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

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

Bacteria↗

The integration of methanogenesis with denitrification and anaerobic ammonium oxidation in an expanded granular sludge bed reactor.

The integration of methanogenesis with denitrification and anaerobic ammonium oxidation (ANAMMOX) was studied in an expanded granular sludge bed (EGSB) reactor in this work. Experimental results from the continuous treatment of wastewater with nitrite and ammonium, which lasted for 107 days, demonstrated that wastewater with high nitrite and ammonium could be anaerobically treated in an expanded granular sludge bed reactor. More than 91% to 97% of COD were removed at up to about 3.9 g COD/(L x d) of COD volumetric loading rate. More than 97% to 100% of nitrite was denitrified at up to about 0.8 g NO2(-) -N/(L x d), which is 16 times higher than that in a conventional activated sludge system with nitrification/denitrification (0.05 gN/(L x d)). No dissimilatory reduction of nitrite to ammonium occurred in the process. However, maximum of about 40% ammonium was found to be lost. Batch tests of 15 days with sludge from the reactor showed that 100% of nitrite was denitrified completely, and about 3% of ammonium was removed when only ammonium (34.3 mg/L) and nitrite (34.3 mg/ L) were added into the sludge suspension medium. Furthermore, about 15% of ammonium amounts were lost with organic COD addition. It suggested that the methanogenesis in the system could enhance ANAMMOX because of intermediate hydrogen produced during methanogenesis.

Bacteria, Anaerobic↗

Sulphate reduction and methanogenesis in the ovine rumen and porcine caecum: a comparison of two microbial ecosystems.

A series of experiments was conducted to study the relationship between methanogenesis and sulphate reduction in ovine rumen and porcine caecum. Effect of 2-bromoethane sulphonate on hydrogen production by digesta suggested that the most important H2-disposal system in the rumen is methanogenesis and that methanogenesis is not predominant H2-disposal system in the porcine caecum. This inference was supported by the difference in predominant H2-utilizers in these two microbial ecosystems; Methanogenic bacteria (MB) were predominant in the rumen and sulphate reducing bacteria (SRB) were predominant in porcine caecum. Free sulphate levels in digesta appear to affect the relationship between MB and SRB. Sulphate levels in the rumen were likely to be insufficient for SRB to outcompete MB.

Animals↗

Zero valent iron as an electron-donor for methanogenesis and sulfate reduction in anaerobic sludge.

Zero valent iron (ZVI) is a reactive media commonly utilized in permeable reactive barriers (PRBs). Sulfate reducing bacteria are being considered for the immobilization of heavy metals in PRBs. The purpose of this study was to evaluate the potential of ZVI as an electron donor for sulfate reduction in natural mixed anaerobic cultures. The ability of methanogens to utilize ZVI as an electron-donor was also explored since these microorganisms often compete with sulfate reducers for common substrates. Four grades of ZVI of different particle sizes (1.120, 0.149, 0.044, and 0.010 mm diameter) were compared as electron donor in batch bioassays inoculated with anaerobic bioreactor sludge. Methanogenesis was evaluated in mineral media lacking sulfate. Sulfate reduction was evaluated in mineral media containing sulfate and the specific methanogenic inhibitor, 2-bromoethane sulfonate. ZVI contributed to significant increases in methane production and sulfate reduction-compared to endogenous substrate controls. The rates of methane formation or sulfate reduction were positively correlated with the surface area of ZVI. The highest rates of 0.310 mmol CH4 formed/mol Fe0.day and 0.804 mmol SO4(2-) reduced/mol Fe0.day were obtained with the finest grade of ZVI (0.01 mm). The results demonstrate that ZVI is readily utilized as a slow-release electron donor for methanogenesis and sulfate reduction in anaerobic sludge; and therefore, has a promising potential in bioremediation applications.

Biological Assay↗

Methanogenesis at low temperatures by microflora of tundra wetland soil.

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 degrees C. Methane was the only end product in balanced microbial community with H2/CO2 as a substrate, besides acetate was produced as an intermediate at temperatures below 10 degrees C. The activity of different microbial groups of methanogenic community in the temperature range of 6-28 degrees C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 degrees C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H2/CO2-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.

