Methanosaeta Thermophila Research Articles

Using archaeal genomics to fight global warming and clostridia to fight cancer

The end of 2006 brought an unusually high number of completely sequenced microbial genomes. The list of new genomes (Table 1) includes three archaea and a large number of environmental, as well as parasitic, bacteria. Two of the newly sequenced genomes represent methanogenic archaea of the Methanosarcinales family that are commonly found in the rice fields. These organisms are key producers of methane whose emission from the rice fields comprises anywhere from 10% to 25% of the total global CH4 emissions into the atmosphere and is a significant source of greenhouse gases (Neue, 1993; West et al., 2006). Reducing methane emission without decreasing the grain yield appears to be feasible and could make an important contribution to the fight against global warming (Denier van der Gon et al., 2002; Sass and Cicerone, 2002). Unfortunately, methane emission appears to be linked to oxygen transport into the root and might be part of the normal functioning of the rice rhizosphere (Nouchi et al., 1990; Inubushi et al., 2001). This makes understanding the physiology of methanogenic archaea inhabiting the rice paddies and their role in rice rhizosphere a particularly urgent task. Methanosaeta thermophila strain PT (previously referred to as Methanosaeta thermoacetophila or Methanothrix thermophila) is an obligately anaerobic moderately thermophilic (optimal temperature 55‐60°C) archaeon that grows exclusively on acetate, metabolizing it into methane. The exclusive reliance of M. thermophila on acetate as the substrate may account for its relatively slow growth and low growth yield. Still, its ability to effectively scavenge low concentrations of acetate allows M. thermophila to compete with faster-growing methanogenic archaea, such as Methanosarcina spp. As a result, at low acetate levels, M. thermophila and other members of the Methanosaeta genus become predominant

Effect of nitrite on a thermophilic, methanogenic consortium from an oil storage tank

Samples from an oil storage tank (resident temperature 40 to 60 degrees C), which experienced unwanted periodic odorous gas emissions, contained up to 2,400/ml of thermophilic, lactate-utilizing, sulfate-reducing bacteria. Significant methane production was also evident. Enrichments on acetate gave sheathed filaments characteristic of the acetotrophic methanogen Methanosaeta thermophila of which the presence was confirmed by determining the PCR-amplified 16S rDNA sequence. 16S rDNA analysis of enrichments, grown on lactate- and sulfate-containing media, indicated the presence of bacteria related to Garciella nitratireducens, Clostridium sp. and Acinetobacter sp. These sulfidogenic enrichments typically produced sulfide to a maximum concentration of 5-7 mM in media containing excess lactate and 10 mM sulfate or thiosulfate. Both the production of sulfide and the consumption of acetate by the enrichment cultures were inhibited by low concentrations of nitrite (0.5-1.0 mM). Hence, addition of nitrite may be an effective way to prevent odorous gas emissions from the storage tank.

Anaerobic microbiology of an alkaline Icelandic hot spring

Samples of sediments and biofilm, defined as biomats or microbial mats, collected from a hot spring in Hveragerdi, Iceland at approximately 55°C and 75°C, were examined for anaerobic microbial activity and bacterial numbers. Generally more bacteria were present in the biomat at 75°C than at 55°C; 9.98×105 and 9.48×103 xylan degrading bacteria/gram volatile solids and 6.89×107 and 0 cellulose degrading bacteria/gram volatile solids were found at 75°C and 55°C, respectively. No proteolytic or lipolytic bacteria were present in the biomat at 75°C, but 1.38×103 proteolytic and 4.48×102 lipolytic cells/gram volatile solids were found in the biomat at 55°C. Acetate, propionate and butyrate were not degraded by the biomat either at 55°C or 75°C. No methanogenic activity was found at 75°C. However, most-probable number enumerations and indirect immunofluorescence using antibody probes against selected methanogenic archaea showed that methanogens were present at this temperature. H2 was the major electron donor for the methanogenic archaea at 55°C. No methane production was observed with acetate as substrate during short time (i.e. 24 h) incubation at 55°C. However, a significant amount of methane appeared after long periods of incubation at this temperature. Indirect immunofluorescence using antibody probes revealed a diverse methanogenic population in the spring. Cells immunologically related to Methanobacterium thermoautotrophicumΔH and Methanobacterium thermoformicicum CB 12 were the dominating methanogenic archaea at both temperatures. In addition, cells immunologically related to Methanoculleus thermophilus strain UCLA were present in high numbers at 75°C. Furthermore, cells immunologically related to Methanosaeta thermophila CALS-1 were found in low numbers in the biomat at 55°C.

