Dataset: Sponge 3day VacuSIP Jan 2022

Incurrent/excurrent water samples were taken from three sponge species from the Florida Keys (USA) over two or three days using a modified version of the vacuSIP (Morganti et al. 2016). Briefly, acid-cleaned and combusted bottles were negatively pressurized and sealed. PEEK tubing lines were connected to the pressurized bottles via a needle, and this pulled seawater from the opening of the sponge ("excurrent") or nearby seawater ("incurrent") at a slow and steady rate to fill the bottles. Nutrient data from the seawater samples were compiled and organized by sponge species and water type (incurrent or excurrent) in an Excel file. Seawater samples from two sponge species (Niphates digitalis and Verongula rigida) were filtered through 0.2-micrometer (µm) polyethersulfone filters, while seawater from the sponge Xestspongia muta was filtered through 0.2 µm Teflon Omnipore filters. Filtrate was then saved for inorganic nutrients and those from X. muta were processed for dissolved organic matter composition using targeted metabolomics analysis. Flow cytometry of phytoplankton and bacteria data was collected from pre-filtered seawater and preserved in 0.5% paraformaldehyde final concentration. Seawater for total organic carbon (TOC) and total nitrogen (TN) was also collected prior to filtration and was acidified to ~pH 2 using concentrated HCl. Metabolomics and TOC analysis were performed at Woods Hole Oceanographic Institution at the Mass Spectrometry Facility.

Dissolved Organic Matter Extraction:
Filtered seawater was processed using PPL solid phase extraction following the protocol by Dittmar et al. (2008). Extracts were dried to nearly completeness, leaving a small viscous drop in the vial. These extracts were then shipped to WHOI for metabolomics analysis.

Targeted Metabolite Analysis by UPLC-MS:
DOM extracts were reconstituted in 200 microliters (μl) MilliQ water with 50 nanograms per milliliter (ng/ml) isotopically-labeled injection standards d2 biotin, d6 succinic acid, d4 cholic acid, and d7 indole 3 acetic acid. We used ultra-performance liquid chromatography (Accela Open Autosampler and Accela 1250 Pump, Thermo Scientific) coupled to a heated electrospray ionization source (H-ESI) and a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Scientific) operated under selected reaction monitoring (SRM) mode. We performed chromatographic separation with a Waters Acquity HSS T3 column (2.1 × 100 millimeters (mm), 1.8 μm) equipped with a Vanguard pre-column and maintained at 40 degrees Celsius (°C). We eluted the metabolites from the column with (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile at a flow rate of 0.5 milliliters per minute (mL min-1), according to the gradient: 0 min, 1% B; 1 min, 1%B; 3 min, 15%B; 6 min, 50%B; 9 min, 95%B; 10 min, 95%B; 10.2 min, 1%B; 12 min, 1%B (total run time = 12 min). Settings for source gases were 55 (sheath), 20 (auxiliary) and 0 (sweep), and these settings are presented in arbitrary units. The heated capillary temperature was 375 °C and the vaporizer temperature was 400 °C. For positive and negative modes, we performed separate autosampler injections of 5 μL each.

Flow Cytometry:
Samples were preserved and stored at -80°C until later batch analysis at the University of Hawaii SOEST Flow Cytometry Facility (www.soest.hawaii.edu/sfcf). Microbial cells were enumerated using flow cytometry (Selph, 2021). In brief, samples (0.1 mL) were thawed in batches, stained with the DNA dye Hoechst 34580 (1 microgram per milliliter (µg/mL) final), then run at 30 microliters per minute (µL min-1) on a Beckman-Coulter CytoFlex S flow cytometer, using lasers emitting at 375 nanometers (nm) (to detect Hoechst), 488 nm (for scatter and chlorophyll parameters), and 561 nm (for phycoerythrin). Resulting listmode files (FCS 3.0) were analyzed using FlowJo software (Becton Dickinson, v. 10.8.2) to distinguish microbial populations based on their fluorescence signals (chlorophyll, phycoerythrin, DNA), as well as forward and right-angle light scatter. Heterotrophic bacteria were distinguished from phytoplankton by their DNA signature and absence of pigment. Prochlorococcus and Synechococcus were separated from larger eukaryotic phytoplankton by their light scatter signatures, as well as their characteristic pigment and DNA signatures. Other phytoplankton (eukaryotes, mostly 2-20 µm pico- and nano-sized cells given the small volume analyzed) had higher light scatter and more chlorophyll fluorescence per cell.

Inorganic nutrients:
Inorganic nutrients included phosphate, nitrate+nitrite, nitrite, ammonia, and silicic acid. The phosphate method is a modification of the molybdenum blue procedure of Bernhardt and Wilhelms (1967), in which phosphate is determined as reduced phosphomolybdic acid employing hydrazine as the reductant. The nitrate + nitrite analysis uses the basic method of Armstrong et al. (1967), with modifications to improve the precision and ease of operation. Sulfanilamide and N-(1-Napthyl)ethylenediamine dihydrochloride react with nitrite to form a colored diazo compound. For the nitrate + nitrite analysis, nitrate is first reduced to nitrite using an OTCR and imidazole buffer as described by Patton (1983). Nitrite analysis is performed on a separate channel, omitting the cadmium reductor and the buffer. The method is based on that of Armstrong et al. (1967) as adapted by Atlas et al. (1971). The addition of an acidic molybdate reagent forms silicomolybdic acid, which is then reduced by stannous chloride. This indophenol blue method is modified from ALPKEM RFA methodology which references Methods for Chemical Analysis of Water and Wastes, March 1984, EPA-600/4-79-020, "Nitrogen Ammonia", Method 350.1 (Colorimetric, Automated Phenate) A detailed description of the continuous segmented flow procedures used can be found in Gordon et. al. (1994).