Enzymology of one-carbon metabolism in methanogenic pathways.

Enzymology of one-carbon metabolism in methanogenic pathways

Mapping of a chromosome replication origin in an archaeon: Response

Structurally and functionally diverse CO dehydrogenases are key components of various energy-yielding pathways in aerobic and anaerobic microbes from the Bacteria and Archaea domains. Aerobic microbes utilize Mo-Fe-flavin CO dehydrogenases to oxidize CO in respiratory pathways. Phototrophic anaerobes grow by converting CO to H2, a process initiating with a CO dehydrogenase that contains nickel and iron-sulfur centers. Acetate-producing anaerobes employ a nickel/iron-sulfur CO dehydrogenase to synthesize acetyl-CoA from a methyl group, CO, and CoA. A similar enzyme is responsible for the cleavage of acetyl-CoA by anaerobic Archaea that obtain energy by fermenting acetate to CH4 and CO2. Acetotrophic sulfate reducers from the Bacteria and Archaea also utilize CO dehydrogenase to cleave acetyl-CoA yielding methyl and carbonyl groups. These microbes obtain energy for growth via a respiratory pathway in which the methyl and carbonyl groups are oxidized to CO2, and sulfate is reduced to sulfide.

We are trying to develop a methane-producing system using indigenous microbes in depleted oil fields as a new microbial enhanced oil recovery process. In particular, we aim to combine a microbial conversion of the residual oil into methane with the geological sequestration of carbon dioxide. The mechanism is as follows: Hydrocarbon-degrading bacteria are harnessed to produce hydrogen and/or acetate from residual oil in the depleted oil reservoir. Then, methane-producing microbes (methanogens) utilize the produced acetate or hydrogen and carbon dioxide, which is injected for geological sequestration, to generate methane. We successfully isolated hydrogen- and methane-producing microbes (hydrogen-producing bacteria and methanogens) from oil fields (Yabase and other oil fields) in Japan. Our analysis of microbial cultures incubated under high temperature and high pressure, the condition similar to in situ petroleum reservoir conditions, revealed that indigenous microbes in the reservoir brine are capable of generating methane by utilizing crude oil and carbon dioxide. Consumption/production rate of gases (methane and carbon dioxide) and acetic acid indicated that the methane production under reservoir conditions is likely mediated through two major pathways; the acetoclastic (acetic-acid utilizing) and the hydrogenotrophic (hydrogen and carbon-dioxide utilizing) pathways. Furthermore, by analyzing methane-producing ability of isolated microbes, we found that the syntrophic cooperation between hydrogen-producing bacteria and methanogens was critical for the methane producing under the reservoir condition. 0%.tures with carbon dioxideent Strikingly, addition of carbon dioxide accelerated methane production of the cultures. The methane production rate of the cultures, in which high concentration (10%) of carbon dioxide was supplied into the head spaces, was 0.30 mmol/L/Day. On the other hand, the cultures without the addition of carbon dioxide showed the methane production rate of 0.12 mmol/L/Day, significantly slower (ca. 40%) than the production rate of the cultures with carbon dioxide. These results suggested that addition (injection) of carbon dioxide into reservoirs might accelerate the microbial methane production. We further investigated the methanogenic communities and pathways in petroleum reservoirs by incubating the reservoir brine from the Yabase oil field, combined with radiotracer experiments and molecular biological analyses. The brine samples were incubated without exogenous-nutrient supplementation under the high-temperature and high-pressure condition (the in-reservoir condition). The radiotracer analysis (using 14C-biocarbonate and 14C-acetate) indicated that the methane production rate of hydrogenotrophic methanogenesis was 50-fold higher than that of acetoclastic methanogenesis, suggesting dominance of methane production by syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis in reservoir. In this study, we assessed the rate of oil biodegradation coupled with methanogenesis by using 14C-labeled toluene and hexadecane as tracers. The analysis revealed that the rate was very low, being only about one thousandth of that of the hydrogenotrophic methanogenesis. We are currently trying to enhance the crude-oil biodegradation for effective conversion of crude oil to methane. Our goal is to establish effective microbial conversion system from residual oil into methane in depleted oil fields as a new EOR technology.

