Abstract:

A mesocosm experiment was carried out in and October of 2015 (Mesocosm#2) using coastal water collected near Galveston Bay, Texas. Four mesocosm treatments were prepared in triplicate for this study: (1) Control (seawater only); (2) WAF (oil + seawater); (3) Chemically-enhanced WAF (CE-WAF; oil + Corexit + seawater); (4) Diluted CE-WAF (1:10 dilution). Initial mesocosm volumes were 79L. Prior to experiment initiation, phytoplankton were collected from coastal Galveston, TX waters and added to each tank to stimulate interactions between phytoplankton, oil, and Corexit. Starting at time-point zero and every 12 hours thereafter, water samples were collected from each mesocosm in clean, opaque, Nalgene bottles. For each timepoint, 150mL of water was filtered onto 47mm, 0.2um filters and DNA was extracted using MP Biomedical FastDNA spin kits. Primers P450fw1 (5’-GTSGGCGGCAACGACACSAC-3’) and P450rv3 (5’-GCASCGGTGGATGCCGAAGCCRAA-3’) were used to obtain PCR products for alkane monooxygenase P450 (van Beilen et al., 2006). PCR products were ligated into competent E. coli cells and cloned using the TOPO TA Cloning Kit (Life technologies; Carlsbad, CA). Plasmid extractions were performed using Qiagen Mini-prep Kits. Sequencing of 7 random clones verified the success of the cloning reaction and exact size and sequence of the targeted PCR products were confirmed with BlastX. A mixture of five P450 clone sequences were used to create a standard for qPCR, four Alphaproteobacteria and one Gammaproteobacteria. qPCR was carried then out for on all DNA samples in triplicate.

Suggested Citation:

Shawn Doyle, Emily Whitaker, Veronica De Pascuale, Jason Sylvan. 2017. p450 gene abundance measurements in the COAST mesocosm study. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7SB43TH

Publications:

Doyle, S. M., Whitaker, E. A., De Pascuale, V., Wade, T. L., Knap, A. H., Santschi, P. H., … Sylvan, J. B. (2018). Rapid Formation of Microbe-Oil Aggregates and Changes in Community Composition in Coastal Surface Water Following Exposure to Oil and the Dispersant Corexit. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.00689

Purpose:

To quantify the abundance of the p450 gene within the mesocosms.

Data Parameters and Units:

Treatment (control, WAF, DCE-WAF, CE-WAF), Replicate (A, B, C), Timepoint, Quantification cycle (Cq), p450 gene abundance (copy number), dilution factor, volume (mL), P450 gene abundance (copy number per mL), Standard curve, Standard gene abundance (copy number), Standard quantification cycle (Cq)

Methods:

The seawater was collected from the same location as that used in TeCOAST Mesocsom, but on October 17 2015, from 8 kilometers off shore south of Galveston (TX) in the Gulf of Mexico. The salinity was 31. The seawater was processed through a charcoal filter to remove large particles and debris. Four treatments were prepared in triplicate. Control tanks were filled with seawater and a plankton mixture. Water accommodated fraction (WAF) of oil was prepared by mixing 25 mL (5 ml ~ every 30 min for 2.5 hrs) of Macondo surrogate oil into 130 L of seawater then mixing for 12 to 24 hrs (Knap et al. 1986; Knap et al. 2016 in preparation). The WAF was then introduced into the WAF mesocosm tanks and filled to 87 L and mixed. From these WAF tanks 6 L was removed for other experiments and analyses (2L for roller tables, 2 L dark/light, 4 L hydrocarbon analyses). In order to make chemically enhanced water accommodated fraction (CEWAF), Corexit was mixed with oil in a ratio of 1:20 and 25 mL of this mixture (5 ml every 30 min for 2.5 hrs) of surrogate oil plus Corexit was added to 130 L of seawater which was mixed for 8 to 24 hrs prior to being transferred to the mesocosm tanks. The CEWAF was then introduced into the CEWAF mesocosm tanks and filled to 96 L and mixed. From these CEWAF tanks 15 L was removed for other experiments and analyses (9 L for the DCEWAF mesocosms, 2L for roller tables, 2 L dark/light, 4 L hydrocarbon analyses). Diluted CEWAF (DCEWAF) was prepared by mixing 9 L of CEWAF with 78 L of the original seawater for a total volume of 87 L. From these DCEWAF tanks 6 L was removed for other experiments and analyses (2L for roller tables, 2 L dark/light, 4 L hydrocarbon analyses). Plankton (≥63 µm) were collected using a net and transferred into polycarbonate bottles. This concentrated plankton mass was introduced to the tanks and stirred (2 L to each final volume 83 L) immediately prior to starting the experiments. The EOE mean concentration of the three mesocosms for the control, WAF, DCEWAF and CEWAF at the start of the experiments were 0 mg/L, 0.26 mg/L, 2.74 mg/L and 41.5 mg/L, respectively. The EOE mean concentration of the three for the control, WAF, DCEWAF and CEWAF mesocosms after 72 hours were 0 mg/L, 0.06 mg/L, 1.03, and 17.3 mg/L, respectively. Estimated Oil Equivalence (EOE) The estimated oil equivalents (EOE) were determined by fluorescence (Wade et al. 2011) using Macondo surrogate oil as a standard to produce calibration curves at 5 to 7 concentrations. Water samples (5 to 20 ml) were extracted with 5 ml of dichloromethane. An aliquot of the extract was placed in a cuvette for fluorescence analyses (Horiba Scientific Aqualog Fluorometer). The EOE were determined from the calibration curve (Wade et al. 2011). Samples with florescence responses that exceeded the calibration curve were diluted so that their florescence was within the calibration range. Samples were taken at the beginning and end of the experiment and at intervals in between and at the same time point as measurements of other parameters during the experiment.

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