Methods:
Sinking aggregates were sampled only at Day 16 for the control treatment (without any addition of oil and Corexit), whereas sinking marine oil snow (MOS) were sampled for the diluted chemically-enhanced water accommodated fraction (DCEWAF) at Day 0.5, 1, 2, 3, 4, 8 and 16. Replicates were sampled for Day 16 of both treatments. The sinking aggregates that settled to the bottom of the tanks were collected, after most of the water was slowly drained from the tank. The slurry was gently filtered onto a 0.4 µm polycarbonate membrane 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 was quickly removed, followed by the freeze-drying step for later EPS analysis. ~2-3 mg of the MOS/MS material was dissolved in 2 mL used for EPS analysis.
Water samples (0.5- 1 L) were collected from each mesocosm tank and filtered through a 0.4 µm polycarbonate membrane to collect suspended particulate matter (SPM) for EPS analysis. The whole filter was soaked at 4 mL nanopure water at 4 ˚C for three days, sonicate for 30 min and the slurry was used for EPS analysis. Protein content in EPS was measured based on a modified bicinchoninic acid (BCA) method (Smith et al., 1985), using the Pierce BCA protein assay kit (Cat. # 23225), with bovine serum albumin (BSA) as the standard. Neutral sugar concentration was determined by 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 et al., 2001). The sum of proteins, carbohydrates and uronic acid, which were expressed as the sum of bovine serum albumin (BSA), glucose and glucuronic acid equivalents, was reported as EPS contents for the colloidal fraction, suspended particulate matter (SPM) and the sinking marine oil snow (MOS)/marine snow (MS). Pre-combusted GF/F filter (0.7 µm, Whatman, Little Chalfont, UK) was used for the collection and analysis of particulate organic carbon (POC) in SPM. In order to collect the colloidal fraction, ~ 150 mL samples from each treatment were collected and then filtered through a 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 extensively diafiltered with nanopure water (18.2 MΩ) and then concentrated to 2 mL for the determination of neutral sugars, uronic acid, and protein contents (Xu et al., 2018a). Organic carbon concentrations in the dissolved phase (< 0.4 µm) and colloidal phase (3 kDa- 0.4 µm) were measured with a Shimadzu TOC-L analyzer (Xu et al., 2018a,b, Xu et al., 2019a, b).
Seawater was collected ~0.5 km offshore south of Galveston (Texas) on May 7, 2019, in the Gulf of Mexico. Water was settled in large tanks to remove large particles and debris before collection. This seawater was used for the control (no oil or dispersant additions) and DCEWAF preparation. A plankton concentrate was collected nearby in Galveston Bay using a mesh size of 63 µm plankton net; 2 L of this “concentrate” was added to all mesocosm treatments, immediately prior to the start of the experiment to make a final volume of 102 L per mesocosm. A PTFE stopcock 10 cm off the bottom of the tank was used for sampling. The oil used was Macondo “surrogate” oil from the Marlin Platform Dorado and the dispersant used was Corexit. The WAF mixture was prepared by mixing 25 mL oil with ~130 L seawater in a stirring baffled recirculating borosilicate glass tanks of 170 L capacity (43 × 88 × 44 cm) and allowed to equilibrate over 24 hours. After that, only the aqueous phase at the bottom layer (no surface slick: the WAF fraction) was removed and 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. At the start of the experiment (time zero), oil concentrations were measured using a Horiba Scientific Aqualog Fluorometer (excitation 254nm; emission 365nm) and expressed as estimated oil equivalent (EOE). Dispersants caused negligible interference to EOE measurement, due to the facts that it was only added at 1/20 of the oil concentration, and also its fluorescence contribution at the wavelength mentioned above is below the background. The EOE at Day 0 in the DCEWAF was determined as 0.54 +/- 0.07 mg/L.
Provenance and Historical References:
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
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
Xu, C., Lin, P., Zhang, S., Sun, L., Xing, W., Schwehr, K. A., Chin, W.-C., Wade, T. L., Knap, A. H., Hatcher, P. G., Yard, A., Jiang, C., Quigg, A., Santschi, P. H. (2019a). The interplay of extracellular polymeric substances and oil/Corexit to affect the petroleum incorporation into sinking marine oil snow in four mesocosms. Science of The Total Environment, 693, 133626. doi:10.1016/j.scitotenv.2019.133626
Xu, C., Chin, W.-C., Lin, P., Chen, H., Chiu, M.-H., Waggoner, D. C., Xing, W., Sun, L., Schwehr, K. A., Hatcher, P. G., Quigg, A., Santschi, P. H. (2019b). Comparison of microgels, extracellular polymeric substances (EPS) and transparent exopolymeric particles (TEP) determined in seawater with and without oil. Marine Chemistry, 215, 103667. doi:10.1016/j.marchem.2019.103667.
View Metadata