Marine Pollution Bulletin 54 (2007) 894904 www.elsevier.

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Biodegradation and environmental behavior of biodiesel mixtures in the sea: An initial study
Jared A. DeMello a, Catherine A. Carmichael a, Emily E. Peacock a, Robert K. Nelson a, J. Samuel Arey b, Christopher M. Reddy a,*
a

Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, United States b Laboratory of Biochemistry and Computational Chemistry, Swiss Federal Institute of Lausanne, Switzerland

Abstract Biodiesel, a mixture of fatty acid methyl esters (FAMEs) derived from animal fats or vegetable oils, is rapidly moving towards the mainstream as an alternative source of energy. However, the behavior of biodiesel, or blends of biodiesel with fossil diesel, in the marine environment have yet to be fully understood. Hence, we performed a series of initial laboratory experiments and simple calculations to evaluate the microbial and environmental fate of FAMEs. Aerobic seawater microcosms spiked with biodiesel or mixtures of biodiesel and fossil diesel revealed that the FAMEs were degraded at roughly the same rate as n-alkanes, and more rapidly than other hydrocarbon components. The residues extracted from these dierent microcosms became indistinguishable within weeks. Preliminary results from physicalchemical calculations suggest that FAMEs in biodiesel mixtures will not aect the evaporation rates of spilled petroleum hydrocarbons but may stabilize oil droplets in the water column and thereby facilitate transport. 2007 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel; Fatty acid methyl esters; Microbial degradation; Weathering; Dispersion; GC GC

1. Introduction With the rapid rise in the price of crude oil and projected decreases in oil supplies, alternative fuels are receiving considerable attention (Hill et al., 2006). One of the most promising alternatives is biodiesel, which is a mixture of fatty acid methyl esters (FAMEs) derived from the transesterication of animal fats and vegetable oils. Please note that some articles refer to biodiesel as the actual fats or oils prior to these reactions (sometimes called straight fats or oils), but in this manuscript, biodiesel refers to only FAMEs. Proponents of biodiesel in the United States as well as other countries tout its ability to enhance engine lubrication, decrease harmful emissions, and minimize the dependence on foreign oil imports (Kemp, 2006). It is also

Corresponding author. Tel.: +1 508 289 2316; fax: +1 508 457 2164. E-mail address: creddy@whoi.edu (C.M. Reddy).

proposed as a partial solution to CO2 emissions contributing to global warming by closing the carbon cycle, i.e., being carbon neutral (Peterson and Hustrulid, 1998). One of Rudolph Diesels interests after developing the diesel engine was for farmers to use vegetable oils as fuels (Pahl, 2005). Initial eorts in the early 1900s to run diesel engines with straight vegetable oil were hindered by the oils high viscosity. This inspired others to chemically modify vegetable oils into mixtures that had lower viscosities, with processes such as transesterication to FAMEs, which was rst patented in 1937. Although, numerous other methods have since been developed (Demirbas, 2005; Demirbas and Kara, 2006), transesterication is now the most commonly used method (Noureddini and Zhu, 1997). When the transesterication is performed with methanol, these reactions convert glycerol-based fats and oils into glycerin and FAMEs. Methanol and the production of FAMEs are the norm because methanol is the least expensive alcohol, although other esters, such as iso-propyl

