bio degradation and environmental behavior of biodiesel

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Marine Pollution Bulletin 54 (2007) 894–904

Biodegradation and environmental behavior of biodiesel mixturesin 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 Statesb 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 themainstream as an alternative source of energy. However, the behavior of biodiesel, or blends of biodiesel with fossil diesel, in the marineenvironment have yet to be fully understood. Hence, we performed a series of initial laboratory experiments and simple calculations toevaluate the microbial and environmental fate of FAMEs. Aerobic seawater microcosms spiked with biodiesel or mixtures of biodieseland fossil diesel revealed that the FAMEs were degraded at roughly the same rate as n-alkanes, and more rapidly than other hydrocarboncomponents. The residues extracted from these different microcosms became indistinguishable within weeks. Preliminary results fromphysical–chemical calculations suggest that FAMEs in biodiesel mixtures will not affect the evaporation rates of spilled petroleum hydro-carbons 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 projecteddecreases in oil supplies, alternative fuels are receiving con-siderable attention (Hill et al., 2006). One of the mostpromising alternatives is biodiesel, which is a mixture offatty acid methyl esters (FAMEs) derived from the trans-esterification of animal fats and vegetable oils. Please notethat some articles refer to biodiesel as the actual fats or oilsprior to these reactions (sometimes called straight fats oroils), but in this manuscript, biodiesel refers to onlyFAMEs. Proponents of biodiesel in the United States aswell as other countries tout its ability to enhance enginelubrication, decrease harmful emissions, and minimize thedependence on foreign oil imports (Kemp, 2006). It is also

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doi:10.1016/j.marpolbul.2007.02.016

* Corresponding author. Tel.: +1 508 289 2316; fax: +1 508 457 2164.E-mail address: [email protected] (C.M. Reddy).

proposed as a partial ‘‘solution’’ to CO2 emissions contrib-uting to global warming by closing the carbon cycle, i.e.,being carbon neutral (Peterson and Hustrulid, 1998).

One of Rudolph Diesel’s interests after developing thediesel engine was for farmers to use vegetable oils as fuels(Pahl, 2005). Initial efforts in the early 1900s to run dieselengines with straight vegetable oil were hindered by theoil’s high viscosity. This inspired others to chemically mod-ify vegetable oils into mixtures that had lower viscosities,with processes such as transesterification to FAMEs, whichwas first patented in 1937. Although, numerous othermethods have since been developed (Demirbas, 2005;Demirbas and Kara, 2006), transesterification is now themost commonly used method (Noureddini and Zhu,1997). When the transesterification is performed withmethanol, these reactions convert glycerol-based fats andoils into glycerin and FAMEs. Methanol and the produc-tion of FAMEs are the norm because methanol is the leastexpensive alcohol, although other esters, such as iso-propyl

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J.A. DeMello et al. / Marine Pollution Bulletin 54 (2007) 894–904 895

esters, have been shown to have better fuel properties thanmethyl esters (Knothe, 2005). The chain length and degreeof unsaturation can vary in animal fats and vegetable oilsfor numerous reasons (Gurr et al., 2002), but transesterifi-cation of either yields primarily a mixture of methylhexadecanoate (C16 FAME), methyl octadecanoate (C18

FAME), and C18 FAME isomers with one, two, or threedouble bonds referred to as C18:1, C18:2, or C18:3 FAMEs,respectively.

Biodiesel is used to formulate a range of mixtures fromB2 (2% biodiesel mixed with 98% fossil diesel) to B100(100% biodiesel). Almost any diesel engine can be run onB2 though B20 and performance characteristics are compa-rable to those of burning 100% fossil diesel (Kaplan et al.,2006). One potential concern with biodiesel mixtures hasbeen whether they degrade seals and fittings in fuel systems.However, in a survey of transportation agencies of 48states in the United States, there were no reports of fuelsystem leaks resulting from biodiesel blends as high asB20 (Humburg et al., 2006).

