21 pages, 6 figures, supplementary information https://doi.org/10.1038/s41564-023-01374-2.-- Data availability: The sequence data generated in this study have been deposited in the EMBL Nucleotide Sequence Database (ENA) database under Bioproject PRJEB35712 (metagenomic and metatranscriptomic raw reads, metagenomic and metatranscriptomic assemblies, metagenomic assembled genomes and single-cell amplified genomes) and in the NCBI Sequence Read Archive (SRA) under Bioproject PRJNA593264 (16S rRNA amplicon reads).-- Code availability: Scripts available at Zenodo (https://doi.org/10.5281/zenodo.7721930.2023), The deep ocean (>200 m depth) is the largest habitat on Earth. Recent evidence suggests sulfur oxidation could be a major energy source for deep ocean microbes. However, the global relevance and the identity of the major players in sulfur oxidation in the oxygenated deep-water column remain elusive. Here we combined single-cell genomics, community metagenomics, metatranscriptomics and single-cell activity measurements on samples collected beneath the Ross Ice Shelf in Antarctica to characterize a ubiquitous mixotrophic bacterial group (UBA868) that dominates expression of RuBisCO genes and of key sulfur oxidation genes. Further analyses of the gene libraries from the ‘Tara Oceans’ and ‘Malaspina’ expeditions confirmed the ubiquitous distribution and global relevance of this enigmatic group in the expression of sulfur oxidation and dissolved inorganic carbon fixation genes across the global mesopelagic ocean. Our study also underscores the unrecognized importance of mixotrophic microbes in the biogeochemical cycles of the deep ocean, This research was facilitated by the New Zealand Antarctic Research Institute (NZARI)-funded Aotearoa New Zealand Ross Ice Shelf Program. Samples for MICRO-CARD-FISH were collected on several research cruises led by M. Simon (Sonne 248 cruise), B. Quéguiner and I. Obernosterer (MobyDick) and L. J. A. Gerringa (Geotraces-1). F.B. was supported by the Austrian Science Fund (FWF) projects OCEANIDES (P34304-B), ENIGMA (TAI534), EXEBIO (P35248) and OCEANBIOPLAST (P35619-B). G.J.H. was supported by the FWF project ARTEMIS (P28781-B21) and I486-B09 and by the ERC under the European Community’s 7th Framework Programme (FP7/2007-2013)/ERC grant agreement no. 268595 (MEDEA project). R.L. was supported by INTERACTOMICS, CTM2015-69936-P, and J.M.G. by project PID2019-110011RB-C32 (Spanish Ministry of Science and Innovation, Spanish State Research Agency, doi: 10.13039/501100011033), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), No

Bulk dark dissolved inorganic carbon (DIC) fixation rates were determined and compared to microbial heterotrophic production in subsurface, meso- and bathypelagic Atlantic waters off the Galician coast (NW Iberian margin). DIC fixation rates were slightly higher than heterotrophic production throughout the water column, however, more prominently in the bathypelagic waters. Microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH) allowed us to identify several microbial groups involved in dark DIC uptake. The contribution of SAR406 (Marinimicrobia), SAR324 (Deltaproteobacteria) and Alteromonas (Gammaproteobacteria) to the dark DIC fixation was significantly higher than that of SAR202 (Chloroflexi) and Thaumarchaeota, in agreement with their contribution to microbial abundance. Q-PCR on the gene encoding for the ammonia monooxygenase subunit A (amoA) from the putatively high versus low ammonia concentration ecotypes revealed their depth-stratified distribution pattern. Taken together, our results indicate that chemoautotrophy is widespread among microbes in the dark ocean, particularly in bathypelagic waters. This chemolithoautotrophic biomass production in the dark ocean, depleted in bio-available organic matter, might play a substantial role in sustaining the dark ocean's food web., Funding for the sampling and analyses was provided by the projects ‘Biodiversidade Funcional do Microplancton nas profundidades mariñas de Galicia’ (BIO-PROF, Ref. 10MMA604024PR, 2010–2012, Xunta de Galicia) and ‘Fuentes de Materia Orgánica y Diversidad Funcional del Microplancton en las aguas profundas del Atlántico Norte’ (MODUPLAN, Plan Nacional I + D+I) to MMV. Additional funding was provided by the Axencia Galega de Innovación (GAIN, Xunta de Galicia) through IEO-GAIN Programme Contracts (Contratos Programa). EGF was supported by the BIO-PROF and MODUPLAN projects. ES received funding by the Austrian Science Fund (FWF) project P27696-B22 and GJH was funded by the Austrian Science Fund (FWF) projects I486-B09 and Z194-B17 and the European Research Council under the European Community's Seventh Framework Program (FP7/2007–2013)/ERC grant agreement No. 268595 (MEDEA project). This work is in partial fulfilment of the requirements for a Ph.D. degree from the Universidade de A Coruña by E.G.-F., Peer reviewed