0025-326X/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2007.02.016

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esters, have been shown to have better fuel properties than methyl esters (Knothe, 2005). The chain length and degree of unsaturation can vary in animal fats and vegetable oils for numerous reasons (Gurr et al., 2002), but transesterication of either yields primarily a mixture of methyl hexadecanoate (C16 FAME), methyl octadecanoate (C18 FAME), and C18 FAME isomers with one, two, or three double bonds referred to as C18:1, C18:2, or C18:3 FAMEs, respectively. Biodiesel is used to formulate a range of mixtures from B2 (2% biodiesel mixed with 98% fossil diesel) to B100 (100% biodiesel). Almost any diesel engine can be run on B2 though B20 and performance characteristics are comparable to those of burning 100% fossil diesel (Kaplan et al., 2006). One potential concern with biodiesel mixtures has been whether they degrade seals and ttings in fuel systems. However, in a survey of transportation agencies of 48 states in the United States, there were no reports of fuel system leaks resulting from biodiesel blends as high as B20 (Humburg et al., 2006). Biodiesel is moving past the novelty stage and closer to mainstream usage. It is available in the United States at over 1000 distributors and also formulated by private consumers or user groups. In addition, companies are now beginning to manufacture diesel engines that are designed to run on biodiesel blends. One example is the 2007 6.7 l Dodge Ram Turbo Diesel Engine. Despite this large swell in usage and signicant anecdotal information by user groups, there has been little peer-reviewed research on the environmental chemistry of this product. Biodiesel engine emissions have been characterized and some toxicity tests have been performed (Jung et al., 2006; Krahl et al., 2005; McCormick and Alleman, 2005; Peterson and Moller, 2005; Smekens et al., 2005; Turrio-Baldassarri et al., 2004). A recent review stressed the need for more research on the health eects of biodiesel exhaust (Swanson et al., 2007). There have been several studies on the microbial degradation of biodiesels (Donofrio, 1996; Floro, 1996; Follis, 1994; Lapinskien_ et al., 2006; Peterson and Moller, 2005; e Zhang et al., 1998), which have shown that they degrade. One of these eorts published gas chromatographic traces to monitor this process qualitatively (Zhang et al., 1998). It is noteworthy that there have been numerous spills of vegetable oils (straight oils) in the sea where microbes are capable of degrading them (Bucas and Saliot, 2002). However, reactions initiated at the double bonds of the fatty acids in these glyercol-based oils have been shown to polymerize making them less available to bacteria even when stimulated with nutrients. In fact, Mudge (1997) hypothesized that some residues of spilled vegetable oils may be more recalcitrant than mineral oils. To expand our current knowledge on the behavior in the marine environment of biodiesel and mixtures of biodiesel with fossil diesel, we performed a series of experiments and calculations. In particular, we amended seawater and autoclaved seawater with 100% fossil diesel, B8, B25, and B100. Individual samples were harvested over the course of 53

days and analyzed by gas chromatography (GC). Our main goals were: (1) to determine the microbial and environmental fate of fossil diesel in seawater cultures when biodieselderived FAMEs (or vice versa) were present; (2) to measure the relative degradation rate of FAMEs compared to components of fossil diesel; (3) to evaluate whether any new indicators or ratios of molecules within these mixtures could be identied so that in future cases they could be used to assess the short and long term fate of a biodiesel spill; (4) to consider other potential environmental processes (e.g., abiotic hydrolysis) that may act on biodiesel in the environment; and (5) to evaluate whether biodieselderived FAMEs may aect the environmental fate and transport of petroleum hydrocarbons when there is a spill. One aspect that we did not study in this manuscript was photochemistry or oxidation of the double bonds on the FAMEs. 2. Methods 2.1. Obtaining and preparing the fuel mixtures used in this experiment The 100% fossil diesel was collected from the cargo hold of the oil barge Bouchard 65, at the time that it spilled oil in Buzzards Bay in October, 1974, and has been previously well characterized (Arey et al., 2005; Peacock et al., 2007). The B100 sample was purchased from Loud Fuel (Falmouth, MA). The biodiesel mixtures, B8 and B25, were prepared by weighing and mixing the 100% fossil diesel and B100. To conrm that the biodiesel mixtures would be the same whether mixed by mass or volume, we measured the densities of the fossil diesel (0.853 g ml1) and B100 (0.869 g ml1) and found them to be similar. To compare commercially available B20 mixtures, we obtained four dierent samples in 2006 from two dierent distributors (referred to as Distributors I and II) in the state of Massachusetts. One sample was obtained from Distributor I. The other three all came from Distributor II. 2.2. Preparation of biodegradation cultures Four experiments were performed in seawater spiked with either 100% fossil diesel fuel, B8, B25, or B100. We employed classic die away procedures (Drenzek et al., 2001) to collect numerous samples each representing their own microcosm. Seawater was collected in two pre-cleaned, 4-l solvent jugs from Vineyard Sound, MA and used for the incubation studies. Both jugs were amended with NH4Cl and K3PO4 so that the nal concentration of nitrogen and phosphorus was 5 and 0.9 mmol l1, respectively. One jug was autoclaved and used as the control jug. We referred to the other jug as the live jug. Glass vials (60-ml volume; 14-cm height, and 2.4-cm inside diameter) were used for each microcosm. Forty milliliters of either seawater or autoclaved seawater were

