Article pubs.acs.org/EF
Effect of Conventional and Alternative Fuels on a Marine Bacterial Community and the Significance to Bioremediation Oscar N. Ruiz,*,† Lisa M. Brown,‡ Richard C. Striebich,‡ Caitlin E. Smart,† Loryn L. Bowen,‡ Jason S. Lee,§ Brenda J. Little,§ Susan S. Mueller,‡ and Thusitha S. Gunasekera‡ †
Air Force Research Laboratory, Aerospace Systems Directorate, Fuels and Energy Branch, Wright-Patterson AFB, Ohio 45433, United States ‡ University of Dayton Research Institute, Dayton, Ohio 45469, United States § Naval Research Laboratory, Stennis Space Center, Mississippi 39559, United States S Supporting Information *
ABSTRACT: Understanding the effect of conventional and alternative fuels on the marine bacterial community is crucial, as it pertains to the impact, biodegradation, and final fate of these fuels in the environment. Metagenomics analysis demonstrated that conventional and alternative fuels promoted the growth of Proteobacteria. Marinobacter and Desulfovibrio were predominant in seawater exposed to conventional jet propellant-5 (JP-5), while Hyphomonas and Rhodovulum were most abundant in seawater with hydroprocessed renewable jet fuel (HRJ) and conventional F-76 diesel, respectively. The phyla Bacteroidetes, Firmicutes, and Lentisphaerae were underrepresented in samples with fuel, and these phyla were largely comprised of unclassified bacteria. Culture-dependent tests isolated several of the same genera detected in high abundance by metagenomics DNA sequencing, including Marinobacter, Rhodovulum, and Halobacillus. Growth studies in fuel and gas chromatography analysis demonstrated that isolates grew in fuel and metabolized hydrocarbons efficiently. The hydrocarbon degradation profile of each bacterium was conserved from conventional to alternative fuels. The study indicated that bacteria must out-compete others to get established and proliferate. Competition between hydrocarbon degraders was an important factor affecting the bioremediation process. This study provides insights into the growth characteristics of hydrocarbon-degrading bacteria and the effects of fuel on marine bacterial communities.
■
INTRODUCTION A wide range of bacteria have been isolated from marine and terrestrial environments contaminated with hydrocarbons.1−5 Bacteria of the phyla Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes are often found in hydrocarbon polluted marine and terrestrial environments.6−11 Gammaproteobacteria, including Marinobacter, Alcanivorax, Pseudomonas, and Acinetobacter were recovered from salt marshes and ocean surface water contaminated with hydrocarbons.12 Studies have shown that biostimulation of indigenous marine bacteria at spill sites can enhance the biodegradation of crude oil.11,13−15 Hydrocarbon fuel can be divided into two classes: conventional and alternative fuels. Conventional fuels are liquid hydrocarbon mixtures such as gasoline, kerosene, jet fuel, and diesel derived from the distillation of nonrenewable crude oil (petroleum). Alternative fuels are liquid hydrocarbon fuels derived from renewable feedstock and, on rare occasions, from unconventional nonrenewable resources (i.e., natural gas and coal) through advanced physicochemical and biological processes.16,17 Unlike biodiesel (fatty acid methyl esters or FAME) and ethanol biofuels, which are not comprised of hydrocarbons, alternative fuels are considered “drop in” fuels, meaning they resemble the corresponding conventional hydrocarbon fuel in composition and physicochemical properties, and may be used directly in vehicles without having to modify any of their systems. While alternative fuels derived from renewable feedstocks may prove more environmentally © XXXX American Chemical Society
friendly than conventional fuels due to a reduced carbon footprint, their complex hydrocarbon composition presents environmental challenges similar to those of conventional fuels, including biodegradability and environmental impact. Because of these factors, it is important to assess the effect of these new fuels on the environment. The metabolic flexibility of bacteria was observed in the crude oil plumes of the Deepwater Horizon blowout in the Gulf of Mexico in 2010, where the indigenous marine microbial population remediated the oil plume.18 While the microbial population in the oil plume was enriched with hydrocarbondegrading bacteria, the microbial diversity was drastically reduced in comparison to the clean surrounding water column.18 Similarly, exposure of seawater to biodiesel (fatty acid methyl ester fuel) substantially altered the microbial community.19 These observations indicate that not every bacterium has the genetic adaptation required to persist and proliferate in environments contaminated with hydrocarbons. Bacteria grow at the interface between fuel and water, secreting emulsifiers such as rhamnolipids to increase the solubility of hydrocarbons in water and facilitate utilization.20−22 While fuel can be an excellent source of carbon, some of its components may be toxic to living cells. Received: October 16, 2015 Revised: December 18, 2015
A
DOI: 10.1021/acs.energyfuels.5b02439 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels
Figure 1. Rarefaction curve showing the effect of different fuels on bacterial diversity. OTUs assigned at 5% distance.
study expands our understanding on the effects of fuel contamination on marine environments and demonstrates that marine bacterial communities contain active metabolic pathways for the degradation of diverse hydrocarbon classes.
