Methods:
Twelve 100L mesocosm tanks were filled with Gulf of Mexico seawater collected from the Flower Gardens National Marine Sanctuary Area (27° 53.4180'N; 94° 2.2020'W) which is located ~120 miles off the coast of Galveston (TX). Four treatments were prepared in triplicate. Control tanks were filled with seawater. 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. Mixing ended 24 hrs after the initial oil addition (Knap et al. 1983; Wade et al. 2017). 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 (2 L light /dark bottles, 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. Mixing ended 24 hrs after the initial oil addition. The CEWAF was then introduced into the CEWAF mesocosm tanks and filled to 96 L and mixed. From these CEWAF tanks 13 L was removed for other experiments and analyses (7 L for the DCEWAF mesocosms, 2 L light/dark, 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 (2 L light/dark, 4 L hydrocarbon analyses). To the water in the 12 mesocosms, nutrients were added (final concentration f/20) and the tanks stirred. Banks of lights were placed behind each of the glass mesocosm tanks and a 12:12 light/dark cycle employed. Sampling commenced and defined as time zero. The estimated oil equivalents (EOE) were determined using Macondo surrogate oil as the calibration standard (Wade et al. 2011) for the fluorescence analyses (Horiba Scientific Aqualog Fluorometer). 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 mesocosms for the in the control, WAF, DCEWAF and CEWAF after 72 hours were 0 mg/L , 0.06 mg/L, 1.03. and 17.3 mg/L, respectively. Raw sequence reads in FASTQ format for each replicate of each treatment (i.e. Control, WAF, CE-WAF, Diluted CE-WAF). The hyper-variable V4 region of the 16S rRNA gene was PCR amplified from the DNA extracts with GoTaq Flexi DNA Polymerase (Promega) using a methodology similar to that described by Caporaso et al. 2011. Each sample was amplified in triplicate 25μL reactions with the following cycling parameters: 95°C for 3 min, 30 cycles of 95°C for 45 s, 50°C for 60 s, and 72°C for 90 s, and a final elongation step at 72°C for 10 min. All reactions were performed using the 515F-806R primer pair (10µM each) which was modified to include Golay barcodes and adapters for Illumina MiSeq sequencing (Caporaso et al. 2012). The primer pair was also modified to include recently published revisions that reduce bias against the Crenarchaeota/Thaumarchaeota archaeal lineages and the SAR11 bacterial clade (Parada et al. 2016; Apprill et al. 2015). Following amplification, triplicate reactions were combined together and run on a 1.5% agarose gel to assess amplification and relative band intensity. Amplicons were then quantified with the QuantiFluor dsDNA System (Promega), pooled at equimolar concentrations, and purified with an UltraClean PCR Clean-Up Kit (MoBio Laboratories). The purified library, along with aliquots of the three sequencing primers, were sent to the Georgia Genomics Facility (Athens, GA, USA) for MiSeq sequencing (v2 chemistry, 2 x 250bp). Illumina MiSeq Sequencing: V2-Chemistry; PE-250.
Provenance and Historical References:
Knap, A.H., Sleeter, T.D., Dodge, R.E., Wyers, S.C., Frith, H.R., and S.R. Smith. (1983). The effects of oil spills and dispersant use on corals: A review and multidisciplinary experimental approach. Oil and Petrochemical Pollution, 1(3): 157-169. doi: 10.1016/S0143-7127(83)90134-5 Wade, T.L., Morales-McDevitt, M., Bera, G., Shi, D., Sweet, S., Want, B., Gold-Bouchot, G., Quigg, A., and A.H. Knap (2017) A method for the production of large volumes of WAF and CEWAF for dosing mesocosms to understand marine oil snow formation. Heliyon, 3(10): e00419, doi: 10.1016/j.heliyon.2017.e00419 Wade, T. L., S. T. Sweet, J. N. Walpert, J. L. Sericano, J. J. Singer, and N. L. Guinasso Jr. (2011) Evaluation of possible inputs of oil from the Deepwater Horizon spill to the Loop Current and associated eddies in the Gulf of Mexico, in Monitoring and Modeling the Deepwater Horizon Oil Spill: A Record-Breaking Enterprise. Geophys. Monogr. Ser., doi:10.1029/2011GM 001095. Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh, P.J., Fierer, N., and R. Knight (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. PNAS, 108(Supplement 1): 4516-4522, doi: 10.1073/pnas.1000080107 Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S.M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J.A., Smith, G. and R. Knight (2012) Ultra-high throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6: 1621-1624, doi: 10.1038/ismej.2012.8 Parada, A.E., Needham, D.M., and J.A. Fuhrman (2016) Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Env. Microbiol. 18(5): 1403-1414, doi: 10.1111/1462-2920.13023 Apprill, A., McNall, S., Parsons, R., and L. Weber (2015) Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat. Microb. Ecol. 75: 129-137, doi: 10.3354/ame01753
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