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.anaerobic methanotrophy ͉ deep biosphere ͉ FISH-secondary ion MS ͉ intact polar lipids ͉ stable carbon isotopes

The subseafloor marine biosphere may be one of the largest reservoirs of microbial biomass on Earth and has recently been the subject of debate in terms of the composition of its microbial inhabitants, particularly on sediments from the Peru Margin. A metagenomic analysis was made by using whole-genome amplification and pyrosequencing of sediments from Ocean Drilling Program Site 1229 on the Peru Margin to further explore the microbial diversity and overall community composition within this environment. A total of 61.9 Mb of genetic material was sequenced from sediments at horizons 1, 16, 32, and 50 m below the seafloor. These depths include sediments from both primarily sulfate-reducing methane-generating regions of the sediment column. Many genes of the annotated genes, including those encoding ribosomal proteins, corresponded to those from the Chloroflexi and Euryarchaeota. However, analysis of the 16S smallsubunit ribosomal genes suggests that Crenarchaeota are the abundant microbial member. Quantitative PCR confirms that uncultivated Crenarchaeota are indeed a major microbial group in these subsurface samples. These findings show that the marine subsurface is a distinct microbial habitat and is different from environments studied by metagenomics, especially because of the predominance of uncultivated archaeal groups.Archaea ͉ Chloroflexi ͉ marine sediment ͉ quantitative PCR

Members of the highly diverse Miscellaneous Crenarchaeotal Group (MCG) are globally distributed in various marine and continental habitats. In this study, we applied a polyphasic approach (rRNA slot blot hybridization, quantitative PCR (qPCR) and catalyzed reporter deposition FISH) using newly developed probes and primers for the in situ detection and quantification of MCG crenarchaeota in diverse types of marine sediments and microbial mats. In general, abundance of MCG (cocci, 0.4 lm) relative to other archaea was highest (12-100%) in anoxic, low-energy environments characterized by deeper sulfate depletion and lower microbial respiration rates (P ¼ 0.06 for slot blot and P ¼ 0.05 for qPCR). When studied in high depth resolution in the White Oak River estuary and Hydrate Ridge methane seeps, changes in MCG abundance relative to total archaea and MCG phylogenetic composition did not correlate with changes in sulfate reduction or methane oxidation with depth. In addition, MCG abundance did not vary significantly (P40.1) between seep sites (with high rates of methanotrophy) and non-seep sites (with low rates of methanotrophy). This suggests that MCG are likely not methanotrophs. MCG crenarchaeota are highly diverse and contain 17 subgroups, with a range of intragroup similarity of 82 to 94%. This high diversity and widespread distribution in subsurface sediments indicates that this group is globally important in sedimentary processes.

1, 4,, 6, 9, 10 that exhibits peaks of cell abundance, and profiles of sulfate and methane 43 suggestive of microbial activity 1 (Figure 1). 44Picogram quantities of total RNA were extracted from 25 grams of Peru Margin sediment 45 from six depths (5, 30, 50, 70, 91, 159 mbsf), consistent with basal levels of microbial activity 46 predicted for this environment 3, 4 . Illumina ® sequencing of total cDNA produced over 1 billion 47 reads, with 50% to 85% of reads mapping to open reading frames that were assigned a functional 48 annotation (Table S1). 49The dominance of transcripts from Firmicutes, Actinobacteria, Alphaproteobacteria, and 50 Gammaproteobacteria (Fig. S1) is consistent with previous cultivation-based, metagenomic, and 51 phylogenetic surveys from Peru Margin subsurface sediment 1, 5, 13, 14 , and suggests these to be 52 some of the most active microbial groups. The abundance of gammaproteobacterial transcripts 53 ( Fig. S1) suggests that they are likely the most active microbial group in the deeper, anoxic, 54 subseafloor sediment at this site. Fungal transcripts were also present in every sample ranging in 55representation from 3% at 70 mbsf to 20% at 5 mbsf. Archaea and Chloroflexi are present in 56 noticeably low abundance, despite their previous detection at this site 6, 13, 15 , suggesting that our 57 approach might miss organisms with lower mRNA expression levels. As such, interpretations of 58 relative abundances should be treated cautiously 16 . Changes in pressure and temperature may 59 have altered gene expression during sampling. However, low representation of heat shock 60 proteins (a proxy for physiological stress response 17 ) in protein coding reads (< 10 -5 %) suggests 61 the physiological state of most microbes was not significantly altered during sample retrieval and 62 storage. 63Dissimilatory sulfate reduction may represent a major form of microbial metabolism and 64 energy production in the sub-seafloor 1, 2, 18 and is indicated by porewater sulfate concentrations at 65Site 1229 1 (Fig. 1). Representation of Dsr transcripts was highest in sediment with sulfate 66 profiles suggestive of biogenic sulfate reduction (Fig. 1) and supports biogeochemical evidence 67 for sulfate reduction at this site 1, 4 . Surprisingly, transcripts coding for dissimilatory nitrate 68 reductases (Nar) were represented throughout the sediment column, despite no measureable 69 nitrate. The origin of nitrate as a substrate in this sediment is unknown, but could potentially be 70 produced as a by-product of anaerobic ammonium oxidation. Once produced, nitrate would 71likely not accumulate to measurable concentrations given the higher free energy yield of nitrate 72 as electron acceptor compared to the dominant electron acceptors in this environment, sulfate 73 and iron. Nitrate reduction appears to be performed predominantly by Alphaproteobacteria and 74Betatproteobacteria at most depths (Fig. 1) and the resulting nitrite is likely reduced by Fungi, 75Gammaproteobacteria, and Firmicutes (Fig. S3). ...