Abstract:

Here we used ELLA (Enzyme Linked Lectin Assay) to determine exopolysaccharide concentrations. ELLA ConA tests specifically for binding to saccharide residues most frequently encountered in biofilms matrices: D-glucose or D-mannose.

Suggested Citation:

Wei-Chun Chin and James (Meng-Hsuen) Chiu. 2017. The effect of Oil and Corexit on Marine Phytoplankton Exopolymeric Substances Release. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7RF5S4X

Publications:

Shiu, R.-F., Vazquez, C. I., Tsai, Y.-Y., Torres, G. V., Chen, C.-S., Santschi, P. H., … Chin, W.-C. (2020). Nano-plastics induce aquatic particulate organic matter (microgels) formation. Science of The Total Environment, 706, 135681. doi:10.1016/j.scitotenv.2019.135681

Shiu, R.-F., Chiu, M.-H., Vazquez, C. I., Tsai, Y.-Y., Le, A., Kagiri, A., … Chin, W.-C. (2020). Protein to carbohydrate (P/C) ratio changes in microbial extracellular polymeric substances induced by oil and Corexit. Marine Chemistry, 223, 103789. doi:10.1016/j.marchem.2020.103789

Kamalanathan, M., Mapes, S., Prouse, A., Faulkner, P., Klobusnik, N. H., Hillhouse, J., Hala, D., & Quigg, A. (2022). Core metabolism plasticity in phytoplankton: Response of Dunaliella tertiolecta to oil exposure. Journal of Phycology, 58(6), 804–814. Portico. https://doi.org/10.1111/jpy.13286

Purpose:

Using EPS released from phytoplankton as a marine environment indicator, if we measure its intensity before and after oil and Corexit are introduced, it would tell us how much impact oil and dispersant have on phytoplankton.

Data Parameters and Units:

Files: DNA amount & Normalized_oil part_[culture collection of marine phytoplankton code].csv - Concentration (v/v) = Concentration of Corexit, CWAF, or WAF (1:10^5, 1:10^4, 1:10^3, 1:10^2, 1:10^1); Corexit1, Corexit2 = UV/Vis intensity (arbitrary units) with given concentration of Corexit in column A in replicate 1 and replicate 2; CWAF1, CWAF2 = UV/Vis Intensity (arbitrary units) with given concentration of CWAF in column A in replicate 1 and replicate 2; WAF1, WAF2 = UV/Vis Intensity (arbitrary units) with given concentration of WAF in column A in replicate 1 and replicate 2. Column K Corexit = DNA concentration of Corexit treatment the order followed by the dilution factor in column A; column K CWAF = DNA concentration of the CWAF treatment the order followed by the dilution rate in column A; column K WAF = DNA concentration of WAF treatment the order followed by the dilution rate in column A. normalized = average ELLA fluorescence for the treatment divided by the average UV/Vis intensity for the treatment where the treatment is the column K label (Corexit, CWAF, WAF) at the concentration reported in column A. Files: ELLA oil_[culutre collection of marine phytoplankton code].csv - Concentration (v/v) = Concentration of Corexit, CWAF, or WAF (1:10^5, 1:10^4, 1:10^3, 1:10^2, 1:10^1); Control 1, Control 2, Control 3 = fluorescence (arbitrary units) in control treatment replicates 1, 2, 3; WAF 1, WAF 2, WAF 3 = fluorescence (arbitrary units) in WAF treatment replicates 1, 2, 3; Corexit 1, Corexit 2, Corexit 3 = fluorescence (arbitrary units) in Corexit treatment replicate 1, 2, 3; CWAF 1, CWAF 2, CWAF 3 = fluorescence (arbitrary units) in CWAF treatment replicate 1, 2, 3. % = average fluorescence for all replicates in a treatment divided by controls times 100. Arbitrary units of fluorescence and UV/Vis intensities When data values were not available, -999 was used. Chemical treatments: 1) Control: to test growth of phytoplankton 2) Water accommodated fraction, WAF 3) Chemically enhanced WAF, CEWAF CCMP code number (Culture Collection of Marine Phytoplankton from National Center for Marine Algae and Microbiota (NCMA): 597: Odontella mobiliensis 646: Phaeodactylum tricornutum 775: Skeletonema grethae 999: Dunaliella tertiolecta TP: Thalassiosira pseudonana Acronyms: ELLA: Enzyme Link Lectin Assay ELLA OIL: oil project measure by ELLA ELLA OIL XXX: particular phytoplankton in oil project measurement; (XXX is for CCMP phytoplankton code) Normalized: means ELLA concentration divided by DNA concentration A number following these 3 chemical file names represents an independent experiment, i.e., like Corexit 2, is the second Corexit treatment experiment independently performed after Corexit 1 AVG: average SDTV: standard deviation Each individual data is using their own control, so you will only see one control there but three chemical treatments. Concentrations used for WAF, Corexit, and CEWAF (volume/volume): 1. Control (Medium only) 2. 1:10^5 3. 1:10^4 4. 1:10^3 5. 1:10^2 6. 1:10 7. Stock

Methods:

Water accommodated fraction, WAF: 1. 1L filtered seawater (0.2um twice) 2. Pasteurized 65C for 4hrs. 3. 200 uL crude oil added to seawater, mixed slowly for 24hrs at room temperature. Chemically enhanced WAF, CEWAF: 1. 1L filtered seawater (0.2um twice) 2. Pasteurized 65C for 4hrs. 3. Mix 10ml crude oil and 500ul Corexit at room temperature for 24 hrs. 4. Add 20ul of step 3 into 1L of step 2 seawater. The final carbon content for both WAF and CWAF will each be 4mg/L. Phytoplankton Odontella mobiliensis, Skeletonema grethae, Phaeodactylum tricornutum, Thalassiosira psaeudonana and one green algae Dunaliella tertiolecta were cultured in L1 medium artificial seawater via 96 wells plate. A series of concentrations of WAF, CEWAF and Corexit were used as indicated above. ConA-HRP (Concanavalin A from Canavalia ensiformis (Jack bean) peroxidase conjugate; Sigma L6397) was used in ELLA to measure polysaccharides. Cells were treated with different chemical treatments, WAF, CEWAF and Corexit, for 24 hrs and washed by PBST (Phosphate-buffered Saline with Tween 20) and PBS (Phosphate-buffered saline), with ConA-HRP. The color was developed using TMB (3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System; Sigma T0440). Then the reaction was stopped by applying 1N H2SO4, read by spectrophotometer in 450nm, 1 s for each well. The pellet containing phytoplankton was analyzed for DNA to use for a proxy for the cell count to normalize ELLA. To quantify DNA, the kit was used from ZR-96 Quick-gDNA kit (ZYMO research, CA USA). Briefly, 4X lysis buffer was used to break phytoplankton cells, which were then applied to the DNA binding column, and eluted using the supplied kit elution buffer. DNA concentrations were measured using Nano Drop ND-1000 (Thermo, CA USA) UV/VIS spectrophotometry. Protocol was adapted from manufacturer kit protocol.

Instruments:

PerlinElmer VICTOR3 fluorescence and luminescence plate reader Nano Drop ND-1000 (Thermo, CA USA) UV/VIS spectrophotometry

Individual Files: