RESUMO

The production and anaerobic oxidation of methane (AOM) by microorganisms is widespread in organic-rich deep subseafloor sediments. Yet, the organisms that carry out these processes remain largely unknown. Here we identify members of the methane-cycling microbial community in deep subsurface, hydrate-containing sediments of the Peru Trench by targeting functional genes of the alpha subunit of methyl coenzyme M reductase (mcrA). The mcrA profile reveals a distinct community zonation that partially matches the zonation of methane oxidizing and -producing activity inferred from sulfate and methane concentrations and carbon-isotopic compositions of methane and dissolved inorganic carbon (DIC). McrA appears absent from sulfate-rich sediments that are devoid of methane, but mcrA sequences belonging to putatively methane-oxidizing ANME-1a-b occur from the zone of methane oxidation to several meters into the methanogenesis zone. A sister group of ANME-1a-b, referred to as ANME-1d, and members of putatively aceticlastic Methanothrix (formerly Methanosaeta) occur throughout the remaining methanogenesis zone. Analyses of 16S rRNA and mcrA-mRNA indicate that the methane-cycling community is alive throughout (rRNA to 230 mbsf) and active in at least parts of the sediment column (mRNA at 44 mbsf). Carbon-isotopic depletions of methane relative to DIC (-80 to -86‰) suggest mostly methane production by CO2 reduction and thus seem at odds with the widespread detection of ANME-1 and Methanothrix. We explain this apparent contradiction based on recent insights into the metabolisms of both ANME-1 and Methanothricaceae, which indicate the potential for methanogenetic growth by CO2 reduction in both groups.

RESUMO

The coupling of subseafloor microbial life to oceanographic and atmospheric conditions is poorly understood. We examined diagenetic imprints and lipid biomarkers of past subseafloor microbial activity to evaluate its response to glacial-interglacial cycles in a sedimentary section drilled on the Peruvian shelf (Ocean Drilling Program Leg 201, Site 1229). Multiple and distinct layers of diagenetic barite and dolomite, i.e., minerals that typically form at the sulfate-methane transition (SMT), occur at much shallower burial depth than the present SMT around 30 meters below seafloor. These shallow layers co-occur with peaks of (13)C-depleted archaeol, a molecular fossil of anaerobic methane-oxidizing Archaea. Present-day, non-steady state distributions of dissolved sulfate also suggest that the SMT is highly sensitive to variations in organic carbon flux to the surface shelf sediments that may lead to shoaling of the SMT. Reaction-transport modeling substantiates our hypothesis that shallow SMTs occur in response to cyclic sediment deposition with a high organic carbon flux during interglacials and a low organic carbon flux during glacial stages. Long diffusion distances expectedly dampen the response of deeply buried microbial communities to changes in sediment deposition and other oceanographic drivers over relatively short geological time scales, e.g., glacial-interglacial periods. However, our study demonstrates how dynamically sediment biogeochemistry of the Peru Margin has responded to glacial-interglacial change and how these changes are now preserved in the geological record. Such changes in subsurface biogeochemical zonation need to be taken into account to assess the role of the subseafloor biosphere in global element and redox cycling.

Assuntos

Sedimentos Geológicos/química , Fenômenos Geológicos , Metano/análise , Modelos Químicos , Oceanografia/métodos , Sulfato de Bário/análise , Biomarcadores/análise , Carbonato de Cálcio/análise , Isótopos de Carbono/análise , Lipídeos/análise , Magnésio/análise , Metano/metabolismo , Oxirredução , Oceano Pacífico , Peru , Fatores de Tempo

RESUMO

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3' end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples.

RESUMO

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5'-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 10(8)/g sediment close to the sediment surface to less than 10(5)/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5-1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40-121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 10(4)/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.