Methanosarcina Barkeri Research Articles

Anaerobic degradation of sulphite evaporator condensate in a fixed-bed loop reactor by a defined bacterial consortium

After elucidating the composition of an anaerobic bacterial enrichment culture treating sulphite evaporator condensate (SEC), an effluent in the pulp and paper industry, we built up stepwise a defined mixed culture to convert the organic constituents of SEC (acetate, methanol, furfural) to methane and CO2. In batch cultures Desulfovibrio furfuralis and Methanobacterium bryantii degraded furfural in the absence of sulphate via inter-species H2 transfer yielding 0.42 mol methane and 1.87 mol acetate/mol furfural degraded. When Methanosarcina barkeri was added to this diculture, acetate was also transformed to methane yielding 0.93 mol methane/mol acetate converted. This consortium (D. furfuralis, Methanobacterium bryantii and Methanosarcina barkeri) degraded furfural in continuous culture (fixed-bed loop reactor) to 92%, but the conversion of acetate was only 67%. The conversion of acetate could be further improved to 86% by adding 10 mm sulphate to the medium. This resulted in a space time yield of 10.9 g chemical oxygen demand (COD)/1 per day for the overall conversion. With a consortium consisting of M. barkeri, Methanobrevibacter arboriphilus, Methanosaeta concilii and D. furfuralis, a synthetic SEC could be degraded at a space time yield of 13.35 g COD/1 per day. This defined culture degraded all the constituents of SEC at an efficiency of almost 90% compared to an enrichment culture under identical conditions.

Hydroxyarchaetidylserine and hydroxyarchaetidyl- myo-inositol in Methanosarcina barkeri: polar lipids with a new ether core portion

Lipids of the methanogenic archaebacterium, Methanosarcina barkeri were analyzed. The lipid content was 5.4% of dry cell and polar lipids comprised 87% of the total lipid. Polar lipids were separated into 14 spots by two-dimensional thin-layer chromatography. These were six phospholipids, seven aminophospholipids and one glycolipid, of which two phospholipids and two aminophospholipids were major constituents. After removal of polar head groups from total lipids, two kinds of glycerol diether core lipids were found. One was 2,3-di- O-phytanyl- sn-glycerol (archaeol) and the other 2- O-(3′-hydroxy-3′, 7′, 11′, 15′-tetramethyl)hexadecyl-3- O-phytanyl- sn-glycerol (hydroxyarhaeol). Those structures were identified on the basis of chemical analysis, fast atom bombardment-mass spectrometry, gas-liquid chromatography-mass spectrometry and 1H- and 13C-NMR spectrometry. The latter was a new core lipid which was different from hydroxyarchaeol of Methanothrix concilii. The hydroxyarchaeol core lipid comprised 60% of polar lipid in M. barkeri. The structures of core lipids are quite different from those previously reported by De Rosa et al. (Biochim. Biophys. Acta (1986) 875, 487–492) concerning M. barkeri lippids. The structures of two major polar lipids, both of which had hydroxyarchaeol as core portions, were elucidated. These lipids were 2- O-(3′-hydroxy)phytanyl-3- O-phytanyl- sn-glycero-1-phosephoserine (hydroxyarchaetidylserine) and 2- O-(3′-hydroxy)phytanyl-3- O-phytanyl- sn-glycero-1-phospho- myo-inositol (hydroxyarchaetidyl- myo-inositol). Archaetidylserine and archaetidylinositol, which had the usual archaeol core portion, were also present as minor polar lipids.

In vitro inhibition of cell growth of MOLT-4 malignant human T-lymphoblasts by coenzyme F 420

The inhibitory effect of methanogenic coenzymes on the proliferation of MOLT-4 human malignant T-lymphoblasts was tested. Furthermore the effects of methanogenic coenzymes on dihydrofolate reductase activity (DHFR) from chicken liver have been examined. The results showed that heat-stable extracts of the hydrogenotrophs Methanobacterium thermoautotrophicum, Methanoculleus thermophilicum and Methanogenium tationis inhibit both proliferation of human T-lymphoblasts and DHFR activity. Heat-stable extract of the methylotroph Methanosarcina barkeri showed neither inhibitory nor stimulatory effects in both test systems. The present study proves coenzyme F 420 to be the active, inhibitory component in methanogenic extracts.

