ALBERT

Description: We investigated seawater microbial abundance, activity and diversity throughthree laboratory-controlled bottle incubations mimicking different mixing scenarios between SGD (either ambient or filtered through 0.1 µm/0.22 µm) and seawater to determine the contribution of SGD to the coastal microbial community. The experiments were conducted with five different treatments (including ambient seawater not exposed to SGD) in triplicates. The first experiment (Exp. 1) was designed to test the relative contribution of brackish discharged groundwater (salinity = 7.9 ppt vs. the ambient salinity of the SEMS of ~39.5 ppt) on the microbial productivity and abundance of reference coastal seawater by mixing different ratios (1, 5, 10 and 20% v:v) of discharged groundwater. Discharged groundwater was collected into acid-cleaned containers on the day the experiment was initiated near Achziv Nature Reserve (33° 3′52 N, 35° 6′14.94 E). The second and third experiments (Exp. 2; Exp.3) were designed to extend Exp. 1 and aimed to specifically investigate how groundwater-derived microorganisms affect the activity and abundance of marine organisms once discharged into the sea. For these experiments, fresh groundwater (FGW) was collected from drilling wells and pumped into 20 L acid-cleaned sample-rinsed carboys the same day the experiment was initiated. At the laboratory, fresh groundwater was either filtered through a 0.1 μm polycarbonate filter (Exp. 2) or serially filtered through 0.22 and 0.1 μm polycarbonate filter (Exp. 3) and the filtrate was added to seawater in different mixing scenarios. Ambient coastal seawater was collected by pumping at the Israel Oceanographic and Limnological Research Institute (IOLR) into acid-cleaned carboys, and mixed with either brackish groundwater (Exp.1) or fresh groundwater (Exp. 2, Exp. 3) at the desired ratios and filtration size. The duration of the experiments was 3-5 days, and samples were taken for the following analyses: chlorophyll a (Exp. 1 & 2, every 24Hr.), dissolved nutrient concentrations (Exp. 2 & 3 T zero and T final), flow cytometry (bacterial and phytoplankton abundance, every 24Hr.), primary and heterotrophic production rates (Exp. 1 & 2, every 24Hr.; Exp. 3 T zero and T final). Currently, little is known about the interactions between groundwater-borne and coastal seawater microbial populations, and groundwater microbes' role upon introduction to coastal seawater populations. Here, we investigated seawater microbial abundance, activity and diversity through laboratory-controlled bottle incubations mimicking different mixing scenarios between SGD (either ambient or filtered through 0.1 µm/0.22 µm) and seawater.

Description: We investigated seawater microbial abundance, activity and diversity in a site strongly influenced by submarine groundwater discharge (SGD). We combined in-situ observations and laboratory-controlled bottle incubations mimicking different mixing scenarios between SGD (either ambient or filtered through 0.1 µm/0.22 µm) and seawater. Three sampling campaigns (August 2020, February 2021 and July 2021) were conducted at a field site, highly influenced by SGD (Achziv, northern Israel), which we recently compared to a reference site (Shikmona) at the oligotrophic Israeli shallow rocky coast. Each field campaign lasted 2-5 days and covered at least 2 tidal cycles. Porewater samples were collected on the shoreline using piezometers (AMS piezometers that reach depths of 〈2 meters) and a portable peristaltic pump. The density (g cm-3), electric conductivity (mS/cm), temperature (°C) and pH, of surface seawater, porewater and groundwater were measured on-site at the time of the sampling. Samples for microbial analysis were collected from the piezometers and divided to aliquots: 1. For community analysis, samples were immediately filtered through polycarbonate 0.2 μm pore size filters, which were kept on ice and transported to the laboratory on the same day. Filter samples were stored frozen (-20°C) until DNA extraction (filtered porewater were kept for dissolved nutrient measurements. After thawing, each filter was cut into small pieces using a sterile scalpel blade, which was placed immediately into PowerSoil DNA bead tubes and extracted with the dNeasy PowerSoil Kit (Qiagen, USA) following the standard protocol. To generate 16S rRNA gene libraries, the V3–V4 hypervariable region of the 16S gene was amplified and sequenced on the Illumina MiSeq platform. Quality-filtered reads were imported into QIIME 2 platform, denoised, dereplicated, clustered and trimmed using the DADA2 plugin. Taxonomic assignment of the ASVs was achieved against the Silva database. The ASV table is provided under "additional metadata". Raw data from Illumina MiSeq sequencing are deposited to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject number PRJNA973031 (will be available upon publication). 2. For Pico-/nano-phytoplankton and heterotrophic prokaryotic abundance, non-filtered samples were chilled on ice and transported to the laboratory on the same day. Samples (1.8 mL) were fixed with glutaraldehyde (final concentration 0.02 % v:v, Sigma-Aldrich 253 G7651), frozen in liquid nitrogen, and later stored at −80°C until analysis. The abundance of autotrophic pico- and nano-eukaryotes, Synechococcus and Prochlorococcus, and other heterotrophic prokaryotes (bacteria and archaea) was determined using an Attune® Acoustic Focusing Flow Cytometer (Applied Biosystems) equipped with a syringe based fluidic system and 488 and 405 nm lasers. To measure heterotrophic prokaryote abundance, a sample aliquot was stained with SYBR Green (Applied Biosystems). 3. Prokaryotic (bacteria and archaea) heterotrophic production was estimated using the 3H-leucine incorporation method. Photosynthetic carbon fixation rates were estimated using the 14C incorporation method.