Showing papers on "Methanosarcina barkeri published in 2016"

Expanding the Diet for DIET: Electron Donors Supporting Direct Interspecies Electron Transfer (DIET) in Defined Co-Cultures

Abstract: Direct interspecies electron transfer (DIET) has been recognized as an alternative to interspecies H2 transfer as a mechanism for syntrophic growth, but previous studies on DIET with defined co-cultures have only documented DIET with ethanol as the electron donor in the absence of conductive materials. Co-cultures of Geobacter metallireducens and Geobacter sulfurreducens metabolized propanol, butanol, propionate, and butyrate with the reduction of fumarate to succinate. G. metallireducens utilized each of these substrates whereas only electrons available from DIET supported G. sulfurreducens respiration. A co-culture of G. metallireducens and a strain of G. sulfurreducens that could not metabolize acetate oxidized acetate with fumarate as the electron acceptor, demonstrating that acetate can also be syntrophically metabolized via DIET. A co-culture of G. metallireducens and Methanosaeta harundinacea previously shown to syntrophically convert ethanol to methane via DIET metabolized propanol or butanol as the sole electron donor, but not propionate or butyrate. The stoichiometric accumulation of propionate or butyrate in the propanol- or butanol-fed cultures demonstrated that M. harundinaceae could conserve energy to support growth solely from electrons derived from DIET. Co-cultures of G. metallireducens and Methanosarcina barkeri could also incompletely metabolize propanol and butanol and did not metabolize propionate or butyrate as sole electron donors. These results expand the range of substrates that are known to be syntrophically metabolized through DIET, but suggest that claims of propionate and butyrate metabolism via DIET in mixed microbial communities warrant further validation.

Methanogens rapidly transition from methane production to iron reduction

Abstract: Methanogenesis, the microbial methane (CH4 ) production, is traditionally thought to anchor the mineralization of organic matter as the ultimate respiratory process in deep sediments, despite the presence of oxidized mineral phases, such as iron oxides. This process is carried out by archaea that have also been shown to be capable of reducing iron in high levels of electron donors such as hydrogen. The current pure culture study demonstrates that methanogenic archaea (Methanosarcina barkeri) rapidly switch from methanogenesis to iron-oxide reduction close to natural conditions, with nitrogen atmosphere, even when faced with substrate limitations. Intensive, biotic iron reduction was observed following the addition of poorly crystalline ferrihydrite and complex organic matter and was accompanied by inhibition of methane production. The reaction rate of this process was of the first order and was dependent only on the initial iron concentrations. Ferrous iron production did not accelerate significantly with the addition of 9,10-anthraquinone-2,6-disulfonate (AQDS) but increased by 11-28% with the addition of phenazine-1-carboxylate (PCA), suggesting the possible role of methanophenazines in the electron transport. The coupling between ferrous iron and methane production has important global implications. The rapid transition from methanogenesis to reduction of iron-oxides close to the natural conditions in sediments may help to explain the globally-distributed phenomena of increasing ferrous concentrations below the traditional iron reduction zone in the deep 'methanogenic' sediment horizon, with implications for metabolic networking in these subsurface ecosystems and in past geological settings.

Secondary Mineralization of Ferrihydrite Affects Microbial Methanogenesis in Geobacter-Methanosarcina Cocultures

