Abstract:

This data set are the chemical characterization of the EDTA (Ethylenediaminetetraacetic acid )-extracted aggregates and EPS (extracellular polymeric substances) in terms of the protein, neutral sugars, and uronic acids with respect to organic carbon. Experiments were conducted with a water accommodated fraction of oil (WAF), simulating conditions below the water/oil interface. Two different oils were compared, because oil characteristics, likely impact incorporation of oil into aggregates. Surrogate oil from the Deepwater Horizon spill in the Gulf of Mexico 2010 (Macondo oil) and the oil spilled in 2015 at Refugio Beach, near Santa Barbara, California referred to as Refugio oil, were used. A total of five roller tank experiments were conducted. Experiments were performed in roller tanks to mimic the continuous sinking of marine snow-sized aggregates through a water column. All experiments were conducted in the dark, simulating the aggregation phase only, not the growth phase of phytoplankton.

Suggested Citation:

Chen Xu, Peter H. Santschi. 2018. Bulk chemistry of aggregates and EPS for roller table experiments for diatom aggregation in the presence or absence of oil. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N780517B

Publications:

Passow, U., Sweet, J., Francis, S., Xu, C., Dissanayake, A., Lin, Y., … Quigg, A. (2019). Incorporation of oil into diatom aggregates. Marine Ecology Progress Series, 612, 65–86. doi:10.3354/meps12881

Purpose:

To investigate the direct impacts of WAF (Macondo or Refugio oil) on the formation of aggregates by a variety of diatom species.

Data Parameters and Units:

File TABLE 1_Experiment number and description.csv details the experimental setups and relates the experiment (exps) numbers as well as other related GRIIDC datasets (R4.x263.188:0002, R4.x263.188:0003, R4.x263.188:0004, R4.x263.000:0005, R4.x263.000:0006) with more detail on each individual experiment; WAF = water accommodated fraction of oil, in control seawater was processed as for WAF, except no oil was added; WAFMac = WAF prepared with Macondo surrogate oil; WAFREf = WAF prepared with Refugio Beach oil; BI = Bacterial Inoculum; EOE = estimated oil equivalence; rpm = rotations per minute. ChemComp of EPS and Agg.csv file includes: Sampling date (MM/DD/YYYY); Experiment # = experiment number; treatment = Control, Macondo, or Refugio; Control = Control treatment containing seawater and plankton community; Macondo surrogate oil = a light sweet crude; Refugio = oil from spill off California coast); replicate = number of replicates; tank # = tank number; sample fraction: Agg or SSW; Agg = Aggregates > 1mm (visually discernible); SSW = surrounding sea water (as opposed to the aggregate phase, Agg.); Amount aliquoted to TAMUG (measured after freeze drying); TAMUG = Texas A&M University at Galveston; DW = dry weight (mg); PON= particulate organic nitrogen (%, wt/ug); POC = particulate organic carbon (%, wt/ug); Volume (mL/tank); EDTA = Ethylenediaminetetraacetic acid (CAS Number‎: ‎60-00-4); OC = organic carbon (ug/mL); CHO = neutral sugars in glucose equivalents; URA = uronic acids in glucuronic acid equivalents; Protein = protein in BSA, eq = Bovine serum albumin equivalent; CHO-C = CHO with respect to organic carbon = CHO * 0.4; URA-C = URA with respect to organic carbon = URA * 0.37; Protein-C = Protein with respect to organic carbon = Protein * 0.53; Protein-C/TCHO-C = protein-C/ (CHO-C + URA-C); EPS = exopolymeric substances. * calculated from concentration in the original WAF and the dilution during set up of experiments; additional oil is contained in aggregates; replicates combined during harvest.

Methods:

