ALBERT

Description: The sources and fate of radiocarbon (14C) in the Dead Sea hypersaline solution are evaluated with 14C measurements in organic debris and primary aragonite collected from exposures of the Holocene Ze’elim Formation. The reservoir age (RA) is defined as the difference between the radiocarbon age of the aragonite at time of its precipitation (representing lakeʼs dissolved inorganic carbon [DIC]) and the age of contemporaneous organic debris (representing atmospheric radiocarbon). Evaluation of the data for the past 6000 yr from Dead Sea sediments reveal that the lakeʼs RA decreased from 2890 yr at 6 cal kyr BP to 2300 yr at present. The RA lies at ~2400 yr during the past 3000 yr, when the lake was characterized by continuous deposition of primary aragonite, which implies a continuous supply of freshwater-bicarbonate into the lake. This process reflects the overall stability of the hydrological-climate conditions in the lakeʼs watershed during the late Holocene where bicarbonate originated from dissolution of the surface cover in the watershed that was transported to the Dead Sea by the freshwater runoff. An excellent correlation (R2=0.98) exists between aragonite ages and contemporaneous organic debris, allowing the estimation of ages of various primary deposits where organic debris are not available.

Description: Submarine groundwater discharge (SGD) is a globally important process supplying nutrients and trace elements to the coastal environment, thus playing a pivotal role in sustaining marine primary productivity. Along with nutrients, groundwater also contains allochthonous microbes that are discharged from the terrestrial subsurface into the sea. 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 in a site strongly influenced by SGD. In addition, through laboratory‐controlled bottle incubations, we mimicked different mixing scenarios between groundwater and seawater. Our results demonstrate that the addition of 0.1 μm filtered groundwater stimulated heterotrophic activity and increased microbial abundance compared to control coastal seawater, whereas 0.22 μm filtration treatments induced primary productivity and Synechococcus growth. 16S rRNA gene sequencing showed a strong shift from a SAR11‐rich community in the control samples to Rhodobacteraceae dominance in the 〈0.1 μm treatment, in agreement with Rhodobacteraceae enrichment in the SGD field site. These results suggest that microbes delivered by SGD may affect the abundance, activity and diversity of intrinsic microbes in coastal seawater, highlighting the cryptic interplay between groundwater and seawater microbes in coastal environments, which has important implications for carbon cycling. Plain Language Summary Submarine groundwater discharge (SGD) is an important process where groundwater flows into the ocean along the coast. When the groundwater mixes with seawater, the microbes from both sources interact with each other, which can impact the diversity, activity, and amount of microbes in the coastal environment. Currently, little is known about how groundwater‐borne microbes affect marine microbial populations. Our research shows that when groundwater microbes are removed before mixing groundwater with seawater, the abundance and activity of certain microbes that consume organic matter significantly increase. Additionally, we noticed a significant difference in the types of microbes present between the sites where SGD occurs versus background (uninfluenced) coastal water, especially in terms of the microbes that consume organic matter. Overall, this study suggests that there is a connection between groundwater and seawater microbes, which can influence the delicate balance between organisms that produce carbon and those that consume it. This has important implications for how carbon cycles globally. Key Points Groundwater discharge into the coastal zone delivers both nutrients and allochthonous microbes Groundwater microbes interact with seawater populations, by which affecting the delicate autotroph‐heterotroph balance Subterranean microbial processes are key drivers of food webs, potentially affecting biogenic carbon fluxes in the ocean

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.

Description: We investigated seawater microbial abundance, activity and diversity in a site strongly influenced by submarine groundwater discharge (SGD). Three sampling campaigns (August 2020, February 2021 and July 2021) were conducted at a field site, highly influenced by SGD (Achziv, northern Israel), Each field campaign lasted 2-5 days and covered at least 2 tidal cycles. Pore-water 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 pore-water 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. 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.