Biodiesel is moving past the novelty stage and closer tomainstream usage. It is available in the United States atover 1000 distributors and also formulated by private con-sumers or user groups. In addition, companies are nowbeginning to manufacture diesel engines that are designedto run on biodiesel blends. One example is the 2007 6.7 lDodge Ram Turbo Diesel Engine. Despite this large swellin usage and significant anecdotal information by usergroups, there has been little peer-reviewed research on theenvironmental chemistry of this product. Biodiesel engineemissions have been characterized and some toxicity testshave 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 researchon the health effects of biodiesel exhaust (Swanson et al.,2007). There have been several studies on the microbial deg-radation of biodiesels (Donofrio, 1996; Floro, 1996; Follis,1994; Lapinskien _e et al., 2006; Peterson and Moller, 2005;Zhang et al., 1998), which have shown that they degrade.One of these efforts published gas chromatographic tracesto monitor this process qualitatively (Zhang et al., 1998).

It is noteworthy that there have been numerous spills ofvegetable oils (straight oils) in the sea where microbes arecapable of degrading them (Bucas and Saliot, 2002). How-ever, reactions initiated at the double bonds of the fattyacids in these glyercol-based oils have been shown to poly-merize making them less available to bacteria even whenstimulated with nutrients. In fact, Mudge (1997) hypothe-sized that some residues of spilled vegetable oils may bemore recalcitrant than mineral oils.

To expand our current knowledge on the behavior in themarine environment of biodiesel and mixtures of biodieselwith fossil diesel, we performed a series of experiments andcalculations. In particular, we amended seawater and auto-claved 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 maingoals were: (1) to determine the microbial and environmen-tal fate of fossil diesel in seawater cultures when biodiesel-derived FAMEs (or vice versa) were present; (2) to measurethe relative degradation rate of FAMEs compared to com-ponents of fossil diesel; (3) to evaluate whether any newindicators or ratios of molecules within these mixturescould be identified so that in future cases they could beused to assess the short and long term fate of a biodieselspill; (4) to consider other potential environmental pro-cesses (e.g., abiotic hydrolysis) that may act on biodieselin the environment; and (5) to evaluate whether biodiesel-derived FAMEs may affect the environmental fate andtransport of petroleum hydrocarbons when there is a spill.One aspect that we did not study in this manuscript wasphotochemistry or oxidation of the double bonds on theFAMEs.

2. Methods

2.1. Obtaining and preparing the fuel mixtures used

in this experiment

The 100% fossil diesel was collected from the cargo holdof the oil barge Bouchard 65, at the time that it spilled oil inBuzzards Bay in October, 1974, and has been previouslywell 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, wereprepared by weighing and mixing the 100% fossil diesel andB100. To confirm that the biodiesel mixtures would be thesame whether mixed by mass or volume, we measured thedensities of the fossil diesel (0.853 g ml�1) and B100(0.869 g ml�1) and found them to be similar.

To compare commercially available B20 mixtures, weobtained four different samples in 2006 from two differentdistributors (referred to as Distributors I and II) in thestate of Massachusetts. One sample was obtained fromDistributor I. The other three all came from Distributor II.

2.2. Preparation of biodegradation cultures

Four experiments were performed in seawater spikedwith either 100% fossil diesel fuel, B8, B25, or B100. Weemployed classic ‘‘die away’’ procedures (Drenzek et al.,2001) to collect numerous samples each representing theirown microcosm.

Seawater was collected in two pre-cleaned, 4-l solventjugs from Vineyard Sound, MA and used for the incuba-tion studies. Both jugs were amended with NH4Cl andK3PO4 so that the final concentration of nitrogen andphosphorus was 5 and 0.9 mmol l�1, respectively. One jugwas autoclaved and used as the ‘‘control’’ jug. We referredto the other jug as the ‘‘live’’ jug.

Glass vials (60-ml volume; 14-cm height, and 2.4-cminside diameter) were used for each microcosm. Forty mil-liliters of either seawater or autoclaved seawater were

896 J.A. DeMello et al. / Marine Pollution Bulletin 54 (2007) 894–904

added to 140 and 70 vials, respectively, providing two livesamples for each control. This design allowed us to collect17 timepoints, each consisting of two live samples and onecontrol for 100% fossil diesel, B8, B25, and B100. Twenty-five microliters of concentrated solutions of either 100%fossil diesel, B8, B25, or B100 dissolved in acetone werespiked into each vial (to yield a total mass of �4.5 mg ofmaterial). The vials were loosely plugged with only auto-claved cotton wool, placed onto a shaker table, coveredwith a large cardboard box to minimize light, and agitatedat 60 revolutions per minute (rpm) for the duration of theexperiment.

