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.     

Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity

The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg²⁺ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.     

Differentiation of Methanosaeta concilii and Methanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.     

Identification of the gene for disaggregatase from Methanosarcina mazei

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates of Methanosarcina mazei to single cells, were determined for three strains of M. mazei (S-6^T, LYC and TMA). The dag genes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genus Methanosarcina and may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed in Escherichia coli and preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed that dag was transcribed to mRNA in M. mazei LYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change of Methanosarcina spp. from aggregate to single cells has been identified.     

Dechlorination of Chloroform by Methanosarcina Strains

Dehalogenation of carbon tetrachloride, chloroform, and bromoform in pure cultures of Methanosarcina sp. strain DCM and Methanosarcina mazei S6 was demonstrated. The initial dechlorination product of chloroform was methylene chloride (dichloromethane), which accumulated transiently to about 70% of the added chloroform; trace amounts of chloromethane were also detected. The amount of chloroform dechlorinated per mole of methane produced was approximately 10 times greater than the ratio observed previously for tetrachloroethene dechlorination by these strains. The production of ¹⁴CO₂ from [¹⁴C]chloroform and the absence of ¹⁴CH₄ imply that processes in addition to reductive dechlorination operate.     

Production and Consumption of H2 during Growth of Methanosarcina spp. on Acetate

Methanosarcina sp. strain TM-1 and Methanosarcina acetivorans produced and consumed H₂ to maintain H₂ partial pressures of 16 to 92 Pa in closed cultures during growth on acetate. Strain TM-1 produced H₂ continuously when H₂ was continuously removed from the culture. The potential physiological significance of H₂ in acetate metabolism to methane is discussed.     

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 and characterization of mutants of mesophilic methanogenic bacteria resistant to analogues of DNA bases and nucleosides

Spontaneous mutants of mesophilic Methanobacterium, Methanobrevibacter and Methanosarcina species resistant to 6-mercaptopurine, 5-fluorouracil, 8-azaguanine, 6-azauracil or 5-fluorodeoxyuridine were isolated. Low level resistant mutants were unstable but highly resistant strains (resistance factor greater than 10-fold) were stable and showed growth characteristics comparable to the parent. Wild type strains showed linear uptake of hypoxanthine and uracil into cells, but guanine uptake was only detected in Methanosarcina mazei. 6-Mercaptopurine-resistant clones of Methanobacterium and Methanobrevibacter species and 8-azaguanine-resistant clones of Methanosarcina mazei showed reduced uptake of hypoxanthine and guanine respectively, but no evidence for altered permeability of 5-fluoro-and 6-azauracil-resistant strains to uracil was obtained. Double resistant mutants of Methanobacterium sp. strain FR-2 were characterised. Although these generally exhibited reduced specific growth rates, several were selected which showed similar growth to the parent.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany - MJC minimum inhibitory concentration - cfu colony forming unit - MP 6-mercaptopurine - FU 5-fluorouracil - FDU 5-fluorodeoxyuridine - AG 8-azaguanine - AU 6-azauracil - DA l-deazaadenosine     

Direct Characterization of Methanogens in Two High-Rate Anaerobic Biological Reactors

The methanogenic flora from two types of turbulent, high-rate reactors was studied by immunologic methods as well as by phase-contrast, fluorescence, and scanning electron microscopy. The reactors were a fluidized sand-bed biofilm ANITRON reactor and an ultrafiltration membrane-associated suspended growth MARS reactor (both trademarks of Air Products and Chemicals, Inc., Allentown, Pa.). Conventional microscopic methods revealed complex mixtures of microbes of a range of sizes and shapes, among which morphotypes resembling Methanothrix spp. and Methanosarcina spp. were noticed. Precise identification of these and other methanogens was accomplished by antigenic fingerprinting with a comprehensive panel of calibrated antibody probes of predefined specificity spectra. The methanogens identified showed morphotypes and antigenic fingerprints indicating their close similarity with the following reference organisms: Methanobacterium formicicum MF and Methanosarcina barkeri W in the ANITRON reactor only; Methanosarcina barkeri R1M3, M. mazei S6, Methanogenium cariaci JR1, and Methanobrevibacter arboriphilus AZ in the MARS reactor only; and Methanobrevibacter smithii ALI and Methanothrix soehngenii Opfikon in both reactors. Species diversity and distribution appeared to be, at least in part, dependent on the degree of turbulence inside the reactor.     

