Multiple Archaeal Groups Mediate Methane Oxidation in Anoxic Cold Seep Sediments
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Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments Victoria J. Orphan*, Christopher H. House†, Kai-Uwe Hinrichs‡, Kevin D. McKeegan§, and Edward F. DeLong*¶ *Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039; †Department of Geosciences, Penn State University, University Park, PA 16802; ‡Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA 02543; and §Department of Earth and Space Sciences, University of California, Los Angeles, CA 90095 Communicated by John M. Hayes, Woods Hole Oceanographic Institution, Woods Hole, MA, April 6, 2002 (received for review January 23, 2002) No microorganism capable of anaerobic growth on methane as the archaeal origin are comprised of at least 6 distinct compounds, sole carbon source has yet been cultivated. Consequently, infor- suggesting their production by more than one source organism mation about these microbes has been inferred from geochemical (5–9). The diversity of archaeal 16S rRNA genes in methane- and microbiological observations of field samples. Stable isotope seep sediments also suggested the presence of multiple groups of analysis of lipid biomarkers and rRNA gene surveys have impli- putative methane-oxidizing Archaea (3, 4, 10, 11). The occur- cated specific microbes in the anaerobic oxidation of methane rence of ANME-1 and ANME-2 in a wide variety seep environ- (AOM). Here we use combined fluorescent in situ hybridization and ments indicates that both archaeal groups may be involved in secondary ion mass spectrometry analyses, to identify anaerobic AOM, and could serve as a source of the 13C-depleted archaeal methanotrophs in marine methane-seep sediments. The results lipids found in methane-seep sediments. provide direct evidence for the involvement of at least two distinct Microscopic evidence has indicated that the archaeal archaeal groups (ANME-1 and ANME-2) in AOM at methane seeps. ANME-2 group is physically associated with a sulfate-reducing Although both archaeal groups often occurred in direct physical bacterial partner (4, 7). In this association, the ANME-2 archaea association with bacteria, they also were observed as monospecific are located in the central core of each microbial aggregate and aggregations and as single cells. The ANME-1 archaeal group more are surrounded by a shell of Desulfosarcina spp. This physical frequently existed in monospecific aggregations or as single fila- association provided direct evidence for microbial consortia that ments, apparently without a bacterial partner. Bacteria associated couple AOM with sulfate reduction. In this case, the archaeon with both archaeal groups included, but were not limited to, close (ANME-2) is thought to mediate ‘‘reverse methanogenesis’’ (7, relatives of Desulfosarcina species. Isotopic analyses suggest 12, 13). The bacterial partner is presumed to couple the oxida- that monospecific archaeal cells and cell aggregates were active tion of incompletely oxidized byproducts of AOM (e.g., H2, in anaerobic methanotrophy, as were multispecies consortia. In acetate or formate; 1, 12, 14) to the reduction of sulfate. total, the data indicate that the microbial species and biotic Combined phylogenetic identification using fluorescent in situ interactions mediating anaerobic methanotrophy are diverse and hybridization (FISH) and secondary ion mass spectrometry complex. The data also clearly show that highly structured ANME- (SIMS) provided isotopic evidence directly implicating ANME- ͞ 2 Desulfosarcina consortia are not the sole entities responsible for 2͞Desulfosarcina consortia in AOM (13). AOM at marine methane seeps. Other microbial groups, including Isotopic analyses of biomarkers suggest that a diversity of ANME-1 archaea, are capable of anaerobic methane consumption microbial groups may be associated with AOM at both methane either as single cells, in monospecific aggregates, or in multispecies seeps (3–9, 13), as well as in subseafloor diffusion-limited consortia. environments (15). In this study the involvement of additional microbial groups in the anaerobic oxidation of methane was arge reservoirs of methane lie buried beneath the seafloor investigated, by using coupled isotopic and phylogenetic analyses Ldissolved in pore fluids, crystallized in solid phase gas at the single-cell level. Our results, which combine phylogenetic hydrates, or as free gas. Typically, little of the methane released and isotopic analyses (FISH–SIMS) of single cells, demonstrate from this enormous natural gas reservoir reaches the oxic water a greater diversity of microbes and microbial associations in column. Instead, it is oxidized to CO2 by microbes living in AOM than has been previously