At various time periods, both live and control sampleswere terminated by adding 10 ml of methyl t-butyl ether(MTBE), which also served as the first extraction step,and then sealed with Teflon-lined caps. The pH was alsomonitored by sampling a small volume of the culture withpH paper (range 0–14). An internal standard, hexacosane(n-C26) was added to each vial (38 lg was added for the100% fossil diesel fuel, B8, and B25 experiments). For theB100, we added 190 lg of n-C26.

Each sample was then recapped, gently inverted, andsonicated for 10 min. The upper layer (MTBE) wasremoved with a Pasteur pipette and delivered to a pre-cleaned 60-ml glass vial. Two subsequent MTBE extrac-tions were performed, each with 11 ml of MTBE. TheMTBE extracts were combined and then dried with anhy-drous sodium sulfate. The dried samples were transferredto 50-ml pear shaped flasks, rotary evaporated to�250 ll, spiked with an external standard, octacosane (n-C28), and then stored until analysis.

2.3. Analysis by one-dimensional gas chromatography

Each extract was analyzed on a Hewlett-Packard 5890Series II gas chromatograph with a cooled injection system(CIS) and flame ionization detector (GC-FID). A 1-llsample was injected splitless into the CIS, which wasprogrammed from 40 �C (0.3-min hold) to 320 �C at720 �C min�1 (5-min hold) and then 350 �C at 720 �C min�1

(4-min hold). Compounds were separated on a gas capillarycolumn (CP-Sil CP, 30 m length, 0.25-mm I.D., 0.25 lmfilm thickness) with H2 as the carrier gas at a constant flowof 5 ml min�1. The GC oven temperature was programmedfrom an initial temperature of 40–120 �C at 30� min�1 andthen from 120 �C to 320 �C at 6� min�1.

The concentrations of individual alkanes, branched alk-anes, and FAMEs as well as bulk total fossil diesel andFAMEs were monitored in this study. As the experimentprogressed, most of the fossil diesel became an unresolvedcomplex mixture (UCM; Farrington and Quinn, 1973).Response factors for the UCM and FAMEs relative tothe internal standard n-C26 were determined from the100% fossil diesel and B100 used in this experiment. Indi-vidual FAMEs were all quantified with the same responsefactor, which has been shown to be accurate to within afew percent (Christie, 1989). We checked the identity of

the individual FAMEs with two standards purchased fromSupelco, FAME Mix Rapeseed oil Lot No. LB-34981 andFAME Mix RM-6 Lot No. LB-37097. For reference, allchromatograms are drawn with the hexane baselineincluded.

