Dataset: Surface water and N.digitalis inhalent/exhalent water sample nutrient and bacterioplankton metadata of Looe Key and Wonderland Reef conducted in 10-15 December 2023

Looe Key Reef, Florida Keys National Marine Sanctuary.  Wonderland Reef, Florida Keys National Marine Sanctuary. Various locations and depths are included in the metadata set.

Inhalant/exhalant water samples were taken from the sponge N. digitalis, at each reef 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 (“exhalent”) or nearby seawater (“inhalant”) at a slow and steady rate to fill the bottles. Surface seawater samples from Looe Key (n=4) and Wonderland Reef (n=2) were collected from each reef’s surface water (“reef”), 0.5 km from the reef (“mid”), and 1.0 km from the reef seaward (“away”). Nutrient data from the seawater samples were compiled and organized by reef, and by distance from the reef in an Excel file. All surface and inhalant/exhalant water samples were filtered through 0.2 µm Teflon Omnipore filters. Filtrate was then saved for nutrients and the rest was acidified and saved for organic nutrient analysis. From this filtrate, inorganic nutrient, fluorescent Dissolved Organic Matter (fDOM), and targeted and untargeted metabolomics data were collected. 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) 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 μl MilliQ water with 50 ng/ml isotopically-labeled injection standards d 2  biotin, d 6  succinic acid, d 4  cholic acid, and d 7  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 mm, 1.8 μm) equipped with a Vanguard pre-column and maintained at 40°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 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 shipped them to the Center for Aquatic Cytometry where they were stored at -80 C until analysis. Picophytoplankton (less than 3 µm) and nanophytoplankton (3-20 µm) were analyzed using a slight modification of the method described in Lomas et al., 2010. Immediately after thawing at room temperature, 300-400 µl of sample was prescreened through 70 µm mesh and run at a flow rate of 1 µl sec -1 . Particles were excited with a 488 nm blue laser and data acquisition was triggered on red fluorescence. Signals were recorded from detectors with bandpass filters for forward scatter (FSC), right angle light scatter (SSC) and fluorescence emission in red (692/80 nm) indicative of chlorophyll a, and orange (593/52 nm) for phycoerythrin. Data files were analyzed from logarithmic dot plots based on fluorescence and characteristic light scattering properties (DuRand & Olson, 1996) using FlowJo 10.6 software (Becton Dickinson & Company, San Jose, CA, USA). Total pico and nano phytoplankton populations were identified based upon cell size and red fluorescence. Phycoerythrin containing cell populations were determined by orange fluorescence. Based upon these gating criteria, the number of cells in each identified population was enumerated and converted to cell abundances using the processed sample volume and adjusted for dilution by preservative.

For total bacteria analysis, samples were thawed, diluted 1:10 with Tris EDTA (TE) Buffer pH 8.0 and stained using a 10x working stock of SYBR Green I Nucleic Acid Stain (Thermofisher Scientific, USA) at room temperature in the dark for 15 min using the protocol of Marie et al. (2005). At a flow rate of 0.5 µl sec -1 , 180 µl of the diluted sample was run. Particles were excited with a 488 nm blue laser and data acquisition was triggered on green fluorescence. Signals were recorded from detectors with bandpass filters for forward scatter (FSC), right angle light scatter (SSC) and fluorescence emission in green (525/35nm). Data files were analyzed from two logarithmic scatter plots based on fluorescence and characteristic light scattering properties. Total bacteria counts were identified based on size and presence of green fluorescence and counts were converted to cell abundances using the volume of sample processed including adjustments for preservation, dilution and staining.

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). 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).

Samples for TOC analysis were stored at 4°C until analysis with a Shimadzu TOC-VCSH total organic carbon analyzer (Longnecker et al., 2015).