SUMMARY The majority of life on Earth—notably, microbial life—occurs in places that do not receive sunlight, with the habitats of the oceans being the largest of these reservoirs. Sunlight penetrates only a few tens to hundreds of meters into the ocean, resulting in large-scale microbial ecosystems that function in the dark. Our knowledge of microbial processes in the dark ocean—the aphotic pelagic ocean, sediments, oceanic crust, hydrothermal vents, etc.—has increased substantially in recent decades. Studies that try to decipher the activity of microorganisms in the dark ocean, where we cannot easily observe them, are yielding paradigm-shifting discoveries that are fundamentally changing our understanding of the role of the dark ocean in the global Earth system and its biogeochemical cycles. New generations of researchers and experimental tools have emerged, in the last decade in particular, owing to dedicated research programs to explore the dark ocean biosphere. This review focuses on our current understanding of microbiology in the dark ocean, outlining salient features of various habitats and discussing known and still unexplored types of microbial metabolism and their consequences in global biogeochemical cycling. We also focus on patterns of microbial diversity in the dark ocean and on processes and communities that are characteristic of the different habitats.

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.

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