A Portable Anaerobic Microbioreactor Reveals Optimum Growth Conditions for the Methanogen Methanosaeta concilii

Conventional studies of the optimum growth conditions for methanogens (methane-producing, obligate anaerobic archaea) are typically conducted with serum bottles or bioreactors. The use of microfluidics to culture methanogens allows direct microscopic observations of the time-integrated response of growth. Here, we developed a microbioreactor (μBR) with ~1-μl microchannels to study some optimum growth conditions for the methanogen Methanosaeta concilii. The μBR is contained in an anaerobic chamber specifically designed to place it directly onto an inverted light microscope stage while maintaining a N₂-CO₂ environment. The methanogen was cultured for months inside microchannels of different widths. Channel width was manipulated to create various fluid velocities, allowing the direct study of the behavior and responses of M. concilii to various shear stresses and revealing an optimum shear level of ~20 to 35 μPa. Gradients in a single microchannel were then used to find an optimum pH level of 7.6 and an optimum total NH₄-N concentration of less than 1,100 mg/liter (<47 mg/liter as free NH₃-N) for M. concilii under conditions of the previously determined ideal shear stress and pH and at a temperature of 35°C.     

Immobilization patterns and dynamics of acetate-utilizing methanogens immobilized in sterile granular sludge in upflow anaerobic sludge blanket reactors

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and mumax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor.     

Acetate and CO2 assimilation by Methanothrix concilii.

Biosynthetic pathways in Methanothrix concilii, a recently isolated aceticlastic methanogen, were studied by 13C-nuclear magnetic resonance spectroscopy. Labeling patterns of amino acids, lipids, and carbohydrates were determined. Similar to other methanogens, acetate was carboxylated to pyruvate, which was further converted to amino acids by various biosynthetic pathways. The origin of carbon atoms in glutamate, proline, and arginine clearly showed that an incomplete tricarboxylic acid cycle operating in the oxidative direction was used for their biosynthesis. Isoleucine was synthesized via citramalate, which is a typical route for methanogens. As with Methanosarcina barkeri, an extensive exchange of the label between the carboxyl group of acetate and CO2 was observed. Lipids predominantly contained diphytanyl chains, the labeling of which indicated that biosynthesis proceeded through mevalonic acid. Labeling of the C-1,6 of glucose from [2-13C]acetate is consistent with a glucogenic route for carbohydrate biosynthesis. Except for the different origins of the methyl group of methionine, the metabolic properties of Methanothrix concilii are closely related to those of Methanosarcina barkeri.     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

Reactor-scale cultivation of the hyperthermophilic methanarchaeon Methanococcus jannaschii to high cell densities.

For the hyperthermophilic and barophilic methanarchaeon Methanococcus jannaschii, we have developed a medium and protocols for reactor-scale cultivation that improved the final cell yield per liter from approximately 0.5 to approximately 7.5 g of packed wet cells ( approximately 1.8 g dry cell mass) under autotrophic growth conditions and to approximately 8.5 g of packed wet cells ( approximately 2 g dry cell mass) with yeast extract (2 g liter(-1)) and tryptone (2 g liter(-1)) as medium supplements. For growth in a sealed bottle it was necessary to add Se to the medium, and a level of 2 microM for added Se gave the highest final cell yield. In a reactor M. jannaschii grew without added Se in the medium; it is plausible that the cells received Se as a contaminant from the reactor vessel and the H(2)S supply. But, for the optimal performance of a reactor culture, an addition of Se to a final concentration of 50 to 100 microM was needed. Also, cell growth in a reactor culture was inhibited at much higher Se concentrations. These observations and the data from previous work with methanogen cell extracts (B. C. McBride and R. S. Wolfe, Biochemistry 10:4312-4317, 1971) suggested that from a continuously sparged reactor culture Se was lost in the exhaust gas as volatile selenides, and this loss raised the apparent required level of and tolerance for Se. In spite of having a proteinaceous cell wall, M. jannaschii withstood an impeller tip speed of 235.5 cms(-1), which was optimal for achieving high cell density and also was the higher limit for the tolerated shear rate. The organism secreted one or more acidic compounds, which lowered pH in cultures without pH control; this secretion continued even after cessation of growth.     

