Abstract:
To test the effects of sunlight on the composition and secretion of exopolymeric substances (EPS) and the subsequent aggregation process, we conducted short-term irradiation experiments in three treatments, i.e., control (coastal seawater of the Gulf of Mexico), water accommodated fraction of oil (WAF), and chemically-enhanced WAF (CEWAF). EPS composition (mainly carbohydrates and proteins) were quantified in the colloidal fractions, and aggregates. In addition, bacterial abundance, live/dead cell ratio, and ambient •OH formation rate were measured under these conditions. In the presence of oil, natural sunlight stimulated polysaccharide secretion, partially due to increased ROS (i.e. •OH) production in the presence of oil. In addition, flow cytometry showed formation of larger sized aggregates in the irradiated WAF treatment, and polysaccharides accumulated into aggregates.
Suggested Citation:
Luni Sun, Chen Xu, Peng Lin, Kathleen A. Schwehr, Antonietta Quigg, Meng-Hsuen Chiu, Wei-Chun Chin, Peter H. Santschi. 2017. The effects of sunlight on the composition of exopolymeric substances affecting the subsequent aggregation process during oil spill and Corexit exposure. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7C53J9B
Data Parameters and Units:
EPS= exopolymeric substances; control= coastal seawater of the Gulf of Mexico; WAF= water accommodated fraction of oil; CEWAF= chemically-enhanced WAF; colloidal= ultrafiltration of the filtrate using Amicon ultra-4 centrifugal filter unit with a 3 kDa cut-off membrane; aggregates= materials collected on filters. The filters were washed three times with mili-Q water, and dried at room temperature overnight. The filters from triplicate samples were combined and re-suspended in 4 mL 1% EDTA solution, followed by the incubation at 4 ºC for 3 h on an orbital shaker at 150 rpm. Excessive EDTA was removed by ultrafiltration, and the sample was concentrated down to 400 µL; DOC=dissolved organic carbon (ppm); DN=dissolved nitrogen (ppm) Dark=no exposure; Light=under irradiation; Dark1=replicate 1 of dark sample; Dark2=replicate 2 of dark sample; Dark3=replicate 3 of dark sample; Dark4=replicate 4 of dark sample: Light1=replicate 1 of light sample; Light2=replicate 2 of light sample; Light3=replicate 3 of light sample; Light4=replicate 4 of light sample; •OH=hydroxyl radical; HPLC=high performance liquid chromatography (no unit); control dead=fluorescence intensity of dead cells in control (RFU); control live=fluorescence intensity of living cells in control (RFU); WAF dead==fluorescence intensity of dead cells in WAF (RFU); WAF live=fluorescence intensity of living cells in WAF (RFU); CEWAF dead=fluorescence intensity of dead cells in CEWAF (RFU); CEWAF live=fluorescence intensity of living cells in CEWAF (RFU); Protein concentration: mg/L Carbohydrate (CHO) concentration: mg/L absorbance: No unit bacterial abundance: cells×10^-10/L pixel number
Methods:
Gulf of Mexico seawater was collected from a NOAA station at Galveston, Texas on May 21 2017. The seawater was pre-treated with a charcoal filter to remove large particles, and with 3µm polycarbonate filters (Millipore) to remove most of the phytoplankton. The total organic carbon and total nitrogen of the filtered seawater were 1.9 ppm and 0.33 ppm respectively. To the seawater we added glucose as a carbon source (424 µM C), ammonium chloride as a nitrogen source (64 µM N) and sodium dihydrogen phosphate as a phosphorus source (4 µM P), in a Redfield ratio. The culture was incubated for 36 h at room temperature (12 h: 12 h=light: dark) to reach a stationary phase. An aliquot of a sample was filtered through 0.2µm filters for ambient ∙OH measurement. To one third of the culture, Macondo surrogate oil (water: oil=4000:1) was added, to another third of the culture we added oil and Corexit (oil: Corexit=20:1). All the samples were stirred for 15 h overnight for mixing The samples with oil and oil/Corexit were subsequently transferred to a separating funnel, and the aqueous phase was collected as WAF and CEWAF, respectively. Experiments were conducted in triplicates. Before irradiation, the control (without the addition of oil), WAF, and CEWAF treatments were filtered through 3µM to remove particles. Oil equivalent concentrations were estimated by extracting with dichloromethane and measuring its fluorescence. 