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added to 140 and 70 vials, respectively, providing two live samples for each control. This design allowed us to collect 17 timepoints, each consisting of two live samples and one control for 100% fossil diesel, B8, B25, and B100. Twentyve microliters of concentrated solutions of either 100% fossil diesel, B8, B25, or B100 dissolved in acetone were spiked into each vial (to yield a total mass of $4.5 mg of material). The vials were loosely plugged with only autoclaved cotton wool, placed onto a shaker table, covered with a large cardboard box to minimize light, and agitated at 60 revolutions per minute (rpm) for the duration of the experiment. At various time periods, both live and control samples were terminated by adding 10 ml of methyl t-butyl ether (MTBE), which also served as the rst extraction step, and then sealed with Teon-lined caps. The pH was also monitored by sampling a small volume of the culture with pH paper (range 014). An internal standard, hexacosane (n-C26) was added to each vial (38 lg was added for the 100% fossil diesel fuel, B8, and B25 experiments). For the B100, we added 190 lg of n-C26. Each sample was then recapped, gently inverted, and sonicated for 10 min. The upper layer (MTBE) was removed with a Pasteur pipette and delivered to a precleaned 60-ml glass vial. Two subsequent MTBE extractions were performed, each with 11 ml of MTBE. The MTBE extracts were combined and then dried with anhydrous sodium sulfate. The dried samples were transferred to 50-ml pear shaped asks, rotary evaporated to $250 ll, spiked with an external standard, octacosane (nC28), and then stored until analysis. 2.3. Analysis by one-dimensional gas chromatography Each extract was analyzed on a Hewlett-Packard 5890 Series II gas chromatograph with a cooled injection system (CIS) and ame ionization detector (GC-FID). A 1-ll sample was injected splitless into the CIS, which was programmed from 40 C (0.3-min hold) to 320 C at 720 C min1 (5-min hold) and then 350 C at 720 C min1 (4-min hold). Compounds were separated on a gas capillary column (CP-Sil CP, 30 m length, 0.25-mm I.D., 0.25 lm lm thickness) with H2 as the carrier gas at a constant ow of 5 ml min1. The GC oven temperature was programmed from an initial temperature of 40120 C at 30 min1 and then from 120 C to 320 C at 6 min1. The concentrations of individual alkanes, branched alkanes, and FAMEs as well as bulk total fossil diesel and FAMEs were monitored in this study. As the experiment progressed, most of the fossil diesel became an unresolved complex mixture (UCM; Farrington and Quinn, 1973). Response factors for the UCM and FAMEs relative to the internal standard n-C26 were determined from the 100% fossil diesel and B100 used in this experiment. Individual FAMEs were all quantied with the same response factor, which has been shown to be accurate to within a few percent (Christie, 1989). We checked the identity of

the individual FAMEs with two standards purchased from Supelco, FAME Mix Rapeseed oil Lot No. LB-34981 and FAME Mix RM-6 Lot No. LB-37097. For reference, all chromatograms are drawn with the hexane baseline included. 2.4. Analysis by comprehensive two-dimensional gas chromatography (GC GC) The 100% fossil diesel, B8, and B25 samples from Day 53 of the experiment were analyzed using comprehensive two-dimensional gas chromatography (GC GC) (Nelson et al., 2006). Due to the considerable sensitivity of GC GC, solutions containing high FAME content (such as neat B8) overwhelmed the system. Hence, we prepared a mixture of B0.5 and analyzed it to compare to the Day 53 samples. Each sample was analyzed with an Agilent 6890 gas chromatograph congured with a 7683 series split/ splitless auto-injector, two capillary gas chromatography columns, a model KT-CLM-ZOE02 loop jet modulator (Zoex Corporation, Lincoln, NE), and a ame ionization detector (GC GC-FID). The samples were analyzed in the splitless injection mode using a 1 ll sample injection volume and the purge vent was opened at 1.0 min. The inlet temperature was 285 C. The rst-dimension column and the loop jet modulator reside in the main oven of the Agilent 6890 gas chromatograph. The second-dimension column is housed in a smaller oven installed adjacent to the main oven. With this conguration, the temperature proles of the rst-dimension column, thermal modulator (hot jet), and the second-dimension column can be independently programmed. The rst-dimension column was a nonpolar 100% dimethyl polysiloxane phase (Restek Rtx-1 Crossbond, 7.5 m length, 0.25 mm I.D., 0.1 lm lm thickness) that was programmed to remain isothermal at 35 C for 5 min and then ramped from 35 C to 235 C at 2.00 C min1. The modulation loop was deactivated fused silica (1.0 m length, 0.10 mm I.D.). The thermal modulator was programmed to remain isothermal at 150 C for 5 min and then ramped from 150 C to 365 C at 2.15 C min1. Second-dimension separations were performed on a 50% phenyl polysilphenylene-siloxane column (SGE BPX50, 2.0 m length, 0.10 mm I.D., 0.1 lm lm thickness) that was programmed to remain isothermal at 42 C for 5 min and then ramped from 42 C to 265 C at 2.23 C min1. The thermal modulator loop pulse frequency was 20.0 s (0.05 Hz), and the pulse width was 350 ms. The carrier gas was H2 at a constant ow rate of 0.9 ml min1. The FID signal was sampled at 100 Hz. 2.5. Dispersion/emulsion experiments The formation of fuel/water emulsions and dispersions can aect the persistence and environmental transport of spilled petroleum products (Patton et al., 1981; Irvine et al., 1999; Lessard and DeMarco, 2000). We performed a simple experiment to test whether biodiesel mixtures