ConspectusGlobal climate change caused by the excessive emission of greenhouse gases has become one of the greatest threats to human survival in the 21st century. Carbon dioxide is the main greenhouse gas on earth and has brought about serious environmental problems nowadays. On the basis of the current situation, it is urgent to reach the peak of carbon dioxide emission and then achieve carbon neutrality via policy support and engineering strategies within advanced materials and technologies. Carbon neutrality requires an appropriate balance between the emission and reduction of carbon dioxide. The emission of carbon dioxide mainly comes from modern industries, and the reduction requires several steps, including capture, conversion, and application. On one hand, it can reduce carbon dioxide emission by promoting the transformation of industrial structure. On the other hand, it is necessary to remove high-level carbon dioxide existing in the atmosphere by physical and chemical methods such as adsorption capture and catalytic conversion.This Account showcases our recent progress on carbon neutrality for the reduction of carbon dioxide through capture and conversion methods within advanced materials and technologies. We mainly focus on the right side of the carbon scale and have made some advances such as moisture-swing chemisorption for carbon dioxide capture, the reduction of oxygen-containing carbon dioxide, and the photothermal catalytic conversion of carbon dioxide. Different from previous studies, our work is about developing materials and techniques for practical applications. First, we have made attempts to develop cheap sorbents with high stability and a high adsorption capacity. Second, we have reported a moisture-swing technique with the capability of directly capturing carbon dioxide from the atmosphere by relying on the humidity variation with low energy consumption. This technique is promising for realizing real-time carbon dioxide capture and utilization, which avoids high-cost storage and transport processes. Third, our work on carbon dioxide utilization focuses on efficient conversion under practical conditions. For instance, we have developed perovskite catalysts for converting carbon dioxide to carbon monoxide in an oxygen-containing environment. Furthermore, core–shell catalysts have been reported for carbon dioxide conversion with a high selectivity of 83% driven by solar energy. In addition, practical applications of captured carbon dioxide have been explored with respect to carbon dioxide-assisted graphene exfoliation, keeping fruit fresh, and crop growth promotion with carbon dioxide gas fertilizer. A future perspective on the challenges and opportunities for carbon neutrality has been provided on the basis of our experimental studies and theoretical predictions. It is expected that this Account will promote tremendous effort in the development of advanced materials and engineering technologies toward the realization of carbon neutrality by the middle of this century.

In many anaerobic environments methane (CH4) is produced by methanogens, with either H2/CO2 or acetate (i.e., the methyl group) as precursors, through what are referred to as hydrogenotrophic and acetoclastic methanogenic pathways respectively. Their relative contribution to total CH4 production can be quantified by determining the stable carbon isotopic fractionation factors for both pathways as well as the isotopic signatures of CO2, CH4, and the methyl group in acetate of the sample. The procedures for measuring carbon isotopic fractionation factors of both methanogenic pathways and isotopic composition of these compounds by isotope ratio mass spectrometry are described in this chapter. The results are very helpful in evaluating the activity of the methanogens involved in each methanogenic pathway as well as those of other biological pathways with different fractionation factors.

Reduction of the disulfide of coenzyme M and coenzyme B (CoMS–SCoB) by heterodisulfide reductases (HdrED and HdrABC) is the final step in all methanogenic pathways. Flavin-based electron bifurcation (FBEB) by soluble HdrABC homologs play additional roles in driving essential endergonic reactions at the expense of the exergonic reduction of CoMS–SCoM. In the first step of the CO2 reduction pathway, HdrABC complexed with hydrogenase or formate dehydrogenase generates reduced ferredoxin (Fdx2-) for the endergonic reduction of CO2 coupled to the exergonic reduction of CoMS–SCoB dependent on FBEB of electrons from H2 or formate. Roles for HdrABC:hydrogenase complexes are also proposed for pathways wherein the methyl group of methanol is reduced to methane with electrons from H2. The HdrABC complexes catalyze FBEB-dependent oxidation of H2 for the endergonic reduction of Fdx driven by the exergonic reduction of CoMS–SCoB. The Fdx2- supplies electrons for reduction of the methyl group to methane. In H2- independent pathways, three-fourths of the methyl groups are oxidized producing Fdx2- and reduced coenzyme F420 (F420H2). The F420H2 donates electrons for reduction of the remaining methyl groups to methane requiring transfer of electrons from Fdx2- to F420. HdrA1B1C1 is proposed to catalyze FBEB-dependent oxidation of Fdx2- for the endergonic reduction of F420 driven by the exergonic reduction of CoMS–SCoB. In H2- independent acetotrophic pathways, the methyl group of acetate is reduced to methane with electrons derived from oxidation of the carbonyl group mediated by Fdx. Electron transport involves a membrane-bound complex (Rnf) that oxidizes Fdx2- and generates a Na+ gradient driving ATP synthesis. It is postulated that F420 is reduced by Rnf requiring HdrA2B2C2 catalyzing FBEB-dependent oxidation of F420H2 for the endergonic reduction of Fdx driven by the exergonic reduction of CoMS–SCoB. The Fdx2- is recycled by Rnf and HdrA2B2C2 thereby conserving energy. The HdrA2B2C2 is also proposed to play a role in Fe(III)-dependent reverse methanogenesis. A flavin-based electron confurcating (FBEC) HdrABC complex is proposed for nitrate-dependent reverse methanogenesis in which the oxidation of CoM-SH/CoB-SH and Fdx2- is coupled to reduction of F420. The F420H2 donates electrons to a membrane complex that generates a proton gradient driving ATP synthesis.

