Microbial Communities in Standing Dead Trees in Ghost Forests are Largely Aerobic, Saprophytic, and Methanotrophic.

Standing dead trees (snags) are recognized for their influence on methane (CH4) cycling in coastal wetlands, yet the biogeochemical processes that control the magnitude and direction of fluxes across the snag-atmosphere interface are not fully elucidated. Herein, we analyzed microbial communities and fluxes at one height from ten snags in a ghost forest wetland. Snag-atmosphere CH4 fluxes were highly variable (- 0.11-0.51 mg CH4 m-2 h-1). CH4 production was measured in three out of ten snags; whereas, CH4 consumption was measured in two out of ten snags. Potential CH4 production and oxidation in one core from each snag was assayed in vitro. A single core produced CH4 under anoxic and oxic conditions, at measured rates of 0.7 and 0.6 ng CH4 g-1 h-1, respectively. Four cores oxidized CH4 under oxic conditions, with an average rate of - 1.13 ± 0.31 ng CH4 g-1 h-1. Illumina sequencing of the V3/V4 region of the 16S rRNA gene sequence revealed diverse microbial communities and indicated oxidative decomposition of deadwood. Methanogens were present in 20% of the snags, with a mean relative abundance of < 0.0001%. Methanotrophs were identified in all snags, with a mean relative abundance of 2% and represented the sole CH4-cycling communities in 80% of the snags. These data indicate potential for microbial attenuation of CH4 emissions across the snag-atmosphere interface in ghost forests. A better understanding of the environmental drivers of snag-associated microbial communities is necessary to forecast the response of CH4 cycling in coastal ghost forest wetlands to a shifting coastal landscape.

Variation in carbon and nitrogen concentrations among peatland categories at the global scale.

Peatlands account for 15 to 30% of the world's soil carbon (C) stock and are important controls over global nitrogen (N) cycles. However, C and N concentrations are known to vary among peatlands contributing to the uncertainty of global C inventories, but there are few global studies that relate peatland classification to peat chemistry. We analyzed 436 peat cores sampled in 24 countries across six continents and measured C, N, and organic matter (OM) content at three depths down to 70 cm. Sites were distinguished between northern (387) and tropical (49) peatlands and assigned to one of six distinct broadly recognized peatland categories that vary primarily along a pH gradient. Peat C and N concentrations, OM content, and C:N ratios differed significantly among peatland categories, but few differences in chemistry with depth were found within each category. Across all peatlands C and N concentrations in the 10-20 cm layer, were 440 ± 85.1 g kg-1 and 13.9 ± 7.4 g kg-1, with an average C:N ratio of 30.1 ± 20.8. Among peatland categories, median C concentrations were highest in bogs, poor fens and tropical swamps (446-532 g kg-1) and lowest in intermediate and extremely rich fens (375-414 g kg-1). The C:OM ratio in peat was similar across most peatland categories, except in deeper samples from ombrotrophic tropical peat swamps that were higher than other peatlands categories. Peat N concentrations and C:N ratios varied approximately two-fold among peatland categories and N concentrations tended to be higher (and C:N lower) in intermediate fens compared with other peatland types. This study reports on a unique data set and demonstrates that differences in peat C and OM concentrations among broadly classified peatland categories are predictable, which can aid future studies that use land cover assessments to refine global peatland C and N stocks.

Beyond the usual suspects: methanogenic communities in eastern North American peatlands are also influenced by nickel and copper concentrations.

Peatlands both accumulate carbon and release methane, but their broad range in environmental conditions means that the diversity of microorganisms responsible for carbon cycling is still uncertain. Here, we describe a community analysis of methanogenic archaea responsible for methane production in 17 peatlands from 36 to 53 N latitude across the eastern half of North America, including three metal-contaminated sites. Methanogenic community structure was analysed through Illumina amplicon sequencing of the mcrA gene. Whether metal-contaminated sites were included or not, metal concentrations in peat were a primary driver of methanogenic community composition, particularly nickel, a trace element required in the F430 cofactor in methyl-coenzyme M reductase that is also toxic at high concentrations. Copper was also a strong predictor, likely due to inhibition at toxic levels and/or to cooccurrence with nickel, since copper enzymes are not known to be present in anaerobic archaea. The methanogenic groups Methanocellales and Methanosarcinales were prevalent in peatlands with low nickel concentrations, while Methanomicrobiales and Methanomassiliicoccales were abundant in peatlands with higher nickel concentrations. Results suggest that peat-associated trace metals are predictors of methanogenic communities in peatlands.

