Showing papers on "Methanosarcina barkeri published in 2018"

Methane Production and Conductive Materials: A Critical Review

Abstract: Conductive materials (CM) have been extensively reported to enhance methane production in anaerobic digestion processes. The occurrence of direct interspecies electron transfer (DIET) in microbial communities, as an alternative or complementary to indirect electron transfer (via hydrogen or formate), is the main explanation given to justify the improvement of methane production. Not disregarding that DIET can be promoted in the presence of certain CM, it surely does not explain all the reported observations. In fact, in methanogenic environments DIET was only unequivocally demonstrated in cocultures of Geobacter metallireducens with Methanosaeta harundinacea or Methanosarcina barkeri and frequently Geobacter sp. are not detected in improved methane production driven systems. Furthermore, conductive carbon nanotubes were shown to accelerate the activity of methanogens growing in pure cultures, where DIET is not expected to occur, and hydrogenotrophic activity is ubiquitous in full-scale anaerobic digesters...

Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri.

Abstract: Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest that H2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature. IMPORTANCE ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.

Electron and Proton Flux for Carbon Dioxide Reduction in Methanosarcina barkeri During Direct Interspecies Electron Transfer.

Abstract: Direct interspecies electron transfer (DIET) is important in diverse methanogenic environments, but how methanogens participate in DIET is poorly understood. Therefore, the transcriptome of Methanosarcina barkeri grown via DIET in co-culture with Geobacter metallireducens was compared with its transcriptome when grown via H2 interspecies transfer (HIT) with Pelobacter carbinolicus. Notably, transcripts for the F420H2 dehydrogenase, Fpo, and the heterodisulfide reductase, HdrABC, were more abundant during growth on DIET. A model for CO2 reduction was developed from these results in which electrons delivered to methanophenazine in the cell membrane are transferred to Fpo. The external proton gradient necessary to drive the otherwise thermodynamically unfavorable reverse electron transport for Fpo-catalyzed F420 reduction is derived from protons released from G. metallireducens metabolism. Reduced F420 is a direct electron donor in the carbon dioxide reduction pathway and also serves as the electron donor for the proposed HdrABC-catalyzed electron bifurcation reaction in which reduced ferredoxin (also required for carbon dioxide reduction) is generated with simultaneous reduction of CoM-S-S-CoB. Expression of genes for putative redox-active proteins predicted to be localized on the outer cell surface was higher during growth on DIET, but further analysis will be required to identify the electron transfer route to methanophenazine. The results indicate that the pathways for electron and proton flux for CO2 reduction during DIET are substantially different than for HIT and suggest that gene expression patterns may also be useful for determining whether Methanosarcina are directly accepting electrons from other extracellular electron donors, such as corroding metals or electrodes.

The deep-subsurface sulfate reducer Desulfotomaculum kuznetsovii employs two methanol-degrading pathways.

Abstract: Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

Necessity of electrically conductive pili for methanogenesis with magnetite stimulation

Abstract: Background Magnetite-mediated direct interspecies electron transfer (DIET) between Geobacter and Methanosarcina species is increasingly being invoked to explain magnetite stimulation of methane production in anaerobic soils and sediments. Although magnetite-mediated DIET has been documented in defined co-cultures reducing fumarate or nitrate as the electron acceptor, the effects of magnetite have only been inferred in methanogenic systems. Methods Concentrations of methane and organic acid were analysed with a gas chromatograph and high-performance liquid chromatography, respectively. The concentration of HCl-extractable Fe(II) was determined by the ferrozine method. The association of the defined co-cultures of G. metallireducens and M. barkeri with magnetite was observed with transmission electron micrographs. Results Magnetite stimulated ethanol metabolism and methane production in defined co-cultures of G. metallireducens and M. barkeri; however, magnetite did not promote methane production in co-cultures initiated with a culture of G. metallireducens that could not produce electrically conductive pili (e-pili), unlike the conductive carbon materials that facilitate DIET in the absence of e-pili. Transmission electron microscopy revealed that G. metallireducens and M. barkeri were closely associated when magnetite was present, as previously observed in G. metallireducens/G. sulfurreducens co-cultures. These results show that magnetite can promote DIET between Geobacter and Methanosarcina species, but not as a substitute for e-pili, and probably functions to facilitate electron transfer from the e-pili to Methanosarcina. Conclusion In summary, the e-pili are necessary for the stimulation of not only G. metallireducens/G. sulfurreducens, but also methanogenic G. metallireducens/M. barkeri co-cultures with magnetite.