Methanogenesis from acetate (aceticlastic methanogenesis or syntrophic acetate oxidation (SAO) coupled with hydrogenotrophic methanogenesis) is the most important step for the biogas process. The major environmental factors influencing methanogenesis are volatile fatty acids, ammonia, pH, and temperature. In our study, the effect of acetate and ammonia concentration on the methanogenic pathway from acetate and on the methanogenic communities was elucidated in two experiments: one where inocula were gradually exposed to increasing concentrations of acetate or ammonia, and another with direct exposure to different ammonia concentrations. The methanogenic pathway was determined by following the production of (14) CH(4) and (14) CO(2) from acetate labeled in the methyl group (C-2). Microbial communities' composition was determined by fluorescence in situ hybridization. Upon acclimatization to acetate and ammonia, thermophilic cultures clearly shifted their acetate bioconversion pathway from SAO with subsequent hydrogenotrophic methanogenesis (mediated by Methanobacteriales spp. and/or Methanomicrobiales spp.) to aceticlastic methanogenesis (mediated by Methanosarcinaceae spp.). On the contrary, acclimatization process resulted in no pathway shift with the mesophilic acclimatized culture. When nonacclimatized thermophilic culture was exposed to high ammonia levels (7 g NH4 +-N L(-1)), aceticlastic Methanosarcinaceae spp. was found to be the dominant methanogen.

A molecular dynamics (MD) simulation was applied to carbon dioxide+trifluoromethylbenzoic acid isomer and carbon dioxide+methylbenzoic acid isomer systems to investigate the interactions between carbon dioxide and the solutes. The pair correlation functions between the carbon dioxide and trifluoromethyl group or methyl group in the solutes were calculated to study the fluorination effect of solvation. As a result, it was found that the interactions between carbon dioxide and trifluoromethyl group in trifluoromethylbenzoic acid isomers were stronger than those between carbon dioxide and the methyl group in methylbenzoic acid isomers. The simulation results had the same tendency as the experimental solubility enhancements and coincided with the trend of the interaction parameters of the Peng-Robinson equation of state that were determined from the solubility data.

Ammonia is a major environmental factor influencing biomethanation in full-scale anaerobic digesters. In this study, the effect of different ammonia levels on methanogenic pathways and methanogenic community composition of full-scale biogas plants was investigated. Eight full-scale digesters operating under different ammonia levels were sampled, and the residual biogas production was followed in fed-batch reactors. Acetate, labelled in the methyl group, was used to determine the methanogenic pathway by following the 14CH4 and 14CO2 production. Fluorescence in situ hybridisation was used to determine the methanogenic communities’ composition. Results obtained clearly demonstrated that syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis was the dominant pathway in all digesters with high ammonia levels (2.8–4.57 g NH4 +-N L−1), while acetoclastic methanogenic pathway dominated at low ammonia (<1.21 g NH4 +-N L−1). Thermophilic Methanomicrobiales spp. and mesophilic Methanobacteriales spp. were the most abundant methanogens at free ammonia concentrations above 0.44 g NH3-N L−1 and total ammonia concentrations above 2.8 g NH4 +-N L−1, respectively. Meanwhile, in anaerobic digesters with low ammonia (<1.21 g NH4 +-N L−1) and free ammonia (<0.07 g NH3-N L−1) levels, mesophilic and thermophilic Methanosaetaceae spp. were the most abundant methanogens.

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

Livestock manure emits reduced sulfur compounds and methane, which affect nature and the climate. These gases are efficiently mitigated by addition of a tannic acid-sodium fluoride combination inhibitor (TA-NaF), and to some extent by acidification. In this paper, TA-NaF treatment was performed on swine manure to study the treatment influence on methanogenic pathways and sulfur transformation pathways in various laboratory experiments. Stable carbon isotope labeling revealed that both untreated and TA-NaF treated swine manures were dominated by hydrogenotrophic methanogenesis. However, in supplementary experiments in wastewater sludge, TA-NaF clearly inhibited acetoclastic methanogenesis, whereas acidification inhibited hydrogenotrophic methanogenesis. In swine manure, TA-NaF inhibited s-amino acid catabolism to a larger extent than sulfate reduction. Conversely, acidification reduced sulfate reduction activity more than s-amino acid degradation. TA-NaF treatment had no significant effect on methanogenic community structure, which was surprising considering clear effects on isotope ratios of methane and carbon dioxide. Halophile sulfate reducers adapted well to TA-NaF treatment, but the community change also depended on temperature. The combined experimental work resulted in a proposed inhibition scheme for sulfur transformations and methanogenic pathways as affected by TA-NaF and acidification in swine manure and in other inocula.

