Dataset Authors

Will Overholt


This dataset contains the results of laboratory experiments performed on samples of shallow sandy sediments from Pensacola Beach, Florida with the goal of determining how beach microbes will respond to Macondo oil buried in sandy sediments. The dataset includes information about oxygen profiles, nitrate concentrations, nitrite concentrations, ammonium concentrations, phosphate concentrations, microbial 16S amplicons, microbial metagenomes, and total petroleum hydrocarbon concentrations.



Dataset contains laboratory measurements of oil degradation, no field sampling involved.

Supplemental Information

The dataset consists of several excel and text files which are named according to their content and are explained below: The excel files “ammonium_data”, “nitrite_data”, “nitrate_data”, and “phosphate_data” contain the following headers -Timepoint (The sampling timepoint label); Type (whether system is a control or experimental treatment); Chamber (replicate of treatment), {Phosphate or nitrate, nitrite, Ammonium, (concentration in micromolar)}; STDEV; Time; Date; Aeration. Cycle (values here represent the aeration cycle for each data point collected).Number; POSIXct_time_EST (Eastern Standard time), and Hours_since_aeration_cycle. The excel files “ammonium_raw_absorbance”, “nitrate_raw_absorbance”, “nitrite_raw_absorbance” and “phosphate” contain the measures of absorbance. The headers are - Treatmentoil = sand from oil treatment chambers; cn = sand from control treatment chambers; cn_control = sand from control chambers incubated with no added acetylene; oil_control = oil sand incubated with no acetylene; cn_killed = autoclave killed control sand; oil_killed = autoclave killed oil sand. The excel files “nitrification_rate_measurements” and “denitrification_rate_measurements” contains the nitrification rates estimated using the allylthiourea block method on sand collected at the end of the experiment, and denitrification rates estimated using the acetylene block assay on sand collected at the end of the experiment respectively. The excel files “RAW_Alkanes” and “RAW_PAHs” contain all the raw data of alkanes and PAHS from the GC/MS and how raw data were converted to concentrations. Abbreviations of PAHs are given in the excel file titled “PAH ID names”. The text file “n2fix_full” contains nitrogen fixation rates estimated from the acetylene reduction assay on sand collected at the end of the experiment. The text file “alkanes_ngg” contains measured alkane concentrations normalized per gram of dry sediment. C12-C37 nalkanes; pristane/phytane branched alkanes (ng/g dry sediment) values are in ng of the compound / dry weight (g). The text file “alkanes_oilonly_normtohopane” contains measured alkane concentrations normalized per ng of HC30-hopane. C12-C37 n-alkanes; pristane/phytane (ng/ng HC-hopane) values are ng compound / ng of HC30 hopane. The text file “pahs_ngg” contains measured PAH concentrations normalized per g of dry sediment. The text file “pah_oilonly_normtohopane” contains measured PAH concentrations normalized per ng of HC30-hopane.|Oil chambers were amended with 5 g weathered oil per kg wet sand. Nitrification rate potentials were estimated using the allylthiourea inhibition assay (Hall, 1984). Triplicate assays were performed for each chamber with and without the inhibitor. Each replicate was performed in a 125 ml Erlenmeyer flask with 50 ml of artificial seawater amended with 10 mM sodium chlorate, 500 µM ammonium sulfate, and control flasks were amended with 20 mg L-1 of allylthiourea. Approximately 5 g of wet sand was added to each flask. All samples were shaken at 140 rpm and 25 °C, and samples were collected, filter sterilized, and frozen every 4 hours for 24 hours. At the end of the assay, all samples were analyzed for nitrite concentrations using the method described in Garcia-Robledo et al., 2014. Nitrification potential was determined by comparing the nitrite produced relative to the allylthiourea controls. Nitrogen-fixation rate potentials were determined using the acetylene reduction assay (Capone, 1993). For each chamber, 5 g of sediment was added to a 12 ml serum vial with 5 ml of filter sterilized, artificial seawater at 36 ppt. Serum vials were sealed with blue bromobutyl stoppers with a 1% acetylene headspace under oxic (atmospheric) conditions. In