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 TNRT, was isolated from an anaerobic, propionate-degradation enrichment culture that was originally established from a ricefield soil sample from Taiwan. Cells were non-motile rods, 2.0–6.5 mm long by 0.3 mm wide.Filamentous (up to about 100 mm) and coccoid (about 1 mm in diameter) cells were also observed in cultures in the late exponential phase of growth. Strain TNRT grew at 20–40 6C (optimally at 37 6C), 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 H2/CO2 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 andthe methanogen-specific marker gene mcrA, strain TNRT was related most closely to Methanolineatarda NOBI-1T; levels of sequence similarities were 94.8 and 86.4%, respectively. The 16S rRNAgene sequence similarity indicates that strain TNRT and M. tarda NOBI-1T represent differentspecies within the same genus. This is supported by shared phenotypic properties, includingsubstrate usage and cell morphology, and differences in growth temperature. Based on thesegenetic and phenotypic properties, strain TNRT is considered to represent a novel species of thegenus Methanolinea, for which the name Methanolinea mesophila sp. nov. is proposed; the type strain is TNRT (5NBRC 105659T5DSM 23604T). 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

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 mm for strain SWAN1T and 0.45–0.85 1.5–35 mm for strain AL-21. The strains used H2/CO2 but not formate or other substrates for methanogenesis, grew optimally around 32–37 6C, 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 (5DSM 25820T5JCM 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

Seasonal Changes In Methanogenesis And Methanogenic Community In Three Peatlands, New York State

Fluctuating environmental conditions can promote diversity and control dominance in com- munity composition. In addition to seasonal temperature and moisture changes, seasonal supply of metabolic substrates selects populations temporally. Here we demonstrate cas- cading 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 (CH4) 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 CH4 production in a fen peatland soil, whereas addition of hydrogen (H2), and simultaneous inhibition of H2-consuming acetogenic bacte- ria with rifampicin, stimulated CH4 production in two acidic bog soils, especially, in autumn and winter. The methanogenic community structure was characterized using T-RFLP analy- ses 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 peat- land, the E1 group (Methanoregulaceae) and members of the Methanosaetaceae were co-dominant, with E1 having a high relative abundance in spring. Change in relative abun- dance profiles among methanogenic groups in response to added metabolic substrates was as predicted. The acetate-amendment increased abundance of Methanosarcinaceae, and H2-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

Carbon Quantity And Quality Drives Variation In Cave Microbial Communities And Regulates Mn(II) Oxidation

Cave ecosystems are carbon limited and thus are particularly susceptible to anthropogenic pollution. Yet, how carbon quality and quantity that can modulate the pathways and amount of Mn cycling in caves remains largely unknown. To explore Mn cycling, baseline bacterial, archaeal, and fungal communities associated with Mn(III/IV) oxide deposits were assessed in both relatively ‘pristine’ and anthropogenically impacted caves in the Appalachian Mountains (USA). Cave sites were then amended with various carbon sources that are commonly associated with anthropogenic input to determine whether they stimulate biotic Mn(II) oxidation in situ. Results revealed patterns between sites that had long-term exogenous carbon loading compared to sites that were relatively ‘pristine,’ particularly among Bacteria and Archaea. Carbon treatments that stimulated Mn(II) oxidation at several sites resulted in signi?cant changes to the microbial communities, indicating that anthropogenic input can not only enhance biotic Mn(II) oxidation, but also shape community structure and diversity. Additional carbon sources amended with copper were incubated at various cave sites to test the role that Cu(II) plays in in situ biotic Mn(II) oxidation. Media supplemented with 100 lM Cu(II) inhibited bacterial Mn(II) oxidation but stimulated fungal Mn(II) oxidation, implicating fungal use of multicopper oxidase (MCO) enzymes but bacterial use of superoxide to oxidize Mn(II). In sites with low C:N ratios, the activity of the Mn(II) oxidizing enzyme manganese peroxidase (MnP) appears to be limited (particularly by MnP-utilizing Basidiomycetes and/or Zygomycetes)

Vertical Profiles Of Methanogenesis And Methanogens In Two Contrasting Acidic Peatlands In Central New York State, USA

Northern acidic peatlands are important sources ofatmospheric methane, yet the methanogens in themare poorly characterized. We examined methanogenicactivities and methanogen populations at differentdepths in two peatlands, McLean bog (MB) and Chicagobog (CB). Both have acidic (pH 3.5–4.5) peatsoils, but the pH of the deeper layers of CB is nearneutral,reflecting its previous existence as a neutralpHfen. Acetotrophic and hydrogenotrophic methanogenesiscould be stimulated in upper samples fromboth bogs, and phylotypes of methanogens using H2/CO2 (Methanomicrobiales) or acetate (Methanosarcinales) were identified in 16S rRNA gene clone librariesand by terminal restriction fragment lengthpolymorphism (T-RFLP) analyses using a novelprimer/restriction enzyme set that we developed. Particularlydominant in the upper layers was a clade intheMethanomicrobiales, called E2 here and the R10or fen group elsewhere, estimated by quantitativepolymerase chain reaction to be present at~108cellsper gram of dry peat. Methanogenic activity was considerablylower in deeper samples from both bogs.The methanogen populations detected by T-RFLP indeeper portions of MB were mainly E2 and the unculturedeuryarchaeal rice cluster (RC)-II group, whereaspopulations in the less acidic CB deep layers wereconsiderably different, and included aMethanomicrobialesclade we call E1-E1¢, as well as RC-I, RC-II,marine benthic group D, and a new cluster that we callthe subaqueous cluster. E2 was barely detectable inthe deeper samples from CB, further evidence for theassociations of most organisms in this group withacidic habitat

