Acetate,methanol and carbon dioxide as substrates for growth of Methanosarcina barkeri

Methanosarcina barkeri grows in defined media with acetate, methanol or carbon dioxide as carbon sources. Methanol is used for methanogenesis at a 5 times higher rate as compared with the other substrates. M. barkeri can use the substrates simultaneously, but due to acidification or alkalification of the medium during growth on methanol or acetate, respectively, growth and methanogenesis may stop before the substrates are exhausted. Growth and methanogenesis on methanol or acetate are inhibited by the presence of an excess of H₂; the inhibition is abolished by the addition of carbon dioxide, which probably serves as an essential source of cell carbon, in the absence of which methano-genesis ceases.     

Influence of sulphur-containing compounds on the growth of Methanosarcina barkeri in a defined medium

Summary Optimal growth of Methanosarcina barkeri occurred in a defined medium containing methanol when 2.5–4 mM sodium sulphide was added giving a concentration of 0.04–0.06 mM dissolved sulphide (HS^–+S^2–. When the sulphide concentration was too low for optimal growth (e.g., 0.1 mM Na₂S added) the addition of the redox resin Serdoxit acted as a sulphide reservoir and caused a significant stimulation of growth. Furthermore it could be demonstrated that iron sulphide, zinc sulphide or L-methionine could also act as sulphur sources while the addition of sodium sulphate to sulphide-depleted media failed to restore growth. The amino acid L-cysteine (0.85 mM) stimulated growth but could not replace Na₂S.Under optimal cysteine-and sulphide concentrations the generation time of this strain was about 7–9 h during growth on methanol, giving a growth yield of about 0.14 g/g methanol consumed. Different M. barkeri strains were also able to grow under these conditions on acetate (30–50 h doubling time) without a significant lag-phase and with complete substrate consumption even though the inoculum was grown on methanol or H₂–CO₂. When methanol and acetate were present as a mixture in the medium both were used simultaneously.     

Isolation and Characterization of a Thermophilic Strain of Methanosarcina Unable to Use H(2)-CO(2) for Methanogenesis

A thermophilic strain of Methanosarcina, designated Methanosarcina strain TM-1, was isolated from a laboratory-scale 55 degrees C anaerobic sludge digestor by the Hungate roll-tube technique. Penicillin and d-cycloserine, inhibitors of peptidoglycan synthesis, were used as selective agents to eliminate contaminating non-methanogens. Methanosarcina strain TM-1 had a temperature optimum for methanogenesis near 50 degrees C and grew at 55 degrees C but not at 60 degrees C. Substrates used for methanogenesis and growth by Methanosarcina strain TM-1 were acetate (12-h doubling time), methanol (7- to 10-h doubling time), methanol-acetate mixtures (5-h doubling time), methylamine, and trimethylamine. When radioactively labeled acetate was the sole methanogenic substrate added to the growth medium, it was predominantly split to methane and carbon dioxide. When methanol was also present in the medium, the metabolism of acetate shifted to its oxidation and incorporation into cell material. Electrons derived from acetate oxidation apparently were used to reduce methanol. H(2)-CO(2) was not used for growth and methanogenesis by Methanosarcina strain TM-1. When presented with both H(2)-CO(2) and methanol, Methanosarcina strain TM-1 was capable of limited hydrogen metabolism during growth on methanol, but hydrogen metabolism ceased once the methanol was depleted. Methanosarcina strain TM-1 required a growth factor (or growth factors) present in the supernatant of anaerobic digestor sludge. Growth factor requirements and the inability to use H(2)-CO(2) are characteristics not found in other described Methanosarcina strains. The high numbers of Methanosarcina-like clumps in sludges from thermophilic digestors and the fast generation times reported here for Methanosarcina TM-1 indicate that Methanosarcina may play an important role in thermophilic methanogenesis.     