parallel, controls included samples without acetylene as well as autoclaved-killed samples. Samples were incubated for 3 weeks in the dark at room temperature. A GC-FID (Greenhouse Gas Monitoring GC, SRI Instruments, Torrance, California, USA) equipped with dual 2 m HayeSep-D columns was used to quantify ethylene production. Samples were measured for ethylene production daily until production was linear. Controls not amended with acetylene did not produce ethylene, as well as the kill controls. Soluble reactive phosphate concentrations were determined using the method outlined by (Murphy and Riley, 1962). Samples were run in triplicate, and duplicate standard curves were generated for each set of analyses. Oxygen concentrations were monitored through ports in the lid of each chamber using a Presens Microx4 needle-microoptode (Presens, Regensburg, Germany). The needle optode was calibrated following the manufacturer’s protocol using sodium sulfite for the 0% solution and bubbling with air for the 100% solution, and the calibration was checked weekly using fresh 0% and 100% solutions. Hydrocarbon extraction: Extraction and analysis of hydrocarbon compounds were performed according to a modified version of EPA method 3510C with accompanying QA/QC protocols. Oil and control treatments were extracted for quantification of total petroleum hydrocarbons (TPHs) as well as the specific hydrocarbon compound classes, aliphatics (n-alkanes C12-C40, and isoprenoids pristine and phytane) and polycyclic aromatic hydrocarbons (PAHs). Extracts were concentrated under a gentle stream of nitrogen using a TurboVap and reconstituted in hexane (100%) for chromatographic analysis. TPHs in the samples was quantified using GC-FID. A one mL of EPH Surrogate Spiking Solution (ISM‌-581X, Lot CL‌-1009, Ultra, Kingstown, RI, USA) containing o-terphenyl and 1-chlorooctadecane was added directly to the separatory funnel before extraction. TPH concentrations were corrected for extraction efficiency based on the recovery of the EPH spiking solution and mass of oil added. Samples (25 ml) were transferred to 250 ml separatory funnels, and 15 ml of dichloromethane was added. Sample glassware containers were rinsed with an additional 15 mL of dichloromethane to collect all residual oil. Separatory funnels were shaken for 2 minutes, and the organic layer was collected in a 60-ml vial. The extraction step was repeated 3x, and 2-4 g of anhydrous sodium sulfate was added to the resulting organic extracts. All solvents used were at the highest purity available and without further purification. All glassware used was previously combusted at 450°C for 4 hours, and rinsed with dichloromethane prior to extraction. An extraction blank was included with each set of samples (10-12 samples) to ensure no contamination from chemicals, glassware and/or laboratory equipment. Aliphatics and aromatics were quantified in a gas chromatograph/mass spectrometric detector (GC/MS) in full scan mode (m/z 50-550). Splitless injections of 1μL of the sample were conducted, and a RXiÒ5sil column (30 m x 0.25 mm x 0.25 μm) was used. Quantitative analysis of aliphatics and PAHs were conducted using the IS method. Samples were spiked before extraction with perdeuterated n-alkane (d50-Tetracosane, Ultra Scientific) and PAHs (Deuterated PAH Mixture: d10-acenaphthene, d10-phenanthrene, d10-fluoranthene, d12-benz(a)anthracene, d12-benzo(a)pyrene, d14-dibenz(ah)anthracene, d14-benzo(ai)perylene; Ultra Scientific). For n-alkanes analysis, GC oven temperature was programmed as 80°C held for 0.5 min, then increased to 320°C at a rate of 10°C min-1 and held for 5.5 min. Injector temperature was set to 280°C. Identification and quantification of n-alkanes (nC12-nC40) and isoprenoids pristine (Pr) and phytane (Phy) were conducted by comparing with a reference standard (Fuel Oil Degradation mixture, Ultra Scientific; C8-C40 Alkane Certification Standard; Supelco) and the spike standard (d50-Tetracosane). For PAHs, a GC oven temperature was programmed as 60°C held for 8 min, then increased to 290°C at a rate of 6°C/min and held for 4 min, then increased to 340°C at a rate of 14°C/min, and held at the upper temperature for 5 min. The temperature of the MS detector was 250°C. Concentrations of PAHs were calculated using response factors by comparison with a known standard