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created dispersions or exhibited emulsion stability dierently from a fossil diesel. Briey, three ml of biodiesel mixtures (100% fossil diesel, B5, B10, B20, B50, B70, and B100) were prepared using the Bouchard 65 cargo and B100. For comparison, we also tested an asphaltene-rich No. 6 fuel oil from the spill of the Bouchard 120 (Nelson et al., 2006). Each fuel was added to a glass vial (60-ml volume; 14-cm height, and 2.4-cm inside diameter) containing 18 ml of seawater collected from Vineyard Sound, MA. The vials were then capped and shaken in the horizontal position at 270 rpm for 4 h. After shaking, the vials were immediately placed upright and repeatedly photographed over a period of 17 h to observe emulsion behavior. 3. Results and discussion In this study, we investigated the microbial degradation of four materials: 100% fossil diesel, B8, B25, and B100. GC-FID chromatograms of each are shown in Fig. 1. The 100% fossil diesel is quite typical, composed of resolved straight-chain and branched alkanes along with many unresolved saturated and aromatic hydrocarbons that elute within the boiling range of dodecane (n-C12) and tetracosane (n-C24) (Fig. 1a). In comparison, the

B100 sample is a simple mixture, composed of C14, C16, C16:1, C18, C18:1, C18:2, and C18:3 FAMEs (Fig. 1d). We prepared the B8 and B25 mixtures from our 100% fossil diesel and B100 solutions in the laboratory (and did not purchase them) (Fig. 1b and c). In the two biodiesel mixtures, all of the FAME compounds are resolved within the fossil diesel range. The only exception is the C14 FAME, which coelutes partially or completely with pristane, depending on their relative concentrations (with the GC-FID conditions used). 3.1. Biodegradation of 100% fossil diesel, B8, and B25 The results of the biodegradation experiments performed in this study must be viewed in the context of the studys experimental conditions. Often the results from laboratory studies are improperly extrapolated into real-world situations (Slater et al., 2005). First, the fuel products were added to seawater that was amended with high concentrations of nutrients under aerobic conditions. Second, the fuel products represented a large source of highly reduced carbon to the natural population of microbes present in the seawater. Last, any eects related to bioavailability due to desorption from particles were eliminated by incubating in sediment-free microcosms (Drenzek et al., 2001). Nevertheless, these experiments permitted us to directly compare microbial degradation of biodiesel versus mixtures composed of biodiesel and fossil diesel. In addition, we only monitored the loss of fossil diesel compounds and FAMEs and did not analyze for any intermediates such as free fatty acids. A time series of chromatograms revealing the loss of the 100% fossil diesel is shown in Fig. 2ae and is consistent with previous studies (Burns and Teal, 1979; Jones et al., 1983; Stout et al., 2002). It allows for the comparison between the degradation of 100% fossil diesel and of B25 shown later in Fig. 3. By day 3 in the 100% fossil diesel experiment, there was already a substantial loss of n-alkanes, indicated by a relative increase in the branched alkanes, norpristane, pristane, and phytane (Fig. 2b). By day 10, the resolved n-alkanes had disappeared completely, leaving behind the more resilient branched alkanes and UCM (Fig. 2c). Continued loss of compounds occurred between days 16 and 53 (Fig. 2d and e) with a remaining residue of pristine, phytane, and an UCM. Throughout this experiment, some losses of low molecular weight compounds up to n-C17 occurred in the control samples (Fig. 2f). However, among these compounds, individual n-alkanes exhibited the same loss rates as isoprenoids having similar volatility (e.g., n-C18 vs. phytane). For this reason, we attribute losses in the control samples to evaporation and not microbial degradation. (Later in this section, employing this ratio to evaluate microbial degradation is described in more detail). A corresponding time-series of chromatograms is shown for the losses of the B25 mixture (Fig. 3ae). Similar trends for B8 were also observed (not shown). Overall, the

