Salinity constraints on subsurface archaeal diversity and methanogenesis in sedimentary rock rich in organic matter

The diversity of microorganisms active within sedimentary rocks provides important controls on the geochemistry of many subsurface environments. In particular, biodegradation of organic matter in sedimentary rocks contributes to the biogeochemical cycling of carbon and other elements and strongly impacts the recovery and quality of fossil fuel resources. In this study, archaeal diversity was investigated along a salinity gradient spanning 8 to 3,490 mM Cl(-) in a subsurface shale rich in CH(4) derived from biodegradation of sedimentary hydrocarbons. Shale pore waters collected from wells in the main CH(4)-producing zone lacked electron acceptors such as O(2), NO(3)(-), Fe(3+), or SO(4)(2-). Acetate was detected only in high-salinity waters, suggesting that acetoclastic methanogenesis is inhibited at Cl(-) concentrations above approximately 1,000 mM. Most-probable-number series revealed differences in methanogen substrate utilization (acetate, trimethylamine, or H(2)/CO(2)) associated with chlorinity. The greatest methane production in enrichment cultures was observed for incubations with salinity at or close to the native pore water salinity of the inoculum. Restriction fragment length polymorphism analyses of archaeal 16S rRNA genes from seven wells indicated that there were links between archaeal communities and pore water salinity. Archaeal clone libraries constructed from sequences from 16S rRNA genes isolated from two wells revealed phylotypes similar to a halophilic methylotrophic Methanohalophilus species and a hydrogenotrophic Methanoplanus species at high salinity and a single phylotype closely related to Methanocorpusculum bavaricum at low salinity. These results show that several distinct communities of methanogens persist in this subsurface, CH(4)-producing environment and that each community is adapted to particular conditions of salinity and preferential substrate use and each community induces distinct geochemical signatures in shale formation waters.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Methanotrophs and Methanogens in Masonry

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH₄ g of stone⁻¹ day⁻¹. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH₄ g of stone⁻¹ day⁻¹ was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.     

Desulfotomaculum and Methanobacterium spp. Dominate a 4- to 5-Kilometer-Deep Fault

Alkaline, sulfidic, 54 to 60°C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO₄²⁻ in the presence of high pH, high concentrations of H₂ and CH₄, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO₄²⁻ reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.     

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H₂S) and methane (CH₄) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H₂S and CH₄ production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H₂S emission was notably high (290.7 ± 72.3 mg S-H₂S liter⁻¹ day⁻¹), whereas emissions of CH₄ remained low (17.9 ± 15.9 mg COD-CH₄ liter⁻¹ day⁻¹). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH₄ emissions increased after 1 year of biofilm growth (327.6 ± 16.6 mg COD-CH₄ liter⁻¹ day⁻¹), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.     

Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs_0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species.     

Rapid Methane Oxidation in a Landfill Cover Soil

Methane oxidation rates observed in a topsoil covering a retired landfill are the highest reported (45 g m⁻² day⁻¹) for any environment. This microbial community had the capacity to rapidly oxidize CH₄ at concentrations ranging from <1 ppm (microliters per liter) (first-order rate constant [k] = −0.54 h⁻¹) to >10⁴ ppm (k = −2.37 h⁻¹). The physiological characteristics of a methanotroph isolated from the soil (characteristics determined in aqueous medium) and the natural population, however, were similar to those of other natural populations and cultures: the Q₁₀ and optimum temperature were 1.9 and 31°C, respectively, the apparent half-saturation constant was 2.5 to 9.3 μM, and 19 to 69% of oxidized CH₄ was assimilated into biomass. The CH₄ oxidation rate of this soil under waterlogged (41% [wt/vol] H₂O) conditions, 6.1 mg liter⁻¹ day⁻¹, was near rates reported for lake sediment and much lower than the rate of 116 mg liter⁻¹ day⁻¹ in the same soil under moist (11% H₂O) conditions. Since there are no large physiological differences between this microbial community and other CH₄ oxidizers, we attribute the high CH₄ oxidation rate in moist soil to enhanced CH₄ transport to the microorganisms; gas-phase molecular diffusion is 10⁴-fold faster than aqueous diffusion. These high CH₄ oxidation rates in moist soil have implications that are important in global climate change. Soil CH₄ oxidation could become a negative feedback to atmospheric CH₄ increases (and warming) in areas that are presently waterlogged but are projected to undergo a reduction in summer soil moisture.     

