Data Files Accompanying: Marine Bacteria Affect Saccharide Enrichment in Sea Spray Aerosol during a Phytoplankton Bloom
Authors:  Elias S. Hasenecz, Thilina Jayarathne, Matthew A. Pendergraft, Mitchell V. Santander, Kathryn J. Mayer, Jon Sauer, Christopher Lee, Wyeth S. Gibson, Samantha M. Kruse, Francesca Malfatti, Kimberly A. Prather, Elizabeth A. Stone
Journal: ACS Earth and Space Chemistry
https://doi.org/10.1021/acsearthspacechem.0c00167

Contact: 
Betsy Stone, betsy-stone@uiowa.edu, Department of Chemistry, The University of Iowa

Cite as:
Hasenecz, E. S.; Jayarathne, T.; Pendergraft, M. A.; Santander, M. V.; Mayer, K. J.; Sauer, J.; Lee, C.; Gibson, W. S.; Kruse, S. M.; Malfatti, F.; Prather, K. A.; Stone, E. A.; 2019 Data from: Marine bacteria affect saccharide enrichment in sea spray aerosol during a phytoplankton bloom. In Center for Aerosol Impacts on Chemistry of the Environment (CAICE) Collection. UC San Diego Library Digital Collections.
DOI: https://doi.org/10.6075/J0R20ZW6


Experimental/analytical/instrumental/computational methods for data collection and creation. This includes information regarding instrument settings, analyte preparation, software and settings utilized for preprocessing of raw data, and computational libraries, software, and initial values utilized:
Microcosm experiment.

In the 2018 Biological Effects on Air Sea Transfer at the Scripps Institution of Oceanography (SIO) in La Jolla, California, USA, three separate phytoplankton blooms were studied. This manuscript focuses on the two phytoplankton blooms that occurred between August 4-15 and August 22-30, referred to as “microcosm 2” and “microcosm 3”, respectively. On day 1 of each microcosm, seawater was collected at the Ellen Browning Scripps Memorial Pier, filtered with a 50 µm mesh, and transferred to a 2,400 L outdoor tank. 

To induce a phytoplankton bloom, f/2 algae growth medium (Proline, Aquatic Eco-Systems, Apopka, FL) and sodium metasilicate nutrients were added. A phytoplankton bloom developed in natural sunlight, then 120 L were transferred to each of three marine aerosol reference tanks (MARTs) to generate sea spray aerosol (SSA). To monitor biological activity in the water, in vivo chlorophyll-a (chl a) was tracked via fluorescence (Turner Designs Aquafluor). While chl a increased, the MARTs were filled with 120 L of the water from the outdoor tank each morning and returned to the outdoor tank at the end of the day. This transfer provided a common source of water (true biological replicates) across the multiple MARTs that was exposed to natural sunlight and reduced potential inhibition of phytoplankton caused by MART plunging and light limitation.  The MART headspace was purged with zero air (Sabio Instruments 1001) before resuming SSA generation and sampling for the day.

Once chl a in the outdoor tank decreased, the water was left in the MARTs and no longer re-mixed with the outdoor tank. The control tank was left unperturbed, while to the HB tank three strains of HB were added: Alteromonas sp. (AltSIO), Psuedoalteromonas ((A)TW7), and Flavobacteria bacterium (BBFL7) at 108 cells per strain per MART. These strains were isolated in the Azam laboratory at SIO from water samples collected from the same location. 

Seawater and SSA sampling/collection
Seawater samples were collected daily from the spigot on the side of each MART at a depth of 8 inches. SSA was collected at ambient RH (65-89%) by a five stage Sioutas Personal Cascade Impactor (PCIS, SKC model 225-370, 50% cut-off aerodynamic diameters: > 2.5, 1.0, 0.5, 0.25, and < 0.25 µm) at 9 L min-1 using clean air as described above. Samples were collected on substrates consisting of 25 mm PTFE substrate (Zefluor, 0.5 µm, PALL Life Sciences) for the top four stages and pre-baked 37 mm quartz fiber filter (QFF, PALL Life Sciences) for the after filter. For each microcosm, three sets of samples were collected for 3-5 days each to try to capture three distinct periods of the phytoplankton bloom. One hour before sampling each day, filters were pulled from cold storage, thawed, impacted for 2-3 hours, and refrozen at -20 °C. Field blanks were obtained before the start of the first and third set for both microcosms. All samples were kept frozen (-20°C) prior to analysis. For heterotrophic bacteria (HB) counts, SSA was collected via impingement into liquid without bubbling (Aerosol Devices Inc., SS110A Universal Spot Sampler in Liquid Spot Sampler configuration).

