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Anaerobic Biodegradation of Alternative Fuels and Associated Biocorrosion of Carbon Steel in Marine Environments Renxing Liang, Deniz Fulya Aktas, Egemen Aydin, Vincent Bonifay, Jan Sunner, and Joseph M Suflita Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06388 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 12, 2016

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Environmental Science & Technology

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Anaerobic Biodegradation of Alternative Fuels and Associated Biocorrosion of Carbon

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Steel in Marine Environments

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Renxing Liang1, Deniz F. Aktas1, Egemen Aydin1,†, Vincent Bonifay1, Jan Sunner1,

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Joseph M. Suflita1*

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Oklahoma, Norman, OK, USA

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*To whom correspondence should be addressed. E-mail: [email protected]

Department of Microbiology and Plant Biology and OU Biocorrosion Center, University of

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Mailing address: 770 Van Vleet Oval, Norman, OK 73069, U.S.A.

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Tel: +1(405) 325-3771; Fax: +1(405) 325-7619.

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†

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Engineering Department, Maslak, 34469, Istanbul, Turkey

Current address: Istanbul Technical University, Faculty of Civil Engineering, Environmental

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Abstract

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Fuels that biodegrade too easily can exacerbate through-wall pitting corrosion of

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pipelines and tanks and result in unintentional environmental releases. We tested the biological

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stability of two emerging naval biofuels (camelina-JP5 and Fisher-Tropsch-F76) and their

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potential to exacerbate carbon steel corrosion in seawater incubations with and without a

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hydrocarbon-degrading sulfate-reducing bacterium. The inclusion of sediment or the positive

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control bacterium in the incubations stimulated a similar pattern of sulfate reduction with

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different inocula. However, the highest rates of sulfate reduction were found in incubations

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amended with camelina-JP5 (57.2±2.2 - 80.8±8.1 µM/day) or its blend with petro-JP5 (76.7±2.4

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µM/day). The detection of a suite of metabolites only in the fuel-amended incubations confirmed

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that alkylated benzene hydrocarbons were metabolized via known anaerobic mechanisms. Most

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importantly, general (r2=0.73) and pitting (r2=0.69) corrosion were positively correlated with

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sulfate loss in the incubations. Thus, the anaerobic biodegradation of labile fuel components

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coupled with sulfate respiration greatly contributed to the biocorrosion of carbon steel. While all

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fuels were susceptible to anaerobic metabolism, special attention should be given to camelina-

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JP5 biofuel due to its relatively rapid biodegradation. We recommend that this biofuel be used

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with caution and that whenever possible extended storage periods should be avoided.

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Introduction

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Over the ensuing decades, world energy demand will likely increase to meet the needs of

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a burgeoning human population.1 Tremendous progress has been made toward the sustainable

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production of alternative fuels from renewable resources to address concerns over finite

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petroleum reserves and the emission of greenhouse gases.2 As the largest energy-consuming

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entity in the United States, the Department of Defense has spearheaded the effort to incorporate

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alternative fuels to reduce the dependence on petroleum fuels and boost green energy

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development.3 For example, it is estimated that 50% of the total energy consumption for US

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Navy will be coming from alternative sources by 2020.4 The fast-growing, alternative fuels

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market will hopefully become cost-competitive to supplement conventional petroleum-based

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reserves.5

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Numerous challenges are associated with adopting alternative fuels to augment or replace

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petroleum-derived fuels 6. Typically, fungible biofuels require various tests to ensure that they

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have physicochemical properties comparable to their petroleum-based counterparts.6-8 However,

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even though fuel performance might meet both explicit and implicit requirements9, the biological

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stability of the emerging alternative fuels 10, 11 and their compatibility with the mainly carbon-

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steel energy infrastructure such as storage tanks and transportation pipelines 12 should not be

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overlooked. Fuel biodeterioration and the biocorrosion (or microbiologically influenced

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corrosion) of the carbon-steel infrastructure can lead to serious economic and environmental

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problems.13 It is noteworthy that naval fuels are often exposed to sulfate-rich seawater in

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compensated fuel ballast systems where anaerobic conditions will eventually develop.14, 15

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Recent studies have demonstrated that sulfate-reducing bacteria can metabolize hydrocarbons in

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conventional petroleum fuels and thereby exacerbate the biocorrosion of carbon steel under