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.

Medium optimization by an orthogonal array design for the growth of Methanosarcina barkeri

Optimization of the medium composition for Methanosarcina barkeri growing on methanol (8 g/ l) was carried out by two different methods, (i) “one-at-a-time” and (ii) analysis of marginal means (ANOMM) in an orthogonal array experimental design. The second method showed to be very effective in the improvement of the medium composition capable of increasing the gas production rate (CH 4 + CO 2) over 1.3 and 2.0 times when compared with the “one-at-a-time” optimized medium and the basal medium, respectively.

Growth characteristics and corrinoid production of Methanosarcina barkeri on methanol-acetate medium

Methanosarcina barkeri strain Fusaro was grown on a mixed substrate medium of methanol and acetate. When 50 mM of acetate was added to the methanol basal medium (250 mM), the rates of methane production, methanol consumption, cell growth and corrinoid production were stimulated 3.2, 2.7, 3.5, and 2.4 times, respectively compared with those in methanol alone. Addition of acetate also has significant influence on corrinoid distribution decreasing the intracellular corrinoid content from 6.8 to 3.0 mg/g dry cell and increasing the extracellular corrinoid concentration from 4.0 to 5.4 mg/ l. The carbon balance analysis for methanogenesis and cellular growth with or without acetate addition revealed that about 50% of the utilized acetate carbon might be incorporated in the cellular materials and the remaining might be oxidized to generate the electrons which stimulate the methanol reduction to methane, accelerating the metabolic activities of the methanogenesis from methanol consequently enhancing the rates of methane and corrinoid production, and cell growth.

In vivo nuclear magnetic resonance studies of the metabolism of methanol and pyruvate by Methanosarcina barkeri

The metabolism of methanol and pyruvate by cells of Methanosarcina barkeri was probed in vivo by NMR taking advantage of the non-invasive characteristics of this technique. Upon administration of substrates, the kinetics of substrate consumption, the product formation and the energetic state of the cells was monitored using carbon-13, phosphorus-31 or proton NMR. The effects of several inhibitors and uncouplers were investigated. Cells supplied with pyruvate developed considerable levels of nucleotide triphosphate; methane production was monitored, as well as CO 2 and H 2 formation. Most of the pyruvate was utilized for the synthesis of valine or intermediates of the valine pathway. The origin of the carbon atom in methane was elucidated using 13C-labelled pyruvate.

The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor

Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6-carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes. A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5-0.8 mol molybdenum, 0.6-0.9 mol pterin, 28 +/- 2 mol non-heme iron and 28 +/- 2 mol acid-labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 mumol.min-1.mg-1 (kcat = 640 s-1) assayed with methylviologen (app. Km = 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.

Sodium bioenergetics in methanogens and acetogens

Recent investigations with Methanosarcina barkeri elucidated the role of sodium ions in the energy metabolism of methanogenic bacteria and provided evidence for a novel mechanism of energy transduction with Na + as the coupling ion. During methanogenesis from methanol, an eletrochemical sodium gradient generated by a Na +/H + antiporter is used as the driving force for the thermodynamically unfavourable oxidation of methanol to the formal redox level of formaldehyde. During methanogenesis from H 2 + CO 2, the reverse reaction, the reduction of formaldehyde to the level of methanol, is accompanied by a primary, electron transport-driven sodium extrusion. Acetogenesis from H 2 + CO 2 as carried out by Acetobacterium woodii is a sodium-dependent process and is accompanied by the generation of a transmembrane sodium gradient with the reduction of formaldehyde to the level of methanol as the sodium-dependent step.

In vivo nuclear magnetic resonance studies of the metabolism of methanol and pyruvate byMethanosarcina barkeri

The metabolism of methanol and pyruvate by cells of Methanosarcina barkeri was probed in vivo by NMR taking advantage of the non-invasive characteristics of this technique. Upon administration of substrates, the kinetics of substrate consumption, the product formation and the energetic state of the cells was monitored using carbon-13, phosphorus-31 or proton NMR. The effects of several inhibitors and uncouplers were investigated. Cells supplied with pyruvate developed considerable levels of nucleotide triphosphate; methane production was monitored, as well as CO2 and H2 formation. Most of the pyruvate was utilized for the synthesis of valine or intermediates of the valine pathway. The origin of the carbon atom in methane was elucidated using 13C-labelled pyruvate.