Dissimilatory Selenate Reduction Potentials in a Diversity of Sediment Types

We measured potential rates of bacterial dissimilatory reduction of ⁷⁵SeO₄²⁻ to ⁷⁵Se⁰ in a diversity of sediment types, with salinities ranging from freshwater (salinity = 1 g/liter) to hypersaline (salinity = 320 g/liter and with pH values ranging from 7.1 to 9.8. Significant biological selenate reduction occurred in all samples with salinities from 1 to 250 g/liter but not in samples with a salinity of 320 g/liter. Potential selenate reduction rates (25 nmol of SeO₄²⁻ per ml of sediment added with isotope) ranged from 0.07 to 22 μmol of SeO₄²⁻ reduced liter⁻¹ h⁻¹. Activity followed Michaelis-Menten kinetics in relation to SeO₄²⁻ concentration (K_m of selenate = 7.9 to 720 μM). There was no linear correlation between potential rates of SeO₄²⁻ reduction and salinity, pH, concentrations of total Se, porosity, or organic carbon in the sediments. However, potential selenate reduction was correlated with apparent K_m for selenate and with potential rates of denitrification (r = 0.92 and 0.81, respectively). NO₃⁻, NO₂⁻, MoO₄²⁻, and WO₄²⁻ inhibited selenate reduction activity to different extents in sediments from both Hunter Drain and Massie Slough, Nev. Sulfate partially inhibited activity in sediment from freshwater (salinity = 1 g/liter) Massie Slough samples but not from the saline (salinity = 60 g/liter) Hunter Drain samples. We conclude that dissimilatory selenate reduction in sediments is widespread in nature. In addition, in situ selenate reduction is a first-order reaction, because the ambient concentrations of selenium oxyanions in the sediments were orders of magnitude less than their K_ms.     

Methane-Derived Carbon in the Benthic Food Web in Stream Impoundments

Methane gas (CH₄) has been identified as an important alternative source of carbon and energy in some freshwater food webs. CH₄ is oxidized by methane oxidizing bacteria (MOB), and subsequently utilized by chironomid larvae, which may exhibit low δ¹³C values. This has been shown for chironomid larvae collected from lakes, streams and backwater pools. However, the relationship between CH₄ concentrations and δ¹³C values of chironomid larvae for in-stream impoundments is unknown. CH₄ concentrations were measured in eleven in-stream impoundments located in the Queich River catchment area, South-western Germany. Furthermore, the δ¹³C values of two subfamilies of chironomid larvae (i.e. Chironomini and Tanypodinae) were determined and correlated with CH₄ concentrations. Chironomini larvae had lower mean δ¹³C values (−29.2 to −25.5 ‰), than Tanypodinae larvae (−26.9 to −25.3 ‰). No significant relationships were established between CH₄ concentrations and δ¹³C values of chironomids (p>0.05). Mean δ¹³C values of chironomid larvae (mean: −26.8‰, range: −29.2‰ to −25.3‰) were similar to those of sedimentary organic matter (SOM) (mean: −28.4‰, range: −29.3‰ to −27.1‰) and tree leaf litter (mean: −29.8 ‰, range: −30.5‰ to −29.1‰). We suggest that CH₄ concentration has limited influence on the benthic food web in stream impoundments.     