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anaerobic conditions.14, 16 Therefore, the biological stability of emerging alternative fuels and

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their potential to impact the biocorrosion of carbon steel should be critically assessed before they

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enter into existing fuel containment and distribution systems. 10

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Typically, the chemical composition of biomass-derived alternative fuels is slightly or

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significantly different from their petroleum counterparts 17 due to different feedstock 2 and

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processing technologies.18 Such differences might impact the biological stability of alternative

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fuels and their potential to corrode the fuel transportation and storage infrastructure.10 For

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example, the first generation biodiesel is primarily composed of fatty acid methyl esters

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(FAMEs).19 Several studies have shown that such first-generation biodiesels are not good fuel

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candidates for military tactical vessels since FAMEs can be readily hydrolyzed and the microbial

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metabolism of the resulting intermediates linked with sulfide production can exacerbate the

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corrosion of carbon steel.15, 20

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Therefore, the US Navy is now considering alternative biofuels 3, 21 mainly including

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hydroprocessed camelina-JP5 and Fischer-Tropsch (FT) diesel fuel FT-F76 from renewable

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sources.3 Typically, these renewable fuels are used experimentally as 50/50 blends with

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petroleum fuel in tactical vehicles.6, 22 Previous studies have shown that the molecular-weight

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range of hydrocarbons in naval biofuels is narrower than that in traditional petroleum fuels.23, 24

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Moreover, FT fuels contain more branched alkanes and fewer aromatic compounds compared to

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petroleum fuels.9 Recent studies have shown that FAME biodiesel and hydroprocessed biofuels

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(camelina-JP5 and algae-F76) are amenable to biodegradation under transient oxygen conditions

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and can accelerate carbon steel corrosion.20, 23, 24 The black corrosion products deposited on

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coupons were mostly mineral sulfides suggesting that majority of metal damage occurred when

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anaerobic conditions prevailed. Therefore, an experimental protocol to evaluate the biostability


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of fuels under anaerobic conditions was recently published.10 We implemented this protocol to, i)

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determine if the findings on fuel/biofuel stability could be extrapolated to other coastal

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seawater/sediment systems, ii) extend the findings to fuels not previously evaluated (e.g.FT-F76),

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and iii) examine the relationship between the degree of corrosion and the utilization of seawater

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sulfate.

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Our findings suggest that camelina-JP5 was more amenable to biodegradation, but FT-

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F76 was relatively stable compared to the petroleum-based counterparts. Most importantly, both

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general and pitting corrosion were positively correlated with the degree of sulfate loss linked to

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the anaerobic metabolism of fuel components. Due to the relatively rapid biodegradation of

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camelina-JP5, caution should be exercised with the use and storage of this hydroprocessed

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biofuel.

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Experimental Section

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Seawaters and Fuels. Coastal seawaters that are typically used to fill carbon-steel ballast tanks

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were used as both medium and inoculum sources. Given the potential differences in microbial

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community composition and water chemistry, three geographically distinct seawaters from Key

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West (KW, Florida, USA), the Gulf of Mexico (GoM, Alabama, USA) and San Diego Bay

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(SDB, California, USA) were collected as previously described and shipped to the University of

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Oklahoma 11. Sediment from SDB was also used in some experiments since it is routinely

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introduced into ship ballast systems and serves as a source of anaerobic microorganisms.25, 26 The

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seawater and sediment samples were stored at 4oC until use. The fuels used (petroleum-F76, FT-

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F76, petroleum-JP5, camelina-JP5) were obtained from Naval Air Systems Command

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(NAVAIR) as cited in23. Fuel blends, 50/50 by volume, were also tested since alternative fuels

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are typically blended with petroleum derived fuels to ensure ideal performance properties.22 In



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addition, a mixture of n-alkanes (C6-C12), whenever required, was introduced as previously

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described.10

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Experimental Design. The biodegradation and biocorrosion assays were performed according to

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a previously published protocol 10 and are summarized in Table S1. The seawater was bubbled

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with N2 for 60 min and then reduced with a sterile stock solution of Na2S (0.05 M). Resazurin

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(0.001%) was added as redox indicator to ensure that obligate anaerobic conditions were

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maintained throughout the incubation. For KW and GoM incubation, reduced anaerobic seawater

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(40 mL) was dispensed inside a well-working anaerobic glove bag into sterile 120 mL serum