Micropepsis pineolensis gen. nov., sp. nov., a mildly acidophilic alphaproteobacterium isolated from a poor fen, and proposal of Micropepsaceae fam. nov. within Micropepsales ord. nov.

An obligately anaerobic, mildly acidophilic, fermentative alphaproteobacterium, designated strain CS4T, was isolated from an acidic, oligotrophic (nutrient poor) poor fen located near Pineola, NC, USA. Cultures contained Gram-negative, slightly curved, non-motile, non-spore-forming, non-prosthecate rods (0.1-0.4 µm in diameter and 0.4-4 µm long, depending, in part, on the growth substrate). Growth optima were 35 °C (range 15-35 °C), pH 5.6 (range of 5.0-6.8), and with 0-50 mM added NaCl (range, 0-100 mM added NaCl). The culture fermented cellobiose, d-glucose, d-mannose, fructose, galactose, glycerol, lactose, maltose, peptone, sucrose, trehalose and xylose. Respiratory growth was not detected. Major fatty acids were C18 : 1ω7c, C19 : 0 cyclo ω8c, C14 : 0 and C16 : 0. The G+C content of the DNA was 61.9±0.3 mol%. The two most closely related species phylogenetically, Rhizomicrobium palustre A48T (AB081581) and Rhizomicrobium electricum Mfc52T (AB365487) shared 94 and 93 % SSU rRNA gene sequence identity, respectively, to that of strain CS4T. Lower SSU rRNA gene sequence identities resulted from pairwise comparisons with members of the order 'Rhizobiales'(85-88 %) or Sphingomonadales(85-86 %). These findings all support the classification of strain CS4T as representative of a novel genus, family and order of Alphaproteobacteria. The type strain of the species pineolensis within the genus Micropepsis, family Micropepsaceae and order Micropepsales is CS4T (=JCM 30711T=ATCC BAA-2724T).

A novel isolate and widespread abundance of the candidate alphaproteobacterial order (Ellin 329), in southern Appalachian peatlands.

Peatlands of all latitudes play an integral role in global climate change by serving as a carbon sink and a primary source of atmospheric methane; however, the microbial ecology of mid-latitude peatlands is vastly understudied. Herein, next generation Illumina amplicon sequencing of small subunit rRNA genes was utilized to elucidate the microbial communities in three southern Appalachian peatlands. In contrast to northern peatlands, Proteobacteria dominated over Acidobacteria in all three sites. An average of 11 bacterial phyla was detected at relative abundance values >1%, with three candidate divisions (OP3, WS3 and NC10) represented, indicating high phylogenetic diversity. Physiological traits of isolates within the candidate alphaproteobacterial order, Ellin 329, obtained here and in previous studies indicate that bacteria of this order may be involved in hydrolysis of poly-, di- and monosaccharides. Community analyses indicate that Ellin 329 is the third most abundant order and is most abundant near the surface layers where plant litter decomposition should be primarily occurring. In sum, members of Ellin 329 likely play important roles in organic matter decomposition, in southern Appalachian peatlands and should be investigated further in other peatlands and ecosystem types.

Nutrient input influences fungal community composition and size and can stimulate manganese (II) oxidation in caves.

Little is known about the fungal role in biogeochemical cycling in oligotrophic ecosystems. This study compared fungal communities and assessed the role of exogenous carbon on microbial community structure and function in two southern Appalachian caves: an anthropogenically impacted cave and a near-pristine cave. Due to carbon input from shallow soils, the anthropogenically impacted cave had an order of magnitude greater fungal and bacterial quantitative-polymerase chain reaction (qPCR) gene copy numbers, had significantly greater community diversity, and was dominated by ascomycotal phylotypes common in early phase, labile organic matter decomposition. Fungal assemblages in the near-pristine cave samples were dominated by Basidiomycota typically found in deeper soils (and/or in late phase, recalcitrant organic matter decomposition), suggesting more oligotrophic conditions. In situ carbon and manganese (II) [Mn(II)] addition over 10 weeks resulted in growth of fungal mycelia followed by increased Mn(II) oxidation. A before/after comparison of the fungal communities indicated that this enrichment increased the quantity of fungal and bacterial cells, yet decreased overall fungal diversity. Anthropogenic carbon sources can therefore dramatically influence the diversity and quantity of fungi, impact microbial community function, and stimulate Mn(II) oxidation, resulting in a cascade of changes that can strongly influence nutrient and trace element biogeochemical cycles in karst aquifers.

