Prokaryotic abundance, activity and community composition were studied in the euphotic, intermediate and deep waters off the Galician coast (NW Iberian margin) in relation to the optical characterization of dissolved organic matter (DOM). Microbial (archaeal and bacterial) community structure was vertically stratified. Among the Archaea, Euryarchaeota, especially Thermoplasmata, was dominant in the intermediate waters and decreased with depth, whereas marine Thaumarchaeota, especially Marine Group I, was the most abundant archaeal phylum in the deeper layers. The bacterial community was dominated by Proteobacteria through the whole water column. However, Cyanobacteria and Bacteroidetes occurrence was considerable in the upper layer and SAR202 was dominant in deep waters. Microbial composition and abundance were not shaped by the quantity of dissolved organic carbon, but instead they revealed a strong connection with the DOM quality. Archaeal communities were mainly related to the fluorescence of DOM (which indicates respiration of labile DOM and generation of refractory subproducts), while bacterial communities were mainly linked to the aromaticity/age of the DOM produced along the water column. Taken together, our results indicate that the microbial community composition is associated with the DOM composition of the water masses, suggesting that distinct microbial taxa have the potential to use and/or produce specific DOM compounds.

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.

The processes mediated by microbial planktonic communities occur along the entire water column, yet the microbial activity and composition have been studied mainly in surface waters. This research examined the vertical variation in bacterial abundance, activity and community composition and structure from surface down to 5000 m depth following a longitudinal transect off the Galician coast (NW Iberian margin, from 43ºN, 9ºW to 43ºN, 15ºW). Community activity and composition changed with depth. The leucine incorporation rates decreased from the euphotic layer to the bathypelagic waters by three orders of magnitude, whereas prokaryotic abundance decreased only by one order of magnitude. The relative abundance of SAR11 and Alteromonas, determined by catalyzed reported deposition fluorescence in situ hybridization (CARD-FISH), decreased with depth. Meanwhile, the contribution of SAR 202 and SAR324 was significantly higher in the deeper layers (i.e. NEADW, North East Atlantic Deep Water and LDW, Lower Deep Water) than in the euphotic zone. Bacterial community structure, assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA), was depth-specific. A distance based linear model (DistLM) revealed that the variability found in bacterial community structure was mainly explained by temperature nitrate, phosphate, dissolved organic matter (DOM) fluorescence, prokaryotic abundance, leucine incorporation and to a lesser extent salinity, oxygen, CDOM absorbance and dissolved organic carbon. Our results displayed a bacterial community structure shaped not only by depth-related physicochemical features but also by DOM quality, indicating that different prokaryotic taxa have the potential to metabolize particular DOM sources.

To study the response of bacteria to different size-fractions of naturally occurring dissolved organic matter (DOM), a natural prokaryotic community from North Atlantic mesopelagic waters (1000 m depth) was isolated and grown in (i) 0.1-μm filtered seawater (CONTROL), (ii) the low-molecular-weight (<1 kDa) DOM fraction (L-DOM), and (iii) the recombination of high- (>1 kDa) and low-molecular-weight DOM fractions (H + L-DOM), to test the potential effect of ultrafiltration on breaking the DOM size continuum. Prokaryotic abundance and leucine incorporation were consistently higher in the H + L-DOM niche than in the L-DOM and CONTROL treatments, suggesting a different interaction with each DOM fraction and the disruption of the structural DOM continuum by ultrafiltration, respectively. Rhodobacterales (Alphaproteobacteria) and Flavobacteriales (Bacteroidetes) were particularly enriched in L-DOM and closely related to the colored DOM (CDOM) fraction, indicating the tight link between these groups and changes in DOM aromaticity. Conversely, some other taxa that were rare or undetectable in the original bacterial community were enriched in the H + L-DOM treatment (e.g., Alteromonadales belonging to Gammaproteobacteria), highlighting the role of the rare biosphere as a seed bank of diversity against ecosystem disturbance. The relationship between the fluorescence of protein-like CDOM and community composition of populations in the H + L-DOM treatment suggested their preference for labile DOM. Conversely, the communities growing on the L-DOM niche were coupled to humic-like CDOM, which may indicate their ability to degrade more reworked DOM and/or the generation of refractory substrates (as by-products of the respiration processes). Most importantly, L- and/or H + L-DOM treatments stimulated the growth of unique bacterial amplicon sequence variants (ASVs), suggesting the potential of environmental selection (i.e., changes in DOM composition and availability), particularly in the light of climate change scenarios. Taken together, our results suggest that different size-fractions of DOM induced niche-specialization and differentiation of mesopelagic bacterial communities.

Understanding the factors that modulate prokaryotic assemblages and their niche partitioning in marine environments is a longstanding challenge in marine microbial ecology. This study analyzes amplicon sequence variant (ASV) diversity and co-occurrence of prokaryotic (Archaea and Bacteria) communities through coastal-oceanic gradients in the NW Iberian upwelling system and adjacent open-ocean (Atlantic Ocean). Biogeographic patterns were investigated in relation with environmental conditions, mainly focusing on the optical signature of the dissolved organic matter (DOM). Alpha- and beta-diversity were horizontally homogeneous [with the only exception of Archaea (∼1700 m depth), attributed to the influence of Mediterranean water, MW], while beta-diversity was significantly vertically stratified. Prokaryotic communities were structured in four clusters (upper subsurface, lower subsurface, intermediate, and deep clusters). Deep (>2000 m) archaeal and bacterial assemblages, and intermediate (500-2000 m) Bacteria (mainly SAR202 and SAR406), were significantly related to humic-like DOM (FDOM-M), while intermediate Archaea were additionally related to biogeochemical attributes of the high-salinity signature of MW. Lower subsurface (100-500 m) Archaea (particularly one ASV belonging to the genus Candidatus Nitrosopelagicus) were mainly related to the imprint of high-salinity MW, while upper subsurface (≤100 m) archaeal assemblages (particularly some ASVs belonging to Marine Group II) were linked to protein-like DOM (aCDOM254). Conversely, both upper and lower subsurface bacterial assemblages were mainly linked to aCDOM254 (particularly ASVs belonging to Rhodobacteraceae, Cyanobacteria, and Flavobacteriaceae) and nitrite concentration (mainly members of Planctomycetes). Most importantly, our analysis unveiled depth-ecotypes, such as the ASVs MarG.II_1 belonging to the archaeal deep cluster (linked to FDOM-M) and MarG.II_2 belonging to the upper subsurface cluster (related to FDOM-T and aCDOM254). This result strongly suggests DOM-mediated vertical niche differentiation, with further implications for ecosystem functioning. Similarly, positive and negative co-occurrence relationships also suggested niche partitioning (e.g., between the closely related ASVs Thaum._Nit._Nit._Nit._1 and _2) and competitive exclusion (e.g., between Thaum._Nit._Nit._Nit._4 and _5), supporting the finding of non-randomly, vertically structured prokaryotic communities. Overall, differences between Archaea and Bacteria and among closely related ASVs were revealed in their preferential relationship with compositional changes in the DOM pool and environmental forcing. Our results provide new insights on the ecological processes shaping prokaryotic assembly and biogeography.