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Research Paper

The Effect of Corexit® 9500A on the Bacterial Community in the Northern Gulf of Mexico Coastal Sediments

Priya Bhattacharya and Joong-Wook Park*

Department of Biological and Environmental Sciences, Troy University, Troy, AL 36082, USA.

*Corresponding author, Joong-Wook Park, email: jwpark@troy.edu

Received 22 March 2018, revised 20 August 2018, accepted 21 August 2018

Publication Date (Web): August 21, 2018

© Frontiers in Science, Technology, Engineering and Mathematics

Abstract

The Deepwater Horizon oil spill resulted in an unprecedented use of the dispersant Corexit® 9500A, which generated concern about the effect of dispersants on the microbiota in the Gulf of Mexico. In this study, sediment collected from two continental shelf sites and two salt marsh sites in the northern Gulf of Mexico were incubated with two different concentrations of Corexit® 9500A for 14 days to investigate the impact of the dispersant on the bacterial community. Our results showed that the effect of 0.02% Corexit® 9500A was negligible, while 0.2% Corexit® 9500A significantly shifted the bacterial community in all tested sediments. Our data demonstrate the concentration of dispersant is a crucial factor affecting the bacterial community in both continental shelf and salt marsh sediments.

Keywords

Dispersant, Corexit, Oil spill, Bacterial community, Continental shelf, Salt marsh

Introduction

The Deepwater Horizon (DWH) oil spill was the world’s largest off-shore oil spill, releasing approximately 4.4 million barrels (700 million liters) of crude oil into the Gulf of Mexico (GOM) (Kourafalou and Androulidakis 2013). Approximately eight million liters of the chemical dispersants were used to treat the DWH oil spill; of which, 5.6 million liters of Corexit® 9500A were used at the wellhead and on the surface (Judson et al. 2010; Kujawinski et al. 2011).

Dispersants are surfactants blended with solvents, which are designed to break up oil slicks (Fiocco and Lewis, 1999). A good dispersant must have HLB (hydrophilic-lipophilic balance) of 10 to 11 on a scale of 0 to 20, where low values tend to dissolve in oil and high values tend to dissolve in water (Canevari 1969; Fiocco and Lewis, 1999). This amphipathic characteristic of dispersants enables them to locate at the oil-water interface and increases the oil-water surface area, which thereby provides more surface for oil-degrading bacteria to act on (Canevari 1969).

The hydrocarbon based Corexit® 9500A is a dispersant designed for high viscosity oils and weathered emulsions (Singer et al. 1996; Ramachandran et al. 2004). Corexit®9500A was reformulated from a water-based Corexit® 9527A (Singer et al. 1996; Ramachandran et al. 2004) and was approved by the US Environmental Protection Agency (US EPA) in 1994. Although Corexit® 9500A was marketed as an environment friendly substitute for Corexit® 9527A, many studies have given some conflicting results. For example, it was reported that the application of Corexit® 9500A led to an increase in oil-degrading bacteria (Bælum et al. 2012; Chakraborty et al. 2012), while other study showed a significant drop in oil-degrading bacteria (Hamdan and Fulmer 2011). Especially, enhanced toxicity of oil with Corexit® 9500A was reported (Ramachandran et al. 2004; USEPA 2014) and our lab’s previous data support this idea by showing the synergistic effect of crude oil with Corexit® 9500A (Al-Jawasim et al. 2015). The conflicting reports regarding Corexit® 9500A toxicity maybe because each component in the dispersants has their own distinctive effect on specific bacterial species (Bruheim et al. 1999). Despite the controversy, it is obvious that the Corexit® 9500A triggers the shift of bacterial community structures.

Bacteria play a crucial role in the functioning of marine sediment ecosystems (Findlay 2010). They are dominant in the marine environments and affect other species as major decomposers and as food sources on the ocean floor (Fuhrman et al. 2006; Krumins et al. 2013). Considering the importance of marine bacteria in the food chain, it is necessary to study how dispersant affects marine bacteria in continental shelf and salt marsh sediments. As a large number of oil rigs in the GOM pose a potential for future oil spills (NOAA 2012; Yáñez-Arancibia et al. 2013), it is highly probable that the dispersant will be applied again in the GOM.