Fig. 1. GC-FID chromatograms of the four neat starting solutions used in the microbial experiments: (a) 100% fossil diesel, (b) B8, (c) B25, and (d) B100. In (a) peaks representing the n-alkanes 1224 are numbered, and F, Np, Pr, and Ph are the isoprenoids farnesane, norpristane, pristane and phytane, respectively. In (d) the FAMEs are labeled in bold. For reference, the hexane baseline is included with all chromatograms.

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Fig. 2. A time-series of GC-FID chromatograms of the 100% fossil diesel experiment (a) live day 0, (b) live day 3, (c) live day 10, (d) live day 16, (e) live day 53, and (f) control day 53. The peak at 24.1 min is the internal standard, n-C26, which was used to normalize the FID signal in each chromatogram.

Fig. 3. A time-series of GC-FID chromatograms of the B25 mixture experiment (a) live day 0, (b) live day 3, (c) live day 10, (d) live day 16, (e) live day 53, and (f) control day 53. The peak at 24.1 min is the internal standard, n-C26, which was used to normalize the signals for each chromatogram.

degradation of the B25 was very similar to the 100% fossil diesel except that the FAMEs were also degraded, so that chromatograms of the weathered 100% fossil diesel and weathered B25 were nearly identical at day 53 (Figs. 2e and 3e). However, careful inspection of chromatograms shows that on day 3 in the B25 sample (Fig. 3b), the n-alkanes are more abundant relative to the branched alkanes compared to those in the 100% fossil diesel sample (Fig. 2b). By day 10, the resulting chromatograms show similar traces for the fossil diesel component, with only lingering FAMEs peaks in the B25 chromatograms (Figs. 2c and 3c). This indicates that the FAMEs and n-alkanes were degraded rst, and all other fossil fuel components followed, unaected by the presence of FAMEs in the starting solution. The slight dierence between the degradation of the 100% fossil diesel and the B25 described above is best illustrated by plotting the n-C18/phytane ratio on each day (also plotted for B8) (Fig. 4ac). Because, n-C18 biodegrades

more quickly than phytane but has a similar volatility, a decrease in the n-C18/phytane ratio indicates biodegradation of n-alkanes. It is historically typical to also make this analysis with n-C17 and pristane (Blumer et al., 1973; Jones et al., 1983), but this distinction was not possible, because C14 FAME co-eluted to varying degrees with pristane. This is evident in our analysis of four commercially available B20 blends shown in Fig. 5. (Please also note in the latter gure how the fossil diesel versus FAMEs content varies in these samples). Hence, this analytical eect should be considered when planning future experiments. The black boxes in Fig. 4 highlight the days on which the n-C18/phytane ratio went essentially to zero, which were day 5 for the 100% fossil diesel, day 6 for the B8, and day 7 for the B25. Together the time-series chromatograms (Figs. 2 and 3) and n-C18/phytane ratio plots (Fig. 4) indicate that the presence of FAMEs may slow the initial biodegradation of n-alkanes in fossil diesel, but this dierence was only

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Fig. 4. The n-C18/phytane ratios for the rst 15 days of the experiment (a) 100% fossil diesel, (b) B8, and (c) B25. Open squares and circles represent live samples (two per day) and lled diamonds represent control samples (one per day). The large square in each plot highlights the day on which the ratio had essentially gone to zero.