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Inhibition of pure cultures of methanogens by benzene ring compounds.

The inhibition of methane production by Methanosaeta concilii GP6, Methanospirillum hungatei GP1, Methanobacterium espanolae GP9, and Methanobacterium bryantii M.o.H. during short-term (6-h) exposure to eight benzene ring compounds was studied. The concentration that caused 50% inhibition of the methane production rate (IC50) was dependent on the species and the toxicant. Pentachlorophenol was the most toxic of the tested compounds, with an IC50 of less than 8 mg/liter for all species except M. hungatei. Abietic acid was the next most toxic compound for all the species, with an IC50 in the range of 21.4 to 203 mg/liter. Sodium benzoate was generally the least toxic, with an IC50 in the range of 1,225 to 32,400 mg/liter. 3-Chlorobenzoate was substantially more toxic (IC50, 450 to 1,460 mg/liter) than benzoate. The inhibition by benzene, phenol, vanillic acid, and toluene was intermediate to that of pentachlorophenol and benzoate. Long-term incubation (days) studies to determine effect on growth indicated that all eight compounds were usually much more toxic than predicted from the short-term data. In these latter studies, there was generally a good correlation in the observed inhibition as determined from growth and methane production.     

Inhibition of pure cultures of methanogens by benzene ring compounds.

The inhibition of methane production by Methanosaeta concilii GP6, Methanospirillum hungatei GP1, Methanobacterium espanolae GP9, and Methanobacterium bryantii M.o.H. during short-term (6-h) exposure to eight benzene ring compounds was studied. The concentration that caused 50% inhibition of the methane production rate (IC50) was dependent on the species and the toxicant. Pentachlorophenol was the most toxic of the tested compounds, with an IC50 of less than 8 mg/liter for all species except M. hungatei. Abietic acid was the next most toxic compound for all the species, with an IC50 in the range of 21.4 to 203 mg/liter. Sodium benzoate was generally the least toxic, with an IC50 in the range of 1,225 to 32,400 mg/liter. 3-Chlorobenzoate was substantially more toxic (IC50, 450 to 1,460 mg/liter) than benzoate. The inhibition by benzene, phenol, vanillic acid, and toluene was intermediate to that of pentachlorophenol and benzoate. Long-term incubation (days) studies to determine effect on growth indicated that all eight compounds were usually much more toxic than predicted from the short-term data. In these latter studies, there was generally a good correlation in the observed inhibition as determined from growth and methane production.     

Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

Methanogenesis in the sediments of Rio Tinto, an extreme acidic river

Río Tinto (Iberian Pyritic Belt, SW Spain) is well known for its low pH (mean pH 2.3), high redox potential (> +400 mV) and high concentration of heavy metals. In this work we describe and analyse the presence of methanogenic archaea in the extreme acidic and oxidizing environment of the Tinto basin. Methane formation was measured in microcosms inoculated with sediments from the Rio Tinto basin. Methanol, formate, volatile fatty acids and lactate stimulated the production of methane. Methane formation was associated with a decrease of redox potential and an increase in pH. Cores showed characteristic well-defined black bands in which a high acetate concentration was measured among the otherwise reddish-brown sediments with low acetate concentration. Methanosaeta concilii was detected in the black bands. In enrichment cultures, M. concilii (enriched with a complex substrate mixture), Methanobacterium bryantii (enriched with H(2)) and Methanosarcina barkeri (enriched with methanol) were identified. Our results suggest that methanogens thrive in micro-niches with mildly acidic and reducing conditions within Rio Tinto sediments, which are, in contrast, immersed in an otherwise extremely acidic and oxidizing environment.     

