Evidence of Active Methanogen Communities in Shallow Sediments of the Sonora Margin Cold Seeps

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.     

Methane Biogeochemistry and Methanogen Communities in Two Northern Peatland Ecosystems,New York State

Rates of methane (CH₄) production vary considerably among northern peat-forming wetlands, and it is not clear whether variability is caused by environmental factors affecting CH₄ production or differences in methanogen communities. We investigated CH₄ production and emission dynamics concomitantly with 16S rRNA gene sequence-based community analysis of Archaea in two contrasting peat-forming northern wetlands, an ombrotrophic bog and a minerotrophic conifer swamp. Individual measurements of CH₄ emissions to the atmosphere followed a lognormal distribution pattern in both sites, and mean rates were 30× greater in the bog site. Rates of CH₄ production measured in vitro were initially 3× greater in the bog than in the conifer swamp; although, after 30 days of incubation, production rates were similar suggesting that in situ environmental conditions limited production in the conifer swamp. Amplified ribosomal DNA restriction analysis (ARDRA) and rarefaction techniques indicated that both sites had similar levels of archaeal richness, with 27 unique taxa in the bog and 23 taxa in the conifer swamp. However, the bog had more pronounced dominance of a few taxa, whereas the conifer swamp had more even distribution among taxa. A 16S rRNA gene sequence-based phylogenetic analysis indicated high levels of diversity with similarity to known methanogenic families Methanosarcinaceae, Methanosaetaceae, Methanobacteriaceae, and likely Methanomicrobiaceae as well as two additional lineages previously characterized as groups of yet uncultivated Euryarchaeota commonly occurring in flooded rice soils. Therefore, sites with low and high rates of CH₄ production supported very diverse methanogenic communities.     

Nitrogen Fertilization Has a Stronger Effect on Soil Nitrogen-Fixing Bacterial Communities than Elevated Atmospheric CO2

Biological nitrogen fixation is the primary supply of N to most ecosystems, yet there is considerable uncertainty about how N-fixing bacteria will respond to global change factors such as increasing atmospheric CO₂ and N deposition. Using the nifH gene as a molecular marker, we studied how the community structure of N-fixing soil bacteria from temperate pine, aspen, and sweet gum stands and a brackish tidal marsh responded to multiyear elevated CO₂ conditions. We also examined how N availability, specifically, N fertilization, interacted with elevated CO₂ to affect these communities in the temperate pine forest. Based on data from Sanger sequencing and quantitative PCR, the soil nifH composition in the three forest systems was dominated by species in the Geobacteraceae and, to a lesser extent, Alphaproteobacteria. The N-fixing-bacterial-community structure was subtly altered after 10 or more years of elevated atmospheric CO₂, and the observed shifts differed in each biome. In the pine forest, N fertilization had a stronger effect on nifH community structure than elevated CO₂ and suppressed the diversity and abundance of N-fixing bacteria under elevated atmospheric CO₂ conditions. These results indicate that N-fixing bacteria have complex, interacting responses that will be important for understanding ecosystem productivity in a changing climate.     

Differentiated Response of Denitrifying Communities to Fertilization Regime in Paddy Soil

The impact of fertilization regimes on sequential denitrifying communities was investigated in a rice paddy field with 17 years continuous fertilization, located in Taoyuan Agro-ecosystem Research Station (110°72″ E, 28°52″ N), China. The diversity, community composition, and size of denitrifying genes of narG, qnorB, and nosZ were determined using molecular tools including terminal restriction fragment length polymorphism, quantitative polymerase chain reaction (qPCR), cloning, and sequencing analysis. Soil samples were collected from the plots with no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). UR and BM caused marked increase in the community size of the denitrifying genes; however, BMR resulted in the highest abundance. The community size of narG was the most affected by the fertilization regimes, while qnorB was the least. Fertilization also induced some shifts in the composition of denitrifying genes, but the responses of different genes varied. However, fertilization regimes caused no significant changes to the diversity of the denitrifying genes. Potential denitrification activity (PDA) was significantly correlated with the abundance of narG and nosZ rather than qnorB, but there were no such correlations between PDA and the composition and diversity of denitrifying communities. Conclusively, long-term fertilization significantly affected denitrifying community size and composition, but not diversity. Among the sequential denitrifying genes, narG was the most, while qnorB was the least sensitive communities to fertilization regimes.     

