Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C(1) metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean's largest habitat.

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.

The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4,6-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up L-aspartic acid (19% versus 10%).

A fraction of the carbon captured by phytoplankton in the sunlit surface ocean sinks to depth as dead organic matter and faecal material. The microbial breakdown of this material in the subsurface ocean generates carbon dioxide. Collectively, this microbially mediated flux of carbon from the atmosphere to the ocean interior is termed the biological pump. In recent decades it has become clear that the composition of the phytoplankton community in the surface ocean largely determines the quantity and quality of organic matter that sinks to depth. This settling organic matter, however, is not sufficient to meet the energy demands of microbes in the dark ocean. Two additional sources of organic matter have been identified: non-sinking organic particles of debated origin that escape capture by sediment traps and exhibit stable concentrations throughout the dark ocean, and microbes that convert inorganic carbon into organic matter. Whether these two sources can together account for the significant mismatch between organic matter consumption and supply in the dark ocean remains to be seen. It is clear, however, that the microbial community of the deep ocean works in a fundamentally different way from surface water communities.In the sunlit surface waters of the ocean, phytoplankton convert carbon dioxide into particulate organic carbon. A significant fraction of this newly produced particulate organic carbon is remineralized-that is, respired back to carbon dioxide-in these surface waters. However, about 1 to 40% of the photosynthetically fixed carbon is exported into the dark realm of the ocean where it is remineralized at substantially slower rates than in surface waters 1 . The resultant increase in dissolved inorganic carbon concentrations towards the interior of the ocean, coined the biological pump, is regulated by food web processes such as grazing.Non-living particles, termed 'marine snow' 2-4 , transport organic matter to depth in the ocean. These particles comprise decaying phytoplankton, faecal matter generated by zooplankton, the most abundant multicellular organisms in the oceanic water column, and aggregates of high-molecular-weight dissolved organic matter released by phytoplankton in the senescent stage of blooms, or as a result of an imbalance in nutrient supply ratios 5,6 . The aggregation of high-molecular-weight dissolved organic matter, largely in the form of © 2013 Macmillan Publishers Limited. All rights reserved * Correspondence and requests for materials should be addressed to G.J.H. gerhard.herndl@univie.ac.at. Competing financial interestsThe authors declare no competing financial interests. Additional informationReprints and permissions information is available online at www.nature.com/reprints. Europe PMC Funders GroupAuthor Manuscript Nat Geosci. Author manuscript; available in PMC 2014 April 02. Published in final edited form as:Nat Geosci. 2013 September 1; 6(9): 718-724. doi:10.1038/ngeo1921. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts polysacchari...

a b s t r a c tMetabolic activity in the water column below the euphotic zone is ultimately fuelled by the vertical flux of organic material from the surface. Over time, the deep ocean is presumably at steady state, with sources and sinks balanced. But recently compiled global budgets and intensive local field studies suggest that estimates of metabolic activity in the dark ocean exceed the influx of organic substrates. This imbalance indicates either the existence of unaccounted sources of organic carbon or that metabolic activity in the dark ocean is being over-estimated. Budgets of organic carbon flux and metabolic activity in the dark ocean have uncertainties associated with environmental variability, measurement capabilities, conversion parameters, and processes that are not well sampled. We present these issues and quantify associated uncertainties where possible, using a Monte Carlo analysis of a published data set to determine the probability that the imbalance can be explained purely by uncertainties in measurements and conversion factors. A sensitivity analysis demonstrates that the bacterial growth efficiencies and assumed cell carbon contents have the greatest effects on the magnitude of the carbon imbalance. Two poorly quantified sources, lateral advection of particles and a population of slowly settling particles, are discussed as providing a means of closing regional carbon budgets. Finally, we make recommendations concerning future research directions to reduce important uncertainties and allow a better determination of the magnitude and causes of the unbalanced carbon budgets.