detectable in the rst week of the experiment. Chromatograms from day 6 of 100% fossil diesel and B8 depicted in Fig. 6a and b, respectively, highlight how quickly the residues in these microcosms became similar. Results from the last day of the experiment (day 53) are shown in Fig. 7 and provide further evidence of the apparent trend revealed in Fig. 6. In addition, the resulting UCM patterns in Fig. 7 are similar to those found in temperate salt marshes contaminated with fossil diesel from spills that occurred in 1969 and 1974 (Reddy et al., 2002; Peacock et al., 2005, 2007). Hence in the event of a biodiesel spill, we predict that FAMEs will be consumed by bacteria. After only a short period, samples from a contaminated area will become indistinguishable from a fossil diesel spill. These factors could hinder eorts focusing on spill source identication and forensic investigations. Phytosterols can be present in FAMEs mixtures produced from rapeseed oil (Plank and Lorbeer, 1994), and hence useful tracers for some B100s. However when we analyzed the B100 used in this study, we did not detect any of these compounds. To further examine and conrm the similarities between 100% fossil diesel, B8, and B25 after 53 days of degradation in the laboratory, these samples were analyzed by GC GC-FID (Fig. 8). This technology produces high resolution chromatographic separations of complex mixtures because each compound is subjected to two dierent sta-

Fig. 5. GC-FID chromatograms from four commercially obtained B20 mixtures from 2006. (a) Distributor I collected in June and (bd) Distributor II collected in June, September, and November, respectively. The inserts in the top left corner focus on the n-C17, C14:0 FAME, and pristane retention windows that are highlighted in gray in the full chromatograms. Please also note the extreme variability in the fossil diesel versus FAMEs content among these samples. In particular, observe the near absence of fossil diesel in the sample collected from Distributor II in September (c), suggesting some type of mixing problem occurred when this blend was prepared. Yet, the same distributor did not have this apparent problem two months later (e) or in previous months (b).

tionary phase selectivities (Gaines et al., 2006). Here, the rst dimension separation uses a non-polar phase to separate each component by volatility dierences, and the second dimension uses a more polar phase to separate rst dimension coeluters by polarity dierences. The resulting two-dimensional chromatograms resolve many more peaks, sorted according to their volatility and polarity properties. A GC GC chromatogram has compound peaks grouped by volatility along the x-axis and by chemical class along the y-axis. For petroleum, this produces separated chemical classes such as alkanes, cycloalkanes, and one-, two-, and multi-ring aromatics, with additional

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3.2. Biodegradation of B100 The FAMEs in the B100 samples were degraded down to $10% of their original mass within three weeks of starting the experiment (data not shown). The controls for the B100 experiment showed no change in FAME mass or composition during the 53 days of experiment. We hypothesize that the B100 residue that remained at the end of the experiment was likely unavailable for degradation, possibly stuck to the high sides of the vial. In addition to monitoring the loss of total FAME mass, we also looked for preferential loss of specic FAMEs in the B100 experiment. We found that C16 FAME was degraded faster than any of the C18 FAMEs and that the degradation rate among the C18 FAMEs did not correspond with the degree of saturation. The latter observation is contrary to that of Miller and Mudge (1997) who observed that several unsaturated C18 FAMEs were degraded more quickly than C16 FAME in experiments that focused on determining the eectiveness of biodiesel on the remediation of crude oil spills in the environment. 3.3. FAMEs hydrolysis rates Although, no losses of FAMEs were observed in the controls for these experiments, esters can hydrolyze abiotically. To conrm our inferences about biodegradation, we considered the plausible half-life of FAME hydrolysis in seawater both at experimental pH values (6.07.0) and natural seawater pH values (7.48.3). While we did not nd measured hydrolysis rate constants of FAMEs in the literature, data are available for ethyl acetate, a close structural analog, and this guided our predictions for FAMEs. Throughout the relevant pH range (6.08.3), the ethyl acetate hydrolysis rate is strongly dominated by the base-catalyzed reaction, relative to acid-catalyzed or neutral hydrolysis, by at least an order of magnitude (Mabey and Mill, 1978). Hence, we assumed that the base-catalyzed pathway likewise controls FAMEs hydrolysis kinetics as well. Using the EPA module, Hydro (Mill et al., 1987), we estimated that basecatalyzed hydrolysis half-lives of FAMEs are 7 years at pH 7, and 70 years at pH 6 (25 C). Thus, we expect that abiotic hydrolysis did not contribute to FAMEs losses in biodegradation experiments presented here. At pH values of 7.4 and 8.3, FAMEs had estimated base-catalyzed hydrolysis half-lives of 3 years and 19 weeks, respectively (25 C). These were considered upper bound rate estimates, since environmental temperatures (520 C) would decrease the actual reaction rates. While these rates of hydrolysis are slower than the microbial degradation rates of FAMEs in this study, abiotic hydrolysis could become more relevant in conditions where microbial degradation is less ideal. 3.4. Evaporation By inspection of the GC-FID chromatograms of the abiotic controls in the biodegradation experiments, we