There are considerable numbers of publications on the DWH oil spill that triggered shift in microbial community in sediments (Liu Z, Liu J 2013; Thomas et al. 2014; Bacosa et al. 2018), salt marsh (Koo et al. 2015), water column (Liu Z, Liu J 2013; Thomas et al. 2014), and beach sand (Kostka et al. 2011; Newton et al. 2013; Kappell et al. 2014; Rodriguez-R et al. 2015; Bacosa et al. 2015; Liu et al. 2016). Additionally, several studies have been published on the impact of Corexit® 9500A on the bacterial community (Hamdan and Fulmer 2011; Chakraborty et al. 2012; Kleindienst et al. 2015), especially a study by Hamdan and Fulmer (2011) reported the responses of bacteria isolated from an oiled GOM beach with different concentrations of Corexit® 9500A. However, no study has examined the impact of different concentrations of Corexit ® 9500A on bacterial communities in the GOM continental shelf and salt marsh sediments. In this study, continental shelf and salt marsh sediments collected from four distinct locations in the northern GOM were used to study the response of their indigenous bacteria after exposure to different concentrations of Corexit® 9500A.

Materials and Methods

Sediment sampling

Two sediment samples were collected from the GOM continental shelf using a Shipek® grab sampler aboard the National Oceanic and Atmospheric Administration (NOAA) ship Gordon Gunter in November 2014 by Jonathan Miller and Ceil Jones of Troy University. The GPS coordinates of two sampling sites, called GOM 1 and GOM 2, are N29.3649°, W88.8853° at a depth of 22 meters (GOM 1) and N29.7786°, W88.4660° at a depth of 32 meters (GOM 2). The third sediment was collected in November 2014 from a salt marsh at Dauphin Island (DI) (N30.2574°, W88.1238° at the surface) by Dr. Stephen Landers of Troy University. The fourth sediment was collected in November 2014 from a salt marsh at Lake Pontchartrain (LP) (N30.1463°, W89.7445° at the surface) by Dr. Kewei Yu of Troy University. These two salt marsh sediments were collected with a shovel at a depth of 0 to 10 cm. The samples were stored at 4 _C prior to analysis. The locations of sampling sites and the DWH oil spill site are shown on Figure 1, which was constructed using ArcGIS with bathymetric data from the USGS site (http://woodshole.er.usgs.gov/pubs/of2005-1071/data/background/bathy_contours).

Microcosm preparation

Twelve sets of microcosms were prepared with the four sediments (GOM 1, GOM 2, DI, and LP), with each given three separate treatments as follows: (1) treated with no Corexit® 9500A as a control, (2) treated with 0.02% Corexit® 9500A, and (3) treated with 0.2% Corexit® 9500A. The concentration was calculated by the volume of Corexit®9500A per weight of sediment sample (v/w). All microcosms were set up in triplicate (4 sediment types x 3 treatments x triplicate = 36 microcosms in total). The Corexit® 9500A was obtained from Nalco/Exxon Energy Chemicals, L.P. (Sugar Land, TX). The microcosms were prepared with 2 grams of sediment and 2 mL of sterilized artificial sea water. The artificial sea water was prepared by dissolving Instant Ocean® Sea Salt (Aquarium Systems, Mentor, Ohio) in deionized water and sterilized by using a 2 µm non-pyrogenic filter. Different salinities were used to mimic the salinity of sampling sites, which were 36‰ for GOM1 and GOM2 microcosms, 24‰ for DI microcosms, and 12‰ for LP microcosms. The 36 microcosms were incubated at 18 _C under static conditions for 14 days.

Figure 1. Location of sampling sites in the Gulf of Mexico. Red dots indicate sampling sites and a yellow rectangle indicates the Deepwater Horizon oil spill site.