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bottles containing pre-weighed coupons. Since n-alkanes are the common hydrocarbons in these

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fuels, a hydrocarbon-degrading bacterium, 5 ml of D. alkanexedens strain ALDCT 27 grown into

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the stationary phase (6.04-8.48×107 cells/mL), was inoculated into 35 ml seawater as a positive

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control. Due to the hydrocarbon biodegradation potential in SDB sediment 28, 10 g of sediment

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was placed in 40 ml seawater for SDB incubations as an additional inoculum source. Carbon-

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steel coupons were suspended by PTFE (polytetrafluoroethylene) coated quartz thread according

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to previous published procedures 10 whenever sediment was included. The headspace of the

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bottles was exchanged three times with N2/CO2 (80:20). The pH of the incubations was initially

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slightly alkaline (7.46-7.80) and other chemical parameters of three seawaters (e.g. the content of

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Cl- and SO42-) were reported previously.11 Fuels (0.1 ml; ~2 g/L) were aseptically added to 40 ml

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seawater by sterile syringe. Fuel-unamended and sterile incubations served as negative controls

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(Table S1). Autoclaved inocula (20 min at 121oC; 20 psi) served as sterile controls. The

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experiment was conducted in triplicate (Table S1) and all incubations were in the dark at ambient

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temperatures (21±2 oC). The data and error bars in all figures represent the mean and standard

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deviation of triplicate or duplicate (sterile control) determinations.


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Corrosion Assay. The carbon-steel (type 1018) coupons were fabricated into round disks (9.53

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mm in diameter; thickness,1 mm) and their chemical composition (wt. %: 0.15–0.20% C, 0.6–

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0.9% Mn, 0.035% maximum S, 0.03% maximum P, and the remainder Fe) was described

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previously.10 Coupons were cleaned with deionized water and then washed with two successive

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acetone treatments as described elsewhere 20. The pre-cleaned coupons were then flushed with

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N2 and sealed in serum bottles under N2 before autoclaving. At the end of experiment, coupons

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were harvested and cleaned according to described ASTM G1-03 (American Society for Testing

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and Materials) standard procedures.29 The cleaned coupons were dried under a stream of N2 and

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stored under N2 prior to weight loss measurements and profilometry analyses. The corresponding

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general corrosion rate (milli-inch per year, 1 mpy = 0.0254 mm/year) was calculated from mass

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loss data according to ASTM Standard G1-03.29

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Analytic Techniques. Dissolved sulfide in KW incubations was determined

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spectrophotometrically by the methylene blue method.30 Samples (0.5 ml) were withdrawn every

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30 days from the incubations by a sterile and gassed syringe to monitor sulfate loss by ion

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chromatography (Dionex model IC-3000, 122 Sunnyvale, CA) as previously described.14

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Fuel Characterization and Metabolite Profiling. To characterize major fuel components,

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petroleum (JP5 and F76) and biomass-based (camelina-JP5 and FT-F76) fuels were mixed with

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ethyl acetate (1:20) and directly analyzed by gas chromatography-mass spectrometry (GC/MS)

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as previously described.14 To identify the metabolites generated during the course of fuel

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biodegradation in KW incubations, approximately 20 ml aqueous sample from each incubation,

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was collected, acidified (~pH 2 with 6 N HCl) and subjected to liquid-liquid extraction with an

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equal volume (20 ml) of ethyl acetate according to the previously described procedures.15 The



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solvent extract was concentrated to approximately 100 µL and derivatized with N,O-

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Bis(trimethylsilyl) trifluoroacetamide (BSTFA) prior to analysis by GC/MS.15

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To assess the kinetics of metabolite flux, as little as 300 µL samples of the GoM

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incubations were sampled over time and stored at - 20 oC. The frozen samples were thawed and

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subsequently acidified to pH ~2 with 4N HCl, extracted with 2 mL ethyl acetate (2 times). The

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organic phase was collected and evaporated to dryness, followed by reconstitution of analytes in

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100 µL isopropanol prior to analysis by high performance liquid chromatography (HPLC,

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Agilent Infinity 1290) and quadrupole time-of-flight (q-TOF) mass spectrometry (MS, Agilent

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6538, UHD Accurate Mass). A similar procedure was used for metabolite profiling in the SDB

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incubations, except that 5 ml of the aqueous phase was initially solvent extracted, concentrated to

