Multilayer approach for characterization of bacterial diversity in a marginal sea: From surface to seabed

Bacteria are the most important microorganisms in the world oceans, accounting for up to 75% of the total biomass. They are responsible for fundamental biogeochemical processes and therefore often used as ecological indicators. In this study, bacteria were quantified by flow cytometry and their diversity assessed by High Throughput Sequencing (HTS) in the southern Adriatic Sea. The most abundant bacterial groups were also quantified by qPCR. The samples were collected from the surface to the seabed over a total of 16 different depths at four stations during the late winter BIOTA (BIO-Tracing Adriatic water masses) cruise conducted in March 2016. The investigated area showed unusual water mass properties and was characterized by a shallow mixed layer, which differed from the usual winter convection conditions, typical of middle-altitude ecosystems and important for the seasonal picoplankton dynamics of this area. Heterotrophic bacteria were separated into HNA (relative High Nucleic Acid content) and LNA (Low Nucleic Acid content) subpopulations with abundances up to 1.8 × 105 and 8.8 × 105 cells mL−1, respectively. HNA dominated at offshore stations reaching their maximum at depths below the euphotic zone. The bacterial community was dominated by Alphaproteobacteria, accounting for >40% of the total sequence reads and were mainly represented by the SAR11 clade (90.84%), followed by Marinimicrobia (18% of the total sequence reads), mainly represented by clade SAR406 (8.44%). Distinctive bacterial groups were found in the euphotic layer (Bacteroidetes and Actinobacteria) and aphotic layer samples (Deltaproteobacteria, Marinimicrobia, Chloroflexi, Acidobacteria and Planctomycetes). Results of the qPCR analyses further confirmed HTS results with highest abundances obtained for Alphaproteobacteria, followed by Gammaproteobacteria and Bacteroidetes. The adopted multiple approach, combining different molecular tools, critically supported by optics and flow cytometry, reveal changes in the bacterial assemblages during the unusual thermohaline conditions observed in 2016 in the southern Adriatic Sea.