, the log concentration of methanogens increased from 5.5 to 9/g (dry weight) during the same time period. Therefore, methanogens increased as a percentage of the total anaerobes from 0.005% at 3.0 weeks to 2.0% at 96 weeks. About 12 doublings of the methanogenic population and 3.3 doublings of the nonmethanogenic population took place from weaning until death. The slow increase in the ratio of methanogens to total anaerobes with age followed the same pattern in cecal contents as found in feces. There were no relationships between animal weights or fecal outputs and the increase in total anaerobe and methanogen concentrations in feces. A possible explanation for the slow increase in the Methanobrevibacter species in Wistar rats with age is a gradual shifting of the use of electrons from the reduction of CO2 to acetate by acetogens to the reduction of CO2 to CH4. The results provide the first evidence for an age-related change in the nonmethanogenic bacteria of the colon and supporting microbiological evidence for physiological studies that have shown age-related increases in colonic methane production in humans.     

Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactate by Selenomonas ruminantium.

A method is described for increasing the production of H2 from glucose or lactate by Selenomonas ruminantium by sequential transfers in media containing pregrown Methanobacterium ruminantium. The methanogen uses the H2 formed by the selenomonad to reduce CO2 to CH4. Analysis of fermentation products from glucose showed that lactate was the major product formed from glucose by S. ruminantium alone. Several sequential transfers in the presence of the methanogen caused a marked decrease in lactate production, which was accompanied by an increase in acetate. When lactate was the fermentation substrate, S. ruminantium alone produced propionate, acetate, and CO2. Addition to the pregrown methanogen in the sequential transfer procedure caused a significant decrease in the production of propionate and an increase in acetate formed from lactate. These results are interpreted in terms of the influence of H2 utilization by the methanogen on the production of H2 versus lactate or propionate from reduced pyridine nucleotides by S. ruminantium.