Characterization of the archaeal community in a minerotrophic fen and terminal restriction fragment length polymorphism-directed isolation of a novel hydrogenotrophic methanogen

Abstract

Minerotrophic fen peatlands are widely distributed in northern latitudes and, because of their rapid turnover of organic matter, are potentially larger sources of atmospheric methane than bog peatlands per unit area. However, studies of the archaeal community composition in fens are scarce particularly in minerotrophic sites. Several 16S rRNA-based primer sets were used to obtain a broad characterization of the archaeal community in a minerotrophic fen in central New York State. A wide archaeal diversity was observed in the site: 11 euryarchaeal and 2 crenarchaeal groups, most of which were uncultured. The E1 group, a novel cluster in the order Methanomicrobiales, and Methanosaetaceae were the codominant groups in all libraries and results of terminal restriction fragment length polymorphism (T-RFLP) analysis. Given its abundance and potential hydrogenotrophic methane contribution, the E1 group was targeted for culture attempts with a low-ionic-strength medium (PM1). Initial attempts yielded Methanospirillum-dominated cultures. However, by incorporating a T-RFLP analysis as a quick selection tool for treatments and replicates, we were able to select an enrichment dominated by E1. Further dilutions to 10(-9) and tracking with T-RFLP yielded a strain named E1-9c. E1-9c is a novel coccoid hydrogenotrophic, mesophilic, slightly acidophilic methanogen and is highly sensitive to Na(2)S concentrations (requires <0.12 mM for growth). We propose E1-9c as the first representative of a novel genus in the Methanomicrobiales order.

FIG. 1.

Phylogenetic analysis of archaeal 16S rRNA gene clones from the MH fen. Members of the order Methanosarcinales include Methanosarcinaceae (MS), Methanosaetaceae (MT); subaqueous cluster (SC); rice cluster I (RC-I); rice cluster II (RC-II). Member of the order Methanomicrobiales include group E1, group E1′, group E2, and Methanospirillaceae (MP). Members of the order Methanobacteriales include Methanobacteriaceae (MB), marine benthic group D (MBD). The tree was constructed by Bayesian analysis using MrBayes software version 3 (see Materials and Methods); posterior probabilities greater than 80 are indicated. Clones from MH fen are indicated by the initials MH, followed by the indication of their primer mixture of origin: 1100 (1af-1100r), 1492r (1af-1492r), and LSU47 (1af-LSU47r).

FIG. 2.

Clone distribution from libraries constructed with different primer combinations. The total number of clones (n) and primer combination (Pc) are indicated for each primer set. The recovered groups are Methanosarcinaceae (MS), Methanosaetaceae (MT), subaqueous cluster (SC), rice cluster I (RC-I), rice cluster II (RC-II), group E1 (E1), group E1′ (E1′), group E2 (E2), Methanospirillaceae (MP), Methanobacteriaceae (MB), marine benthic group D (MBD), rice cluster IV (RC-IV), and rice cluster VI (RC-VI).

FIG. 3.

CH₄ production by peat slurries from MH fen. Samples were nonamended statically grown (white circles) or statically grown and amended with 1 mM acetate (black circles), shaken and amended with rifampin (black triangles), or shaken and amended with H₂-CO₂ and rifampin (white triangles). Points represent the means ± standard deviations for three samples.

FIG. 4.

T-RFLP analysis of peat samples and enrichments using the 1Af-1100r primer set and HhaI-Sau96 restriction enzymes. T-RFLP profiles (A) were standardized to a total of 100 relative fluorescence units (RFU), and the peaks were matched with their corresponding groups are presented in a single-column format (B and C). Initial (panel 1. Init) or nonincubated samples are grouped with incubated slurries (A and B) amended with rifampin only (panel 2. rif) or H₂-CO₂/rifampin (panel 3. H₂-CO₂/rif). Enrichment culture (C) profiling was done with the highest dilution reached for of each E1-targeted culture transfer: transfer 1 dilution, 10⁻⁵ (T1-5); transfer 2 dilution, 10⁻⁶ (T2-6); transfer 5 dilution, 10⁻⁹ (T5-9). Other enrichment attempts that yielded Methanospirillum (Mthsp) and Methanobacterium (Mthbc) and related organisms were also included. MS, Methanosarcinaceae; MT, Methanosaetaceae; SC, subaqueous cluster; RC-I, rice cluster I; RC-II, rice cluster II; E1, group E1; E1′, group E1′; E2, group E2; MP, Methanospirillaceae; MB, Methanobacteriaceae.

FIG. 5.

Microscopy examination of E1-9c cells. (A) Phase-contrast microscopy; (B) phase-contrast and fluorescence microscopy showing the autofluorescence of cells illuminated with light near 420 nm; (C) FISH, using the 915-Arch probe; (D) negative stain electron microscopy.

FIG. 6.

Physiological characterization of E1-9c. Effect of temperature on growth and methanogenesis (A); effect of pH on growth and methanogenesis (B); effect of sulfide (Na₂S) on methanogenesis (C). The asterisk represents a set of tubes with H₂S gas addition instead of Na₂S (as H₂S gas was initially used for the isolation of E1-9c). Points represent the means ± standard deviations for three samples.