A Survey of Deepwater Horizon (DWH) Oil-Degrading Bacteria from the Eastern Oyster Biome and Its Surrounding Environment

ORIGINAL RESEARCH ARTICLE published: 09 April 2014 doi: 10.3389/fmicb.2014.00149 A survey of deepwater horizon (DWH) oil-degrading bacteria from the Eastern oyster biome and its surrounding environment Jesse C.Thomas IV 1†, Denis Wafula1†, Ashvini Chauhan1*, Stefan J. Green 2,3 , Richard Gragg1 and Charles Jagoe1,4 1 Environmental Biotechnology Laboratory, School of the Environment, Florida Agricultural and Mechanical University, Tallahassee, FL, USA 2 DNA Services Facility, University of Illinois at Chicago, Chicago, IL, USA 3 Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA 4 NOAA Environmental Cooperative Science Center, School of the Environment, Florida Agricultural and Mechanical University, Tallahassee, FL, USA Edited by: The deepwater horizon (DWH) accident led to the release of an estimated 794,936,474 L Andreas Teske, University of North of crude oil into the northern Gulf of Mexico over an 85 day period in 2010, resulting in Carolina at Chapel Hill, USA the contamination of the Gulf of Mexico waters, sediments, permeable beach sands, Reviewed by: coastal wetlands, and marine life. This study examines the potential response of the TorstenThomas, The University of New South Wales, Australia Eastern oyster’s microbiome to hydrocarbon contamination and compares it with the Xiang Xiao, Shanghai Jiao Tong bacterial community responses observed from the overlaying water column (WC) and University, China the oyster bed sediments. For this purpose, microcosms seeded with DWH crude oil *Correspondence: were established and inoculated separately with oyster tissue (OT), mantle fluid (MF), Ashvini Chauhan, Environmental overlaying WC, and sediments (S) collected from Apalachicola Bay, FL, USA. Shifts in the Biotechnology Laboratory, School of the Environment, Florida Agricultural microbial community structure in the amended microcosms was monitored over a 3-month and Mechanical University, Suite period using automated ribosomal intergenic spacer region analysis, which showed that 305B, FSH Science Research Center, the microbiome of the OT and MF were more similar to the sediment communities than Tallahassee, FL 32307, USA e-mail: [email protected] those present in the overlaying WC. This pattern remained largely consistent, regardless of the concentration of crude oil or the enrichment period. Additionally, 72 oil-degrading †Present address: Jesse C. Thomas, Department of bacteria were isolated from the microcosms containing OT, MF,WC, and S and identified Environmental Health Sciences, using 16S ribosomal RNA gene sequencing and compared by principal component analysis, University of Georgia, 150 East Green which clearly showed that the WC isolates were different to those identified from the Street, Athens, GA 30602, USA; Denis Wafula, College of Pharmacy, sediment. Conversely, the OT and MF isolates clustered together; a strong indication The University of New Mexico, that the oyster microbiome is uniquely structured relative to its surrounding environment. Albuquerque, NM 87131, USA When selected isolates from the OT, MF,WC, and S were assessed for their oil-degrading potential, we found that the DWH oil was biodegraded between 12 and 42%, under the existing conditions. Keywords: deepwater horizon oil spill, oyster, bacteria, biodegradation, hydrocarbon INTRODUCTION the 1989 Exxon Valdez accident in Alaska and more recently, the Since the start of the second industrial revolution, the use of fossil deepwater horizon (DWH) spill illustrate how oil hydrocarbons fuels including petroleum hydrocarbons has increased dramati- can cause acute (e.g., lethality) and chronic (e.g., reduced growth cally (Mojib et al., 2011). The world’s total oil consumption is and foraging success, reduced fecundity, increased levels of defor- expected to rise to over 96.1 million barrels/day by 2015 and mities, and abnormal social behavior) impacts to coastal ecosystem up to 113.3 million barrels/day by 2030 [United States Energy services (Peterson, 1991, 2001; Peterson et al., 2003; Hazen et al., Information Administration (US-EIA), 2011a]. As of 2010, the 2010; Kostka et al., 2011; Henkel et al., 2012; Whitehead et al., United States alone consumed 19.15 million barrels/day of refined 2012). Assessing impacts of oil spills requires a comprehensive petroleum products and biofuels; which is approximately 22% of understanding of ecosystem services, including the water filtration the world’s total petroleum consumption (United States Energy capacity and nursery areas that are typically provided by oyster reef Information Administration (US-EIA), 2011b). However, despite habitats (Peterson and Heck, 1999; French McCay et al., 2003). stimulating growth in areas such as agriculture and