2.4. Analysis by comprehensive two-dimensional gaschromatography (GC · GC)

The 100% fossil diesel, B8, and B25 samples from Day53 of the experiment were analyzed using comprehensivetwo-dimensional gas chromatography (GC · GC) (Nelsonet al., 2006). Due to the considerable sensitivity ofGC · GC, solutions containing high FAME content (suchas neat B8) overwhelmed the system. Hence, we prepared amixture of B0.5 and analyzed it to compare to the Day 53samples. Each sample was analyzed with an Agilent 6890gas chromatograph configured with a 7683 series split/splitless auto-injector, two capillary gas chromatographycolumns, a model KT-CLM-ZOE02 loop jet modulator(Zoex Corporation, Lincoln, NE), and a flame ionizationdetector (GC · GC-FID). The samples were analyzed inthe splitless injection mode using a 1 ll sample injectionvolume and the purge vent was opened at 1.0 min. The inlettemperature was 285 �C. The first-dimension column andthe loop jet modulator reside in the main oven of theAgilent 6890 gas chromatograph. The second-dimensioncolumn is housed in a smaller oven installed adjacent tothe main oven. With this configuration, the temperatureprofiles of the first-dimension column, thermal modulator(hot jet), and the second-dimension column can be inde-pendently programmed. The first-dimension column wasa nonpolar 100% dimethyl polysiloxane phase (RestekRtx-1 Crossbond, 7.5 m length, 0.25 mm I.D., 0.1 lm filmthickness) that was programmed to remain isothermal at35 �C for 5 min and then ramped from 35 �C to 235 �C at2.00 �C min�1. The modulation loop was deactivated fusedsilica (1.0 m length, 0.10 mm I.D.). The thermal modulatorwas programmed to remain isothermal at 150 �C for 5 minand then ramped from 150 �C to 365 �C at 2.15 �C min�1.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 film thickness) thatwas programmed to remain isothermal at 42 �C for 5 minand then ramped from 42 �C to 265 �C at 2.23 �C min�1.The thermal modulator loop pulse frequency was 20.0 s(0.05 Hz), and the pulse width was 350 ms. The carriergas was H2 at a constant flow rate of 0.9 ml min�1. TheFID signal was sampled at 100 Hz.

2.5. Dispersion/emulsion experiments

The formation of fuel/water emulsions and dispersionscan affect the persistence and environmental transport ofspilled petroleum products (Patton et al., 1981; Irvineet al., 1999; Lessard and DeMarco, 2000). We performeda simple experiment to test whether biodiesel mixtures


created dispersions or exhibited emulsion stability differ-ently from a fossil diesel. Briefly, three ml of biodiesel mix-tures (100% fossil diesel, B5, B10, B20, B50, B70, andB100) were prepared using the Bouchard 65 cargo andB100. For comparison, we also tested an asphaltene-richNo. 6 fuel oil from the spill of the Bouchard 120 (Nelsonet al., 2006). Each fuel was added to a glass vial (60-ml vol-ume; 14-cm height, and 2.4-cm inside diameter) containing18 ml of seawater collected from Vineyard Sound, MA.The vials were then capped and shaken in the horizontalposition at 270 rpm for 4 h. After shaking, the vials wereimmediately placed upright and repeatedly photographedover a period of 17 h to observe emulsion behavior.

3. Results and discussion

In this study, we investigated the microbial degradationof 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 ofresolved straight-chain and branched alkanes along withmany unresolved saturated and aromatic hydrocarbonsthat elute within the boiling range of dodecane (n-C12)and tetracosane (n-C24) (Fig. 1a). In comparison, the

Fig. 1. GC-FID chromatograms of the four neat starting solutions used inthe microbial experiments: (a) 100% fossil diesel, (b) B8, (c) B25, and (d)B100. In (a) peaks representing the n-alkanes 12–24 are numbered, and F,Np, Pr, and Ph are the isoprenoids farnesane, norpristane, pristane andphytane, respectively. In (d) the FAMEs are labeled in bold. For reference,the hexane baseline is included with all chromatograms.

B100 sample is a simple mixture, composed of C14, C16,C16:1, C18, C18:1, C18:2, and C18:3 FAMEs (Fig. 1d). We pre-pared the B8 and B25 mixtures from our 100% fossil dieseland B100 solutions in the laboratory (and did not purchasethem) (Fig. 1b and c). In the two biodiesel mixtures, all ofthe FAME compounds are resolved within the fossil dieselrange. The only exception is the C14 FAME, which co-elutes partially or completely with pristane, depending ontheir relative concentrations (with the GC-FID conditionsused).

3.1. Biodegradation of 100% fossil diesel, B8, and B25

The results of the biodegradation experiments per-formed in this study must be viewed in the context of thestudy’s experimental conditions. Often the results from lab-oratory studies are improperly extrapolated into real-worldsituations (Slater et al., 2005). First, the fuel products wereadded to seawater that was amended with high concentra-tions of nutrients under aerobic conditions. Second, thefuel products represented a large source of highly reducedcarbon to the natural population of microbes present inthe seawater. Last, any effects related to bioavailabilitydue to desorption from particles were eliminated by incu-bating in sediment-free microcosms (Drenzek et al.,2001). Nevertheless, these experiments permitted us todirectly compare microbial degradation of biodiesel versusmixtures composed of biodiesel and fossil diesel. In addi-tion, we only monitored the loss of fossil diesel compoundsand FAMEs and did not analyze for any intermediatessuch as free fatty acids.