mixture (16-unsubstituited EPA Priority Pollutants and selected congeners: Ultrascientific US‌-106N PAH mix, NIST 1491a) and the spike standard. When no commercial reference standard was available, compounds were quantitated using the response factor for an isomer. Therefore, the concentrations determined for many of the alkylated PAHs were semiquantitative. Recoveries were generally within QA/QC criteria of 70-120%. Total genomic DNA was extracted from 0.25 g of sand in triplicate from each chamber at each major sediment sampling point with a MoBIO PowerSoil DNA Extraction kit following the manufacturers protocol. For the 16S SSU rRNA gene amplicon libraries, the triplicate extracts were amplified using the Earth Microbiome Project primers (515F / 806R) with a Fluidigm Access Array kit common sequence to allow secondary indexing, as has previously been described (Caporaso et al., 2012; Overholt et al., 2018). The triplicate PCR products for each sample were pooled in equimolar amounts, and the resultant mix was indexed. Multiplexed samples were pooled at equimolar ratios and paired-end sequenced (2x250 bp) on an Illumina MiSeq (Green et al., 2015). For the metagenomic libraries, the triplicate extractions were pooled in equimolar ratios. The DNA libraries were prepared using the Illumina Nextera XT DNA library prep kit according to manufacturer’s instruction for dual-indexed libraries except the protocol was terminated after isolation of cleaned double-stranded libraries. Library concentrations were determined by fluorescent quantification using a Qubit HS DNA kit and Qubit 2.0 fluorometer (ThermoFisher Scientific) according to manufacturer’s instructions and samples run on a High Sensitivity DNA chip using the Bioanalyzer 2100 instrument (Agilent) to determine library insert sizes. An equimolar mixture of each library was sequenced on an Illumina HiSEQ 2500 instrument located in the School of Biology, Georgia Institute of Technology for 300 cycles (2 x 150 bp paired end rapid run). Adapter trimming and demultiplexing of sequenced samples was carried out by the instrument. Adapter sequences were further screened using cutadapt, and quality control was performed with SolexaQ++ with the following settings: [solexa dynamictrim -h 25] & [solexa lengthsort -l 50 -c].|Gas chromatography-flame ionization detector (GC-FID) was used to quantify ethylene production and PAHs. Gas chromatograph/mass spectrometric detector (GC/MS) in full scan mode (m/z 50-550) was used to measure Aliphatics and aromatics. Library concentrations were determined with a Bioanalyzer 2100 instrument (Agilent). Presens Microx4 needle-microoptode was used to monitor Oxygen concentrations.|||Capone, D. G. 1993. Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. In P. F. Kemp, J. J. Cole, B. F. Sherr, and E. B. Sherr (Eds.), Handbook of methods in aquatic microbial ecology (pp. 621–631). 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. and Gormley, N. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME Journal, 6(8), 1621–1624. doi:10.1038/ismej.2012.8 Hall, G. H. (1984). Measurement of nitrification rates in lake sediments: Comparison of the nitrification inhibitors nitrapyrin and allylthiourea. Microbial Ecology, 10(1), 25–36. doi:10.1007/bf02011592 Murphy, J., & Riley, J. P. (1962). A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta, 27, 31–36. doi:10.1016/s0003-2670(00)88444-5


To determine how beach microbes respond to Macondo oil buried in sandy sediments under laboratory controlled conditions. The focus was upon determining populations susceptible to oil toxicity, parameters that control population succession, and niche availability for competing populations.


subtidal sediments, permeable sediments, oil biodegradation, aliphatics, nitrogen cycle, Deepwater Horizon, microbial community response, biogeochemical cycles, Polycyclic aromatic hydrocarbon (PAH), Total petroleum hydrocarbon (TPH), microbial metagenomes




April 2019

Point of Contact


Joel E. Kostka


Georgia Institute of Technology / School of Earth and Atmospheric Sciences


Will Overholt


Georgia Institute of Technology / School of Earth and Atmospheric Sciences

Funding Source




Rights Information

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This work is licensed under a Creative Commons Public Domain Dedication 1.0 License.