Data Parameters and Units:
The excel files “Bottom Aggregate”, "Colloidal Organic Matter”, and “Suspended Particulate Matter” contains: Mesocosm (three different mesocosm experiment; M3, M4, and M5); Day; Treatment (either a control, water accommodated fraction (WAF), chemically-enhanced WAF (CEWAF), or diluted CEWAF (DCEWAF)), neutral sugar (mg-glucose equivalent/L), protein (mg-BSA equivalent/L), URA (mg-glucuronic acid/L), EPS production (mg/L), and Protein-C/TCHO-C.
The excel file "Efficiency rev" contains: Mesocosm (three different mesocosm experiment; M3, M4, and M5); Day; Treatment (either a control, WAF, CEWAF, or DCEWAF), particle yield (mg), %, OC, D14C, Petro-carbon (%), non-petro-carbon (%), petro-carbon sedimentation efficiency, and non-petro-carbon sedimentation efficiency.
SPM = suspended particulate matter; EPS = extracellular polymeric substances; TCHO = total carbohydrates (neutral sugars + uronic acids); eq. = organic carbon equivalents with respect to respective standard (glucose for neutral sugar; BSA for protein; glucuronic acid for uronic acid); BSA = bovine serum albumin; URA = uronic acid.
M3 = mesocosm 3 = GOMOO, a mesocosm using Gulf Of Mexico Open Ocean water;
M4=mesocosm 4= GOMCOAST, a mesocosm using Gulf Of Mexico COASTal water;
M5=mesocosm 5= LTMOSE, a Long Term Marine Oil Snow Experiment
Methods:
1. Mesocosm experimental set up: Three mesocosm experiments (M2, M3 and M4) were conducted in October 2015 (M2) and July 2016 (M3 and M4) respectively. For each, controls (only seawater, no oil added) and oil treatments, as water accommodated fraction (WAF) of Macondo oil surrogate, were prepared in triplicate. For M2, seawater was collected near-shore (~0.5 km) south of Galveston (Texas) in the Gulf of Mexico at 29.27°N, 94.81°W and settled in large tanks to remove large particles and debris before collection. WAF was prepared by mixing 25 ml oil with 130 L seawater in stirring baffled recirculating borosilicate glass tanks of 170 L capacity and allowed to equilibrate over 24 hours (specific details are in Wade et al. (2017)). After that, only the aqueous phase in the bottom layer was added to the mesocosm tanks. CEWAF was made by adding dispersant to the oil at a ratio of oil‐to‐dispersant of 20:1. The DCEWAF was prepared by diluting the CEWAF with seawater. A plankton concentrate was collected nearby in Galveston Bay using a 63 µm plankton net, in order to obtain the natural phytoplankton and their associated bacterial consortia. 2 L of this “concentrate” was added to both the WAF and Control to make a final volume of ~ 80 L. For M3, seawater was collected ~174 km off the coast of Texas near the Flower Garden Banks National Marine Preserve in the Gulf of Mexico (27.88°N, 94.03°W, salinity: 30.77 ppt; pH: 8.38, temperature: 30.8 ˚C), a site representing an open ocean location. For M4, a coastal site was chosen (~20 km from shore) at 29.37°N, 93.38°W (Salinity: 31.13 ppt, pH: 8.02 Temperature: 30.5 ˚C) in the Gulf of Mexico. Unlike from M2, nutrients (N, P and Si) were added at f/20 concentrations to all tanks of M3 and M4, but not the natural microbial “concentrate”. In M4, additional treatment was adopted in which a silicon tube was filled with crude oil and inserted into the mesocosm tank (Bera et al. 2018). The oil was allowed to passively diffuse into the mesocosm tank 24 hrs prior to the start of the experiment (“Si-WAF” treatment). M5 was conducted on May 22 to June 8, 2017, using the same conditions that had been used in M2, with the difference being the longer duration of the experiment (M5, 16-day and M2, 4-day).
2. Collection of sinking MOS/marine snow (MS), suspended particulate matter (SPM), colloidal organic matter (COM): The MOS/MS formation in all 12 mesocosm tanks was followed visually and recorded by the camera. On Day 2 and Day 4 distinct MOS events occurred in the Control, WAF and DCEWAF treatments and the MOS/MS that had settled to the bottom of the tanks was collected using a 100 mL syringe attached with a long stainless steel needle (length: 7.6 cm, needle gauge:12, OD= 2.77 mm and ID: 2.16 mm). This was a MOS/Marine Snow (MS) slurry and aliquots were gently filtered onto a polycarbonate membrane (pore size: 0.4 µm) and rinsed three times with 15 mL of nanopure water (18.2 MΩ). The material retained on the filter (> 0.4 µm) was then re-suspended in nanopure water and the filter quickly removed. The samples were then freeze-dried for later analysis.
Water samples were also collected on Day 1, 2, 3 and 4 from each mesocosm. Aliquots were then filtered through a 0.4 µm polycarbonate membrane to collect SPM for EPS extraction and analysis, and a pre-combusted GF/F filter (0.7 µm, Whatman, Littlechalfont, UK) and analyzed for organic carbon (OC) determination.
One each time point of the four-day experiment, ~ 150 mL samples from each treatment were collected and then filtered through the Flipmate 100 System (0.4 µm polyethersulphone, Environmental Express, USA). Aliquots of the filtrate (< 0.4 µm) were further ultrafiltered through an Amicon Ultra-15 centrifugal filter unit with a 3 kDa cut-off membrane (Millipore, USA). The retentate (3 kDa- 0.4 µm) was then diafiltered with nanopure water (18.2 MΩ) extensively and then concentrated to 2 mL for all further analysis. OC concentrations of the dissolved phase (< 0.4 µm), colloidal phase (3 kDa- 0.4 µm) and truly-dissolved phase (< 3 kDa) were measured with a Shimadzu TOC-L analyzer.