A time series of chromatograms revealing the loss of the100% fossil diesel is shown in Fig. 2a–e and is consistentwith previous studies (Burns and Teal, 1979; Jones et al.,1983; Stout et al., 2002). It allows for the comparisonbetween the degradation of 100% fossil diesel and of B25shown later in Fig. 3. By day 3 in the 100% fossil dieselexperiment, there was already a substantial loss of n-alk-anes, indicated by a relative increase in the branched alk-anes, norpristane, pristane, and phytane (Fig. 2b). By day10, the resolved n-alkanes had disappeared completely,leaving behind the more resilient branched alkanes andUCM (Fig. 2c). Continued loss of compounds occurredbetween days 16 and 53 (Fig. 2d and e) with a remainingresidue of pristine, phytane, and an UCM. Throughout thisexperiment, some losses of low molecular weight com-pounds up to n-C17 occurred in the control samples(Fig. 2f). However, among these compounds, individualn-alkanes exhibited the same loss rates as isoprenoids hav-ing similar volatility (e.g., n-C18 vs. phytane). For this rea-son, we attribute losses in the control samples toevaporation and not microbial degradation. (Later in thissection, employing this ratio to evaluate microbial degra-dation is described in more detail).

A corresponding time-series of chromatograms is shownfor the losses of the B25 mixture (Fig. 3a–e). Similar trendsfor B8 were also observed (not shown). Overall, the

Fig. 2. A time-series of GC-FID chromatograms of the 100% fossil dieselexperiment (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 internalstandard, n-C26, which was used to normalize the FID signal in eachchromatogram.

Fig. 3. A time-series of GC-FID chromatograms of the B25 mixtureexperiment (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 internalstandard, n-C26, which was used to normalize the signals for eachchromatogram.


degradation of the B25 was very similar to the 100% fossildiesel except that the FAMEs were also degraded, so thatchromatograms of the weathered 100% fossil diesel andweathered B25 were nearly identical at day 53 (Figs. 2eand 3e). However, careful inspection of chromatogramsshows that on day 3 in the B25 sample (Fig. 3b), the n-alk-anes are more abundant relative to the branched alkanescompared to those in the 100% fossil diesel sample(Fig. 2b). By day 10, the resulting chromatograms showsimilar traces for the fossil diesel component, with only lin-gering FAMEs peaks in the B25 chromatograms (Figs. 2cand 3c). This indicates that the FAMEs and n-alkanes weredegraded first, and all other fossil fuel components fol-lowed, unaffected by the presence of FAMEs in the startingsolution.

The slight difference between the degradation of the100% fossil diesel and the B25 described above is best illus-trated by plotting the n-C18/phytane ratio on each day (alsoplotted for B8) (Fig. 4a–c). Because, n-C18 biodegrades

more quickly than phytane but has a similar volatility, adecrease in the n-C18/phytane ratio indicates biodegrada-tion of n-alkanes. It is historically typical to also make thisanalysis with n-C17 and pristane (Blumer et al., 1973; Joneset al., 1983), but this distinction was not possible, becauseC14 FAME co-eluted to varying degrees with pristane. Thisis evident in our analysis of four commercially availableB20 blends shown in Fig. 5. (Please also note in the latterfigure how the fossil diesel versus FAMEs content variesin these samples). Hence, this analytical effect should beconsidered when planning future experiments. The blackboxes in Fig. 4 highlight the days on which the n-C18/phy-tane ratio went essentially to zero, which were day 5 for the100% 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 thepresence of FAMEs may slow the initial biodegradationof n-alkanes in fossil diesel, but this difference was only