Acetates↗

Energetics of methanogenesis studied in vesicular systems.

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

Adenosine Triphosphate↗

Polycyclic aromatic hydrocarbon (PAH) degradation coupled to methanogenesis.

Baltimore Harbor (Baltimore, MD) sediments were utilized to initiate anaerobic enrichment cultures with polycyclic aromatic hydrocarbons (PAHs) in the absence of supplementary electron acceptors. Cultures amended with naphthalene and phenanthrene exhibited sustained, transferable degradation of the PAHs. Bromoethanesulfonic acid, a selective inhibitor of methanogenesis, inhibited the degradation of 200 microM: naphthalene and phenanthrene; molecular characterization based on 16S rRNA sequences confirmed that methanogenic Archaea were eliminated, thus providing evidence that methanogenesis is involved in the degradation pathway.

Alkanesulfonates↗

A stable isotope titration method to determine the contribution of acetate disproportionation and carbon dioxide reduction to methanogenesis.

A novel stable isotope titration approach was developed to determine the contributions to total methane production made by CO(2) reduction and the disproportionation of acetate in anoxic environments. (13)CH(4), (12)CH(4), (13)CO(2) and (12)CO(2) production rates were measured in the head space of replicate anaerobic microcosms titrated with increasing amounts of (13)C-labelled substrates. The contribution of CO(2) reduction was calculated from the linear relationship between ratios of labelled and total CH(4) production and ratios of labelled and total CO(2) after the addition of (13)C-bicarbonate. In the case of acetoclastic methanogenesis rates of (13)CH(4) and (12)CH(4) production were fitted to a model based on an assumption that the relationship between the concentration of (13)C-labelled acetate and the rates of labelled and unlabelled methane production followed Michaelis-Menten kinetics. A comparison of the raw data with the model supported the assumption and provided both an estimate of the contribution of acetate to methane production and an estimate of the size of the indigenous acetate pool without the need to measure acetate directly. The method was applied to a freshwater sediment in the English Lake District where it was found that 66.3% (se 4.9) of methane production was due to acetate disproportionation and 28.9% (se 1.9) of methane production resulted from CO(2) reduction. This is in agreement with theoretical predictions and other empirical measurements of methanogenesis.

Acetates↗

Occurrence of methanogenesis during start-up of a full-scale synthesis gas-fed reactor treating sulfate and metal-rich wastewater.

The start-up of a full-scale synthesis gas-fed gas-lift reactor treating metal and sulfate-rich wastewater was investigated. Sludge from a pilot-scale reactor was used to seed the full-scale reactor. The main difference in design between the pilot- and full-scale reactor was that metal precipitation and sulfate reduction occurred in the same reactor. After 7 weeks the full-scale reactor achieved the sulfate conversion design rate of 15 kg/m3day. Zinc sulfide precipitation inside the reactor did not interfere with obtaining a high rate of sulfate reduction. 16S rRNA gene analysis demonstrated that the bacterial communities in both reactors were dominated by the sulfate-reducing genus Desulfomicrobium. Archaeal communities of both reactors were dominated by the methanogenic genus Methanobacterium. Most Probable Number (MPN) counts confirmed that heterotrophic Sulfate-Reducing Bacteria (SRB) were dominant (10(11) -10(12) cells/g VSS) compared to homoacetogens (10(5) -10(6) cells/g VSS) and methanogens (10(8) -10(9) cells/g VSS). Methanogenesis was not suppressed during start-up of the full scale-reactor, despite the predominance of SRB, which have a lower hydrogen threshold. Due to the short sludge retention time (4-7 days) competition for hydrogen is determined by Monod kinetics, not hydrogen thresholds. As the kinetic parameters for SRB and methanogens are similar, methanogenesis may persist which results in a loss of hydrogen.

Archaea↗

Toxic effect of surfactants and probable products of their biodegradation on methanogenesis in an anaerobic microbial community.

