Microbial diveristy of experimental sediments with grass leachate after the Deepwater Horizon oil spill
Soil, sediment, and water samples were collected from two neighboring marsh sites (Coastal Waters Consortium sites PS03a and PS06) near the city of Port Sulphur, Louisiana, in October 2013. Replicate cores were collected at 5 meters inland on the marsh and at 1-2 meters off the edge of the marsh (in water), and cores were sectioned to obtain depths 0-1 cm and 9-10 cm. Seawater was collected at both sites and was sterilized by passing through a 0.2 µm Millipore (GF/F+ PVDF) filter. Spartina alterniflora stems, shoots, and roots were collected at both sites for labile dissolved organic matter leachate creation through tyndallization. All samples were stored on ice until return to the laboratory, then stored at 4 oC prior to the start of the experimental microcosms. Batch microcosms were created in triplicate in sterile serum bottles (Wheaton) with 1.5 g of homogenized soil (inland marsh) or sediment (off-marsh) from each depth and location and 15 mL of filtered seawater. Bottles were sealed with sterile butyl rubber caps and aluminum seals (Wheaton). Microcosms were spiked with 1.5 mL of concentrated S. alterniflora leachate sourced from each site. Live control microcosms were created following the same protocol, but were not enriched with leachate. Sterilized control microcosms (“dead” controls) were created by autoclaving bottles, water, and soils or sediments at 121 oC for 60 minutes after sealing and prior to adding leachate. Total nucleic acids were extracted from original marsh soil or sediment and at the end of the experiment at the end at 28 days. Extraction was done using methods modified from Somerville et al. (1989; Appl Environ Microbiol, 55(3): 548-554) and Zhou et al. (1996, Appl Environ Microbiol, 62(2): 316-322) that incorporated sucrose lysis buffer with lysozyme (1 mg/ml), a solution of 5X proteinase K/CTAB/SDS, and overnight incubation at 55oC while shaking at 100 rpm. After protein precipitation, nucleic acids were precipitated in isopropanol overnight at -20oC prior to washing with ethanol and purification. Pyrosequencing of the V1-V3 region of 16S rRNA genes was done using a 454 GS FLX Titanium at Molecular Research LP (Shallowater, TX).