DNA extraction and polymerase chain reaction (PCR)

Total DNA was extracted from 0.5 grams of sediment using the PowerSoil™ DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) and the DNA samples were stored at -20 _C prior to analysis. PCR was conducted to amplify the V3 region of bacterial 16S rRNA gene using a 341F primer (5'- CCT ACG GGA GGC AGC AG -3') with GC clamp (5'- CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G -3) and a 534R primer (5'- ATT ACC GCG GCT GCT GG -3') (Muyzer et al. 1993). The total PCR mix per sample was 50 µL, which contained 40 µL of deionized water, 5 µL of 10x Green Taq PCR buffer, 0.25 mM of dNTP, 10 pmol of forward and reverse primers, 2 µL of template DNA, and 1 U of Green Taq DNA polymerase (GenScript, Piscataway, NJ). A DNA thermal cycler (GeneAmp PCR System 2700, Applied Biosystems, Foster City, CA) was used to carry out the PCR process at an initial temperature of 94 °C for five minutes, followed by 35 cycles of 94 °C for 20 seconds, 55 °C for 45 seconds, and 72 °C for 45 seconds. A final extension of 72 °C was performed for seven minutes and the resulting PCR products were confirmed by an agarose gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE)

Eight percent polyacrylamide gel was used to separate the PCR products with denaturing gradient of 40% and 60% by a BioRad DCodeTM universal mutation detection system (Bio-Rad Laboratories, Hercules, CA) in 1 x TAE buffer. The PCR product was mixed with 2x DGGE dye, 40 µL of the mixture was loaded on the DGGE gel, and the electrophoresis was carried out at 60 V for 12 hours at 60 °C. Thereafter the DGGE gel was stained with ethidium bromide for 15 minutes and photographed on a UV transilluminator (Fisher Scientific, Pittsburgh, PA).

DGGE gel images analysis

The DGGE images were analyzed using the PyElph software (Pavel and Vasile 2012) to generate dendrograms by comparing the different DGGE band patterns of each lane. Briefly, the gel image was loaded by the PyElph software, lanes and DGGE bands were manually selected, and the matrix patterns (presence of bands and their positions in each lane) were used to cluster similar lanes together. In this study, UPGMA (Unweighted Pair Group Method with Arithmetic Mean) was used to cluster lanes and construct dendrograms (Drummond and Rodrigo 2000). Lane clustering and dendrogram constructions were automatically computed by the PyElph software 

(A)

(B)

Figure 2. DGGE profile of the GOM1 microcosms (A) and a corresponding UPGMA dendrogram (B). The values on the horizontal lines in the dendrogram stand for distances among samples in percentages. Original: untreated sediments at day 0; M: custom marker; numbers 1, 2, and 3 represents the triplicates.

(A)

(B)

Figure 3. DGGE profile of the GOM2 microcosms (A) and a corresponding UPGMA dendrogram (B). The values on the horizontal lines in the dendrogram stand for distances among samples in percentages. Original: untreated sediments at day 0; M: custom marker; numbers 1, 2, and 3 represents the triplicates.

(A) 

(B)

Figure 4. DGGE profile of the LP microcosms (A) and a corresponding UPGMA dendrogram (B). The values on the horizontal lines in the dendrogram stand for distances among samples in percentages. Original: untreated sediments at day 0; M: custom marker; numbers 1, 2, and 3 represents the triplicates.

(A)

(B)

Figure 5. DGGE profile of the DI microcosms (A) and a corresponding UPGMA dendrogram (B). The values on the horizontal lines in the dendrogram stand for distances among samples in percentages. Original: untreated sediments at day 0; M: custom marker; numbers 1, 2, and 3 represents the triplicates.

