Article pubs.acs.org/est
Influence of Petroleum Deposit Geometry on Local Gradient of Electron Acceptors and Microbial Catabolic Potential Gargi Singh,† Amy Pruden,*,† and Mark A. Widdowson† †
Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States S Supporting Information *
ABSTRACT: A field survey was conducted following the Deepwater Horizon blowout and it was noted that resulting coastal petroleum deposits possessed distinct geometries, ranging from small tar balls to expansive horizontal oil sheets. A subsequent laboratory study evaluated the effect of oil deposit geometry on localized gradients of electron acceptors and microbial community composition, factors that are critical to accurately estimating biodegradation rates. One-dimensional top-flow sand columns with 12-h simulated tidal cycles compared two contrasting geometries (isolated tar “balls” versus horizontal “sheets”) relative to an oil-free control. Significant differences in the effluent dissolved oxygen and sulfate concentrations were noted among the columns, indicating presence of anaerobic zones in the oiled columns, particularly in the sheet condition. Furthermore, quantification of genetic markers of terminal electron acceptor and catabolic processes via quantitative polymerase chain reaction of dsrA (sulfate-reduction), mcrA (methanogenesis), and cat23 (oxygenation of aromatics) genes in column cores suggested more extensive anaerobic conditions induced by the sheet relative to the ball geometry. Denaturing gradient gel electrophoresis similarly revealed that distinct gradients of bacterial communities established in response to the different geometries. Thus, petroleum deposit geometry impacts local dominant electron acceptor conditions and may be a key factor for advancing attenuation models and prioritizing cleanup.
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INTRODUCTION Explosion of the Deepwater Horizon rig in April 2010 resulted in a spill of about 5 million barrels of petroleum,1 leading to contamination of more than ∼1050 km of the Gulf of Mexico coastline.2 The vast expanse of this spill, extensive application of dispersants, and warm climate of the Gulf region together imposed unique conditions on the released petroleum, presenting an unprecedented challenge in modeling and predicting the fate of petroleum hydrocarbons in marine and coastal environments. As became apparent during the outfall of the disaster, sensitive and accurate models of petroleum fate and persistence are urgently needed to efficiently guide and prioritize cleanup efforts, especially when vulnerable coastal ecosystems are at risk. Whereas petroleum hydrocarbons themselves are a rich source of organic carbon and are readily biodegradable,3 particularly under aerobic conditions, their extreme hydrophobicity and low solubility4−7 complicates accurate predictions © 2012 American Chemical Society
of natural attenuation rates. One potentially critical factor governing persistence in coastal environments that has generally been overlooked in models is geometry, or the 3-D conformation, of the oil deposits. Geometry of the contaminant source is recognized as an emerging factor impacting the fate of nonaqueous phase liquids (NAPLs) in the subsurface.8−10 NAPL geometry dictates the surface area, which in turn drives dissolution rates as well as the available area for microbial colonization.11 Furthermore, taking the local hydrogeologic conditions into account, NAPL geometry can impact recharge, potentially posing a localized obstruction to flow.12 Thus, “dead zones” exhausted of essential Received: Revised: Accepted: Published: 5782
January 30, 2012 April 30, 2012 May 10, 2012 May 10, 2012 dx.doi.org/10.1021/es300393r | Environ. Sci. Technol. 2012, 46, 5782−5788
Environmental Science & Technology
Article
Sand Column Design and Operation. Duplicate sand columns were set up for each of the experimental conditions: spherical tar balls (ball) and disk-shaped tar layer (sheet), and compared to a no-oil control (blank). The blank column served as a point of reference of the microbial community structure that developed in the absence of oil. The clear PVC columns (10-cm diameter and 30-cm length) were equipped with a side drainage valve at the bottom (Figure 1). Equivalent mass (49.5 g) of
nutrients and electron acceptors may form in proximity to the NAPL. This local condition can have a profound impact on the biodegradation potential and thus the ultimate fate and persistence of the NAPL. In the case of oil spills, an organic carbon source3,4 suddenly becomes abundant, and therefore mineral nutrient13,14 and electron acceptor15−17 availabilities become the limiting factors. For these reasons, geometry is poised to be a key driver of microbial colonization,18,19 fate, and persistence of oil deposited in the coastal environment. The Deepwater Horizon spill brought to light a range of petroleum deposit geometries that can wash ashore following a spill. Field observations suggest that as oil traveled ashore it interacted with sand and other particulate matter to deposit stable geometries. Although a range of geometries were observed in the present study, quasi-spherical deposits and horizontally layered sheets represent two extremes of the spectrum. It was hypothesized that these two basic forms impose unique local conditions on nutrient and electron acceptor availability, and thus drive distinct microbial community characteristics in the vicinity of the petroleum deposit. Although geometry has not previously been invoked in oil spill persistence modeling, prior studies have noted that the combination of the beach geomorphic factors and hydraulics can act to limit availability of dissolved oxygen and other nutrients critical to petroleum biodegradation.20,21 The purpose of this study was to provide proof of concept of the effect of petroleum deposit geometry on genetic markers of terminal electron acceptor and catabolic processes and the corresponding microbial community composition. Contrasting geometries of spherical tar balls and a sheet molded from equivalent mass of Deepwater Horizon oil were compared to a no-oil control in one-dimensional columns to isolate the effect of the oil deposits and their geometries under controlled conditions. Denaturing gradient gel electrophoresis (DGGE) combined with quantitative polymerase chain reaction (qPCR) quantification of key functional genes related to methanogenesis (mcrA), sulfate reduction (dsrA), and catechol pathway (cat23) indicative of aerobic aromatic catabolism22,23 of column cores provided a snapshot of the impact of geometry on the microbial community structure. Overall this study suggests that petroleum deposit geometry may be a critical factor that should be considered in accurately modeling the ultimate fate and persistence of oil spills and could be of value for prioritizing cleanup of future spills.