重要环境因子对小球藻去除污水中氮磷的影响

对小球藻去除污水中氮磷的性能进行了研究,考察了初始氮磷浓度、氮磷比、光照条件和pH等因素对其去除效率的影响。结果表明,在初始氮磷浓度分别在35 mg/L和7 mg/L以下时去除率接近100%;氮磷比为5∶1和10∶1,小球藻第4 d基本完全去除了污水中的氨态氮,而氮磷比对小球藻的除磷能力没有显著影响;在初始氨态氮或总磷浓度相同的条件下,光照条件(L/D为24 h∶0 h和12 h∶12 h)对氮磷去除效果的无明显差异性（P＞0.05）,而随着氮磷浓度的增加,连续光照条件逐渐展现出去除氮磷的优势;小球藻在pH 7～8范围内对氨态氮的去除率最佳,pH 5～7范围内,对总磷的去除率最佳。     

Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

The effect of methanogen growth on mineral substrates: will Ni markers of methanogen‐based communities be detectable in the rock record?

Methanogens, thought to be present on early Earth, have a high requirement for Ni, suggesting that Ni utilization could be a potential biosignature for methanogens if enhanced Ni extraction from surrounding minerals accompanies methanogenic growth. To test the potential for such Ni extraction from minerals by methanogens, Ni release from Ni‐containing silicate glass was measured in Ni‐free growth medium in the presence of the methanogen Methanothermobacter thermoautotrophicus (average pH ∼7.0) and observed to be higher than an abiotic control (average pH ∼6.8). However, batch dissolution experiments and a siderophore assay indicate that cell exudates such as siderophores, low molecular weight organic acids, or lysates accompanying cell death are not responsible for the observed increase in Ni release rate. In addition, scanning electron microscopy (SEM) shows little to no evidence of direct microbe–mineral interactions such as biofilms or pitting. Instead, comparison with abiotic experiments suggests that changes in pH due to CO₂ uptake may be responsible for enhanced dissolution in the presence of metabolizing cells. These results document that methanogens may not preferentially extract Ni from surrounding minerals although they may indirectly affect mineral reaction rates that are pH sensitive. Thus identifiable Ni biosignatures may not exist in the rock record to document the presence of methanogens on early Earth or Mars.     

Genome sequence of a mesophilic hydrogenotrophic methanogen Methanocella paludicola, the first cultivated representative of the order Methanocellales

We report complete genome sequence of a mesophilic hydrogenotrophic methanogen Methanocella paludicola, the first cultured representative of the order Methanocellales once recognized as an uncultured key archaeal group for methane emission in rice fields. The genome sequence of M. paludicola consists of a single circular chromosome of 2,957,635 bp containing 3004 protein-coding sequences (CDS). Genes for most of the functions known in the methanogenic archaea were identified, e.g. a full complement of hydrogenases and methanogenesis enzymes. The mixotrophic growth of M. paludicola was clarified by the genomic characterization and re-examined by the subsequent growth experiments. Comparative genome analysis with the previously reported genome sequence of RC-I(MRE50), which was metagenomically reconstructed, demonstrated that about 70% of M. paludicola CDSs were genetically related with RC-I(MRE50) CDSs. These CDSs included the genes involved in hydrogenotrophic methane production, incomplete TCA cycle, assimilatory sulfate reduction and so on. However, the genetic components for the carbon and nitrogen fixation and antioxidant system were different between the two Methanocellales genomes. The difference is likely associated with the physiological variability between M. paludicola and RC-I(MRE50), further suggesting the genomic and physiological diversity of the Methanocellales methanogens. Comparative genome analysis among the previously determined methanogen genomes points to the genome-wide relatedness of the Methanocellales methanogens to the orders Methanosarcinales and Methanomicrobiales methanogens in terms of the genetic repertoire. Meanwhile, the unique evolutionary history of the Methanocellales methanogens is also traced in an aspect by the comparative genome analysis among the methanogens.     