Experimental set up and sample collection: Five roller tank experiments (exps.) were conducted to investigate the direct impacts of the WAF on the aggregation of diatoms (Table 1). Roller tanks allow the formation and continuous sinking of aggregates without these touching container walls, once solid body rotation is established (Ploug et al. 2010). Each experiment consisted of a different diatom exposed to a control and to WAF treatments (Table 1). The WAFMac or WAFRef were prepared with Macondo surrogate oil (Marlin Platform, Dorado source oil; 0.865 g/mL) and Refugio Beach oil (0.946 g/mL), respectively. Dense cultures in late exponential/ early stationary phase were mixed with WAF or in case of the control, with pasteurized oil-free filtered seawater, prepared identically to WAF, except that no oil was added. The seawater, collected from the UCSB seawater system, stems from 800 m off-shore and 18 m depth and passes through a series of gravity sand filters. The general set-up was the same in all experiments. Roller tanks were filled bubble-free and incubated in the dark on roller tables at the respective growth temperatures of the cultures (Table 1). Low light levels were used during observations and harvest of exps. 1 and 2, and only low green light was used in exps. 3, 4, and 5. Green light does not allow photosynthesis for most diatoms, and was meant to simulate dark conditions more effectively, while allowing monitoring of cultures and handling of samples. In exps. 4 and 5 an inoculum of a natural bacteria community was also added. Sampling for exp. 3 was the most comprehensive, with 4 replicate tanks per treatment, whereas exps. 1, 2, 4 and 5 each involved duplicate tanks per treatment. After 3-14 days (Table 1), when marine snow-sized aggregates had formed and did not visually change from day to day, all treatments were harvested. For logistical reasons the second replicate or the third and fourth replicates of each treatment were harvested a day later in exps. 2 and 3, respectively. After careful removal of tanks from rolling tables, aggregates > 1mm (visually discernible) were manually collected and analyzed. This fraction will be called aggregate phase (Agg). Collection techniques of aggregates depended on aggregate size and stability. Individual intact aggregates were collected with a cut off pipette, a known discrete number of aggregates were collected via syringe, or at times, aggregated material could only be collected in bulk, without associating aggregate numbers with the collected aggregate slurry, because aggregates were too abundant or too fragile for individual collection. After all aggregates were removed, the remainder of the material was mixed and subsampled. This fraction that contained aggregates < 1mm and un-aggregated cells will be called the surrounding seawater (SSW) fraction. Sampling of aggregates inadvertently also collected some SSW. A correction for this was only possible when the total aggregate volume was determined. However, particle concentrations in aggregates were so high, compared to those in SSW, that this intrinsic sampling error was negligible. Particulate and dissolved substances were analyzed in the SSW fraction, whereas the Agg fraction was only analyzed for particulates, with the exception of EOE (estimated oil equivalent), which was, when possible also analyzed in the aggregate fraction. Functionally oil droplets were part of the particulate phase, as they were collected on filters. Subsamples from each fraction were collected to analyze particulate organic carbon and nitrogen (POC and PON), transparent exopolymer particles (TEP), EDTA-extractable EPS and cell numbers. Results were usually normalized per tank to make Agg and SSW fractions directly comparable with each other and allow estimates on the fraction of material that was aggregated. When possible (exps. 2, 3, 5), sinking velocities of individual aggregates were determined as a function of the aggregate size. Cultures, WAF and bacterial inoculum: Cultures of Skeletonema grethae CCMP 775, Skeletonema grethae CCMP 776, Odontella aurita CCMP 816, Chaetoceros sp. (isolated Nov. 2014; see Passow et al. 2017), and Thalassiosira pseudonana CCMP 1335, all diatoms, were grown at 70 µmol m-2 s-1 using modified f/2 media, with reduced macro-nutrients. The seawater for making media came from the UCSB seawater intake, and was filtered twice (0.2 µm) and then UV treated for > 30 minutes before use. End concentrations of nutrients in the media were 58.9 µM nitrate, 3.6 µM phosphate, and 53.5 µM silicic acid. Trace metals and vitamins were added according to the original f/2 recipe (Guillard 1975). Cultures were grown at 13°C or 22°C (Table 1) under a 12 hrs: 12 hrs light: dark cycle and used for experiments in late exponential/ early senescent phase. To make WAF, filtered seawater was pasteurized at 65˚C for 4-5 hours, oil added (1% vol.: vol.) to a glass (exps. 2-5) or Teflon (exp. 1) container and stirred with a sterile stir bar in the dark at room temperature for 24 hours. After a two hour resting