Figure 1. Schematic of sand columns comparing sheet (left) and ball (right) oil deposit geometries. Depths subsampled from cores relative to the oil deposits are indicated and were subject to further microbial community analysis (Figures 4 and 5; Table 1; Figures S4−S7; Table S6).
field-collected Deepwater Horizon oil samples that had washed ashore were molded into the respective geometries and emplaced during the construction of each column. These served both as a source of oil and inoculum for this study. The columns with the sheet geometry contained a single deposit (5-cm diameter by 1.5 cm height) while columns with spherical deposits contained 17 balls approximately 1.5 cm in diameter. Oil deposits were positioned at a depth between 7.5 and 10 cm within the 20 cm of sand. The highly uniform, coarse sand (Spectrum Chemicals & Laboratory Products, Gardena, CA; average diameter >0.5 mm and uniformity coefficient =1.088) was washed with distilled water and baked at 440 °C for 16 h prior to packing the columns (in accordance with ASTM standard D2974-07) in order to minimize background organics, which could interfere with the aim of isolating the effect of the oil. The average porosity of the sand column was 0.3. The experiments were performed in absence of light at a constant temperature of 22 °C. Sand Column Maintenance and Sacrifice. Columns were recharged daily with 50 mL of artificial seawater24 (93.33 mg/L N, 45.55 mg/L P), which was prepared fresh daily, autoclaved, and cooled before adding carbonates. The average concentration of sulfate in the prepared media was 1.92 ± 0.02 g/L and that of dissolved oxygen was 6.6 ± 0.2 mg/L. Seawater is deficient in nitrate and iron,25 and therefore these were not amended to the media as potential electron acceptors. A 24-h cycle was initiated by top-filling the columns with 50 mL of artificial seawater in a nearly instantaneous manner. At the end of 12 h, effluent was drawn from the bottom of the column to provide an integrated sample representative of the entire column. Dissolved oxygen (DO) in the effluent was measured immediately upon sample collection using an Orion DO probe and a portion of sample was stored without headspace at 4 °C for weekly analysis of sulfate. Sheet column B developed a leak on the 22nd day of the study and was noted to drain water within nearly 4 h of initiating the high tide phase.
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EXPERIMENTAL SECTION Field Observation and Sampling. A field survey was conducted west to east from West Point Island to Gulf Shores, AL (Figure S1), on July 27 and 28, 2010, to document the variety and distribution of various geometries of petroleum deposits. Grab samples of seawater, oil (representing both tar ball and sheet deposits), and paired clean and visibly contaminated beach sand (5−15 cm depth, within moist layer) were collected, placed on ice, and transported to 4 °C storage at the laboratory within 48 h. Oil samples were eluted in methylene chloride and analyzed by gas chromatography−mass spectrometry (GC-MS) and GC with selected ion monitoring (GC-SIM) to profile the hydrocarbon composition. n-Alkanes and aromatics associated with weathered petroleum, such as chrysene and phenantharenes, were targeted. GC with total ion chromatography (GC-TIC) and GC-SIM were also conducted in-kind as an independent analysis by Dr. Roger Prince of Exxon Mobile. 5783
dx.doi.org/10.1021/es300393r | Environ. Sci. Technol. 2012, 46, 5782−5788
Environmental Science & Technology
Article
default selecting the complexity by Akaike information criterion. Significance of qPCR results for mcrA, dsrA, and cat23 was determined by Friedman rank sum test. Spatial correlation between samples from the same column was also tested to determine whether samples from the same column could be treated independently, and was not observed to be a significant for any column type. Hierarchical clustering was done on the basis of Bray−Curtis similarity of square root transformed intensity for each band in DGGE image using Primer E.33 A p-value