Leachate and gaseous emissions from initial phases of landfilling mechanically and mechanically-biologically treated municipal solid waste residuals

In this study, the behaviour, and leachate and gaseous emissions during the initial phases of landfilling mechanically (M) and mechanically-biologically (MB) treated municipal solid waste residuals in northern climatic conditions was compared using two landfill lysimeters (112 m3). The results demonstrate that the strong acid phase of M residuals degradation lasts at least 2 years, while in the MB residuals the acid phase lasts only a few months. The SCOD and NH4-N concentrations varied 20-100g/l and 600-1800 mg/l in M leachate and 1-4 g/l and 100-400mg/l in MB leachate, respectively. The leaching of SCOD was approximately 40-fold (24.2 and 0.6 kg/t TS) and leaching of NH4-N approximately 5-fold (356 and 60 g/t TS) from the M than MB residuals; thus the effect of biological stabilisation was more marked on the leaching of SCOD than of NH4-N. Moreover gas (methane, carbon dioxide and nitrous oxide) emissions were several-fold higher from the M than MB residuals.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

The genome characteristics and predicted function of methyl-group oxidation pathway in the obligate aceticlastic methanogens, Methanosaeta spp

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, (13)C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F(420)H(2) was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis.     

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge

The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobacterium formicicum at pH 6.5 was studied. The latter three strains were resistant to greater than 60 g/L of the volatile fatty acids and to greater than 10 g/L of NH3 N. Methanospirillum hungatei was somewhat more sensitive with 50% inhibition of methanogenesis occurring at 4.2 g/L NH3 N, 27 g/L butyrate, and 41 g/L propionate. All strains were very sensitive to both copper (1-5 mg/L) and zinc (1-10 mg/L), but much more resistant to nickel. Zinc and copper concentrations 30 to 270 times higher were required to cause inhibition of Msp. hungatei incubated in sewage sludge compared with buffer, indicating a strong protective environment was afforded the methanogens against heavy metal toxicity in the sludge.     

Isolation and characterization of Methanomethylovorans hollandica gen. nov., sp. nov., isolated from freshwater sediment, a methylotrophic methanogen able to grow on dimethyl sulfide and methanethiol.

A newly isolated methanogen, strain DMS1(T), is the first obligately anaerobic archaeon which was directly enriched and isolated from a freshwater sediment in defined minimal medium containing dimethyl sulfide (DMS) as the sole carbon and energy source. The use of a chemostat with a continuous DMS-containing gas stream as a method of enrichment, followed by cultivation in deep agar tubes, resulted in a pure culture. Since the only substrates utilized by strain DMS1(T) are methanol, methylamines, methanethiol (MT), and DMS, this organism is considered an obligately methylotrophic methanogen like most other DMS-degrading methanogens. Strain DMS1(T) differs from all other DMS-degrading methanogens, since it was isolated from a freshwater pond and requires NaCl concentrations (0 to 0.04 M) typical of the NaCl concentrations required by freshwater microorganisms for growth. DMS was degraded effectively only in a chemostat culture in the presence of low hydrogen sulfide and MT concentrations. Addition of MT or sulfide to the chemostat significantly decreased degradation of DMS. Transient accumulation of DMS in MT-amended cultures indicated that transfer of the first methyl group during DMS degradation is a reversible process. On the basis of its low level of homology with the most closely related methanogen, Methanococcoides burtonii (94.5%), its position on the phylogenetic tree, its morphology (which is different from that of members of the genera Methanolobus, Methanococcoides, and Methanohalophilus), and its salt tolerance and optimum (which are characteristic of freshwater bacteria), we propose that strain DMS1(T) is a representative of a novel genus. This isolate was named Methanomethylovorans hollandica. Analysis of DMS-amended sediment slurries with a fluorescence microscope revealed the presence of methanogens which were morphologically identical to M. hollandica, as described in this study. Considering its physiological properties, M. hollandica DMS1(T) is probably responsible for degradation of MT and DMS in freshwater sediments in situ. Due to the reversibility of the DMS conversion, methanogens like strain DMS1(T) can also be involved in the formation of DMS through methylation of MT. This phenomenon, which previously has been shown to occur in sediment slurries of freshwater origin, might affect the steady-state concentrations and, consequently, the total flux of DMS and MT in these systems.