Abstract: The transformation of ferrihydrite to stable iron oxides over time has important consequences for biogeochemical cycling of many metals and nutrients. The response of methanogenic activity to the presence of iron oxides depends on the type of iron mineral, but the effects of changes in iron mineralogy on methanogenesis have not been characterized. To address these issues, we constructed methanogenic cocultures of Geobacter and Methanosarcina strains with different ferrihydrite mineralization pathways. In this system, secondary mineralization products from ferrihydrite are regulated by the presence or absence of phosphate. In cultures producing magnetite as the secondary mineralization product, the rates of methanogenesis from acetate and ethanol increased by 30.2% and 135.3%, respectively, compared with a control lacking ferrihydrite. Biogenic magnetite was proposed to promote direct interspecies electron transfer between Geobacter and Methanosarcina in a manner similar to that of c-type cytochrome and thus facilitate methanogenesis. Vivianite biomineralization from ferrihydrite in the presence of phosphate did not significantly influence the methanogenesis processes. The correlation between magnetite occurrence and facilitated methanogenesis was supported by increased rates of methane production from acetate and ethanol with magnetite supplementation in the defined cocultures. Our data provide a new perspective on the important role of iron biomineralization in biogeochemical cycling of carbon in diverse anaerobic environments. IMPORTANCE It has been found that microbial methanogenesis is affected by the presence of iron minerals, and their influences on methanogenesis are associated with the mineralogical properties of the iron minerals. However, how changes in iron mineralogy affect microbial methanogenesis has not been characterized. To address this issue, we constructed methanogenic cocultures of Geobacter and Methanosarcina strains with different ferrihydrite mineralization pathways. The experimental results led to two contributions, i.e., (i) the transformation of iron minerals might exert an important influence on methanogenesis under anaerobic conditions and (ii) both biogenic and chemical magnetite can accelerate syntrophic ethanol oxidization between Geobacter metallireducens and Methanosarcina barkeri. This study sheds new light on the important role of iron biomineralization in the biogeochemical cycling of carbon in diverse anaerobic environments, particularly in iron-rich natural and agricultural wetland soils.

Didehydroaspartate Modification in Methyl-Coenzyme M Reductase Catalyzing Methane Formation

Abstract: All methanogenic and methanotrophic archaea known to date contain methyl-coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl-coenzyme M to methane. This enzyme contains the nickel porphinoid F430 as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post-translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high-resolution X-ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine-tuning the active site to increase the catalytic efficiency.

Effect of Nickel Levels on Hydrogen Partial Pressure and Methane Production in Methanogens

Abstract: Hydrogen (H2) consumption and methane (CH4) production in pure cultures of three different methanogens were investigated during cultivation with 0, 0.2 and 4.21 μM added nickel (Ni). The results showed that the level of dissolved Ni in the anaerobic growth medium did not notably affect CH4 production in the cytochrome-free methanogenic species Methanobacterium bryantii and Methanoculleus bourgensis MAB1, but affected CH4 formation rate in the cytochrome-containing Methanosarcina barkeri grown on H2 and CO2. Methanosarcina barkeri also had the highest amounts of Ni in its cells, indicating that more Ni is needed by cytochrome-containing than by cytochrome-free methanogenic species. The concentration of Ni affected threshold values of H2 partial pressure (pH2) for all three methanogen species studied, with M. bourgensis MAB1 reaching pH2 values as low as 0.1 Pa when Ni was available in amounts used in normal anaerobic growth medium. To our knowledge, this is the lowest pH2 threshold recorded to date in pure methanogen culture, which suggests that M.bourgensis MAB1 have a competitive advantage over other species through its ability to grow at low H2 concentrations. Our study has implications for research on the H2-driven deep subsurface biosphere and biogas reactor performance.

The auxiliary [4Fe–4S] cluster of the Radical SAM heme synthase from Methanosarcina barkeri is involved in electron transfer

Abstract: The heme synthase AhbD catalyzes the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme during the alternative heme biosynthesis pathway occurring in sulfate-reducing bacteria and archaea. AhbD belongs to the family of Radical SAM enzymes and contains two [4Fe–4S] clusters. Whereas one of these clusters is required for substrate radical formation, the role of the second iron–sulfur cluster is not known. In this study, the function of the auxiliary cluster during AhbD catalysis was investigated. Two single cluster variants of AhbD from M. barkeri carrying either one of the two clusters were created. Using these enzyme variants it was shown that the auxiliary cluster is not required for substrate binding and formation of the substrate radical. Instead, the auxiliary cluster is involved in a late step of AhbD catalysis most likely in electron transfer from the reaction intermediate to a final electron acceptor. Moreover, by using alternative substrates such as coproporphyrin III, Cu-coproporphyrin III and Zn-coproporphyrin III for the AhbD activity assay it was observed that the central iron ion of the porphyrin substrate also participates in the electron transfer from the reaction intermediate to the auxiliary [4Fe–4S] cluster. Altogether, new insights concerning the completely uncharacterized late steps of AhbD catalysis were obtained.