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dryness, and re-dissolved in 0.5 ml isopropanol. Samples were analyzed using HPLC-q-TOF MS

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in negative ionization mode. The injection volume was 5 µL and the instrument was operated

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under conditions reported elsewhere 31 with some modifications. An Acquity HSS C18 SB

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(2.1x100 mm, 1.8 µm) analytical column was used for chromatographic separation. Mobile

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phase A was 10% HPLC-grade acetonitrile in HPLC-grade water and mobile phase B was

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HPLC-grade acetonitrile. At the beginning of the run, percentage of mobile phase B was 15% for

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3 minutes. It was linearly increased to 95% in 22 minutes and kept at this level for 10 minutes.

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Then, it was decreased to 5% in 1 minute and kept at this level for 6 minutes to re-equilibrate the

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column. The flow rate was 400 µL/min and the column temperature was maintained at 40°C.

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Triplicate injections were performed in sequence followed by a blank before the analysis of the

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next sample. The acquired data were processed using Agilent B.06.00 MassHunter Qualitative

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Analysis software. Signature metabolites, such as alkylsuccinates and alkylbenzylsuccinates,


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were identified based on the exact mass and on their retention times verified by authentic

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standards 31.

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Biofilm Characterization and Profilometry. For KW incubations, coupons were harvested and

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immediately imaged using a Canon EOS Rebel T3 camera to visualize corrosion products

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deposited on metal surfaces. To characterize potential biofilms formed on corroded metal

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surfaces, the corroded coupons stored under N2 were further examined using high resolution

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Field-Emission Scanning Electron Microscopy (FE-SEM) (Zeiss NEON 40 EsB,Carl Zeiss,

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Oberkochen, Germany) as previously described.32 Meanwhile, the elemental composition of

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corrosion products was characterized to confirm the likely deposition of iron sulfide using the

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coupled Energy Dispersive Spectroscopy (EDS) at 10 kV.

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The cleaned coupons after exposure were scanned using a Nanovea non-contact optical

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profilometer PS50 (MicroPhotonics, Inc, Irvine, CA) with a 4 µm step size as described

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previously 10 to acquire 3-dimentional images. Ultra MountainsMap Topography XT6.2 software

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(MicroPhotonics, Inc, Irvine, CA) was employed to process the topography data and quantify the

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number of pits. For KW and GoM incubations, pits were defined as the points that are 20 µm

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below the mean plane (average depth of all measured points) and with an equivalent (the

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diameter of an irregular region whose outer circumference equals a circular disk) greater than 50

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µm. To better reveal the severe pitting corrosion in SDB incubations, pits were defined as having

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a vertical depth of 50 µm below the mean plane and an equivalent diameter greater than 25 µm.

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Results

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Fuel Characterization. A better understanding of the chemical composition of various biomass-

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and petroleum-derived fuels can shed some light on their susceptibility to anaerobic

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biodegradation and their propensity to exacerbate metal corrosion. To that end, gas



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chromatographic profiles of biofuels (FT-F76 and camelina-JP5) and their petroleum

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counterparts (F76 and JP5) are depicted in Figure S1. The hydrocarbon composition of the

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petroleum and biomass-derived fuels are distinguishable with respect to abundance and

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complexity of the resolvable peaks in in the total ion chromatograms (Figure S1). Of course,

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mass spectral response factors vary for dissimilar classes of hydrocarbons and direct

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comparisons are not intended. That point notwithstanding, the relative abundance of major

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resolvable hydrocarbons in the fuels can be compared (Figure S2) by integrating the peak areas

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associated with particular classes of hydrocarbons. Thus, short chain n-alkanes (C8-C12) are

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detectable in all fuels, but vary from 2.2% to 12% total peak area in FT-F76 and JP5,

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respectively. Longer chain n-alkanes (≥ 13 carbons) are relatively more abundant in petroleum-

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F76 (19.9%) than in any of the other fuels (2.8-7.9%)). Similarly, the relative abundance of

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aromatic hydrocarbons in petroleum fuels (F76 and JP5) was much greater than the

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corresponding measure in the biofuels. It is also apparent that the branched alkanes (iso-alkanes)

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are far more pronounced in camelina-JP5 and FT-76 than in their petroleum counterparts. In

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particular, the branched alkane fraction in FT-F76 is estimated at 50.6% and this might

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reasonably be expected to impact the biological stability of this fuel.