3. Extraction of EPS from sinking MOS and SPM (Xu et al. 2018a): Freeze-dried sinking MOS/MS (~5mg) was re-suspended in 10 mL of a 1% EDTA solution in 20 mL glass scintillation vials. The MOS/MS slurries were then incubated at 4 °C for three hours on an orbital shaker at 150 rpm. The particles were removed using a Flipmate 100 System, while the filtrate was retained to be further ultrafiltered using an Amicon Ultra-4 Centrifugal Filter Unit with a 3 kDa cut-off membrane (Millipore, USA). EPS released from the MOS/MS, which was in the colloidal fraction (3 kDa- 0.4 µm), was concentrated to a final volume of 2 mL and analyzed for carbohydrates and protein (Xu et al. 2010; Xu et al. 2011b). The sum of these expressed as glucose and bovine serum albumin equivalents, respectively, is reported herein as “1% EDTA extractable EPS”. Such EDTA extracts are operationally-defined and are not necessarily exclusively composed of EPS, thus may also contain another minor bio- or geopolymers, such as humic substances. EPS extraction of the SPM followed the same procedure except the whole polycarbonate membrane was extracted into the EDTA solution. A procedural blank was included to correct for any interferences that may come from the membrane itself.
4. Colorimetric determination of protein, carbohydrate, and uronic acid concentrations: Protein content in EPS was measured based on a modified bicinchoninic acid method (Smith et al. 1985) using the Pierce protein assay kit, with bovine serum albumin as the standard. Carbohydrate concentration was determined using the anthrone method (Morris 1948), with glucose as the standard. Uronic acid was estimated by adding sodium borate (75 mM) in concentrated sulfuric acid and m-hydroxydiphenyl, with glucuronic acid as the standard (Hung and Santschi 2001).
5. Particulate organic carbon (POC), δ13C, and Δ14C determination: Particulate organic carbon (POC) concentration in the sinking MOS and SPM were measured using a Perkin Elmer CHNS/O 2400 analyzer, after an acid-fuming step to eliminate inorganic carbon (Xu et al. 2011a; Xu et al. 2010). The collected samples of sinking MOS which were acid fumed were sent to the National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) facility for Δ14C analysis (Xu et al. 2018b).
Provenance and Historical References:
Bera, G., Parkerton, T., Redman, A., Turner, N. R., Renegar, D. A., Sericano, J. L., & Knap, A. H. (2018). Passive dosing yields dissolved aqueous exposures of crude oil comparable to the CROSERF (Chemical Response to Oil Spill: Ecological Effects Research Forum) water accommodated fraction method. Environmental Toxicology and Chemistry. doi:10.1002/etc.4263
Hung, C.-C., & Santschi, P. H. (2001). Spectrophotometric determination of total uronic acids in seawater using cation-exchange separation and pre-concentration by lyophilization. Analytica Chimica Acta, 427(1), 111–117. doi:10.1016/s0003-2670(00)01196-x
Morris, D. L. (1948). Quantitative Determination of Carbohydrates With Dreywood’s Anthrone Reagent. Science, 107(2775), 254–255. doi:10.1126/science.107.2775.254
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76–85. doi:10.1016/0003-2697(85)90442-7
Wade, T.L., Morales-McDevitt, M., Bera, G., Shi, D., Sweet, S., Wang, B., Gold-Bouchot, G., Quigg, A. and Knap, A.H. (2017). A method for the production of large volumes of WAF and CEWAF for dosing mesocosms to understand marine oil snow formation. Heliyon, 3(10), e00419. doi:10.1016/j.heliyon.2017.e00419
Xu, C., S. Zhang, C.-Y. Chuang, K. A. Schwehr, and P. H. Santschi. (2010). Chemical characterization of strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II). 2010 Ocean Sciences Meeting.
Xu, C., Santschi, P.H., Hung, C.C., Zhang, S., Schwehr, K.A., Roberts, K.A., Guo, L., Gong, G.C., Quigg, A., Long, R.A. and Pinckney, J.L. (2011a). Controls of 234Th removal from the oligotrophic ocean by polyuronic acids and modification by microbial activity. Marine Chemistry, 123(1-4), 111–126. doi:10.1016/j.marchem.2010.10.005
Xu, C., Zhang, S., Chuang, C., Miller, E. J., Schwehr, K. A., & Santschi, P. H. (2011b). Chemical composition and relative hydrophobicity of microbial exopolymeric substances (EPS) isolated by anion exchange chromatography and their actinide-binding affinities. Marine Chemistry, 126(1-4), 27–36. doi:10.1016/j.marchem.2011.03.004
Xu, C., Zhang, S., Beaver, M., Lin, P., Sun, L., Doyle, S.M., Sylvan, J.B., Wozniak, A., Hatcher, P.G., Kaiser, K. and Yan, G. (2018a). The role of microbially-mediated exopolymeric substances (EPS) in regulating Macondo oil transport in a mesocosm experiment. Marine Chemistry, 206, 52–61. doi:10.1016/j.marchem.2018.09.005
Xu, C., Zhang, S., Beaver, M., Wozniak, A., Obeid, W., Lin, Y., Wade, T.L., Schwehr, K.A., Lin, P., Sun, L. and Hatcher, P.G. (2018b). Decreased sedimentation efficiency of petro- and non-petro-carbon caused by a dispersant for Macondo surrogate oil in a mesocosm simulating a coastal microbial community. Marine Chemistry, 206, 34–43. doi:10.1016/j.marchem.2018.09.002
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