transportation, As of 2000, estuaries in the Gulf of Mexico region contributed petroleum fuels are a major source of environmental pollution 83% of the total Eastern oysters (Crassostrea virginica) harvested by way of accidental spills, particularly in estuarine and marine in the United States (U.S. National Marine Fisheries Service1). As ecosystems (Paine et al., 1996; Newman and Unger, 2003). a keystone species in the Gulf, oysters and their reef assemblages When coastal ecosystems are impacted by contaminants, serve as critical habitat for commercially important seafood, while important functions (e.g., fishery production, biofiltration) may be affected (Livingston, 1984; Nixon, 1995). Oil spills such as 1www.st.nmfs.gov www.frontiersin.org April 2014 | Volume 5 | Article 149 | 1 Thomas et al. Biodegradation of crude oil by oyster microbiome also performing critical ecosystem services such as water filtration et al., 1990), and may also supply the bivalve host with vita- and sequestration of excess nitrogen (Raj,2008; Roosi-Snook et al., mins and amino acids that serve as growth factors- as shown 2010; Piehler and Smyth, 2011; Grabowski et al., 2012). The value in the Pacific vesicomyid clam- Calyptogena magnifica found at of this habitat, both ecologically and economically, however, may cold seeps (Newton et al., 2007). Moreover, certain symbiotic be greatly reduced by pollution, including accidental oil spills. bacteria can even protect their host from pathogens by either It should however, be noted that the Gulf of Mexico waters producing antimicrobial agents, or by growing in high densi- are continuously exposed to a background level of hydrocarbons ties that prevents colonization by other strains (Pujalte et al., vented by numerous hydrocarbon seeps, which emit hydrocar- 1999). More recently, King et al. (2012) used pyrosequencing to bon gas into the overlaying water column (WC) as bubble plumes reveal substantial differences between stomach and gut micro- which are often coated with a thin layer of oil (MacDonald et al., biomes of oysters from Lake Caillou in Louisiana. These authors 2002; Leifer and MacDonald, 2003; Solomon et al., 2009). Accord- found that bacteria belonging to Chloroflexi, Mollicutes, Planc- ing to some estimates, seepage in the Gulf of Mexico, from over 200 tomycetes, and Spartobacteria likely comprise a major core of active seeps, contributes oil at a high rate of 0.4–1.1 × 108 L/year the oyster stomach microbiome, whereas, Chloroflexi, Firmicutes, (Mitchell et al., 1999). As a corollary, this “natural” oil likely α-proteobacteria, and Verrucomicrobia were more abundant in the primes the environment continuously, by selectively enriching gut. native microorganisms with the ability to metabolize and min- Despite the existing information on the nature of oyster- eralize some of this oil. Therefore, it is very likely that a suite of associated microbial communities, not much is known on their native hydrocarbon-degrading bacteria exist in different niches of ability to degrade oil hydrocarbons. Therefore, the purpose of this the Gulf of Mexico, and as reported previously responded rapidly study was to not only to assess the oyster microbiome, which in to the large volume of oil released by the DWH spill (Hazen itself necessitates further studies because it is a largely understud- et al., 2010; Redmond and Valentine, 2012; Dubinsky et al., 2013; ied microhabitat, but also to enrich, isolate and compare the oil Gutierrez et al., 2013). biodegradation efficacy of oyster-associated bacteria. A significant body of information exists on the pathways and the nature of oil-degrading microbes from a variety of diverse envi- MATERIALS AND METHODS ronments, including soils and aquatic ecosystems (Margesin and SITE DESCRIPTION AND SAMPLE COLLECTION Schinner, 2001; Aitken et al., 2004; Hamamura et al., 2005; Hazen This study was conducted on oysters, water, and sediment et al., 2010; Kostka et al., 2011), but virtually nothing is known (S) samples collected from Dry Bar, the most productive on the nature and ability of microbes that can degrade oil from bar, in Apalachicola Bay, Florida (29◦ 40.474N, 085◦03.497W). the oyster reefs or within the oyster itself. In this report, we have Apalachicola Bay is a relatively pristine estuary, well mixed investigated the propensity of a largely understudied ecosystem- by freshwater from the Apalachicola-Chattahoochee-Flint (ACF) the Eastern oyster (Crassostrea virginica) microbiome – to degrade river system and oceanic Gulf tides (Chauhan et al., 2009). The the Gulf crude oil. Previous studies have shown that the oyster bay produces 90% of Florida oysters, the third highest catch of biome consists of a diverse collection of heterotrophic bacteria shrimp, a rich supply of brown shrimp,

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