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Sulfate Reduction in Fuel/Seawater Incubations. None of the test fuels or blends stimulated

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sulfate loss in KW (240 d) or GoM (300d) seawater incubations relative to the fuel-unamended

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or sterile controls (Figure S3). When D. alkanexedens strain ALDCT was used as a positive

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control bacterium, increased sulfate loss rates (Figure S4) in KW (16.7±5.2-23.4±0.6 µM/day)

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and GoM incubations (4.9±1.1-11.5±3.5 µM/day) relative to the fuel-unamended controls

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(KW:11.5±3.3 µM/day; GoM:3.7±0.4 µM/day) was noted. We presumed that the sulfate loss

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(0.2-3.1 mM, substracted from fuel-unamended controls) in the incubations was ascribed to


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oxidation of certain fuel components catalyzed by sulfate reducing bacteria. In a consistent

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fashion, we also measured the dissolved sulfide (0.3±0.17 - 0.59±0.07 mM) content in KW

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incubations and found that it was 2-6 times higher than in the sterile controls (Figure S5A).

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However, the amounts of dissolved sulfide were far less than the stoichiometrically expected

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quantities (2.1-4.8 mM) based on the degree of sulfate loss. The precipitation of iron sulfide

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minerals and the escaping H2S in the headspace might explain the unbalance sulfur in the

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systems.

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The highest rate of sulfate removal was observed in KW (45.8±3.5 - 80.8±8.1 µM/day)

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and GoM (23.3±3.4 - 76.7±2.4 µM/day) incubations when both D. alkanexedens strain ALDCT

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and a mixture of C6-C12 n-alkanes were used as experimental amendments (Figure 1). Despite

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the precipitation of dissolved sulfide through reaction with released iron in the systems, sulfide

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accumulation in the aqueous phase was from 1.31±0.26 mM to 5.09±1.74 mM (Figure S5B) in

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KW incubations, or 3 - 15 times more than the fuel-unamended incubations. The sulfate loss

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associated with the fuel amendments in KW seawater was higher than that measured in the fuel-

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unamended controls (3.7±0.4 - 11.5±3.3 µM/day) or the n-alkane-amended incubation (16.6±0.7

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- 39.4±0.2 µM/day). The higher rates of sulfate depletion might be attributed to the

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biodegradation of particular fuel components regardless of the fuel type. In both KW and GoM

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incubations, camelina-JP5 or its blend with its petroleum counterpart tended to stimulate the

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highest sulfate depletion rates relative to other fuels (Figure 1).

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The SDB incubations (without a D. alkanexedens amendment) presented a different

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scenario where seawater and sediment were chronically exposed to hydrocarbons 28. Previous

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studies have demonstrated that enrichments from SDB sediment can metabolize n-alkanes and

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polycyclic aromatic hydrocarbons under sulfate-reducing conditions 28, 33. In this regard, SDB



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sediment can represent a promising inoculum to accelerate the inherently slow sulfate reduction

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rates observed in seawater only incubations, most likely due to the lack of suitable hydrocarbon-

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degrading, sulfate reducing bacteria. Despite these differences, a very similar pattern of sulfate

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reduction (Figure 1) was observed in the SDB incubations relative to the other seawater

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incubations amended with the positive control bacterium and alkanes. The SDB sulfate depletion

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rates associated with fuel amendment (29.8±4.8 - 57.2±2.2 µM/day) were generally higher than

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the fuel-unamended control (26.7±0.8 µM/day). However, incubations amended with FT-F76 or

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its blend exhibited only baseline rates of sulfate loss (25.8±4.5 - 26.2±1.6 µM/day). Like the

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other incubations, the highest sulfate depletion rate was associated with camelina-JP5 in the SDB

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incubations. Such consistent observations in three marine incubations suggested that this

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hydroprocessed biofuel might harbor constituents that are more amenable to anaerobic

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biodegradable than other fuel options. No significant sulfate loss could be measured in any of the

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sterile controls with the three seawaters (Figure 1, S3 and 4).