Orthogonal Protein Translation Using Pyrrolysyl-tRNA Synthetases for Single- and Multiple-Noncanonical Amino Acid Mutagenesis

Abstract: To date, the two systems most extensively used for noncanonical amino acid (ncAA) incorporation via orthogonal translation are based on the Methanococcus jannaschii TyrRS/tRNA CUA Tyr and the Methanosarcina barkeri/Methanosarcina mazei PylRS/tRNA CUA Pyl pairs. Here, we summarize the development and usage of the pyrrolysine-based system for orthogonal translation, a process that allows for the recombinant production of site-specifically labeled proteins and peptides. Via stop codon suppression in Escherichia coli and mammalian cells, genetically encoded biomolecules can be equipped with a great diversity of chemical functionalities including click chemistry handles, post-translational modifications, and photocaged sidechains.

Combining culture-dependent and culture-independent molecular methods for the isolation and purification of a potentially novel anaerobic species from pit mud in a Chinese liquor distillery

Abstract: Strain S12–27–1-3-5 (a potentially novel anaerobic species) with a 16S rRNA sequence homology of <97% was isolated and purified from pit mud by combining culture-dependent and culture-independent molecular methods, such as cloning of 16S rRNA, amplified rRNA restriction analysis, and denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the 16S rRNA gene indicated that strain S12–27–1-3-5 was related to Aminobacterium mobile strain ILE-3 DSM 12262T and Aminobacterium colombiense strain DSM 12261T (95 and 96% similarity value, respectively). The results verified that cloning of the 16S rRNA was efficient to identify whether a potentially new bacterial taxon existed in impure isolates and that the DGGE method was a powerful tool for screening the target bacteria and for identifying duplicate strains. Therefore, the application of the culture-independent molecular methods for the isolation and purification of a potentially novel species was effective. Strain S12–27–1-3-5 (= DSM 27871 = JCM 19605 = CICC 10731T) was an anaerobic amino acid-degrading bacterium. The results of fermentation experiments demonstrated that strain S12–27–1-3-5 produced volatile fatty acids (VFAs) and the presence of Methanosarcina barkeri enhanced the generation of VFAs, which contribute to the aroma composition of Chinese liquor. This work could enrich the species resources and promote the development and utilization of an uncultured species. Copyright © 2016 The Institute of Brewing & Distilling

Compound bacterium capable of quickly removing propionic acid accumulation as well as preparation method and application of compound bacterium

Abstract: The invention provides a preparation method of a compound bacterium capable of quickly removing propionic acid accumulation. The preparation method comprises the following steps: (1) taking effluent of a dextranum wastewater anaerobic digester as an original inoculums, and sodium propionate as a carbon source, and performing inoculated culture till the degradation rate of propionic acid in a culture reaches 50-80% and the content of methane is 30-50%; (2) transferring the object obtained in the step (1) to a fresh culture medium for culture, and adding the following bacteria into every ml of a culture solution of the object obtained in the step (1): (2.5-5)*10 Pelotomaculum schinkii, (1-2.5)*10 Pelotomaculum propionicicum, (1-3)*10 Syntrophobacter wolinii, (0.4-1.4)*10 Methanospirillum hungatei, (0.5-1.4)*10 Methanoculleus palmolei, (0.5-1.4)*10 Methanoculleus bourgensis, (0.5-2.5)*10 Methanosarcina barkeri and (1-3)*10 Methanosarcina mazei; (3) performing airtight culture on the object obtained in the step (2) to obtain the compound bacterium capable of quickly removing propionic acid accumulation. The invention further provides application of the compound bacterium to the anaerobic digestion technology, particularly to biogas fermentation. The compound bacterium can quickly and efficiently remove propionic acid accumulation, and greatly improve the anaerobic digestion efficiency and stability.

Contributions and Future Recommendations

Abstract: Rapid depletion of fossil fuel-based resources has caused energy and environmental concerns and encourages production of renewable chemicals and fuels from CO2 consuming microorganisms.