Methanobacterium paludis sp. nov. and a novel strain of Methanobacterium lacus isolated from northern peatlands.

Two mesophilic, hydrogenotrophic methanogens, designated strains SWAN1T and AL-21, were isolated from two contrasting peatlands: a near circumneutral temperate minerotrophic fen in New York State, USA, and an acidic boreal poor fen site in Alaska, USA, respectively. Cells of the two strains were rod-shaped, non-motile, stained Gram-negative and resisted lysis with 0.1% SDS. Cell size was 0.6×1.5-2.8 µm for strain SWAN1T and 0.45-0.85×1.5-35 µm for strain AL-21. The strains used H2/CO2 but not formate or other substrates for methanogenesis, grew optimally around 32-37 °C, and their growth spanned through a slightly low to neutral pH range (4.7-7.1). Strain AL-21 grew optimally closer to neutrality at pH 6.2, whereas strain SWAN1T showed a lower optimal pH at 5.4-5.7. The two strains were sensitive to NaCl with a maximal tolerance at 160 mM for strain SWAN1T and 50 mM for strain AL-21. Na2S was toxic at very low concentrations (0.01-0.8 mM), resulting in growth inhibition above these values. The DNA G+C content of the genomes was 35.7 mol% for strain SWAN1T and 35.8 mol% for strain AL-21. Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains are members of the genus Methanobacterium. Strain SWAN1T shared 94-97% similarity with the type strains of recognized species of the genus Methanobacterium, whereas strain AL-21 shared 99% similarity with Methanobacterium lacus 17A1T. On the basis of phenotypic, genomic and phylogenetic characteristics, strain SWAN1T (=DSM 25820T=JCM 18151T) is proposed as the type strain of a novel species, Methanobacterium paludis sp. nov., while strain AL-21 is proposed as a second strain of Methanobacterium lacus.

Ubiquitous dissolved inorganic carbon assimilation by marine bacteria in the Pacific Northwest coastal ocean as determined by stable isotope probing.

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM (13)C-NaHCO(3), doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.

Seasonal changes in methanogenesis and methanogenic community in three peatlands, new york state.

Fluctuating environmental conditions can promote diversity and control dominance in community composition. In addition to seasonal temperature and moisture changes, seasonal supply of metabolic substrates selects populations temporally. Here we demonstrate cascading effects in the supply of metabolic substrates on methanogenesis and community composition of anaerobic methanogenic archaea in three contrasting peatlands in upstate New York. Fresh samples of peat soils, collected about every 3 months for 20 months and incubated at 22 ± 2°C regardless of the in situ temperature, exhibited potential rates of methane (CH(4)) production of 0.02-0.2 mmol L(-1) day(-1) [380-3800 nmol g(-1) (dry) day(-1)). The addition of acetate stimulated rates of CH(4) production in a fen peatland soil, whereas addition of hydrogen (H(2)), and simultaneous inhibition of H(2)-consuming acetogenic bacteria with rifampicin, stimulated CH(4) production in two acidic bog soils, especially, in autumn and winter. The methanogenic community structure was characterized using T-RFLP analyses of SSU rRNA genes. The E2 group of methanogens (Methanoregulaceae) dominated in the two acidic bog peatlands with relatively greater abundance in winter. In the fen peatland, the E1 group (Methanoregulaceae) and members of the Methanosaetaceae were co-dominant, with E1 having a high relative abundance in spring. Change in relative abundance profiles among methanogenic groups in response to added metabolic substrates was as predicted. The acetate-amendment increased abundance of Methanosarcinaceae, and H(2)-amendment enhanced abundance of E2 group in all peat soils studied, respectively. Additionally, addition of acetate increased abundance of Methanosaetaceae only in the bog soils. Variation in the supply of metabolic substrates helps explain the moderate diversity of methanogens in peatlands.

Methanolinea mesophila sp. nov., a hydrogenotrophic methanogen isolated from rice field soil, and proposal of the archaeal family Methanoregulaceae fam. nov. within the order Methanomicrobiales.