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Metabolite Profiling. When D. alkanexedens and a mixture of C6-C12 n-alkanes were used as

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experimental variables, a series of new peaks were detected during the GC/MS analysis of the

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KW incubations containing either petroleum-JP5 or camelina-JP5 relative to sterile controls

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(Figure S6). The retention time of the peaks and their mass spectral profile allowed them to be

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identified as the alkylsuccinate metabolites that would be expected from the anaerobic

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metabolism of C6-C12 n-alkanes in the incubation mixture (Figure S6). Other alkylsuccinates,

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beyond the range associated with the C6-C12 n-alkanes or even different classes of hydrocarbon

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substrates (e.g. alkylbenzylsuccinates from alkylbenzenes) were below detection limits. Such a

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destructive sampling and limited profiling effort provides only a single snapshot of the

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metabolites detectable at the conclusion of the experiment.


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However, the analyses of subsamples by q-TOF allowed for a better assessment of

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metabolite formation and utilization with higher resolution and greater sensitivity. The kinetics

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of alkylsuccinate production and consumption was obtained from frozen samples (300 µL) that

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were periodically obtained from the GoM incubations (Figure S7 and S8) amended with D.

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alkanexedens and a mixture of C6-C12 n-alkanes. For instance, a suite of alkylsuccinates

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containing 10 to 16 carbon atoms were detected after 150 d of incubation and increased with

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time in most cases (Figure S7). The detection of these particular alkylsuccinates in the GoM

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incubations confirmed that the C6 to C12 n-alkanes were at least being metabolized via fumarate

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addition reactions. Similarly, in incubations amended with both petroleum- and camelina-JP5,

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the same series of alkylsuccinates were accumulated up to 90 d of incubation and then were

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consumed to undetectable levels by the end of the incubation (Figure S8). The transitory nature

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of these signature metabolites suggested that sampling at several time points is critical for the

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interpretation of metabolite profiles.

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In contrast to the KW and GoM incubations, a different series of metabolites were

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detected in the SDB experiments without an exogenous amendment of n-alkanes. In this case,

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alkylbenzylsuccinates with carbon atoms ranging from C13 to C16 were identified in incubations

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amended with petroleum fuels (Figure 2 and S9). However, only C13 alkylbenzylsuccinate

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(Figure 2 and S10) was identified in the incubation containing camelina-JP5 and none was

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detected in incubations amended with FT-F76 or its blend. As is typical for mass spectral

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analysis, the precise isomeric form of the alkylbenzylsuccinates produced during the course of

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these incubations cannot reliably be deduced. Nonetheless, the production of

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alkylbenzylsuccinates attests to the anaerobic metabolism of fuel components (i.e.,

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alkylbenzenes) by fumarate addition reactions.



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Biofilm and Corrosion Product Deposition. Carbon-steel coupons exposed to the fuel/seawater

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mixtures turned black with time due to the deposition of a precipitate (Figure S11). Generally,

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coupons in non-sterile incubations containing fuel had a visually thicker layer of corrosion

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product deposition relative to the corresponding sterile- or fuel-unamended controls. Energy

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dispersive spectroscopy revealed that the major elements in the corrosion product deposits were

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iron and sulfur (Figure S12). Numerous morphologically distinct bacterial cells were observed to

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be deeply embedded in the corrosion product (Figure S13) that was deposited on the metal

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coupon surface.

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Characterization of Corrosion. The general corrosion rate, calculated based on the degree of

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coupon weight loss over the course of the KW and GoM incubations, are shown in Figure 3.

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Relatively modest corrosion rates were measured in KW (0.53±0.03 - 1.14±0.08 mpy) and GoM

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(0.33±0.01 - 0.72±0.03 mpy) incubations containing only seawater and fuel (Figure 3A). These

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rates were only marginally higher than the corresponding sterile and fuel-unamended controls

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(Figure 3A). The profilometry analyses of these coupons revealed that no substantive localized

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or pitting corrosion was evident in the non-sterile or the sterile incubations (Figure S14 and S15).

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The number of pits in coupons exposed to various fuels was slightly higher than the

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corresponding sterile controls but comparable to the fuel-unamended incubations (Figure 4A).

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The amendment of D. alkanexedens to the fuel/seawater incubations stimulated corrosion

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rates in KW and GoM incubations (Figure 3B) relative to comparable incubations containing

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only seawater as the inoculum (Figure 3A). Generally, the rates of corrosion were between

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0.98±0.2 and 1.34±0.01 mpy in incubations amended with different fuels (Figure 3B), which

338

were slightly higher than the sterile (

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