Limitations And Benefits Of ARISA Intra-Genomic Diversity Fingerprinting

Monitoring diversity changes and contamination in mixed cultures and simple microcosms is challenged by fast community structure dynamics, and the need for means allowing fast, cost-efficient and accurate identification of microorganisms at high phylogenetic resolution. The method we explored is a variant of Automated rRNA Intergenic Spacer Analysis based on Intra-Genomic Diversity Fingerprinting (ARISA-IGDF), and identifies phylotypes with multiple 16S–23S rRNA gene Intergenic Transcribed Spacers. We verified the effect of PCR conditions (annealing temperature, duration of final extension, number of cycles, group-specific primers and formamide) on ARISA-IGD fingerprints of 44 strains of Shewanella. We present a digitization algorithm and data analysis procedures needed to determine confidence in strain identification. Though using stringent PCR conditions and group-specific primers allow reasonably accurate identification of strains with three ARISA-IGD amplicons within the 82–1000 bp size range, ARISA-IGDF is best forphylotypes with=4 unambiguously different amplicons. This method allows monitoring the occurrence of culturable microbes and can be implemented in applications requiring high phylogenetic resolution, reproducibility, low cost and high throughput such as identifying contamination and monitoring the evolution of diversity in mixed cultures and low diversity microcosms and periodic screening of small microbial culture libraries

Enrichment Of Peat Yields Novel Methanogens: Approaches For Obtaining Uncultured Organisms In The Age Of Rapid Sequencing

Methanogens are among the oldest forms of life on Earth and are detectable in a wide range of environments, but our knowledge of their overall diversity and functioning is limited. Peatlands in particular host a broad range of methanogens that contribute large amounts of methane to the atmosphere, but are largely under-represented in pure cultures. Here, we anaerobically enriched peat with common growth substrates, supplements and antibiotics to identifying novel methanogen sequences and potential growth conditions. Over 3 years, we obtained 28 new mcrA sequences from taxa that have remained previously uncultured and undescribed beyond distantly related clones or sequences detected in environmental samples. Evidence suggests that the novel methanogens, representing five of the seven known orders, were capable of growing on H2 as well as acetate and at temperatures ranging from 6 to ca. 22°C. Methods involving the use of ampicillin proved useful, although obtaining high methane production in the absence of H2 was difficult. Our results also indicate that many methanogens may rely on bacterial symbionts (commonly Clostridium spp.). Such enrichment approaches represent a useful intermediary between maker-gene detection and isolation, allowing us to broaden our understanding of methanogen physiological ecology while potentially providing valuable sequence data

Genome of Methanoregula Boonei 6A8 Reveals Adaptations To Oligotrophic Peatland Environments

Analysis of the genome sequence of Methanoregula boonei strain 6A8, an acidophilic methanogen isolated from an ombrotrophic (rain-fed) peat bog, has revealed unique features that likely allow it to survive in acidic, nutrient-poor conditions. First, M. boonei is predicted to generate ATP using protons that are abundant in peat, rather than sodium ions that are scarce, and the sequence of a membrane-bound methyltransferase, believed to pump Na+ in all methanogens, shows differences in key amino acid residues. Further, perhaps re?ecting the hypokalemic status of many peat bogs, M. boonei demonstrates redundancy in the predicted potassium uptake genes trk, kdp and kup, some of which may have been horizontally transferred to methanogens from bacteria, possibly Geobacter spp. Overall, the putative functions of the potassium uptake, ATPase and methyltransferase genes may, at least in part, explain the cosmopolitan success of group E1/E2 and related methanogenic archaea in acidic peat bogs

Aurantimonas Manganoxydans, Sp. Nov. And Aurantimonas Litoralis, Sp. Nov.: Mn(II) Oxidizing Representatives Of A Globally Distributed Clade Of Alpha-Proteobacteria From The Order Rhizobiales

Several closely related Mn(II)-oxidizing alpha-Proteobacteriawere isolated from very different marine environments: strainSI85-9A1 from the oxic/anoxic interface of a stratified Canadianfjord, strain HTCC 2156 from the surface waters off the Oregoncoast, and strain AE01 from the dorsal surface of a hydrothermalvent tubeworm. 16S rRNA analysis reveals that these isolatesare part of a tight phylogenetic cluster with previously characterizedmembers of the genus Aurantimonas. Other organisms withinthis clade have been isolated from disparate environments such assurface waters of the Arctic and Mediterranean seas, a deep-seahydrothermal plume, and a Caribbean coral. Further analysis ofall these strains revealed that many of them are capable of oxidizing dissolved Mn(II) and producing particulate Mn(III/IV) oxides.Strains SI85-9A1 and HTCC 2156 were characterized further. Despite sharing nearly identical 16S rRNA gene sequences with the previously described Aurantimonas coralicida,whole genome DNADNA hybridization indicated that their overall genomic similarity is low. Polyphasic phenotype characterization further supported distinguishing characteristics among these bacteria. Thus SI85-9A1 and HTCC 2156 are described as two new species within the family ‘Aurantimonadaceae’: Aurantimonas manganoxydanssp. nov. and Aurantimonas litoralis sp. nov. This clade of bacteria is widely distributed around the globe and may be important contributors to Mn cycling in many environments. Our results highlight the difficulty in utilizing 16S rRNA-based approaches to investigate the microbial ecology of Mn(II) oxidation

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 13C-NaHCO3, 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