Methanosaeta harundinacea sp. nov., a novel acetate-scavenging methanogen isolated from a UASB reactor

Two methanogenic strains, 8AcT and 6Ac, were isolated from an upflow anaerobic sludge blanket reactor treating beer-manufacture wastewater in Beijing, China. Cells of strains 8AcT and 6Ac were rod-shaped (0.8-1.0 x 3-5 microm) and non-motile, occurring singly or in pairs; however, at high cell density the cells were arranged in long chains within a common sheath. The two strains used acetate exclusively for growth and methane production. The specific growth rate of strain 8AcT was 0.030 h(-1) when growing in acetate (20 mM) at 37 degrees C. The temperature range for growth was 25-45 degrees C, with the fastest growth at 34-37 degrees C. The pH range for growth and methane production was 6.5-9.0, with the fastest growth at pH 7.2-7.6. The G+C content of genomic DNA of strain 8AcT was 55.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that the novel strains clustered with Methanosaeta species; the 16S rRNA gene sequence similarities between strain 8AcT and Methanosaeta concilii DSM 3013 and 'Methanosaeta thermophila' DSM 6194 were 92.5 and 87.3 %, respectively. The sequence similarity levels of mcrA, the gene encoding the alpha-subunit of methyl-coenzyme M reductase, and of the deduced amino acids of mcrA, between strain 8AcT and Methanosaeta concilii DSM 3671T were 36 and 78.9 %, respectively. Based on the phylogenetic and phenotypic analyses, the novel species Methanosaeta harundinacea sp. nov. is proposed, with strain 8AcT (= JCM 13211T = CGMCC 1.5026T) as the type strain.

PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules

Methane is produced by various methanogenic bacteria present in upflow anaerobic sludge blanket (UASB) bioreactors. Methane can be used to predict and improve UASB bioreactor efficiency. The methanogen population in the granules can be influenced by the composition of the substrate. The aim of this study was to fingerprint and identify the methanogens present in three different types of UASB granules that had been used to treat winery, brewery and peach-lye canning effluents. This was done using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. The DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions of the DGGE bands that did not correspond well to the bands of the known species were sequenced and compared to sequences available on GenBank using the Blastn search option. The aligned DNA sequences were used to construct a phylogenetic tree. Based on the data obtained, a DGGE marker was constructed which was used to provide a quick method to identify the Archaeal members of the microbial consortium in UASB granules.

Carbon and hydrogen isotope fractionation by moderately thermophilic methanogens

A series of laboratory studies were conducted to increase understanding of stable carbon (13C/12C) and hydrogen (D/H) isotope fractionation arising from methanogenesis by moderately thermophilic acetate- and hydrogen-consuming methanogens. Studies of the aceticlastic reaction were conducted with two closely related strains of Methanosaeta thermophila. Results demonstrate a carbon isotope fractionation of only 7‰ (α = 1.007) between the methyl position of acetate and the resulting methane. Methane formed by this process is enriched in 13C when compared with other natural sources of methane; the magnitude of this isotope effect raises the possibility that methane produced at elevated temperature by the aceticlastic reaction could be mistaken for thermogenic methane based on carbon isotopic content. Studies of H2/CO2 methanogenesis were conducted with Methanothermobacter marburgensis. The fractionation of carbon isotopes between CO2 and CH4 was found to range from 22 to 58‰ (1.023 ≤ α ≤ 1.064). Greater fractionation was associated with low levels of molecular hydrogen and steady-state metabolism. The fractionation of hydrogen isotopes between source H2O and CH4 was found to range from 127 to 275‰ (1.16 ≤ α ≤ 1.43). Fractionation was dependent on growth phase with greater fractionation associated with later growth stages. The maximum observed fractionation factor was 1.43, independent of the δD-H2 supplied to the culture. Fractionation was positively correlated with temperature and/or metabolic rate. Results demonstrate significant variability in both hydrogen and carbon isotope fractionation during methanogenesis from H2/CO2. The relatively small fractionation associated with deuterium during H2/CO2 methanogenesis provides an explanation for the relatively enriched deuterium content of biogenic natural gas originating from a variety of thermal environments. Results from these experiments are used to develop a hypothesis that differential reversibility in the enzymatic steps of the H2/CO2 pathway gives rise to variability in the observed carbon isotope fractionation. Results are further used to constrain the overall efficiency of electron consumption by way of the hydrogenase system in M. marburgensis, which is calculated to be less than 55%.

Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor.

A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities. Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C. The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors. The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique. In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide. Cell numbers of glutamate-degrading organisms increased 400-fold over the first year. In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation. After 2 years of reactor operation the predominant organisms were isolated and characterized. Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively. The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively. The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii. It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide. Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens.

Hydrogen production by methanogens under low-hydrogen conditions.