Carbon flow from volcanic CO2 into soil microbial communities of a wetland mofette

Effects of extremely high carbon dioxide (CO₂) concentrations on soil microbial communities and associated processes are largely unknown. We studied a wetland area affected by spots of subcrustal CO₂ degassing (mofettes) with focus on anaerobic autotrophic methanogenesis and acetogenesis because the pore gas phase was largely hypoxic. Compared with a reference soil, the mofette was more acidic (ΔpH ∼0.8), strongly enriched in organic carbon (up to 10 times), and exhibited lower prokaryotic diversity. It was dominated by methanogens and subdivision 1 Acidobacteria, which likely thrived under stable hypoxia and acidic pH. Anoxic incubations revealed enhanced formation of acetate and methane (CH₄) from hydrogen (H₂) and CO₂ consistent with elevated CH₄ and acetate levels in the mofette soil. ¹³CO₂ mofette soil incubations showed high label incorporations with ∼512 ng ¹³C g (dry weight (dw)) soil⁻¹ d⁻¹ into the bulk soil and up to 10.7 ng ¹³C g (dw) soil⁻¹ d⁻¹ into almost all analyzed bacterial lipids. Incorporation of CO₂-derived carbon into archaeal lipids was much lower and restricted to the first 10 cm of the soil. DNA-SIP analysis revealed that acidophilic methanogens affiliated with Methanoregulaceae and hitherto unknown acetogens appeared to be involved in the chemolithoautotrophic utilization of ¹³CO₂. Subdivision 1 Acidobacteriaceae assimilated ¹³CO₂ likely via anaplerotic reactions because Acidobacteriaceae are not known to harbor enzymatic pathways for autotrophic CO₂ assimilation. We conclude that CO₂-induced geochemical changes promoted anaerobic and acidophilic organisms and altered carbon turnover in affected soils.     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas)

Even if many Vibrio spp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulating Vibrio populations in a salinity gradient along the Swedish coastline, we combined Vibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The total Vibrio abundance ranged from 4 × 10³ to 9.6 × 10⁴ cells liter⁻¹, with the highest abundances in the more saline waters of the Skagerrak Sea. Several Vibrio populations were present throughout the salinity gradient, with abundances of single populations ranging from 5 × 10² to 7 × 10⁴ cells liter⁻¹. Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives of V. anguillarum and V. aestuarianus genotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genus Vibrio may also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. A V. cholerae/V. mimicus population was detected in more than 50% of the samples, with abundances exceeding 10³ cells liter⁻¹, even in the cold (annual average water temperature of around 5°C) and low-salinity (2 to 4‰) samples from the Bothnian Bay (latitude, 65°N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potential Vibrio pathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Gammaproteobacterial Methanotrophs Dominate Cold Methane Seeps in Floodplains of West Siberian Rivers

A complex system of muddy fluid-discharging and methane (CH₄)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH₄ flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH₄ h⁻¹, while some seeps emitted up to 5.54 g CH₄ h⁻¹. The δ¹³C value of methane released from these seeps varied between −71.1 and −71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situ temperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH₄ ml⁻¹ day⁻¹) were measured in mud samples. Fluorescence in situ hybridization detected 10⁷ methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding particulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosoma group, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH₄ sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.     

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3⁺ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103⁺ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103⁺ DCs from Il10 ^−/−, Tlr2 ^−/−, and Myd88 ^−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103⁺ DCs failed to induce IL-10 production from co-cultured Il27ra ^−/− T cells. B. breve treatment of Tlr2 ^−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103⁺ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4⁺ T cells from wild-type mice, but not Il10 ^−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.     

Effects of Winter Cover Crops Straws Incorporation on CH4 and N2O Emission from Double-Cropping Paddy Fields in Southern China

