Shifts in methanogenic archaea communities and methane dynamics along a subtropical estuarine land use gradient.

In coastal aquatic ecosystems, prokaryotic communities play an important role in regulating the cycling of nutrients and greenhouse gases. In the coastal zone, estuaries are complex and delicately balanced systems containing a multitude of specific ecological niches for resident microbes. Anthropogenic influences (i.e. urban, industrial and agricultural land uses) along the estuarine continuum can invoke physical and biochemical changes that impact these niches. In this study, we investigate the relative abundance of methanogenic archaea and other prokaryotic communities, distributed along a land use gradient in the subtropical Burnett River Estuary, situated within the Great Barrier Reef catchment, Australia. Microbiological assemblages were compared to physicochemical, nutrient and greenhouse gas distributions in both pore and surface water. Pore water samples from within the most urbanised site showed a high relative abundance of methanogenic Euryarchaeota (7.8% of all detected prokaryotes), which coincided with elevated methane concentrations in the water column, ranging from 0.51 to 0.68 µM at the urban and sewage treatment plant (STP) sites, respectively. These sites also featured elevated dissolved organic carbon (DOC) concentrations (0.66 to 1.16 mM), potentially fuelling methanogenesis. At the upstream freshwater site, both methane and DOC concentrations were considerably higher (2.68 µM and 1.8 mM respectively) than at the estuarine sites (0.02 to 0.66 µM and 0.39 to 1.16 mM respectively) and corresponded to the highest relative abundance of methanotrophic bacteria. The proportion of sulfate reducing bacteria in the prokaryotic community was elevated within the urban and STP sites (relative abundances of 8.0%- 10.5%), consistent with electron acceptors with higher redox potentials (e.g. O2, NO3-) being scarce. Overall, this study showed that ecological niches in anthropogenically altered environments appear to give an advantage to specialized prokaryotes invoking a potential change in the thermodynamic landscape of the ecosystem and in turn facilitating the generation of methane-a potent greenhouse gas.

Temporal and spatial impact of Spartina alterniflora invasion on methanogens community in Chongming Island, China.

Methane production by methanogens in wetland is recognized as a significant contributor to global warming. Spartina alterniflora (S. alterniflora), which is an invasion plant in China's wetland, was reported to have enormous effects on methane production. But studies on shifts in the methanogen community in response to S. alterniflora invasion at temporal and spatial scales in the initial invasion years are rare. Sediments derived from the invasive species S. alterniflora and the native species Phragmites australis (P. australis) in pairwise sites and an invasion chronosequence patch (4 years) were analyzed to investigate the abundance and community structure of methanogens using quantitative real-time PCR (qPCR) and Denaturing gradient gel electrophoresis (DGGE) cloning of the methyl-coenzyme M reductase A (mcrA) gene. For the pairwise sites, the abundance of methanogens in S. alterniflora soils was lower than that of P. australis soils. For the chronosequence patch, the abundance and diversity of methanogens was highest in the soil subjected to two years invasion, in which we detected some rare groups including Methanocellales and Methanococcales. These results indicated a priming effect at the initial invasion stages of S. alterniflora for microorganisms in the soil, which was also supported by the diverse root exudates. The shifts of methanogen communities after S. alterniflora invasion were due to changes in pH, salinity and sulfate. The results indicate that root exudates from S. alterniflora have a priming effect on methanogens in the initial years after invasion, and the predominate methylotrophic groups (Methanosarcinales) may adapt to the availability of diverse substrates and reflects the potential for high methane production after invasion by S. alterniflora.

Methanocaldococcus villosus sp. nov., a heavily flagellated archaeon that adheres to surfaces and forms cell-cell contacts.

A novel chemolithoautotrophic, hyperthermophilic methanogen was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, north of Iceland. Based on its 16S rRNA gene sequence, the strain belongs to the order Methanococcales within the genus Methanocaldococcus, with approximately 95 % sequence similarity to Methanocaldococcus jannaschii as its closest relative. Cells of the novel organism stained Gram-negative and appeared as regular to irregular cocci possessing more than 50 polar flagella. These cell appendages mediated not only motility but also adherence to abiotic surfaces and the formation of cell-cell contacts. The new isolate grew at 55-90 °C, with optimum growth at 80 °C. The optimum NaCl concentration for growth was 2.5 % (w/v), and the optimal pH was 6.5. The cells gained their energy exclusively by reduction of CO(2) with H(2). Selenate, tungstate and yeast extract stimulated growth significantly. The genome size was determined to be in the range 1.8-2.0 kb, and the G+C content of the genomic DNA was 30 mol%. Despite being physiologically nearly identical to the other members of the genus Methanocaldococcus, analysis of whole-cell proteins revealed significant differences. Based on the results from phylogenetic, morphological and protein analyses, we conclude that the novel strain represents a novel species of the genus Methanocaldococcus, for which the name Methanocaldococcus villosus sp. nov. is proposed (type strain KIN24-T80(T)  = DSM 22612(T)  = JCM 16315(T)).

Identification of Archaeal population in the granular sludge of an UASB reactor treating sewage at low temperatures.