Hydrocarbon fuels may contain aromatics, cyclic hydrocarbons, branched and normal alkanes, and additives which often are toxic to microorganisms.9,23,24 Hence, bacteria that grow in fuel have evolved mechanisms such as the formation of biofilms, secretion of biosurfactants, regulation of efflux pumps and porins, and hydrocarbon catabolism for protection against toxic compounds. 25,26 Important nutrients such as nitrogen, phosphorus, and iron are often in low availability in oilcontaminated environments. The up-regulation of multiple iron uptake genes including pyoverdin and pyochelin in P. aeruginosa during growth in Jet-A fuel was reported.26 Due to the diversity of hydrocarbon compounds in alternative and conventional fuels, no single bacterium can metabolize every compound. Bacteria selectively consume hydrocarbon components within fuel with many showing specificity for n-alkanes and simple aromatics.10,27−29 The goal of this study is to understand how fuels with different hydrocarbon composition affect the bacterial community in a sample of coastal seawater from Key West, Florida. Members of the fuel-enriched population were isolated, identified, and characterized for their ability to grow and metabolize fuel. The effect of competition between hydrocarbon degraders was evaluated. Culture-independent metagenomic analysis was used to identify and quantify bacteria before and after exposure to conventional and alternative fuels of different compositions.4,30,31 Bacteria were isolated from the fuel-seawater enrichments by culturing. Their growth rate in fuel and hydrocarbon degradation profile were determined by gas chromatography−mass spectrometry (GC-MS) to confirm the hydrocarbon bioremediation potential. The fuel-degrading isolates were used in fuel growth assays to test the degradability of several conventional and alternative fuels. Finally, fuels of different hydrocarbon composition were evaluated to see how they affected a consortium of hydrocarbon degraders. This
■
EXPERIMENTAL SECTION
Conventional and Alternative Fuels. Conventional fuels U.S. military petroleum marine diesel (F-76), petroleum jet propellant 5 (JP-5), petroleum jet propellant 8 (JP-8), and commercial jet fuel (JetA) were used in this study. Jet-A, JP-8, and JP-5 are all kerosene jet fuel of similar composition and characteristics. What makes JP-8 and JP-5 different from Jet-A is the addition of specific additive packages that may serve to improve lubricity, reduce corrosion, prevent the formation of ice and dissipate static charges. Also, JP-5 has a higher flash point than Jet-A and JP-8 so it can be used in shipboard aircrafts. Alternative fuels camelina-derived hydro-processed renewable JP-5 (HRJ-5), algal-derived hydro-processed renewable F76 diesel (HRD), and catalytic hydrothermal conversion diesel (CHCD) were used in this study. In addition, JP-5 and HRJ-5 were blended in a 50/50 volume percent ratio (v/v) to produce 50/50 JP-5/HRJ-5 (Blend) which was also tested. These fuels were selected because they are representative of middle-range distillate fuels commonly used in civilian and military transportation. Seawater Exposure to Fuel. Oligotrophic coastal seawater containing sediment was collected from Key West, FL, and used as inoculum and growth medium.32 Throughout this paper, the natural seawater with sediment is abbreviated as NKWSW. During metagenomic analysis (Figures 1−3, and Figure S1, Supporting Information), the coastal seawater inoculum was separated into the liquid fraction (KWSW) and the solid sediment fraction (KW sediment) to facilitate DNA extraction and characterization of bacterial populations. Seven chambers were constructed to contain seawater and fuel combinations. The chambers (Tank Depot, Pompano Beach, FL) were rectangular and constructed from heavy gauge, chemical and fuel resistant, opaque black cross-linked polyethylene plastic. A schematic representation of the fuel/seawater exposure chamber is provided in Figure S2, Supporting Information. NKWSW (4.5 L) with fuel (3 L), B
DOI: 10.1021/acs.energyfuels.5b02439 Energy Fuels XXXX, XXX, XXX−XXX
Article
Energy & Fuels
remove short sequence reads (