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

Fig. 5. GC-FID chromatograms from four commercially obtained B20mixtures from 2006. (a) Distributor I collected in June and (b–d)Distributor II collected in June, September, and November, respectively.The inserts in the top left corner focus on the n-C17, C14:0 FAME, andpristane retention windows that are highlighted in gray in the fullchromatograms. Please also note the extreme variability in the fossil dieselversus FAMEs content among these samples. In particular, observe thenear absence of fossil diesel in the sample collected from Distributor II inSeptember (c), suggesting some type of mixing problem occurred when thisblend was prepared. Yet, the same distributor did not have this apparentproblem two months later (e) or in previous months (b).


detectable in the first week of the experiment. Chromato-grams from day 6 of 100% fossil diesel and B8 depictedin Fig. 6a and b, respectively, highlight how quickly the res-idues in these microcosms became similar. Results from thelast day of the experiment (day 53) are shown in Fig. 7 andprovide further evidence of the apparent trend revealed inFig. 6. In addition, the resulting UCM patterns in Fig. 7are similar to those found in temperate salt marshes con-taminated with fossil diesel from spills that occurred in1969 and 1974 (Reddy et al., 2002; Peacock et al., 2005,2007). Hence in the event of a biodiesel spill, we predictthat FAMEs will be consumed by bacteria. After only ashort period, samples from a contaminated area willbecome indistinguishable from a fossil diesel spill. Thesefactors could hinder efforts focusing on spill source identi-fication and forensic investigations. Phytosterols can bepresent in FAMEs mixtures produced from rapeseed oil(Plank and Lorbeer, 1994), and hence useful tracers forsome B100s. However when we analyzed the B100 usedin this study, we did not detect any of these compounds.

To further examine and confirm the similarities between100% fossil diesel, B8, and B25 after 53 days of degradationin the laboratory, these samples were analyzed byGC · GC-FID (Fig. 8). This technology produces high res-olution chromatographic separations of complex mixturesbecause each compound is subjected to two different sta-

tionary phase selectivities (Gaines et al., 2006). Here, thefirst dimension separation uses a non-polar phase to sepa-rate each component by volatility differences, and the sec-ond dimension uses a more polar phase to separate firstdimension coeluters by polarity differences. The resultingtwo-dimensional chromatograms resolve many morepeaks, sorted according to their volatility and polarityproperties. A GC · GC chromatogram has compoundpeaks grouped by volatility along the x-axis and by chem-ical class along the y-axis. For petroleum, this producesseparated chemical classes such as alkanes, cycloalkanes,and one-, two-, and multi-ring aromatics, with additional

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 isoprenoidsfarnesane, norpristane, pristane and phytane, respectively. Please note thateach 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 arethe isoprenoids norpristane, pristane and phytane, respectively. Pleasenote that each chromatogram was not normalized to the internal standard,n-C26.


groupings showing homologous series within each class. Asshown in Fig. 8b–d, the day 53 samples for 100% fossil die-sel, B8, and B25 were quite similar, even when scrutinizedwith the higher sensitivity of GC · GC, providing addi-tional evidence that it would difficult to distinguish a bio-diesel spill from 100% fossil diesel after initial weathering.This also indicates that the presence or absence of FAMEsdid not alter the composition of the UCMs that are shownin Fig. 7.

3.2. Biodegradation of B100

The FAMEs in the B100 samples were degraded downto �10% of their original mass within three weeks of start-ing the experiment (data not shown). The controls for theB100 experiment showed no change in FAME mass orcomposition during the 53 days of experiment. We hypoth-esize that the B100 residue that remained at the end of theexperiment was likely unavailable for degradation, possiblystuck to the high sides of the vial. In addition to monitor-ing the loss of total FAME mass, we also looked for pref-erential loss of specific FAMEs in the B100 experiment. Wefound that C16 FAME was degraded faster than any of theC18 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 andMudge (1997) who observed that several unsaturated C18

FAMEs were degraded more quickly than C16 FAME inexperiments that focused on determining the effectivenessof biodiesel on the remediation of crude oil spills in theenvironment.