Fig. 6. GC-FID chromatograms from day 6 of the live experiment of (a) 100% fossil diesel and (b) B8. In (a) F, Np, Pr, and Ph are the isoprenoids farnesane, norpristane, pristane and phytane, respectively. Please note that each chromatogram was not normalized to the internal standard, n-C26.

Fig. 7. GC-FID chromatograms from day 53 of the live experiment of (a) 100% fossil diesel, (b) B8, and (c) B25. Where shown, Np, Pr, and Ph are the isoprenoids norpristane, pristane and phytane, respectively. Please note that each chromatogram was not normalized to the internal standard, n-C26.

groupings showing homologous series within each class. As shown in Fig. 8bd, the day 53 samples for 100% fossil diesel, B8, and B25 were quite similar, even when scrutinized with the higher sensitivity of GC GC, providing additional evidence that it would dicult to distinguish a biodiesel spill from 100% fossil diesel after initial weathering. This also indicates that the presence or absence of FAMEs did not alter the composition of the UCMs that are shown in Fig. 7.

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Fig. 8. GC GC-FID chromatograms (a) B0.5 prepared in the laboratory and live samples from day 53 for (b) 100% fossil diesel, (c) B8, and (d) B25. The C16 and C18 FAMEs in (a) have First and Second Dimension Retention Times of 60 min and 3.5 s and 62 min and 3.4 s, respectively. The peaks marked n-C26 and n-C28 in (b) are the internal and external standards, respectively.

inferred that FAMEs will not aect the evaporation rates of fossil diesel components. In the abiotic controls, fossil diesel hydrocarbons evaporated at a similar rate for both the 100% fossil diesel and biodiesel mixtures. To corroborate this observation, we estimated activity coecients of fossil diesel hydrocarbon compounds in simulated fossil diesel and biodiesel mixtures using the Universal QuasiChemical Functional Group Activity Coecient (UNIFAC) model (Gmehling et al., 1998). Our results indicated that the activity coecients of petroleum hydrocarbons are not aected by the presence of FAMEs in a fossil diesel mixture, and this is consistent with experimental evidence in previous work (Yuan et al., 2005). Consequently, we expect that evaporation rates of petroleum hydrocarbons

will not increase or decrease in the presence of FAMEs; this is consistent with the results of our study. This assessment should be claried in the context of observations of Miller and Mudge (1997), who suggest that the physical mobility of heavy oil mixtures may be enhanced by FAMEs amendment. By adding FAMEs to a spilled crude oil, Miller and Mudge lowered the viscosity of the putative mixture, consequently the ow properties and physical transport of the crude oil in the environment were enhanced. This could indirectly accelerate the weathering rate of a heavy oil, by increasing the distribution and exposure of the oil in the environment. But in cases such as biodiesel, where the mixture viscosity is not signicantly altered by the presence of FAMEs, our results suggest that