D-Glucose-1-C14, D-arabinose-1-C14, and L-arabinose-1-C14were dissimilated anaerobically by Aerobacter aerogenes. The major products (2,3-butanediol, ethanol, acetic acid, lactic acid, formic acid, and carbon dioxide) were isolated and the location of C14determined. The products from glucose were all labeled, mainly in the methyl groups, in agreement with the hypothesis that they were derived from methyl-labeled pyruvate formed by the reactions of the classical Embden–Meyerhof scheme for glycolysis. The products from both pentoses appeared to have been formed from pyruvate labeled in both the methyl and carboxyl groups with twice as much C14in the methyl group as in the carboxyl group. This result may be explained quantitatively by a hypothesis assuming complete conversion of pentose to triose via a heptulose.

D-Glucose-1-C14, D-arabinose-1-C14, and L-arabinose-1-C14were dissimilated anaerobically by Aerobacter aerogenes. The major products (2,3-butanediol, ethanol, acetic acid, lactic acid, formic acid, and carbon dioxide) were isolated and the location of C14determined. The products from glucose were all labeled, mainly in the methyl groups, in agreement with the hypothesis that they were derived from methyl-labeled pyruvate formed by the reactions of the classical Embden–Meyerhof scheme for glycolysis. The products from both pentoses appeared to have been formed from pyruvate labeled in both the methyl and carboxyl groups with twice as much C14in the methyl group as in the carboxyl group. This result may be explained quantitatively by a hypothesis assuming complete conversion of pentose to triose via a heptulose.

Rapidly increasing levels of atmospheric carbon dioxide and their damaging impact on the global climate system raise doubts about the sustainability of the fossil resource based energy system. Meanwhile, raising living standards and increasing global population lead to an ever growing need for energy. Renewable energy sources are believed to present a solution to these problems with the sheer abundance of solar energy showing particular promise to fulfill the world's energy needs. However, for large scale application of solar energy to be possible, the problem of its storage has to be addressed. The insufficient flexibility of present-day storage technologies has led to the quest for producing solar fuels, centering on hydrogen as a fuel in a prospective hydrogen economy. Nevertheless, the gaseous state, low volumetric energy density and explosive nature of hydrogen makes it a challenging fuel for practical applications. Using solar energy to produce carbon-based liquid fuels solves these challenges, closes the anthropogenic carbon cycle and allows for the continued utilization of existing infrastructures. A promising method for the production of such fuels consists in the photoelectrochemical and electrochemical conversion of carbon dioxide. In this thesis, both methods are investigated using molecular homogeneous catalysts and heterogeneous systems. The photoelectrochemical reduction of carbon dioxide on TiO2-protected Cu2O photocathodes was investigated using a rhenium bipyridyl catalyst in solution. Important charge transport limitations were encountered, which could be overcome by the addition of protic additives to the electrolyte. Improving on this result, the molecular catalyst was covalently immobilized on the TiO2 surface of the photocathode by modifying the bipyridyl ligand with a phosphonate binding group. A nanostructure of TiO2 was needed to support sufficient catalyst to sustain the photocurrent generated by the Cu2O photoelectrode. The complete device showed photocurrents exceeding 2.5 mA cm-2 and large faradaic efficiency for the production of CO. Moving toward heterogeneous catalysis, the promotion of the CO2 to CO conversion reaction on silver surfaces by imidazolium cations was investigated. Replacing the imidazolium C2 proton with a phenyl substituent led to an enhancement of the co-catalytic effect. Replacing the C4 and C5 protons with methyl groups, however, suppressed the catalysis-promoting effect of the imidazolium salt for different C2 substituents and led to new insights into the role of imidazolium. The unassisted solar-driven splitting of CO2 into CO and O2 was demonstrated using water as electron source. This was achieved by the use of a porous gold cathode and an IrO2 anode, driven by three methylammonium lead iodide perovskite solar cells in series. Extended operation over 18 h was shown, achieving a solar to CO efficiency exceeding 6.5 %. Atomic layer deposition (ALD) modification of CuO nanowire cathodes with SnO2 was investigated, leading to striking impacts on the catalytic selectivity of this system. In an aqueous electrolyte, bare CuO led to the production of a wide spectrum of products, which was modified to the production of CO with high selectivity upon ALD modification. By exploiting the oxygen evolving activity of SnO2-coated CuO, a low cost bifunctional system was constructed, achieving sustained solar-driven production of CO with up to 13.4% efficiency.