Flow cytometry

To quantify picoplankton abundances, 1 ml samples of water from each pooled treatment (T0; n=3 per treatment) and from each incubation bottle (n=4 per treatment) at both T24 and T48 was collected in a sterile cryovial and fixed with paraformaldehyde (1% final volume), incubated in the dark at 4ºC for 1-hr, and then frozen at -80ºC. The fixed samples were used for enumeration of total bacterial cells (Prochlorococcus, Synechococcus, picoeukaryotic cells, and unpigmented cells (heterotrophic bacteria and archaea)) via flow cytometry at Bigelow Laboratory for Ocean Sciences. Flow cytometry analysis was completed using a Bio-Rad ZE5 (Hercules, California, USA) with a 488 nm laser activated. Samples were thawed and pre-screened through 70 µm mesh and diluted 1:10 with Tris EDTA (TE) Buffer pH 8.0. Cells were then stained using 10x working stock DNA stain SYBRGreen I (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol of Marie et al. (2005). A total of 180 µl of diluted sample was run at a flow rate of 0.5 µl sec -1. Particles were excited with the 488 nm blue later and data acquisition was triggered by green fluorescence. Data signals were recorded by detectors with three bandpass filters including forward scatter (FSC), right angle light scatter (SSC), and fluorescence emission in green (525/35nm). To minimize noise, samples were subgated in FSC-405. Data files logarithmic scatter plots of fluorescent and light scattering properties were analyzed and total bacterial counts were identified based on size and presence of the green fluorescence. All counts were converted to cell abundance based on sample volume (including adjustments for preservation, dilution, and staining).

Biochemical Analyses

Total Organic Carbon (TOC) and Total Nitrogen (TN)

For TOC and TN analyses, 20 ml of water was collected from the pooled treatments prior to their distribution into individual incubation bottles (T0: reef metabolome, n=3; sponge metabolome, n=3) and for each bottle at the end of the experiment (T48: n=4 per treatment) in acid-washed, combusted 40-ml amber EPA vials. Samples were immediately acidified to pH 3 using 12M trace-metal grade hydrochloroic acid (HCL, OptimaTM, Fisher Chemical, Fisher Scientific, Hampton, NH, USA) and stored in the dark at 4ºC. The acidified samples were processed at Woods Hole Oceanographic Institute using a Shimadzu TOC-L analyzer (Longnecker et al. 2015). Additionally,

Filtering for Biochemical Analyses

All other biochemical analyses were completed only on filtered water samples from the pooled treatments at T0 (reef metabolome, n=1 and sponge metabolome, n=2) and T48 incubation bottles (n=4 per treatment). Approximately 680 ml of water from each incubation bottle was individually passed through a 0.22 µm, 47 mm PTFE filter using peristalsis as previous detailed except for a slightly slower filtering rate (30 ml/min) to avoid bursting the microbial cells. Following filtration, the PTFE filters were placed in individual sterile cryovials and stored at -80ºC.

Inorganic macronutrients

For inorganic macronutrient analyses (PO43-, NO3-, Silicate, NO2, NH4+), ~25 ml of the 0.22 µm filtrate from each sample was collected in an acid-washed 30 ml break-proof Nalgene© HDPE bottle (Thermo Fisher Scientific, Waltham, MA, USA), frozen and stored at -20ºC before being shipped to College of Earth, Ocean, and Atmospheric Sciences at Oregon State University for inorganic macronutrient analyses. Phosphate (PO43-) and ammonium (NH4+) were measured using a Technicon Auto Analyzer II (SEAL Analytical Inc., Mequon, Wisconsin, USA) and silicic acid (i.e., silicate), nitrate (NO3-) and nitrate+nitrite (NO3- + NO2-) were measured using an Alpkem RFA 300 colorimetric autoanalyzer (Alpkem, Kranj, Slovenia). Analytical methods and data processing were completed as described in Gordon et al. (1994), but a brief summary of each analysis is provided. The PO43- method was modified from the molybdenum blue procedure (Bernhardt and Wilhelms 1967), in which PO43- is determined as reduced phosphomolybdic acid employing hydrazine as the reductant. The indophenol blue method, modified from ALPKEM RFA methodology, was used for the measurement of NH4+. Silicic acid was measured using the methodology of Atlas et al. (1971) in which the addition of an acidic molybdate reagent forms silicomolybdic acid that is then reduced by stannous chloride. Lastly, NO3- and NO3-+ NO2- were measured based on the methods of Atlas et al. (1967) with modifications to improve precision. Sulfanilamide and N-(1-Napthyl)ethylenediamine dihydrochloride react with NO2- to form a diazo compound. For the NO3- + NO2- analysis, NO3- is first reduced to NO2- using an OTCR and imidazole buffer as described by Patton (1983). The NO2- analysis is performed on a separate channel, omitting the cadmium reductor and the buffer.