period the WAF was harvested from the bottom layer using a bottom spigot (exps. 3, 4, 5) or teflon tubing inserted through the interface to the bottom of the container (exps. 1, 2). The upper oily layer was discarded, and WAF mixed with diatom cultures (Table 1). Fresh WAF was prepared for each experiment and experiments started immediately after WAF preparation. Seawater was treated identically to generate “control-WAF” for controls, except that no oil as added. The bacteria inoculum was prepared by collecting raw seawater water off the steps at the Marine Science Institute, UC Santa Barbara and pre-filtering it sequentially through 5.0 µm and 2.0 µm filters. Freshly produced bacterial inoculum was added into tanks in exps. 4 and 5 (Table 1). EDTA-extraction: Composition of attached EDTA-extractable extracellular polymeric substances (EPS) were determined in the Agg fractions of exps. 1, 2, 3, and 5, and the SSW fraction of exp. 5. Subsamples of the Agg and SSW fractions were shipped on ice overnight to Galveston to carry out further analysis. Aggregate slurries of exps. 1, 2 and 3 were filtered onto 0.4 µm polycarbonate filters and rinsed three times with 5 mL of Milli-Q water (18.2 MΩ·cm) to remove salt. The material retained on the filter was re-suspended in 25 mL Milli-Q water, the filter discarded, and the material freeze-dried. One aliquot was used to determine the POC/PON content (for reference purpose), the second, was used for the extraction of EPS. In exp. 5 (Control and WAFMac treatments), 10 mL of the Agg was filtered onto the 0.4 µm polycarbonate membrane, and the whole membrane used for EPS extraction, due to the limited amount of sample that could be weighed for EPS extraction. Additionally, ~0.5 L of SSW from exp. 5 was filtered through pre-combusted GF/F to measure POC/PON concentration and another ~0.5 L of SSW was filtered onto a 0.4 µm polycarbonate membrane filter for EPS extraction. To extract EPS, 2 - 5 mg of the freeze dried (exps. 1, 2,3) or filtered (exp.5) material was re-suspended in 10 mL of 1 or 2% EDTA (Ethylenediaminetetraacetic) solution in pre-combusted 20 mL glass scintillation vials. The EDTA binds Ca2+, thus releasing EPS that is bound via Ca-bridging, into the solution. A procedural blank was included in order to correct for any interferences from the membrane. The samples were then incubated at 4°C for 3 hours on an orbital shaker at 150 rpm. After the incubation, the particles were removed using a Flipmate 100 System (0. 45 µm PES Environmental Express, USA). Excess EDTA was removed by ultrafiltering the resulting filtrate (< 0.45 µm) via Amicon Ultra-4 Centrifugal Filter Unit with a 3 kDa cut-off membrane (Millipore, USA). The retentate (3 kDa- 0.45 µm), which included the ETDA extracted EPS was concentrated to 1 mL for further chemical analysis. Aliquots of this extract were used for the determination of organic carbon using a Shimadzu TOC-L analyzer, as well as for analysis of neutral carbohydrate, protein and the uronic acids. Neutral carbohydrate content was determined by the anthrone method (Morris 1948), with glucose as the standard. Uronic acid was estimated by adding sodium borate (75 mM) to concentrated sulfuric acid, heating at 100 ⁰C for 10 min and then adding m-hydroxydiphenyl, with glucuronic acid as the standard (Hung & Santschi 2001). The detection limit was 0.136 mg/L. Protein content was measured using the Pierce protein assay kit based on a modified bicinchoninic acid assay method (Smith et al. 1985). Bovine serum albumin was used as the standard and the detection limit was 0.04 mg/L. EPS extractions from Agg were normalized to POC in exps. 1 and 2, and based on the volume fraction used for extraction in relation to total volume per tank in exp 5. The carbon content of 40%, 37% and 53% were assumed for neutral sugars, uronic acids and proteins, respectively, to calculate the carbon contributions of these constituents to EDTA extractable organic carbon.

Provenance and Historical References:

Hung C-C, Santschi PH (2001) Spectrophotometric determination of total uronic acids in seawater using cation-exchange separation and pre-concentration by lyophilization. Analytica Chimica Acta 427:111-117 Morris D (1948) Quantitative Determination of Carbohydrates with Dreywoods Anthrone Reagent. Science 107 Passow U, Sweet J, Quigg A (2017) How the dispersant Corexit impacts the formation of sinking marine oil snow. Marine Pollution Bulletin Ploug H, Terbrüggen A, Kaufmann A, Wolf-Gladrow D, Passow U (2010) A novel method to measure particle sinking velocity in vitro, and its comparison to three other in vitro methods. Limnology and Oceanography: Methods 8:386-393 Smith P, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M, Fujimoto E, Goeke N, Olson B, Klenk D (1985) Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150:76-85 Guillard, R. R. (1975). Culture of phytoplankton for feeding marine invertebrates. In Culture of marine invertebrate animals (pp. 29-60). Springer, Boston, MA.

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