Residue management in cropping systems is believed to improve soil quality. However, the effects of residue management on methane (CH₄) and nitrous oxide (N₂O) emissions from paddy field in Southern China have not been well researched. The emissions of CH₄ and N₂O were investigated in double cropping rice (Oryza sativa L.) systems with straw returning of different winter cover crops by using the static chamber-gas chromatography technique. A randomized block experiment with three replications was established in 2004 in Hunan Province, China, including rice–rice–ryegrass (Lolium multiflorum L.) (Ry-R-R), rice–rice–Chinese milk vetch (Astragalus sinicus L.) (Mv-R-R) and rice–rice with winter fallow (Fa-R-R). The results showed that straw returning of winter crops significantly increased the CH₄ emission during both rice growing seasons when compared with Fa-R-R. Ry-R-R plots had the largest CH₄ emissions during the early rice growing season with 14.235 and 15.906 g m⁻² in 2012 and 2013, respectively, when Ry-R-R plots had the largest CH₄ emission during the later rice growing season with 35.673 and 38.606 g m⁻² in 2012 and 2013, respectively. The Ry-R-R and Mv-R-R also had larger N₂O emissions than Fa-R-R in both rice seasons. When compared to Fa-R-R, total N₂O emissions in the early rice growing season were increased by 0.05 g m⁻² in Ry-R-R and 0.063 g m⁻² in Mv-R-R in 2012, and by 0.058 g m⁻² in Ry-R-R and 0.068 g m⁻² in Mv-R-R in 2013, respectively. Similar result were obtained in the late rice growing season, and the total N₂O emissions were increased by 0.104 g m⁻² in Ry-R-R and 0.073 g m⁻² in Mv-R-R in 2012, and by 0.108 g m⁻² in Ry-R-R and 0.076 g m⁻² in Mv-R-R in 2013, respectively. The global warming potentials (GWPs) from paddy fields were ranked as Ry-R-R>Mv-R-R>Fa-R-R. As a result, straw returning of winter cover crops has significant effects on increase of CH₄ and N₂O emission from paddy field in double cropping rice system.     

Layered Structure of Bacterial and Archaeal Communities and Their In Situ Activities in Anaerobic Granules

The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH₄, H₂, pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH₄, H₂, pH, and ORP revealed that acid and H₂ production occurred in the upper part of the granule, below which H₂ consumption and CH₄ production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H₂ was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH₄ in the inner layer. We determined the effective diffusion coefficient for H₂ in the anaerobic granules to be 2.66 × 10⁻⁵ cm² s⁻¹, which was 57% in water.     

Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH₃Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH₃Cl; then it catalyzed methyl transfer from CH₃Cl, CH₃Br, or CH₃I to the following acceptor ions (in order of decreasing efficacy): I⁻, HS⁻, Cl⁻, Br⁻, NO₂⁻, CN⁻, and SCN⁻. Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N₂O, and Hg²⁺ to affect the methyltransferase suggests significant differences. During CH₃Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS⁻, a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH₃Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899–2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.     

Cattle Manure Enhances Methanogens Diversity and Methane Emissions Compared to Swine Manure under Rice Paddy

Livestock manures are broadly used in agriculture to improve soil quality. However, manure application can increase the availability of organic carbon, thereby facilitating methane (CH₄) production. Cattle and swine manures are expected to have different CH₄ emission characteristics in rice paddy soil due to the inherent differences in composition as a result of contrasting diets and digestive physiology between the two livestock types. To compare the effect of ruminant and non-ruminant animal manure applications on CH₄ emissions and methanogenic archaeal diversity during rice cultivation (June to September, 2009), fresh cattle and swine manures were applied into experimental pots at 0, 20 and 40 Mg fresh weight (FW) ha⁻¹ in a greenhouse. Applications of manures significantly enhanced total CH₄ emissions as compared to chemical fertilization, with cattle manure leading to higher emissions than swine manure. Total organic C contents in cattle (466 g kg⁻¹) and swine (460 g kg⁻¹) manures were of comparable results. Soil organic C (SOC) contents were also similar between the two manure treatments, but dissolved organic C (DOC) was significantly higher in cattle than swine manure. The mcrA gene copy numbers were significantly higher in cattle than swine manure. Diverse groups of methanogens which belong to Methanomicrobiaceae were detected only in cattle-manured but not in swine-manured soil. Methanogens were transferred from cattle manure to rice paddy soils through fresh excrement. In conclusion, cattle manure application can significantly increase CH₄ emissions in rice paddy soil during cultivation, and its pretreatment to suppress methanogenic activity without decreasing rice productivity should be considered.