Methanogenesis from Ethanol by Defined Mixed Continuous Cultures

Methanogenesis from ethanol by defined mixed continuous cultures was studied. Under sulfate-free conditions, a Desulfovibrio strain was used as the ethanol-degrading species producing acetic acid and hydrogen. In a two-membered mutualistic coculture, the hydrogen was converted to methane by a Methanobacterium sp. and pH was maintained at neutrality by the addition of alkali. Introduction of a third species, the acetate-utilizing Methanosarcina mazei, obviated the need for external pH control. Methanogenesis by the co-and triculture was studied at various dilution rates in the steady state. The mutualistic coculture performed like a composite single species, as predicted from the theory of mutualistic interactions. Coupling between the mutualistic coculture and the acetate-utilizing methanogen was less tight. Increasing the dilution rate destabilized the triculture; at low dilution rates, instability was soon recovered, but at higher dilution rates imbalance between the rates of production and removal of acetic acid led to a drop in pH. Flocs formed in the triculture. An annulus of the Methanobacterium sp. and Desulfovibrio sp. was retained around the Methanosarcina sp. by strands of material probably derived from the Methanosarcina sp.     

Two new phospholipids,hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine,from the Archaea Methanosarcina barkeri

The structures of two new ether phospholipids of the methanogenic Archaea, Methanosarcina barkeri, were determined as hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine by means of chemical, chromatographic and enzymatic analyses, and fast atom bombardment-mass spectrometry. These lipids are hydroxy diether analogs of phosphatidylglycerol and phosphatidylethanolamine, respectively, with β-hydroxyarchaeol (2-O-(3′-hydroxy)phytanyl-3-O-phytanyl-sn-glycerol) as a core lipid. In addition, two other ether phospholipids, usual archaetidylglycerol and archaetidylethanolamine, were also identified in the organism. The stereochemical structure of the unalkylated glycerophosphate of hydroxyarchaetidylglycerol and archaetidylglycerol was determined as sn-glycerol-3-phosphate by use of sn-glycerol-3-phosphate dehydrogenase. The stereochemical configuration of the glycerophosphoglycerol backbone of these lipids was a mirror image of that of diacylphosphatidylglycerol from the organisms of the domains Bacteria and Eucarya, and it was shared with extremely halophilic Archaea. These four phospholipids, in addition to five lipids that had already been reported, accounted for 88% of the total polar lipids of this organism.     

Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as ‘mazei-T'' and ‘mazei-WC''. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.     

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.     

Effect of Substrate Concentration on Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosarcina barkeri and M. acetivorans and in Rice Field Soil

Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH₄ production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (ɛ) associated with consumption of total acetate (ɛ_ac), consumption of acetate-methyl (ɛ_ac-methyl) and production of CH₄ (ɛ_CH4) were an ɛ_ac of −30.5‰, an ɛ_ac-methyl of −25.6‰, and an ɛ_CH4 of −27.4‰ for M. barkeri and an ɛ_ac of −35.3‰, an ɛ_ac-methyl of −24.8‰, and an ɛ_CH4 of −23.8‰ for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an ɛ_ac of −18.7‰, an ɛ_ac-methyl of −16.9‰, and an ɛ_CH4 of −20.8‰. However, in rice field soil as well as in the pure cultures, values of ɛ_ac and ɛ_ac-methyl decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The ɛ values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.Methane (CH₄) is the most abundant reduced gas in the earth''s atmosphere and is an important greenhouse gas with a high global-warming potential (7). It is presently a matter of discussion whether the contribution of CH₄ to the greenhouse effect will increase in the future (3, 23). This has made it necessary and more urgent to understand natural processes that lead to the production of CH₄.Methanogenesis, the microbial formation of CH₄, is the final step in the degradation of organic matter in anoxic environments like natural wetlands, lake sediments, and flooded rice fields. The most important precursors for the production of CH₄ are acetate (equation 1) and CO₂ (equation 2) with the following reactions (8): (1) (2)Acetate is the most important substrate since it contributes more than 67% to microbial methanogenesis during anoxic degradation of polysaccharides. In methanogenic environments only two genera of archaea, Methanosaeta and Methanosarcina, are capable of using acetate (2). While Methanosaeta can be considered a specialist that uses only acetate, Methanosarcina can use a wide range of substrates besides acetate, for example, H₂/CO₂, methanol, methylamines, and methylated sulfides. Among methanogens, Methanosarcinaceae also display the largest environmental distribution. They can be found in freshwater sediments and soil, marine habitats, landfills, and animal gastrointestinal tracts (46).Additionally, differences between Methanosarcina and Methanosaeta were found for isotope fractionation of stable carbon. The fractionation factor (α) or, equivalently, the enrichment factor (ɛ) during acetoclastic methanogenesis in Methanosarcina barkeri strains typically ranges from an α of 1.021 to 1.027 or an ɛ of −27‰ to −21‰ (14, 27, 48), whereas isotope fractionation in Methanosaeta spp. is weaker, i.e., an α of 1.007 (ɛ = −7‰) for Methanosaeta thermophila (43) and an α of 1.010 (ɛ = −10‰) for Methanosaeta concilii (34). It is suggested that the two archaeal genera differ in isotope fractionation due to differences in their biochemical activation of acetate to acetyl-coenzyme A (acetyl-CoA) (34). However, isotopic data for acetoclastic methanogens are rare. For instance, all data for Methanosarcina refer to only one species, namely M. barkeri.Hence, in this study we investigated whether differences in carbon isotope fractionation within the genus Methanosarcina occur. Therefore, we determined isotope ratios of stable carbon in cultures of the acetoclastic species M. barkeri and Methanosarcina acetivorans. Second, we were interested if these data, obtained from pure cultures, could also be applied to understand natural environments. For that reason, we determined isotope fractionation during acetoclastic methanogenesis in the model system rice field soil. Furthermore, we discuss the effect of substrate concentration on carbon isotope fractionation and the importance of monitoring isotope fractionation during the course of acetate consumption.     

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     

Kinetics of Acetate Utilization by Two Thermophilic Acetotrophic Methanogens: Methanosarcina sp. Strain CALS-1 and Methanothrix sp. Strain CALS-1

The kinetics of acetate utilization were examined for washed concentrated cell suspensions of two thermophilic acetotrophic methanogens isolated from a 58°C anaerobic digestor. Progress curves for acetate utilization by cells of Methanosarcina sp. strain CALS-1 showed that the utilization rate was concentration independent (zero order) above concentrations near 3 mM and that acetate utilization ceased when a threshold concentration near 1 mM was reached. Acetate utilization by cells of Methanothrix sp. strain CALS-1 was concentration independent down to 0.1 to 0.2 mM, and threshold values of 12 to 21 μM were observed. Typical utilization rates in the concentration-independent stage were 210 and 130 nmol min⁻¹ mg of protein⁻¹ for the methanosarcina and the methanothrix, respectively. These results are in agreement with a general model in which high acetate concentrations favor Methanosarcina spp., while low concentrations favor Methanothrix spp. However, acetate utilization by these two strains did not follow simple Michaelis-Menton kinetics.     

Trimethylamine and methylamine as growth substrates for rumen bacteria andMethanosarcina barkeri

Trimethylamine and methylamine were found to be used as methanogenic substrates byMethanosarcina barkeri or by bacteria found in low dilutions of rumen contents. When these substrates were used as the only added carbon and nitrogen source, up to 80% of the theoretical amount of methane production was obtained.Methanosarcina were enumerated from rumen contents at 10⁵–10⁶ bacteria/ml. Pure cultures of the various major rumen bacterial species, includingMethanobacterium ruminantium strain M1, were not able to utilize these substrates as energy and/or nitrogen sources. It is suggested that, in the rumen, trimethylamine and methylamine are primarily degraded byMethanosarcina, resulting in release of ammonia which then can be utilized by other rumen bacteria.     

Geranylgeranyl Reductase and Ferredoxin from Methanosarcina acetivorans Are Required for the Synthesis of Fully Reduced Archaeal Membrane Lipid in Escherichia coli Cells

Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.     

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.     

Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10⁷ per g of dry soil for the H₂-CO₂-utilizing methanogens and of up to 10⁶ for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H₂-CO₂, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.