Results and Discussion

The DGGE band patterns representing bacterial communities showed that the controls (0% Corexit® 9500A-treated microcosms) were similar to 0.02% Corexit® 9500A-treated microcosms, while different from 0.2% Corexit® 9500A-treated microcosms after 14 days of incubation (Figures.  2A, 3A, 4A, and 5A). The UPGMA dendrograms analyzed by the PyElph software also demonstrated that the DGGE profiles of controls and 0.02% Corexit® 9500A microcosms were grouped together, while those of 0.2% Corexit® 9500A lied outside (Figures.  2B, 3B, 4B, and 5B). This trend is consistent with all four microcosms prepared with continental shelf and salt marsh sediments. Our data suggest that the effect of 0.02% Corexit® 9500A treatment was negligible, but 0.2% Corexit® 9500A greatly affect the bacterial communities in both continental shelf and salt marsh sediments. In a study by Hamdan and Fulmer (2011), dilutions of Corexit® 9500A of 1:10 and 1:100 (w/v) resulted in the extinction of all bacteria isolated from the oiled beach samples; a 1:1,000 (w/v) dilution inhibited the growth of most isolated bacteria; and 1:10,000 and 1:100,000 (w/v) dilutions did not affect the growth of most isolated bacteria. Assuming a marginal concentration of Corexit® 9500A that impact the most isolated bacteria is a 1:1,000 dilution, their result is similar to ours since the dilution of 1:1,000 (which equals 0.1%) lies between 0.2% and 0.02%. However, Hamdan and Fulmer’s research tested the bacteria isolated from the oiled Louisiana beach, therefore it is difficult to claim that the trend also applies to other areas in the GOM. We used the continental shelf and salt marsh sediments collected from four different GOM locations and showed 0.2% of Corexit® 9500A triggers the bacterial community shift in all tested sediments.Mackay et al. (1984) recommended a conservative dispersant to oil ratio (DOR) of 1:200 (which equals 0.5%), but studies usually use a higher DOR (Fiocco and Lewis 1999; Chandrasekar et al. 2006; Mukherjee and Wrenn 2010). Practical applications of DOR vary from 1:1 to 1:100, but 1:20 is generally used (STAR 2006; ITOPF 2011; IPIECA and IOGP 2015). Additionally, the practical surface application of dispersant tends to be higher than recommended to ensure the dispersant reaches the target area to overcome the effect of wind during spraying (Mackay et al. 1984; ITOPF 2011). It was reported that the DOR used for the DWH oil spill was over 1% (8 million liters to 700 million liters) (Kujawinski et al. 2011; Chakraborty et al. 2012; Kourafalou and Androulidakis 2013; Kleindienst et al. 2015). Considering our result that the bacterial community shifted at 0.2% of dispersant, the applied Corexit® 9500A affected the bacterial communities in the GOM continental shelf and salt marsh.

The bacterial community was analyzed by DGGE in this research, while metagenomic pyrosequencing can be a better option in terms of accuracy and sensitivity. The cost of pyrosequencing is considerably high, especially when the number of samples are large (48 samples including 12 untreated sediments at day 0 were analyzed here). This research was initiated to examine whether there is any impact of different concentrations of Corexit® 9500A on bacterial communities in four GOM sediments. Therefore, the DGGE analysis was a sufficient and cost-effective method for this study.

Conclusions

This study examined the effects of different concentrations of Corexit® 9500A on the indigenous bacterial communities in four GOM sediments collected from continental shelfs and salt marshes. The results showed that a concentration of 0.02% Corexit® 9500A had little impact on bacterial community, while significant changes in the bacterial community structures were observed at a concentration of 0.2% Corexit® 9500A. Bacteria are major players in marine ecosystems serving as food sources and decomposers (Findlay 2010; Fuhrman et al. 2015). Some marine bacteria are also important for their oil-degradation ability. Considering the importance of bacteria in marine ecosystems and the presence of a large numbers of oil rigs in the GOM, further research is necessary to understand types of bacteria that are affected by dispersants.

Acknowledgements

This research was made possible by Troy University Faculty Development Research Grants and a grant from the Gulf of Mexico Research Initiative. Data are publically available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearch initiative.org (doi: 10.7266/N72B8W1Q). We thank the National Marine Fisheries Service / National Ocean and Atmospheric Administration (NMFS /NOAA) crew and scientists on NOAA ship Gordon Gunter, Jonathan Miller and Ceil Jones for their assistance with sample collections. Dr. Stephen C. Landers and Dr. Kewei Yu at Troy University generously provided sediment samples for this study.

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Citation:

Priya Bhattacharya and Joong-Wook Park* (2018) The Effect of Corexit® 9500A on the Bacterial Community in the Northern Gulf of Mexico Coastal Sediments, Frontiers in Science, Technology, Engineering and Mathematics, Volume 2, Issue 2, 105-115