Surfactants used in household and various industries, are rather toxic; therefore, the accumulation of these compounds in the environment through wastewaters has challenged the problem of their biodegradation. In this research, an attempt was made to assess the toxic effect of various surfactants and the likely products of their biodegradation on the acetoclastic methanogens of an anaerobic microbial community. Among the substances investigated, cationic surfactants were found to be most toxic to methanogens: 154 mg/l alkamon DS and 345 mg/l catamin AB induced a 50% inhibition of methanogenesis. Toxicity studies of some aromatic and cyclic compounds, as the probable products of biodegradation of alkylbenzene sulfonate surfactants, showed that methanogenesis in the microbial community under study are rather tolerant to high concentrations of these compounds.

Anaerobiosis↗

The unique biochemistry of methanogenesis.

Methanogenic archaea have an unusual type of metabolism because they use H2 + CO2, formate, methylated C1 compounds, or acetate as energy and carbon sources for growth. The methanogens produce methane as the major end product of their metabolism in a unique energy-generating process. The organisms received much attention because they catalyze the terminal step in the anaerobic breakdown of organic matter under sulfate-limiting conditions and are essential for both the recycling of carbon compounds and the maintenance of the global carbon flux on Earth. Furthermore, methane is an important greenhouse gas that directly contributes to climate changes and global warming. Hence, the understanding of the biochemical processes leading to methane formation are of major interest. This review focuses on the metabolic pathways of methanogenesis that are rather unique and involve a number of unusual enzymes and coenzymes. It will be shown how the previously mentioned substrates are converted to CH4 via the CO2-reducing, methylotrophic, or aceticlastic pathway. All catabolic processes finally lead to the formation of a mixed disulfide from coenzyme M and coenzyme B that functions as an electron acceptor of certain anaerobic respiratory chains. Molecular hydrogen, reduced coenzyme F420, or reduced ferredoxin are used as electron donors. The redox reactions as catalyzed by the membrane-bound electron transport chains are coupled to proton translocation across the cytoplasmic membrane. The resulting electrochemical proton gradient is the driving force for ATP synthesis as catalyzed by an A1A0-type ATP synthase. Other energy-transducing enzymes involved in methanogenesis are the membrane-integral methyltransferase and the formylmethanofuran dehydrogenase complex. The former enzyme is a unique, reversible sodium ion pump that couples methyl-group transfer with the transport of Na+ across the membrane. The formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation of methanofuran. Furthermore, the review addresses questions related to the biochemical and genetic characteristics of the energy-transducing enzymes and to the mechanisms of ion translocation.

Adenosine Triphosphate↗

Effects of mixtures of lauric and myristic acid on rumen methanogens and methanogenesis in vitro.

AIMS: To identify the most effective mixture of non-esterified lauric (C12) and myristic (C14) acid in suppressing ruminal methanogenesis, and to investigate their effects on the methanogenic population. METHODS AND RESULTS: C12/C14 mixtures were incubated with rumen fluid using the Hohenheim gas test apparatus. Methane production and the numbers of Archaea declined with an increasing proportion of C12. With a 2 : 1 proportion of C12/C14, the maximum methane-suppressing effect (96%) was achieved similar to that with C12 alone. The proportions of the individual methanogenic orders of total methanogens were altered by varying the C12/C14 ratio. CONCLUSIONS: Although C14 alone had no effect on methanogenesis, C14 enhanced the methane-suppressing effect of C12 in certain mixtures. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support strategies for an environment-friendly ruminant nutrition as it was demonstrated that part of the less palatable C12 could be replaced by C14 without losing its methane-suppressing potential.

Animals↗

Properties of a novel series of inhibitors of rumen methanogenesis; in vitro and in vivo experiments including growth trials on 2,4-bis (trichloromethyl)-benzo [1, 3]dioxin-6-carboxylic acid.