Genetic, biochemical, and molecular characterization of methanosarcina barkeri mutants lacking three distinct classes of hydrogenase

Abstract: The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2-CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech Δfrh and Δech Δfrh Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420 The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.IMPORTANCE Methanogenic archaea are key players in the global carbon cycle due to their ability to facilitate the remineralization of organic substrates in many anaerobic environments. The consequences of biological methanogenesis are far-reaching, with impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. The data presented here clarify the in vivo function of hydrogenases during methanogenesis, which in turn deepens our understanding of this unique form of metabolism. This knowledge is critical for a variety of important issues ranging from atmospheric composition to human health.

Potential for Methanosarcina to Contribute to Uranium Reduction during Acetate-Promoted Groundwater Bioremediation

Abstract: Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.

Methane-linked mechanisms of electron uptake from cathodes by Methanosarcina barkeri

Abstract: The Methanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes by Methanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here we confirm the ability of M. barkeri to perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g. hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g. hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production in M. barkeri. Our electrochemical measurements of wild-type and mutant strains point to a novel and extracellular-enzyme-free mode of electron uptake able to take up electrons at potentials lower than -498 mV vs. SHE (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest that M. barkeri can perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent) of electrode interactions on cathodes including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Abstract: Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

The Hydrogen Economy of Methanosarcina barkeri: Life in the Fast Lane.

Abstract: Two recent studies (T. D. Mand, G. Kulkarni, and W. W. Metcalf, J. Bacteriol 200:e00342-18, 2018, https://doi.org/10.1128/JB.00342-18, and G. Kulkarni, T. D. Mand, and W. W. Metcalf, mBio 9:e01256-18, 2018, https://doi.org/10.1128/mBio.01256-18) analyzed an impressive array of hydrogenase-deficient mutant strains of Methanosarcina barkeri not only to describe H2-based growth but also to demonstrate the conservation of energy with intracellular hydrogen cycling, a novel strategy for creating a proton motive force to support ATP synthesis.

Genetic, biochemical, and molecular characterization of Methanosarcina barkeri mutants lacking three distinct classes of hydrogenase

Abstract: The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2/CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to wild-type, produced methane and CO2 in the expected 3:1 ratio, but had a ca. 33% slower growth rate. Thus, hydrogenases play a significant, but non-essential, role during growth on this substrate. As previously observed, mutants lacking Ech fail to grow on methanol/H2 unless supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech/Δfrh and Δech/Δfrh/Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of reducing equivalents needed for biosynthesis. Quantification of methane and CO2 produced from methanol by resting cell suspensions of various mutants supports this conclusion. Based on global transcriptional profiles, none of the hydrogenases are upregulated to compensate for loss of the others. However, transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420. The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.

Energy conservation via hydrogen cycling in the methanogenic archaeon Methanosarcina barkeri

Abstract: Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest H2 is a preferred, but non-essential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature. Importance: Adenosine triphosphate (ATP) is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.

Synthetic methanogenic communities reveal differential impact of ecological perturbations on aceto- and hydrogeno-trophic methanogens

Abstract: Synthetic microbial communities provide reduced microbial ecologies that can be studied under defined conditions. Here, we use this approach to study the interactions underpinning anaerobic digestion communities and involving the key microbial populations of a sulfate reducer (Desulfovibrio vulgaris), and aceto- (Methanosarcina barkeri) and hydrogeno-trophic (Methanococcus maripaludis) methanogens. We create all possible mixed culture combinations of these species and analyse the stability and productivity of each system over multiple sub-culturings and under different sulfate levels, mimicking ecological perturbation in the form of strong electron acceptor availability. We find that all three species can co-exist in the absence of sulfate, and that system productivity (in form of methane production from lactate) increases by almost two-fold compared to co-cultures. With increasing sulfate availability, co-existence is perturbed and both methanogenic populations display a diminishing trend. Interestingly, we find that, despite the continued presence of acetate in the system, the acetotrophic methanogens are more readily disrupted by sulfate perturbation. We show that this is due to a shift in M. barkeri metabolism towards increased co-utilisation of hydrogen with acetate, which we verified through experiments on mono cultures and mass balance calculations in co-cultures. We conclude that hydrogen is a key factor for both hydrogeno- and aceto-trophic methanogenesis and can influence these populations differentially under the common ecological perturbation of strong electron acceptor availability. These findings will help engineering of larger synthetic communities for specific applications in biodegradation and understanding complex anaerobic digestion communities found in animal guts, sediments, and bioreactors.