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biodegradation and evaporation of petroleum hydrocarbons will be similar to that observed in fossil diesel spills. We also conclude that the FAMEs themselves will likely biodegrade before evaporating from fuel mixtures in a typical environmental setting. Both our UNIFAC calculations and experimental data (Yuan et al., 2005) conrm that FAMEs are ideal solutes in fossil diesel. This implies that the GC retention time of each FAME on a non-polar stationary phase indicates its volatility from fossil diesel relative to other hydrocarbon compounds in the mixture (Arey et al., 2005). GC-FID chromatograms show that all of the FAMEs studied here elute later than, and hence are less volatile than, n-C17 (Fig. 1). Our mass transfer calculations (Schwarzenbach et al., 2003) and eld observations (Wolfe et al., 1994) both suggest that compounds less volatile than n-C17 usually require months or more to evaporate from oil spills in typical environmental conditions. 3.5. Dispersions and emulsions In our dispersion and emulsion experiments, FAMEs appeared to increase the stability of small oil droplets in water under turbulent conditions, and this may have implications for the transport, weathering rate, and ecological impact of spilled biodiesel. In these experiments, physical agitation produced temporary dispersions (small oil droplets in the water phase) in the cases of both fossil diesel and biodiesel. For the fossil diesel, surface tension and buoyancy forces caused the small oil droplets to re-aggregate into the oil phase within 510 min after the four hour agitation period, as was visually indicated by a relatively clear and colorless water phase. The B5 and B10 biodiesel mixtures displayed similar behavior to the fossil diesel. By comparison, the water phase adjoining the Bouchard 120 heavy fuel oil claried within seconds. However the B20, B50, B70, and B100 mixtures still exhibited an opaque, milky white dispersion in the aqueous phase after 18 min. At 17 h, the semi-stable dispersion had cleared from the water phase in all samples. The results indicate that, at sucient FAME amendment levels, FAMEs stabilize oil droplets in the water phase by decreasing the oilwater surface tension and therefore reducing oil droplet re-aggregation. This result is consistent with chemical intuition. Based on their structural resemblance to surfactants, FAMEs may form ordered associations at the oilwater interface (Israelachvili, 1991) and thereby decrease the interfacial surface tension. As a result, small oil droplets initially formed by agitation are stabilized in the presence of FAMEs, and therefore these droplets experience longer lifetimes in the water phase before reaggregating into larger globules and rising to the surface. In eect, FAMEs amendment may increase the incorporation of oil droplets into the water column during turbulent sea surface events. This may facilitate downward transport of oil into the water column and therefore worsen contamination impacts on aquatic and benthic organisms. Additionally, oil stabilization by FAMEs may increase

rates of petroleum hydrocarbon dissolution into the water column, due to the increased surface area to volume ratio of smaller oil droplets. However, water currents may distribute dispersed and dissolved oil over a larger region and thereby decrease the local severity of coastal oiling and accelerate oil weathering (Lessard and DeMarco, 2000). Finally, dispersants apparently decrease oil toxicity to wildlife (Otitoloju, 2005). Hence, it is unclear whether the dispersant eects of FAMEs exacerbate or diminish the overall ecological impacts of petroleum hydrocarbons. It is worth noting that previous studies usually focus attention on water emulsication into the oil phase (Fingas, 1995), rather than oil dispersion into the water phase as we discuss here. Although, our mixtures showed some differences in their ability to incorporate water in the oil phase, this eect lasted only seconds for all fossil diesel and biodiesel samples. 4. Conclusions In this study, we conducted a preliminary investigation of the biodegradation and behavior of biodiesel under controlled conditions. Our results provide a baseline for future work. From the biodegradation experiments, we observed that the FAMEs were degraded at a similar rate as the nalkanes, and certainly more quickly than other fossil diesel components. Hence, in the event of a biodiesel mixture spill, we predict that the FAMEs will be consumed by bacteria, and samples from a contaminated area may be indistinguishable from a conventional fossil diesel spill after only a short period. At pH values of 7.4 and 8.3, FAMEs had estimated base-catalyzed hydrolysis half-lives of 3 years and 19 weeks, respectively. Experiments and theoretical evidence both suggest that FAMEs will not aect the rate of evaporation of petroleum hydrocarbons. In addition, FAMEs themselves will likely biodegrade before evaporating from spilled biodiesel. The physical properties of FAMEs may alter the environmental behavior of petroleum hydrocarbons. Dispersion experiments suggest that FAMEs will stabilize biodiesel oil droplets in the water column, and this may inuence the transport, weathering rate, and ecological impact of spilled biodiesel. By stabilizing small oil droplets in the water column, FAMEs might also enhance dissolution rates of conventional hydrocarbons. Acknowledgements This work was supported by funds from the National Science Foundation (IIS-0430835), the Department of Energy (DE-FG02-06ER15775), and an Oce of Naval Research Young Investigator Award (N00014-04-010029). We thank Professor James Quinn (University of Rhode Island), Dr. John Farrington (WHOI), Ms. Leah Houghton (WHOI), Mr. Bruce Tripp (WHOI), and Dr. Gerhard Knothe (USDA) for their assistance.

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