A novel mesophilic, hydrogenotrophic methanogen, designated strain TNR(T), was isolated from an anaerobic, propionate-degradation enrichment culture that was originally established from a rice field soil sample from Taiwan. Cells were non-motile rods, 2.0-6.5 µm long by 0.3 µm wide. Filamentous (up to about 100 µm) and coccoid (about 1 µm in diameter) cells were also observed in cultures in the late exponential phase of growth. Strain TNR(T) grew at 20-40 °C (optimally at 37 °C), at pH 6.5-7.4 (optimally at pH 7.0) and in the presence of 0-25 g NaCl l(-1) (optimally at 0 g NaCl l(-1)). The strain utilized H(2)/CO(2) and formate for growth and produced methane. The G+C content of the genomic DNA was 56.4 mol%. Based on sequences of both the 16S rRNA gene and the methanogen-specific marker gene mcrA, strain TNR(T) was related most closely to Methanolinea tarda NOBI-1(T); levels of sequence similarities were 94.8 and 86.4 %, respectively. The 16S rRNA gene sequence similarity indicates that strain TNR(T) and M. tarda NOBI-1(T) represent different species within the same genus. This is supported by shared phenotypic properties, including substrate usage and cell morphology, and differences in growth temperature. Based on these genetic and phenotypic properties, strain TNR(T) is considered to represent a novel species of the genus Methanolinea, for which the name Methanolinea mesophila sp. nov. is proposed; the type strain is TNR(T) ( = NBRC 105659(T) = DSM 23604(T)). In addition, we also suggest family status for the E1/E2 group within the order Methanomicrobiales, for which the name Methanoregulaceae fam. nov. is proposed; the type genus of family is Methanoregula.

Methanoregula boonei gen. nov., sp. nov., an acidiphilic methanogen isolated from an acidic peat bog.

A novel acidiphilic, hydrogenotrophic methanogen, designated strain 6A8(T), was isolated from an acidic (pH 4.0-4.5) and ombrotrophic (rain-fed) bog located near Ithaca, NY, USA. Cultures were dimorphic, containing thin rods (0.2-0.3 µm in diameter and 0.8-3.0 µm long) and irregular cocci (0.2-0.8 µm in diameter). The culture utilized H(2)/CO(2) to produce methane but did not utilize formate, acetate, methanol, ethanol, 2-propanol, butanol or trimethylamine. Optimal growth conditions were near pH 5.1 and 35 °C. The culture grew in basal medium containing as little as 0.43 mM Na(+) and growth was inhibited completely by 50 mM NaCl. To our knowledge, strain 6A8(T) is one of the most acidiphilic (lowest pH optimum) and salt-sensitive methanogens in pure culture. Acetate, coenzyme M, vitamins and yeast extract were required for growth. It is proposed that a new genus and species be established for this organism, Methanoregula boonei gen. nov., sp. nov. The type strain of Methanoregula boonei is 6A8(T) (=DSM 21154(T) =JCM 14090(T)).

Characterization of acid-tolerant H/CO-utilizing methanogenic enrichment cultures from an acidic peat bog in New York State.

Two methanogenic cultures were enriched from acidic peat soil using a growth medium buffered to c. pH 5. One culture, 6A, was obtained from peat after incubation with H(2)/CO(2), whereas culture NTA was derived from a 10(-4) dilution of untreated peat into a modified medium. 16S rRNA gene clone libraries from each culture contained one methanogen and two bacterial sequences. The methanogen 16S rRNA gene sequences were 99% identical with each other and belonged to the novel "R-10/Fen cluster" family of the Methanomicrobiales, whereas their mcrA sequences were 96% identical. One bacterial 16S rRNA gene sequence from culture 6A belonged to the Bacteroidetes and showed 99% identity with sequences from methanogenic enrichments from German and Russian bogs. The other sequence belonged to the Firmicutes and was identical to a thick rod-shaped citrate-utilizing organism isolated from culture 6A, the numbers of which decreased when the Ti (III) chelator was switched from citrate to nitrilotriacetate. Bacterial clones from the NTA culture clustered in the Delta- and Betaproteobacteria. Both cultures contained thin rods, presumably the methanogens, as the predominant morphotype, and represent a significant advance in characterization of the novel acidiphilic R-10 family methanogens.

Isolation of a novel acidiphilic methanogen from an acidic peat bog.

Acidic peatlands are among the largest natural sources of atmospheric methane and harbour a large diversity of methanogenic Archaea. Despite the ubiquity of methanogens in these peatlands, indigenous methanogens capable of growth at acidic pH values have resisted culture and isolation; these recalcitrant methanogens include members of an uncultured family-level clade in the Methanomicrobiales prevalent in many acidic peat bogs in the Northern Hemisphere. However, we recently succeeded in obtaining a mixed enrichment culture of a member of this clade. Here we describe its isolation and initial characterization. We demonstrate that the optimum pH for methanogenesis by this organism is lower than that of any previously described methanogen.