Tungstate does not support synthesis of active formylmethanofuran dehydrogenase in Methanosarcina barkeri

Cell extracts of Methanosarcina barkeri grown on methanol in media supplemented with molybdate exhibited a specific activity of formylmethanofuran dehydrogenase of approximately 1 U (1 mol/min)/mg protein. When the growth medium was supplemented with tungstate rather than with molybdate, the specific activity was only 0.04 U/mg. Despite this reduction in specific activity growth on methanol was not inhibited. An inhibition of both growth and synthesis of active formylmethanofuran dehydrogenase was observed, however, when H₂ and CO₂ were the energy substrates. The results indicate that, in contrast to Methanobacterium wolfei and Methanobacterium thermoautotrophicum, M. barkeri possesses only a molybdenum containing formylmethanofuran dehydrogenase and not in addition a tungsten isoenzyme.     

Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Methanosarcina barkeri strain 227 produced ethane during growth on H₂/CO₂ when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A₆₀₀ 0.46, protein = 64 g/ml) on H₂/CO₂, thoroughly flushed with H₂/CO₂, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10^-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H₂ produced 6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H₂ and/or methanol produced 1.6 nmoles per tube. Cultures of Methanococcus deltae strains LH and RC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced 5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl₄ and Br–CH₂–CH₂–SO _inf3 ^sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.     

Investigation of the trophic relations between anaerobic microorganisms from an underground gas repository during methanol utilization

Joint cultivation of the dominant strains of acetogenic, sulfate-reducing and methanogenic microorganisms isolated from water samples of the North Stavropol underground gas storage facility (UGSF) was carried out for revealing their probable trophic relationships. It was shown that acetogenic strains Eubacterium limosum AG12 and Sporomusa sphaeroides AG8-2 growing on methanol could form a considerable pool of hydrogen, which may support development of hydrogenotrophic cultures, the methanogen Methanobacterium formicicum MG134, or the sulfate reducer Desulfovibrio desulfuricans SR12. Growth of this sulfate-reducing strain was not stimulated under joint cultivation with Methanosarcina barkeri MGZ3 on methanol, probably due to its inability to take up low hydrogen concentrations observed during methanosarcina development. The results show that acetogens in the UGSF system are the most important consumers of methanol and hydrogen and after exhaustion of the latter and switching over to methanol utilization they can supply hydrogen to other microorganisms, including methanogens and sulfate reducers. The role of methanosarcina in the UGSF increases as the hydrogen and CO₂ reserves are exhausted, and methanogenesis on methanol becomes the main way of its destruction.     

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp

When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 10⁸ to 10⁹ free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl₂ (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl₂. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.     

Complete degradation of carbohydrate to carbon dioxide and methane by syntrophic cultures of Acetobacterium woodii and Methanosarcina barkeri

Methanosarcina barkeri (strain MS) grew and converted acetate to CO₂ and methane after an adaption period of 20 days. Growth and metabolism were rapid with gas production being comparable to that of cells grown on H₂ and CO₂. After an intermediary growth cycle under a H₂ and CO₂ atmosphere acetateadapted cells were capable of growth on acetate with formation of methane and CO₂. When acetate-adapted Methanosarcina barkeri was co-cultered with Acetobacterium woodii on fructose or glucose as substrate, a complete conversion of the carbohydrate to gases (CO₂ and CH₄) was observed.Abbreviation CMC carboxymethyl cellulose     

Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia

Methanosarcina frisia accumulates phosphate up to 14% of its dry weight. The phosphate is stored as long-chain polyphosphates as shown by ³¹P-NMR investigations. Further results show that the accumulation of phosphates is substrate-dependent. In the presence of H₂ and CO₂ as the only carbon and energy source 180 mg of PO _inf4 ^sup3- /g protein were accumulated, whereas 260 mg PO _inf4 ^sup3- /g protein were accumulated in the presence of methanol. This is far more than necessary for the maintenance of essential metabolic pathways. In addition, the ³¹P-NMR studies show the occurrence of cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. The role of the phosphate metabolites in cell metabolism are discussed.Abbreviations M. Methanosarcina - CCP cyclic 2,3-diphosphoglycerate     

Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes.     