Dissolved combined neutral sugars (DCNS)

Dissolved combined neutral sugars (DCNS) were measured from frozen 20 µl aliquots of 0.22 µm filtered seawater (collected in acid-washed, combusted 40-ml amber EPA vials and stored in the dark at 4ºC) at the Complex Carbohydrate Research Center, University of Georgia. Each sample was first desalted and hydrolyzed. For desalting, each sample was loaded onto a gravity column, containing forty milliliters of mixed ion exchange resins (AG 501-X8, 20-50 mesh, Bio-Rad), that was packed and prewashed with 200 ml (5x bed volume) of nano-pure water. Samples were then eluted with 120 ml (3x bed volume) of nano-pure water. The resulting flow-through and wash solution were lyophilized. Following lyopholization, the recovered materials were hydrolyzed wth 2 ml of 2 N TFA at 100ºC until high-performance anion exchange chromatography (HPAEC) analysis. By employing a specific HPAEC program, as detailed below, the neutral monosaccharides can be separated allowing the measurement of carbohydrates in each sample.

Monosaccharide standards, including fucose (Fuc), rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal), xylose (Xyl), mannose (Man), and fructose (Frc), were hydrolyzed in the same manner and at the same time as the samples. Three concentrations of the standard mixture were prepared serially to establish a calibration equation. The quantity of each residue in the sample was calculated by linear interpolation of respective residue area units into the calibration equation.

Monosaccharides from each sample were analyzed by HPAEC with pulsed amperometric detection (HPAEC-PAD) using a DIONEX ICS3000 system (Thermo Fisher Scientific) equipped with a gradient pump and an electrochemical detector. The carbohydrates were separated by a Dionex CarboPac PA20 (3x150mm) analytical column with an amino trap column and eluted with degassed 12 mM NaOH. Injections were made every 40 min. Under the HPAEC conditons, Xyl and Man cannot be separated (Borch and Kirchman 1997) and are presented as combined results. Samples were analyzed in triplicate and mean values were reported as ng ml-1 based on the volume analyzed.

Fluorescent dissolved organic matter (fDOM)

Flourescent dissolved organic matter (fDOM) was measured from cool (4ºC) aliquots of 0.22 µm filtered seawater (collected in acid-washed, combusted 40-ml amber EPA vials and stored in the dark at 4ºC) from each sample following the methods detailed in Nelson et al. (2015). Samples were analyzed using a Horiba Aqualog scanning fluorometer with 150 W Xe excitation lamp, Peltier-cooled CCD emission detector, and a simultaneous absorbance spectrometer (Horiba Scientific, Piscataway, New Jersey, USA). Samples were brought to room temperature and loaded into DIW-leached and rinsed quartz cuvettes (1 cm diameter). Excitation-emission matrices (EEMs), 3D contour plots of excitation and emission fluorescence, were measured for each sample. Analysis began and ended with 4 DIW-filled cuvettes as blanks. EEMs were processed with a MATLAB script (https://github.com/zquinlan/fDOMmatlab/script.m) that employs parallel factor analysis (PARAFAC) to identify peaks that correspond to previously characterized fDOM components (Coble 1996, Nelson et al. 2015).

Targeted metabolomics

For targeted metabolomics analysis, the remaining filtrate from each sample (~600 ml) was acidified to pH ~3 using 12M trace-metal grade hydrochloric acid (HCL) and the volume was recorded. Note that for targeted metabolomics analysis only one T0 sample from each treatment was analyzed. Extracellular metabolites were concentrated and extracted from each acidified filtrate using solid phase extraction (SPE) with 1g/6cc BondElut PPL cartridges (Agilent, Santa Clara, CA, USA) using a vacuum manifold (Waters Corporation, Milford, MA, USA) following the protocols from Kido Soul et al. (2015) and Fiore et al. (2017). Briefly, the acidified filtrate from each sample is passed through acid-washed FEP tubing and a pre-conditioned (with 100% HPLC-grade methanol) 1g/6cc BondElut PPL cartridges (Agilent, Santa Clara, CA, USA). Following filtration, each cartridge is wrapped in combusted aluminum foil, placed in a labeled, sterile Whirl-Pak bag and frozen at -80ºC. Frozen cartridges were shipped to Appalachian State University where the extraction process was completed by rinsing each thawed cartridge with 4 volumes of 0.1 M HCL followed by a gentle 5 min drying cycle using vacuum pressure and finally eluted into acid-washed, combusted 8-ml glass vials with 6 ml of 100% methanol. Using combusted glass pipettes, the methanol extracts were transferred to acid-washed, combusted 8-ml amber EMP vials and dried down to a single droplet using vacuum centrifuged then stored at -20ºC.

Targeted metabolite analysis was performed at Woods Hole Oceanographic Institute using UPLC (Accela Open Autosampler and Accela 1250 Pump, Thermo Scientific) coupled with a heated electrospray ionization source (H-ESI) and a triple stage quadrupole mass spectrometer (TSQ Vantage, Thermo Fisher Scientific) in selective reaction monitoring (SRM) mode per methods detailed in Kido Soule et al. (2015). The raw XCalibur files were converted to mzML files using msConvert (Chambers et al. 2012) and processed with MAVEN (Melamud et al. 2010) to obtain calibration curves based on the integrated peak area generated for each metabolite. To calculate environmental concentrations the concentration of the metabolite was divided by the volume of the original acidified filtrate passed through the PPL cartridge. Lastly, metabolites that met the threshold detection and quantification limits for the targeted analysis were corrected for extraction efficiency by dividing their environmental concentrations by their published extraction efficiency in sea water (Johnson et al. 2017).

907866_v1_picoplankton.csv