Hydrogen production was studied in four species of methanogens (Methanothermobacter marburgensis, Methanosaeta thermophila, Methanosarcina barkeri, and Methanosaeta concilii) under conditions of low (sub-nanomolar) ambient hydrogen concentration using a specially designed culture apparatus. Transient hydrogen production was observed and quantified for each species studied. Methane was excluded as the electron source, as was all organic material added during growth of the cultures (acetate, yeast extract, peptone). Hydrogen production showed a strong temperature dependence, and production ceased at temperatures below the growth range of the organisms. Addition of polysulfides to the cultures greatly decreased hydrogen production. The addition of bromoethanesulfonic acid had little influence on hydrogen production. These experiments demonstrate that some methanogens produce excess reducing equivalents during growth and convert them to hydrogen when the ambient hydrogen concentration becomes low. The lack of sustained hydrogen production by the cultures in the presence of methane provides evidence against "reverse methanogenesis" as the mechanism for anaerobic methane oxidation.

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.

Rejection of the species Methanothrix soehngeniiVPand the genus MethanothrixVPas nomina confusa, and transfer of Methanothrix thermophilaVPto the genus MethanosaetaVPas Methanosaeta thermophila comb. nov. Request for an Opinion

We request an Opinion of the Judicial Commission regarding rejection of the species Methanothrix soehngenii VPHuser, Wuhrmann and Zehnder 1983, 439, and the genus Methanothrix VPHuser, Wuhrmann and Zehnder 1983, 439, as nomina confusa because their descriptions were based on the characterization of an impure type strain, strain OpfikonT. We also propose the transfer of Methanothrix thermophila VP Kamagata et al. 1992, 465, to the genus Methanosaeta as Methanosaeta thermophila comb. nov.

Anaerobic microbiology of an alkaline Icelandic hot spring

Samples of sediments and biofilm, defined as biomats or microbial mats, collected from a hot spring in Hveragerdi, Iceland at approximately 55°C and 75°C, were examined for anaerobic microbial activity and bacterial numbers. Generally more bacteria were present in the biomat at 75°C than at 55°C; 9.98×10 5 and 9.48×10 3 xylan degrading bacteria/gram volatile solids and 6.89×10 7 and 0 cellulose degrading bacteria/gram volatile solids were found at 75°C and 55°C, respectively. No proteolytic or lipolytic bacteria were present in the biomat at 75°C, but 1.38×10 3 proteolytic and 4.48×10 2 lipolytic cells/gram volatile solids were found in the biomat at 55°C. Acetate, propionate and butyrate were not degraded by the biomat either at 55°C or 75°C. No methanogenic activity was found at 75°C. However, most-probable number enumerations and indirect immunofluorescence using antibody probes against selected methanogenic archaea showed that methanogens were present at this temperature. H 2 was the major electron donor for the methanogenic archaea at 55°C. No methane production was observed with acetate as substrate during short time (i.e. 24 h) incubation at 55°C. However, a significant amount of methane appeared after long periods of incubation at this temperature. Indirect immunofluorescence using antibody probes revealed a diverse methanogenic population in the spring. Cells immunologically related to Methanobacterium thermoautotrophicum ΔH and Methanobacterium thermoformicicum CB 12 were the dominating methanogenic archaea at both temperatures. In addition, cells immunologically related to Methanoculleus thermophilus strain UCLA were present in high numbers at 75°C. Furthermore, cells immunologically related to Methanosaeta thermophila CALS-1 were found in low numbers in the biomat at 55°C.

Methanogenesis from acetate by cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix thermophila CALS-1

High rates of methanogenesis from acetate and ATP were observed from cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix (Methanosaeta) thermophila strain CALS-1 when cultures were grown in a pH auxostat fed with acetic acid. Specific methanogenic activities ranged from 50-300 nmol min-1 (mg protein)-1, which was comparable to those for whole cells. In contrast to results with Methanosarcina spp., the reaction did not require high levels of H2 in the headspace. CO was inhibitory to methanogenesis from acetate. The inhibition by CO and the lack of effect of H2 on methanogenesis from acetate resemble previous results with whole cells of CALS-1. Protein concentrations in extracts > 5 mg/ml were required for good activity, and the optimum temperature for the methanogenesis was near 65° C. ATP was required in substrate quantities and was converted mainly to AMP. The maximum CH4/ATP stoichiometry obtained was near 1.0, consistent with acetate activation using an acetyl-CoA synthetase mechanism that converts ATP to AMP and pyrophosphate. Methanogenesis in extracts was inhibited by bromoethane sulfonate and cyanide, indicating the involvement of methylcoenzyme M methylreductase and a carbon monoxide dehydrogenase complex with methanogenesis from acetate. These results are consistent with acetyl-coenzyme A (CoA) as the form of activated acetate involved in methanogenesis from acetate in strain CALS-1, but no activity could be obtained from extracts using acetyl-CoA as a substrate.