Sample preparation and analysis of HB
HB abundances were obtained by flow cytometry at The Scripps Research Institute (TSRI) Flow Core Facility. All samples were prepared by pipetting samples into cryogenic vials and preserved using 10% electron microscopy grade glutaraldehyde. Samples were then incubated at 4°C for 10 minutes followed by flash freezing in liquid nitrogen and then storing at -80°C. Samples analyzed via flow cytometry (BIO-RAD, ZE5 Cell Analyzer) for HB were first diluted (1:10) in 1×TE buffer (pH 8), then were stained with SYBR Green I at room temperature for 10 min (at a 10:4 dilution of the commercial stock) in the dark. HB populations were discriminated based on their signature in the FL1 (488 nm laser, green fluorescence) versus SSC specific cytograms. 

Sample preparation and analysis of enzyme activities
Enzyme activities were measured on seawater samples using fluorogenic substrate analogues at saturating concentrations (24 µM).  Leucine protease, serine protease, oleate lipase, stearate lipase, and alkaline phosphatase activities were measured with L-leucine-7-amino-4-methylcoumarin hydrochloride, L-serine-7-amido-4-methylcoumarin hydrochloride, 4-methylumbelliferone oleate, 4-methylumbelliferone stearate, and 4-methylumbelliferone phosphate, respectively.  Fluorescence of the enzymatic release of the fluorophores 4-methylumbelliferone and 7-amino-4-methylcoumarin was measured with a BioTek Synergy H1 multi-mode microplate reader at excitation/emission wavelengths of 360(40)/460(40) nm.  Aliquots of seawater were pipetted into the wells of a 96 well microtiter plate, with each well containing one fluorogenic substrate.  Fluorescence was measured initially and after 45 minute incubation in the dark at in situ temperature.
   
Sample preparation for analysis of saccharides and sodium.
Aliquots of seawater samples were subjected to ultrafiltration accomplished using pre-cleaned (rinsed with ultrapure (UP; 18.2 MΩ*cm, Thermo Barnstead EasyPure II) water, ethanol, UP water immediately before use) commercially available centrifuge tubes containing filter units centrifuged in series (Allegra X-30R centrifuge with a SX4400 swinging bucket rotor). The filtration size cuts were 200 nm (polyether sulfone, PES; PALL Corporation Microsep Advance), 6 nm (100 kDa, PES; Sartorius Vivaspin 6), and 2 nm (3 kDa, PES; Millipore Sigma Amicon Ultra - 4) that were chosen to yield truly dissolved organic matter (<200 nm), two fractions of colloidal dissolved organic matter (2-6 and 6-200 nm), and particulate organic matter (>200 nm). Size cuts based on molecular weights are approximately determined based on the minimum radius.
 
Low mass loadings necessitated that filters were combined into two extractions that enabled analysis of saccharides in submicron (<1 µm) and supermicron (>1 µm) SSA. PTFE substrates were pre-wet with 50 µL acetone, then extracted into UP water by 30:40:30 minutes of shaking-sonication-shaking. A 4.0 mL extraction volume was used for submicron SSA samples, while 3.0 mL was used for supermicron SSA. Unfiltered and ultrafiltrated seawater, as well as SSA extracts were hydrolyzed with 0.1 M trifluoroacetic acid (TFA) at 100 °C for 12 hours then filtered with a 0.45 µm filter (polypropylene, Whatman). 
Quantification of saccharides and sodium.

SSA extracts as well as size-fractionated seawater samples (total, <200 nm, <6 nm, and <2 nm) were analyzed via high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Dionex, ICS 5000). Briefly, saccharides were separated using a 0.480 mL min-1 isocratic flow of 27.5 mM sodium hydroxide (Fisher) with a Dionex AminoTrap guard column and CarboPac PA20 analytical column. Nine saccharides were identified and quantified using standards—rhamnose, mannose, ribose (Across Organics), fucose (Alfa Aesar), galactose (Fisher), arabinose, xylose, fructose (Sigma Aldrich), and glucose (TCI)—with seven-point calibration curves. Size resolved concentrations of saccharides in seawater were calculated. Sodium in SSA extracts and filtered seawater was quantified using ion-exchange chromatography with conductivity detection (Dionex, ICS 5000) following a previously described method. 

Enrichment is quantified with enrichment factors (EFs) according to equation 1,
EF_saccharide=([saccharide]_SSA/[Na^+ ]_SSA)/([saccharide]_seawater/[Na^+ ]_seawater )		(Eq. 1)
where an EF > 1 indicates enrichment and an EF < 1 indicates depletion with respect to sodium concentrations in seawater and SSA.