Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru

Abstract

Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane-carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100-2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.

Fig. 1.

A 16S rRNA archaeal phylogenetic tree based on maximum likelihood distances of ≈900 nucleotide positions (16S rRNA positions 23–914). Bootstrap numbers are based on 200 resamplings. Sequences were obtained from four sediment samples from each of the four SMTZs encountered during ODP Leg 201: 1227D-37.8 (ODP Hole-depth in hole in meters below seafloor), 1229D-29.4, 1229D-86.8, and 1230A-11.0. Closely related sequence clusters are represented by single sequences, annotated with the number of near-identical 16S rRNA clones that they represent and their GenBank accession numbers. Sequences are color coded by habitat to illustrate the diverse environmental range of these uncultured archaeal lineages.

Fig. 2.

Microscopic images of archaeal cells (A–D) and chromatograms of gas and liquid chromatographic analyses of archaeal lipids (E). (A–D) Environmental scanning electron microscopy (ESEM) images of individual cell targets in the following samples: 1227D-34.4 (A) (ODP Hole-depth in hole in meters below seafloor), 1229D-29.8 (B), 1229D-86.8 (C), and 1230C-9.1 (D). (Scale bars: 1 μm.) Insets show FISH images of the same cells. All ESEM analyses showed small cells, most <1 μm in diameter. All images have been subjected to postexposure software enhancement to increase brightness and contrast. (E Lower) Positive ion base-peak HPLC-MS chromatogram of a fraction resulting from consecutive trapping of archaeal IPLs by preparative HPLC-MS of sample 1229D-87.1. Red labeled peaks designate archaeal glycerol-dialkyl-glycerol-tetraethers (GDGTs) with smaller quantities of coeluting archaeal glyceroldialkylethers (structures shown; ∗, tentatively identified as GDGT derivative). (Upper) Total ion current GC-MS chromatogram of isoprenoid hydrocarbons released from IPLs by ether cleavage and subsequent reduction (δ¹³C of individual compounds is reported in Table 3).

Fig. 3.

δ¹³C values of sedimentary methane (filled triangles; linearly interpolated from sample depth used for gas analysis to depth of samples used for FISH and IPL analysis; endpoints of bars indicate actual δ values of methane), DIC (green squares; linearly interpolated, as above), archaeal cells determined by FISH–secondary ion MS (filled red circles; weighted mean of all samples for each site; bars show the error on that mean; mean squares weighted deviation ranges were 0.1–1.5), and archaeal IPLs (open red circles; circle shows the weighted mean δ¹³C of all IPL-derived archaeal hydrocarbons; line shows the range of individual compounds). Brown bars designate δ¹³C of TOC. Gray-shaded samples stem from sediments above the SMTZ. Sample names: ODP Hole-depth in hole in meters below seafloor.