Effect of low temperature on up-flow anaerobic sludge bed (UASB) reactor performance treating raw sewage was investigated in terms of the variations in methanogenic diversity using the 16S rRNA based Fluorescence In-Situ Hybridization (FISH) technique. The diversity of microorganisms present in the anaerobic granular sludge and the structure of the granules operated at 13 degrees C have been investigated using FISH combined with CSLM (Confocal Scanning Laser Microscopy). According to FISH results, archaeal cells representing methanogens were found intensively dominant in the bottom sampling port of the UASB reactor and acetoclastic Methanosaeta was the abundant methanogen. Other methanogens such as Methanosarcina and Methanobacterium like species were also observed. The abundance of originally mesophilic Methanosaeta-related Archaea under low temperature at all sampling days revealed the microbial adaptation to psychrophilic conditions. This might be attributed to the enzymatic alterations in Methanosaeta cells originating from seed sludge, which were exposed to sub-mesophilic temperatures at start-up and then to psychrophilic conditions during gradual decreases of temperature. According to CSLM observation, even though the sludge retained in the reactor kept its granular form as a whole, the majority of the granules had a tendency to be partly broken and they lost their rigidity when raw sewage was fed following synthetic sewage. Besides, Methanosaeta related species prevailing in seed sludge have noticeably lost their long filamentous forms and deteriorated during raw sewage feeding. Members of the order Methanobacteriales constituted the major hydrogenothrophic methanogens present in the psychrophilic UASB reactor, whereas the other hydrogenothrophic methanogens--members of the order Methanococcales and Methanogenium relatives--were absent.

Structure and function of methanogens along a short-term restoration chronosequence in the Florida Everglades.

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.

Analysis of the archaeal sub-seafloor community at Suiyo Seamount on the Izu-Bonin Arc.

A sub-surface archaeal community at the Suiyo Seamount in the Western Pacific Ocean was investigated by 16S rRNA gene sequence and whole-cell in situ hybridization analyses. In this study, we drilled and cased holes at the hydrothermal area of the seamount to minimize contamination of the hydrothermal fluid in the sub-seafloor by penetrating seawater. PCR clone analysis of the hydrothermal fluid samples collected from a cased hole indicated the presence of chemolithoautotrophic primary biomass producers of Archaeoglobales and the Methanococcales-related archaeal HTE1 group, both of which can utilize hydrogen as an electron donor. We discuss the implication of the microbial community on the early history of life and on the search for extraterrestrial life.

Geochemical and microbiological evidence for a hydrogen-based, hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field.

Subsurface microbial communities supported by geologically and abiologically derived hydrogen and carbon dioxide from the Earth's interior are of great interest, not only with regard to the nature of primitive life on Earth, but as potential analogs for extraterrestrial life. Here, for the first time, we present geochemical and microbiological evidence pointing to the existence of hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) dominated by hyperthermophilic methanogens beneath an active deep-sea hydrothermal field in the Central Indian Ridge. Geochemical and isotopic analyses of gaseous components in the hydrothermal fluids revealed heterogeneity of both concentration and carbon isotopic compositions of methane between the main hydrothermal vent (0.08 mM and -13.8 per thousand PDB, respectively) and the adjacent divergent vent site (0.2 mM and -18.5 per thousand PDB, respectively), representing potential subsurface microbial methanogenesis, at least in the divergent vent emitting more 13C-depleted methane. Extremely high abundance of magmatic energy sources such as hydrogen (2.5 mM) in the fluids also encourages a hydrogen-based, lithoautotrophic microbial activity. Both cultivation and cultivation-independent molecular analyses suggested the predominance of Methanococcales members in the superheated hydrothermal emissions and chimney interiors along with the other major microbial components of Thermococcales members. These results imply that a HyperSLiME, consisting of methanogens and fermenters, occurs in this tectonically active subsurface zone, strongly supporting the existence of hydrogen-driven subsurface microbial communities.

Methanotorris formicicus sp. nov., a novel extremely thermophilic, methane-producing archaeon isolated from a black smoker chimney in the Central Indian Ridge.

A novel extremely thermophilic, methane-producing archaeon was isolated from a black smoker chimney at the Kairei field in the Central Indian Ridge. Cells of this isolate were irregular cocci with several flagella; motility was not observed. Growth was observed between 55 and 83 degrees C (optimum of 75 degrees C; 30 min doubling time) and between pH 6.0 and 8.5 (optimum of pH 6.7). The isolate was a strictly anaerobic, methanogenic autotroph capable of using hydrogen and carbon dioxide as sole energy and carbon sources. Formate was utilized as an alternative energy source. The G+C content of the genomic DNA was 33.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate was most closely related to Methanotorris igneus strain Kol 5T. The isolate, however, could be genetically differentiated from this species by DNA-DNA hybridization analysis and on the basis of its physiological properties. The name Methanotorris formicicus sp. nov. is proposed for this isolate; the type strain is Mc-S-70T (=JCM 11930T=ATCC BAA-687T).

Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs.

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90 degrees C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H(2), acetate, and CO(2) as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.