240 mL samples were then irradiated in quartz flasks for 40 min in a water bath by circulating water of a temperature of 25 ± 2 ºC around the flasks. The samples in glass flasks wrapped with aluminum foil were used as dark controls. After irradiation, oil equivalent mean concentrations, bacterial abundance, aggregates size, the ratio of living/dead cells, and EPS composition of the colloidal phase and aggregates were measured. Water samples were fixed with formaldehyde at a final concentration of 2%, and stored at 4⁰C. An aliquot of a sample (500 µL) was stained with 4’6-diamidino-2-phenylindole (DAPI) at a final concentration of 5 µg/ mL. Staining of the cells was allowed for about 20 min in the dark followed by filtration onto a black polycarbonate filter (0.2 µm, Millipore). The filter was then transferred into a glass slide and embedded in nondrying and very low-fluorescing immersion oil (Type N Immersion liquid, Leica). The stained cells were visualized under a Leica DM 2000 LED epifluorescence microscope equipped with an A4 filter cube (excitation 360, emission 470). The live/dead cell ratio was determined with the help of Live/Dead bacteria viability kit (Invitrogen, L7007, USA) according to the manufacturer’s instruction. Firstly, an aliquot of cells were stained with SYTO 9 green fluorescent nucleic acid stain which stains both live and dead cells. Secondly, a separate aliquot of cells were stained with Propidium Iodide (PI), which only stains the dead cells. The fluorescence was measured after 15 min incubation at excitation/emission 485/530 nm for SYTO 9 and at 485/630 nm for PI. The ratio yielded the proportion of live to dead cells. After irradiation, the samples were filtered through 3µM filter and then 0.4µm filter. Dissolved organic carbon (DOC) and dissolved nitrogen (DN) concentration were measured for the filtrate (<0.4µm). The colloidal phase was collected by ultrafiltration of the filtrate using Amicon ultra-4 centrifugal filter unit with a 3 kDa cut-off membrane. Polysaccharide (CHO) concentrations were determined by the anthrone method, and protein contents by the Lowry Protein Assay Kit (Pierce, 23240, USA). URA concentrations were only measurable in the colloidal phase, however. The filters were washed three times with mili-Q water, and dried at room temperature overnight. The filters from triplicate samples were combined and re-suspended in 4 mL 1% EDTA solution, followed by the incubation at 4 ºC for 3 h on an orbital shaker at 150 rpm. Excessive EDTA was removed by ultrafiltration, and the concentrated sample (down to 400 µL) was analyzed for protein and carbohydrate. The probe benzene was added to the control, WAF, and CEWAF prior to irradiation. The benzene can react with ∙OH to form phenol. After irradiation, the phenol concentration was measured by HPLC, and the ∙OH formation rate was estimated by the phenol formation rate. DOC and DN of the filtrate were measured on a Shimadzu TOC-L analyzer. The filtrate was diluted 2-3 times and acidified to pH <3. The filtrate was measured on the instrument. Absorbance for protein and CHO concentration: samples were measured in the well plate, the data was collected every 1 nm wavelength. Fluorescence intensity for oil concentrations: samples were measured in quartz cuvette; with excitation 322 nm, emission 376 nm. Fluorescence for live/cell ratio: SYTOX-9 (for both live and dead) and Propidium Ioidide (PI) (only dead cells) at excitation/emission 485/530 nm and at 485/630 nm after 15 mins of incubation. ∙OH (i.e. phenol) concentration: samples were injected on to HPLC. Mobile phase: 35 % acetonitrile in water (adjusted pH to 2.5); column: Agilent Eclipse Plus C18 (3.5µm, 3×150 mm); injection volume: 150 µL. Bacterial abundance: cell numbers were count using epifluorescence microscope; with excitation 360 nm, emission 470 nm. Random images were captured using a Leica DFC295 digital color camera connected to a Leica Application Suite X software. Images were saved and analyzed by ImageJ (https://imagej.nih.gov/ij/).
Instruments:
absorbance: Biotech-Epoch UV-vis spectrometer fluorescence for oil concentration: Shimadzu RF-5301PC fluorescence for live/dead ratio: Cytation 5 imaging reader by Biotek epifluorescence microscope: Leica DM 2000 LED epifluorescence microscope equipped with an A4 filter cube DOC, DN: Shimadzu TOC-L analyzer HPLC: Waters
Error Analysis:
DOC, DN: automatic by instrument, coefficient of variation set to 3% or less. Absorbance for protein and CHO concentration: errors were calculated from triplicates
Provenance and Historical References:
Manteca, A., Fernandez, M., & Sanchez, J. (2005). A death round affecting a young compartmentalized mycelium precedes aerial mycelium dismantling in confluent surface cultures of Streptomyces antibioticus. Microbiology, 151(11), 3689-3697.
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