Nitrogenase in the archaebacterium Methanosarcina barkeri 227

The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20- to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. The component 1 analog had a molecular size of approximately 250 kDa, as estimated by gel filtration, and sodium dodecyl sulfate-polyacrylamide gels revealed two predominant bands with molecular sizes near 57 and 62 kDa, consistent with an alpha 2 beta 2 tetramer as in eubacterial component 1 proteins. For the component 2 analog, a molecular size of approximately 120 kDa was estimated by gel filtration, with a subunit molecular size near 31 kDa, indicating that the component 2 protein is a tetramer, in contrast to eubacterial component 2 proteins, which are dimers. Rates of C2H2 reduction by the nearly pure subunits were 1,000 nmol h-1 mg of protein-1, considerably lower than those for conventional Mo nitrogenases but similar to that of the non-Mo non-V nitrogenase from Azotobacter vinelandii. Strain 227 nitrogenase reduced N2 at a higher rate per electron than it reduced C2H2, also resembling the non-Mo non-V nitrogenase of A. vinelandii. Ethane was not produced from C2H2. NH4+ concentrations as low as 10 microM caused a transient inhibition of C2H2 reduction by strain 227 cells. Antiserum against component 2 Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.

Sodium bioenergetics in methanogens and acetogens

Recent investigations with Methanosarcina barkeri elucidated the role of sodium ions in the energy metabolism of methanogenic bacteria and provided evidence for a novel mechanism of energy transduction with Na+ as the coupling ion. During methanogenesis from methanol, an eletrochemical sodium gradient generated by a Na+/H+ antiporter is used as the driving force for the thermodynamically unfavourable oxidation of methanol to the formal redox level of formaldehyde. During methanogenesis from H2+ CO2, the reverse reaction, the reduction of formaldehyde to the level of methanol, is accompanied by a primary, electron transport-driven sodium extrusion. Acetogenesis from H2+ CO2 as carried out by Acetobacterium woodii is a sodium-dependent process and is accompanied by the generation of a transmembrane sodium gradient with the reduction of formaldehyde to the level of methanol as the sodium-dependent step.

Formylmethanofuran: Tetrahydromethanopterin formyltransferase from Methanosarcina barkeri Identification of N5-formyltetrahydromethanopterin as the product

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified from methanol grown Methanosarcina barkeri to apparent homogeneity and characterized with respect to its molecular and kinetic properties. The enzyme was found to be very similar to the formyltransferase from H 2/CO 2 grown Methanobacterium thermoautotrophicum. It also catalyzed the formation of N 5-formyltetrahydromethanopterin rather than of N 10-formyltetrahydromethanopterin from formylmethanofuran and tetrahydromethanopterin.

The molybdenum cofactor of formylmethanofuran dehydrogenase from Methanosarcina barkeri is a molybdopterin guanine dinucleotide

The molybdenum cofactor of formylmethanofuran dehydrogenase from methanol-grown Methanosarcina barkeri was isolated as the [difcarboxami-domethyl)]-derivative. The alkylated factor showed an absorption spectrum and chemical properties identical to those recently reported for the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides. By treatment with nucleotide pyrophosphatase the factor was resolved into two components, which were identified as [di(carboxamidomethyl)]-molybdopterin and GMP by their absorption spectra, their retention times on Lichrospher RP-18, and by their conversion to dephospho-[di(carboxamidomcthyl)]-molybdopterin and guanosine. respectively, in the presence of alkaline phosphatase. The GMP-moiety was sensitive to pcriodate, identifying it as the 5'-isomer. These results demonstrate that the molybdenum cofactor isolated from formylmethanofuran dehydrogenase contains the phosphoric anhydride of molybdopterin and 5'-GMP.

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS)

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO 2 and CH 4 at a specific rate of 50 nmol·min −1·mg −1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridiwn pasteurianum. The apparent K m for ferredoxin from M. barkeri was determined to be 2.5 μM. A ferredoxin dependence was also found for the formation of CO 2, H 2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H 2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.

Novel, acid-labile, hydroxydiether lipid cores in methanogenic bacteria.

Polar ether lipids extracted from 15 methanogenic bacteria, representative of seven genera, were screened by nuclear magnetic resonance and thin layer chromatography for the presence of hydroxyl groups on the C20-phytanyl moieties. Major amounts of hydroxydiether core lipid were confirmed for Methanosaeta concilii and discovered in two Methanosarcina species, Methanococcus voltae, and tentatively in several Methanobacterium species. Signals at 1.24 and 1.8-1.9 ppm in 1H NMR spectra are characteristic of Methanosaeta concilii lipids hydroxylated on carbon-3 (sn-3 chain). Related signals, which were shifted slightly, appeared in spectra of the polar lipids extracted from both Methanosarcina species. Following mild hydrolysis to remove the polar head groups, only two chromatographically distinct core lipids were found in significant amounts in Methanosarcina barkeri (and Methanosarcina mazei) consisting of 43% 2,3-di-O-phytanyl-sn-glycerol (C20,20-diether) and 57% C20,20-hydroxydiether. This latter core lipid differed from the hydroxydiether from M. concilii by hydroxylation, on carbon-3, of the phytanyl chain in ether linkage to the sn-2 carbon of glycerol. The structural assignment was based on identification of the novel hydroxydiether core and its methylation products by 1H NMR, 13C NMR, and mass spectroscopy. The hydroxy core lipid degraded to various products during standard methanolic HCl and sulfuric acid procedures, including a methoxy derivative (methanolic HCl) and the 3-mono-O-phytanyl-sn-glycerol.

N5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

1. Summary N5N10-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri. The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin (Km=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F420 (Km=12 μM) at a specific rate (Vmax) of 2200 μmol min−1 · mg protein−1 (Kcat=1320 s−1). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH2=H4MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri.

Conformational study of the chromosomal protein MC1 from the archaebacterium Methanosarcina barkeri

Methanogen chromosomal protein MC1 is a polypeptide of 93 amino acid residues ( M r 10757) which represents the major protein associated with the DNA of the archaebacterium Methanosarcina barkeri and can protect DNA against thermal denaturation. The conformation of protein MC1 has been investigated by means of predictive methods, infrared spectroscopy, circular dichroism and tryptophan fluorescence studies. Protein MC1 has a low amount of α-helix but contains antiparallel β-sheet strands. The larger hydrophobic cluster which contains tryptophan at position 61 appears buried in the protein. Addition of salts induces the unfolding of the protein and makes the tryptophan indole ring more rigid. With respect to its primary structure and its conformation, protein MC1 appears radically different from the chromosomal DNA-binding protein II (also called HU-type protein) in eubacteria.

Enhanced cytochrome formation and stimulate methanogenesis rate by the increased ferrous concentrations inMethanosarcina barkericulture

The concentration of Fe2+ in the methanol minimum medium was found to be a stimulating factor on the formation of cytochromes, the excretion of hemes and the rate of methane formation by Methanosarcino barkeri cells. Overproduced hemes due to the high Fe2+ concentration were released into the medium as free forms. When Fe2+ concentration was employed more than 30 μM for the medium, the contents of cytochrome b and c in the cells seemed to be saturated at 40 nmol/g cell. Under the same condition, not only membrane-bound but also soluble cytochromes were detected in the ultracentrifuged fractions from the cells grown on 35 μM of Fe2+

Enhanced cytochrome formation and stimulate methanogenesis rate by the increased ferrous concentrations in Methanosarcina barkeri culture

The concentration of Fe 2+ in the methanol minimum medium was found to be a stimulating factor on the formation of cytochromes, the excretion of hemes and the rate of methane formation by Methanosarcino barkeri cells. Overproduced hemes due to the high Fe 2+ concentration were released into the medium as free forms. When Fe 2+ concentration was employed more than 30 μM for the medium, the contents of cytochrome b and c in the cells seemed to be saturated at 40 nmol/g cell. Under the same condition, not only membrane-bound but also soluble cytochromes were detected in the ultracentrifuged fractions from the cells grown on 35 μM of Fe 2+