1. A procedure for measuring methane production by rumen contents incubated anaerobically in vitro is described. Assessments of methane production in vivo, in both sheep and cattle, were made by withdrawal of rumen contents and measuring their capacity to produce methane in vitro. 2. Many members of a series of 6-substituted 2,4-bis (trichloromethyl)-benzo[1,3]dioxins were potent inhibitors of methanogenesis by rumen contents in vitro. The most potent compound inhibited methane production by 70% or more at a concentration of 1 microgram/ml (approximately 2.5 mumol/l). 3. Two compounds, namely the 6-carboxylic acid (ICI 13409) and the 6-carboxamide (ICI 43586), caused a large inhibition of methanogenesis sustained for many hours, following a single intrarumen injection in sheep or cattle. Inhibition was maintained for long periods by single daily dosing directly into the rumen or by dietary administration. 4. In a 28-week growth trial in beef cattle inclusion of ICI 13409 in the concentrate element of the diet, at a level of 6 mg/kg body-weight, improved live-weight gain by 8.0% (P less than 0.05) with respect to untreated animals whilst reducing food intake by 5.0% (P less than 0.05). Smaller and not statistically-significant effects were seen with this compound at 3 mg/kg body-weight and with the antibiotic monensin (Romensin; Elanco PLC). All treatments significantly improved the retention of dietary energy into the carcass, offal and intestinal tracts of the trial animals and significantly reduced the quantity of methane eructed into expired gases.

Animals↗

Stimulation of methanogenesis in a laboratory scale UASB reactor treating domestic sewage by Fe(0) application.

The effects of application of zero valence Fe (Fe(0)) on the anaerobic digestion of sewage was investigated using two laboratory scale UASB reactors. One reactor had Fe(0) addition in a container found midway along the recycling loop. The other one was a control reactor. In a test run period of 76 days, the Fe(0) application significantly increased the CH, yield by 8.7% and decreased the effluent COD concentration by 21.0% relative to the control reactor. A decrease of the H, concentration of biogas and the CODs/CODt ratio in effluent by Fe(0) application were observed. The obtained results imply that the methanogenesis and COD removal efficiency of the UASB reactor were stimulated by Fe(0) application. The higher performance of the reactor with Fe(0) application arises from the integrated functions of Fe(0) or its ionic state as donor of H2, macronutrient, and flocculant. This study showed that the supply of Fe(0) to a UASB can improve the methanogenesis and the overall COD removal of a UASB reactor treating low-strength domestic waste water.

Bacteria, Anaerobic↗

Kinetics of LCFA inhibition on acetoclastic methanogenesis, propionate degradation and beta-oxidation.

Kinetics of long-chain fatty acids (LCFAs) inhibition on acetoclastic methanogenesis, propionate degradation and beta-oxidation were studied with granular sludge under mesophilic batch conditions. Mathematical expressions used for reaction rates were as shown below: [formula: see text]. The simulated results revealed that the methane production rates from acetate decreased with an increase in both concentration and the number of double bonds of LCFAs. The concentrations of oleate (C18:1), linoleate (C18:2), palmitate (C16:0), and stearate (C18:0) were 0.54 mM, 0.11 mM 1.62 mM, and 2.58 mM, respectively, at which the methane production rates from acetate dropped 10%, and 3.10 mM, 0.72 mM, 5.71 mM, and 5.37 mM, respectively, at which the rates dropped 50%. The inhibitory effects of LCFAs on propionate degradation showed a similar tendency with acetoclastic methanogenesis; however, were less severe. The concentrations of oleate, linoleate, palmitate, and stearate were 1.02 mM, 0.18 mM, 2.34 mM, and 1.92 mM, respectively, at which the propionate degradation rates dropped 10%, and 4.38 mM, 1.17 mM, 5.88 mM, and 5.18 mM, respectively, at which the rates dropped 50%. The observed maximum beta-oxidation rates of oleate, linoleate, palmitate, and stearate were 0.21 mmol (gVSS)(-1) d(-1), 0.09 mmol (gVSS)(-1) d(-1), 0.12 mmol (gVSS)(-1) d(-1), and 0.08 mmol (gVSS)(-1) d(-1), respectively. The lag-phase times in beta-oxidation were also dependent on LCFA concentrations. The concentrations of oleate, linoleate, palmitate, and stearate, at which the lag-phase times became 5 days, were 5.93 mM, 2.24 mM, 4.02 mM, and 2.81 mM, respectively.

Fatty Acids↗

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

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

Breath Tests↗

Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190.

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.

Archaea↗

Selective inhibition of reactions involved in methanogenesis and fatty acid production on rice roots.

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.

Journal Article↗