reported. anoxic marine sediments. The amount of methane consumed by anaerobic oxidation of methane (AOM) each year is approxi- Methods mately equivalent to 5 to 20% of the total annual methane flux Sample Collection. Methane-seep sediments from Eel River Basin to the atmosphere (1, 2). Geochemical evidence, isotopic data, (ERB) were collected in October 1999 and August 2000 by MICROBIOLOGY and microbial process measurements all indicate that AOM is ROV-operated push cores. High levels of methane and sulfide directly coupled to the reduction of sulfate in marine sediments. in pore waters in Eel River Basin sediments foster the growth of The specific microbes, biochemical pathways, and physiological chemolithotrophic microorganisms. Sediments underlying che- interactions regulating AOM however, are not well understood. mosynthetic clams (Calyptogena pacifica, sample PC-21) and Recent studies combining stable isotopic analyses of lipid sulfur-oxidizing filamentous bacterial mats (sample PC-45) were biomarkers with 16S rRNA gene surveys have identified several used for FISH–SIMS studies described here. Sediments were candidate microbial groups involved in AOM (3, 4). Initially, processed in 3-cm-thick sections as described (13) and subdi- 13C-depleted archaeal and bacterial biomarkers were discovered 13 vided for chemical porewater analysis (measured on site) or in marine methane-seep sediments (3–5). Several of these C- molecular studies. Samples for FISH or nucleic acid extractions depleted lipid biomarkers were characteristic of specific types of were frozen in liquid nitrogen or stored in ethanol͞PBS methanogenic archaea. In the same samples (3, 4), two archaeal Ϫ ͞ (1:1, vol/vol) at 20°C (PBS is 10 mM Na2HPO4 130 mM NaCl, phylogenetic groups (ANME-1 and ANME-2) were found to be pH 7.2). abundant among the recovered rRNA gene clones. Subse- quently, 13C-depleted archaeal lipid biomarkers have been re- covered from other anoxic, methane-rich marine sedimentary Abbreviations: AOM, anaerobic oxidation of methane; FISH, fluorescent in situ hybridiza- habitats, and have been shown to be heterogeneous in both their tion; SIMS, secondary ion mass spectrometry; ERB, Eel River Basin. structure and isotopic composition (6–9). Lipid biomarkers of ¶To whom reprint requests may be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.072210299 PNAS ͉ May 28, 2002 ͉ vol. 99 ͉ no. 11 ͉ 7663–7668 Downloaded by guest on October 1, 2021 Sample Processing for FISH and FISH–SIMS. Sediment samples pro- SIMS instrument), and target cells were then mapped in refer- cessed in three different ways were all successfully used for ence to the score mark. Digital images of individual cells and cell FISH–SIMS. Small aliquots of methane-seep sediment (0.5 g) aggregates hybridizing with group specific oligonucleotide were fixed overnight at 4°C in 3.7% formaldehyde in PBS, probes were captured by using a Spot SP100 cooled, charge washed 2 times with PBS, and stored (Ϫ20°C) in ethanol: PBS coupled device camera (Diagnostic Instruments, Sterling (1:1, vol/vol). Alternatively, sediments were stored at Ϫ20°Cin Heights, MI). Images were recorded by using both epifluroes- ethanol͞PBS (1:1, vol/vol) without the formaldehyde fixation cence and phase microscopy with 80ϫ and 10ϫ objectives. step. In a third procedure, sediments frozen at Ϫ80°C for up to Images were then analyzed in ADOBE PHOTOSHOP 6.0, and po- 2 years were thawed in ethanol: PBS (1:1, vol/vol) and used in the tential targets for analysis with the SIMS instrument were whole-cell FISH assays. Successful assays were obtained by using located and highlighted on the image. samples prepared by each of these procedures. Before process- ing, sediments were diluted 1:20 (vol/vol) in PBS to a final SIMS Analysis. Carbon isotopic compositions (12C͞13C) of indi- volume of 0.5 ml, sonicated on ice with a Vibra Cell sonicating vidual cells and cell aggregates previously identified with FISH wand (Sonics and Materials, Danbury, CT) for 20 s at an were determined by using the UCLA CAMECA IMS 1270 ion Ϫ amplitude setting of 30, and overlaid on top of a pre-established microprobe as described (13). Secondary C2 ions were ejected Percoll gradient (see below). from the surface of the target biomass by using a 10- to 15-m cesium ion beam and analyzed by using a multicollector arrange- Separation of Cells from Sediment–Percoll Density Gradients and ment consisting of two electron multipliers (12C13CϪ detected 12 Ϫ Filtration. Percoll gradients were established before sample ad- on-axis and C2 detected off axis). For this study, a relatively dition. Percoll was initially diluted 1:1 (vol/vol) with filter- low intensity primary Csϩ beam was used (0.03 nanoampere; nA) sterilized PBS and 10-ml aliquots were distributed into Sorvall to allow for