我国产甲烷古菌研究进展与展望

产甲烷古菌广泛分布在湿地、水稻田、动物瘤胃、油藏、海洋和热液等缺氧环境,在全球碳素循环、气候变化和清洁能源生产等领域发挥着重要作用,一直是国内外的研究热点。本文简要回顾了我国产甲烷古菌的研究进展,重点阐述了产甲烷古菌的资源与分类、生理生化、分子生物学、生态学功能和应用等方面的研究进展,并展望了产甲烷古菌的未来研究趋势。     

Nitrogen-Fixing Activity in Peat Soils from a Raised Bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014–0.022 mg N/(kg h). Incorporation of the ¹⁵N₂ isotope into organic compounds of peat soil was 2.71–8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10- to 20-cm layer of soil and much lower in the upper (under green moss) and deeper (20- to 30-cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18–26 h. The maximum nitrogenase activity (3.5–3.8 mg N/(kg h)), observed after 60–70 h, coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity, without a lag period, to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35°C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0–2.5 × 10⁶ cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Methanogen population in a marine biofilm corrosive to mild steel

This study was conducted to analyze the methanogen population in a corrosive marine biofilm based on 16S rDNA analysis, using a PCR-cloning-sequencing approach. There were 80 methanogen clones developed from the PCR-amplified DNA extracted from the biofilm on the mild steel surface. All clones were categorized into one of five operational taxonomy units (OTUs). Two OTUs (comprising 57 clones) were affiliated with the acetotrophic Methanosaeta genus; the remaining three OTUs (23 clones) were affiliated with the hydrogenotrophic genera of Methanogenium, Methanoplanus and Methanocalculus. The hydrogenotrophic methanogens could directly cause metal corrosion through cathodic depolarization, whereas the acetotrophic methanogens grew syntrophically with corrosion-causing sulfate-reducing bacteria, as observed by fluorescent in situ hybridization, and thus contribute indirectly to metal corrosion.     

Pressure-Enhanced Activity and Stability of a Hyperthermophilic Protease from a Deep-Sea Methanogen

We describe the properties of a hyperthermophilic, barophilic protease from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This enzyme is the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. The partially purified enzyme has a molecular mass of 29 kDa and a narrow substrate specificity with strong preference for leucine at the P1 site of polypeptide substrates. Enzyme activity increased up to 116(deg)C and was measured up to 130(deg)C, one of the highest temperatures reported for the function of any enzyme. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125(deg)C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barophilic and thermophilic behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously (J. F. Miller et al., Appl. Environ. Microbiol. 54:3039-3042, 1988).     

Methanogen communities along a primary succession transect of mire ecosystems

Peat accumulating mires are important sources of the greenhouse gas methane. Methane emissions and methanogenic Archaea communities have been shown to differ between fens and bogs, implying that mire succession includes an ecological succession in methanogen communities. We investigated methane production and the methanogen communities along a chronosequence of mires (ca. 100-2,500 years), which consisted of five sites (1-5) located on the land-uplift coast of the Gulf of Bothnia. Methane production was measured in a laboratory incubation experiment. Methanogen communities were determined by amplification of a methyl coenzyme M-reductase (mcr) gene marker and analyzed by terminal-restriction fragment length polymorphism. The terminal-restriction fragment length polymorphism fingerprinting resulted in 15 terminal restriction fragments. The ordination configuration of the terminal restriction fragments data, using nonmetric multidimensional scaling, showed a clear gradient in the methanogen community structure along the mire chronosequence. In addition, fingerprint patterns of samples from the water table level and 40 cm below differed from one another in the bog site (site 5). Methane production was negligible in the three youngest fen sites (sites 1-3) and showed the highest rates in the oligotrophic fen site (site 4). Successful PCR amplification using mcr gene primers revealed the presence of a methanogen community in all five sites along the study transect.     

Spatial Distribution of Algal Assemblages in a Temperate Lowland Peat Bog

Samples of phytobenthos were collected during three different seasons in 2005 along a linear transect of a lowland peat bog at various spatial scales (10 cm, 1 m, 10 m) to investigate the seasonal dynamics, diversity, and factors influencing the spatial patterns of microalgal communities. Non‐metric multidimensional scaling (NMDS), similarity percentage (SIMPER) analyses, ANOSIM, Mantel tests and diversity indices were used to analyze the data. Seasonal dynamics were exhibited by an increase in diversity, and a decrease in dominance from May to October, with significant differences in species composition. Mantel tests showed the significant influence of distance, microhabitat type, and conductivity on maintaining the similarity of species composition on scales of 1 m and 10 m. The small‐scale processes (colonization and niche differentiation), microhabitat type, geographic distance and conductivity were found to be the main factors influencing the distribution of algal assemblages. We conclude that these factors are related to winter disturbance, and the consequent colonization and subsequent niche differentiation. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Isolation and Characterization of a Novel Thermophilic, Freshwater Methanogen