3.3. FAMEs hydrolysis rates

Although, no losses of FAMEs were observed in the con-trols for these experiments, esters can hydrolyze abiotically.To confirm our inferences about biodegradation, we consid-ered the plausible half-life of FAME hydrolysis in seawaterboth at experimental pH values (6.0–7.0) and natural sea-water pH values (7.4–8.3). While we did not find measuredhydrolysis rate constants of FAMEs in the literature, dataare available for ethyl acetate, a close structural analog,and this guided our predictions for FAMEs. Throughoutthe relevant pH range (6.0–8.3), the ethyl acetate hydrolysisrate is strongly dominated by the base-catalyzed reaction,relative to acid-catalyzed or neutral hydrolysis, by at leastan order of magnitude (Mabey and Mill, 1978). Hence,we assumed that the base-catalyzed pathway likewise con-trols FAMEs hydrolysis kinetics as well. Using the EPAmodule, Hydro (Mill et al., 1987), we estimated that base-catalyzed hydrolysis half-lives of FAMEs are 7 years atpH 7, and 70 years at pH 6 (25 �C). Thus, we expect thatabiotic hydrolysis did not contribute to FAMEs losses inbiodegradation experiments presented here. At pH valuesof 7.4 and 8.3, FAMEs had estimated base-catalyzed hydro-lysis half-lives of 3 years and 19 weeks, respectively (25 �C).These were considered upper bound rate estimates, sinceenvironmental temperatures (5–20 �C) would decrease theactual reaction rates. While these rates of hydrolysis areslower than the microbial degradation rates of FAMEs inthis study, abiotic hydrolysis could become more relevantin conditions where microbial degradation is less ideal.

3.4. Evaporation

By inspection of the GC-FID chromatograms of theabiotic controls in the biodegradation experiments, we

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. TheC16 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 markedn-C26 and n-C28 in (b) are the internal and external standards, respectively.


inferred that FAMEs will not affect the evaporation ratesof fossil diesel components. In the abiotic controls, fossildiesel hydrocarbons evaporated at a similar rate for boththe 100% fossil diesel and biodiesel mixtures. To corrobo-rate this observation, we estimated activity coefficients offossil diesel hydrocarbon compounds in simulated fossildiesel and biodiesel mixtures using the Universal Quasi-Chemical Functional Group Activity Coefficient (UNI-FAC) model (Gmehling et al., 1998). Our results indicatedthat the activity coefficients of petroleum hydrocarbons arenot affected by the presence of FAMEs in a fossil dieselmixture, and this is consistent with experimental evidencein previous work (Yuan et al., 2005). Consequently, weexpect 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 assess-ment should be clarified in the context of observations ofMiller and Mudge (1997), who suggest that the physicalmobility of heavy oil mixtures may be enhanced byFAMEs amendment. By adding FAMEs to a spilled crudeoil, Miller and Mudge lowered the viscosity of the putativemixture, consequently the flow properties and physicaltransport of the crude oil in the environment wereenhanced. This could indirectly accelerate the weatheringrate of a heavy oil, by increasing the distribution and expo-sure of the oil in the environment. But in cases such as bio-diesel, where the mixture viscosity is not significantlyaltered by the presence of FAMEs, our results suggest that


biodegradation and evaporation of petroleum hydrocar-bons will be similar to that observed in fossil diesel spills.

We also conclude that the FAMEs themselves will likelybiodegrade before evaporating from fuel mixtures in a typ-ical environmental setting. Both our UNIFAC calculationsand experimental data (Yuan et al., 2005) confirm thatFAMEs are ideal solutes in fossil diesel. This implies thatthe GC retention time of each FAME on a non-polar sta-tionary phase indicates its volatility from fossil diesel rela-tive to other hydrocarbon compounds in the mixture (Areyet al., 2005). GC-FID chromatograms show that all of theFAMEs studied here elute later than, and hence are lessvolatile than, n-C17 (Fig. 1). Our mass transfer calculations(Schwarzenbach et al., 2003) and field observations (Wolfeet al., 1994) both suggest that compounds less volatile thann-C17 usually require months or more to evaporate from oilspills in typical environmental conditions.