Effect of H2-CO2 on Methanogenesis from Acetate or Methanol in Methanosarcina spp

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H₂-CO₂ and mixtures of H₂-CO₂ and acetate or methanol was examined. The growth yield of strain 227 on H₂-CO₂ in complex medium was 8.4 mg/mmol of methane produced. Growth in defined medium was characteristically slower, and cell yields were proportionately lower. Labeling studies confirmed that CO₂ was rapidly reduced to CH₄ in the presence of H₂, and little acetate was used for methanogenesis until H₂ was exhausted. This resulted in a biphasic pattern of growth similar to that reported for strain 227 grown on methanol-acetate mixtures. Biphasic growth was not observed in cultures on mixtures of H₂-CO₂ and methanol, and less methanol oxidation occurred in the presence of H₂. In M. mazei the aceticlastic reaction was also inhibited by the added H₂, but since the cultures did not immediately metabolize H₂, the duration of the inhibition was much longer.     

Genomically and biochemically accurate metabolic reconstruction of Methanosarcina barkeri Fusaro,iMG746

Methanosarcina barkeri is an Archaeon that produces methane anaerobically as the primary byproduct of its metabolism. M. barkeri can utilize several substrates for ATP and biomass production including methanol, acetate, methyl amines, and a combination of hydrogen and carbon dioxide. In 2006, a metabolic reconstruction of M. barkeri, iAF692, was generated based on a draft genome annotation. The iAF692 reconstruction enabled the first genome-Scale simulations for Archaea. Since the publication of the first metabolic reconstruction of M. barkeri, additional genomic, biochemical, and phenotypic data have clarified several metabolic pathways. We have used this newly available data to improve the M. barkeri metabolic reconstruction. Modeling simulations using the updated model, iMG746, have led to increased accuracy in predicting gene knockout phenotypes and simulations of batch growth behavior. We used the model to examine knockout lethality data and make predictions about metabolic regulation under different growth conditions. Thus, the updated metabolic reconstruction of M. barkeri metabolism is a useful tool for predicting cellular behavior, studying the methanogenic lifestyle, guiding experimental studies, and making predictions relevant to metabolic engineering applications.     

Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme

A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H₂-CO₂, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Isolation of Extracellular Cobalt-free Corrinoid from Methanosarcina barkeri

Cobalt-free corrinoids (CFCs) were isolated from Methanosarcina barkeri Fusaro cells growing on a methanol minimum medium. The methanogen cells excreted a trace of CFCs (9.1 μg/I) into the culture medium when cobalt-deficient methanol medium was used. Several CFCs were separated by column chromatographies on ion exchangers and paper electrophoresis, where a major CFC showed a similar characteristic to that of nucleotide-free corrinoid, Factor B (cobinamide), suggesting to be hydrogenobinamide. By chemical insertion of Co^{2 +}, Cu^{2 +}, and Zn²⁺ into CFCs, the corresponding corrinoid and its metal analogues were observed. Bioassay using Escherichia coli 215 revealed that the major CFC (a yellow product obtained after alkaline treatment) and its copper and zinc analogues were inactive as cobalamin but were active as antimetabolites of cobalamin. However, the CFC greatly stimulated the cell growth of M. barkeri grown under cobalt-deficient conditions.     

Utilization of Methanol plus Hydrogen by Methanosarcina barkeri for Methanogenesis and Growth

Methanosarcina barkeri grew on methanol plus H₂. Both substrates were consumed in equimolar amounts. Growth was strictly dependent on the presence of acetate, which was required for the biosynthesis of cellular constituents. Only about 0.4% of the methane produced originated from acetate. By using deuterated methanol, it was demonstrated that methanogenesis from this compound under H₂ did not occur via oxidation of methanol to CO₂ and subsequent reduction but by direct reduction with H₂. Growth yields with methanol plus H₂ and with methanol alone were not significantly different: 2.8 g of cells per mol of methanol in mineral medium and 4.6 g of cells per mol of methanol in complex medium, respectively. Growth of M. barkeri on methanol plus H₂ depended strictly on the presence of sodium ions in the medium. In the presence of 50 mM K⁺ the K_s for Na⁺ was 5 mM.