A novel thermophilic, coccoid methanogen isolated from nonthermal freshwater sediments is described. Hydrogen plus carbon dioxide and formate were substrates for methanogenesis, and methane production was stimulated by yeast extract, Casamino Acids, and tryptose. Growth also occurred autotrophically. Elevated levels of sodium chloride were not required for maximum growth and were inhibitory above 2%. The minimum doubling time occurred at 57°C, and the upper and lower limits for methane production were 62 and 26°C, respectively. The optimum pH for growth was between 7.0 and 7.5. Inhibitory antibiotics included metronidazole, anisomycin, chloramphenicol, and lasalocid. Colonies were circular, dark yellow, shiny, and convex with entire edges. Cells were 1 to 2.5 μm in diameter, nonmotile, occurring singly or in pairs, and fimbriated. Cells were lysed by pronase or trypsin digestion, glass-distilled water, and 1% sodium dodecyl sulfate. Electron micrographs of thin sections showed a monolayered cell wall ca. 20 nm thick. The DNA base ratio was 49.2 mol% guanine plus cytosine. The whole cell protein pattern differed from that of other named coccoid methanogens.     

Isolation and Characterization of a Moderately Halophilic Methanogen from a Solar Saltern

A moderately halophilic methanogenic bacterium was enriched with trimethylamine and isolated from the sediment of a solar salt pond (total dissolved solids of pond water, 250 g/liter; pH 7.5). The isolate (strain SF1, DSM 3243) was an irregular coccus which stained gram negative, with a diameter of 1 μm and a thin monolayered cell wall. The organism grew singly, in pairs, and in irregular clumps. Colonies were tannish yellow, circular, with entire edges, and about 1 mm in diameter within 1 week. Only methylamines or methanol was used for growth and methanogenesis. Most rapid growth (doubling time, 10.2 h) occurred at a temperature of 37°C and a pH of 7.4. The optimum NaCl concentration was 2.1 M. Yeast extract or rumen fluid was required. The isolate was lysed by sodium dodecyl sulfate (0.1 g/liter) and was sensitive to chloramphenicol. The G+C content of the DNA was 41 (±1) mol%.     

Impact of Long-Term Fertilization on the Composition of Denitrifier Communities Based on Nitrite Reductase Analyses in a Paddy Soil

The effect of long-term fertilization on soil-denitrifying communities was determined by measuring the abundance and diversity of the nitrite reductase genes nirK and nirS. Soil samples were collected from plots of a long-term fertilization experiment started in 1990, located in Taoyuan (110°72″ E, 28°52″ N), China. The treatments were no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). The abundance, diversity, and composition of the soil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nirK and nirS genes. There was a pronounced difference in the community composition and diversity of nirK-containing denitrifiers responding to the long-term fertilization regimes; however, less variation was observed in communities of nirS-containing denitrifiers, indicating that denitrifiers possessing nirK were more sensitive to the fertilization practices than those with nirS. In contrast, fertilization regimes had similar effects on the copy numbers of nirK and nirS genes. The BMR treatment had the highest copy numbers of nirK and nirS, followed by the two mineral fertilization regimes (UR and BM), and the lowest was in the NF treatment. Of the measured soil parameters, the differences in the community composition of nirK and the abundance of nir denitrifiers were highly correlated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on the community structure of nirK populations only, and total organic carbon was the dominant factor in relation to the variations of nir community sizes.     

Effect of Agricultural Management on Nematode Communities in a Mediterranean Agroecosystem

The effects of agricultural management on the soil nematode community were investigated in a field study at depths of 0 to 10 cm and 10 to 20 cm during a peanut (Arachis hypogaea) growing season in Israel. Nineteen nematode families and 23 genera were observed. Rhabditidae, Cephalobus, Eucephalobus, Aphelenchus, Aphelenchoides, Tetylenchus, Tylenchus, Dorylaimus, and Discolaimus were the dominant family and genera. Ecological measures of soil nematode community structure, diversity, and maturity indices were assessed and compared between the managed (by fertilization, irrigation, and pesticide application) and unmanaged fields. The total number of nematodes at a 10-cm depth during peanut-sowing, mid-season, and harvest periods was higher in the treated (managed) plot than in the control (unmanaged) plot. Bacterivores and fungivores were the most abundant trophic groups in both plots and both depths. The relative abundance of each group averaged 60.8 to 67.3% and 11.5 to 19.6% of the nematode community, respectively. Plant parasites and omnivores-predators at the 0 to 10-cm depth were much less abundant than any other two groups in our experimental plots. During the growing season, except the harvest period, populations of plant parasites and omnivores-predators at the 10 to 20-cm depth were lower in the treated plot than in the control plot. Maturity index (MI), plant-parasite index (PPI), and ratio of fungivores and bacterivores to plant parasites (WI) were found to be more sensitive indicators than other ecological indices for assessing the response of nematode communities to agricultural management in an Israeli agroecosystem.