3.5. Dispersions and emulsions

In our dispersion and emulsion experiments, FAMEsappeared to increase the stability of small oil droplets inwater under turbulent conditions, and this may have impli-cations for the transport, weathering rate, and ecologicalimpact of spilled biodiesel. In these experiments, physicalagitation produced temporary dispersions (small oil drop-lets in the water phase) in the cases of both fossil dieseland biodiesel. For the fossil diesel, surface tension andbuoyancy forces caused the small oil droplets to re-aggre-gate into the oil phase within 5–10 min after the four houragitation period, as was visually indicated by a relativelyclear and colorless water phase. The B5 and B10 biodieselmixtures displayed similar behavior to the fossil diesel. Bycomparison, the water phase adjoining the Bouchard 120

heavy fuel oil clarified 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 thewater phase in all samples. The results indicate that, atsufficient FAME amendment levels, FAMEs stabilize oildroplets in the water phase by decreasing the oil–water sur-face tension and therefore reducing oil droplet re-aggrega-tion. This result is consistent with chemical intuition. Basedon their structural resemblance to surfactants, FAMEsmay form ordered associations at the oil–water interface(Israelachvili, 1991) and thereby decrease the interfacialsurface tension. As a result, small oil droplets initiallyformed by agitation are stabilized in the presence ofFAMEs, and therefore these droplets experience longerlifetimes in the water phase before reaggregating into largerglobules and rising to the surface.

In effect, FAMEs amendment may increase the incorpo-ration of oil droplets into the water column during turbu-lent sea surface events. This may facilitate downwardtransport of oil into the water column and therefore worsencontamination impacts on aquatic and benthic organisms.Additionally, oil stabilization by FAMEs may increase

rates of petroleum hydrocarbon dissolution into the watercolumn, due to the increased surface area to volume ratioof smaller oil droplets. However, water currents may dis-tribute dispersed and dissolved oil over a larger regionand thereby decrease the local severity of coastal oilingand accelerate oil weathering (Lessard and DeMarco,2000). Finally, dispersants apparently decrease oil toxicityto wildlife (Otitoloju, 2005). Hence, it is unclear whetherthe dispersant effects of FAMEs exacerbate or diminishthe overall ecological impacts of petroleum hydrocarbons.It is worth noting that previous studies usually focus atten-tion on water emulsification into the oil phase (Fingas,1995), rather than oil dispersion into the water phase aswe discuss here. Although, our mixtures showed some dif-ferences in their ability to incorporate water in the oilphase, this effect lasted only seconds for all fossil dieseland biodiesel samples.

4. Conclusions

In this study, we conducted a preliminary investigationof the biodegradation and behavior of biodiesel under con-trolled conditions. Our results provide a baseline for futurework. From the biodegradation experiments, we observedthat the FAMEs were degraded at a similar rate as the n-alkanes, and certainly more quickly than other fossil dieselcomponents. Hence, in the event of a biodiesel mixturespill, we predict that the FAMEs will be consumed by bac-teria, and samples from a contaminated area may be indis-tinguishable from a conventional fossil diesel spill afteronly a short period.

At pH values of 7.4 and 8.3, FAMEs had estimatedbase-catalyzed hydrolysis half-lives of 3 years and 19weeks, respectively. Experiments and theoretical evidenceboth suggest that FAMEs will not affect the rate of evapo-ration of petroleum hydrocarbons. In addition, FAMEsthemselves will likely biodegrade before evaporating fromspilled biodiesel.

The physical properties of FAMEs may alter the envi-ronmental behavior of petroleum hydrocarbons. Disper-sion experiments suggest that FAMEs will stabilizebiodiesel oil droplets in the water column, and this mayinfluence the transport, weathering rate, and ecologicalimpact of spilled biodiesel. By stabilizing small oil dropletsin the water column, FAMEs might also enhance dissolu-tion rates of conventional hydrocarbons.

Acknowledgements

This work was supported by funds from the NationalScience Foundation (IIS-0430835), the Department ofEnergy (DE-FG02-06ER15775), and an Office of NavalResearch Young Investigator Award (N00014-04-01-0029). We thank Professor James Quinn (University ofRhode Island), Dr. John Farrington (WHOI), Ms. LeahHoughton (WHOI), Mr. Bruce Tripp (WHOI), and Dr.Gerhard Knothe (USDA) for their assistance.


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