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Journal of Contaminant Hydrology 96 (2008) 17 31 www.elsevier.com/locate/jconhyd

A strategy for aromatic hydrocarbon bioremediation under anaerobic conditions and the impacts of ethanol: A microcosm study
Yu Dao Chen a,, James F. Barker b , Lai Gui b
a

Department of Resource and Environmental Engineering, Guilin University of Technology, Guilin, Guangxi, 541004, China b Department of Earth Science, University of Waterloo, Waterloo, Ont., Canada N2L 3G1 Received 15 October 2006; received in revised form 7 September 2007; accepted 21 September 2007 Available online 29 September 2007

Abstract Increased use of ethanol-blended gasoline (gasohol) and its potential release into the subsurface have spurred interest in studying the biodegradation of and interactions between ethanol and gasoline components such as benzene, toluene, ethylbenzene and xylene isomers (BTEX) in groundwater plumes. The preferred substrate status and the high biological oxygen demand (BOD) posed by ethanol and its biodegradation products suggests that anaerobic electron acceptors (EAs) will be required to support in situ bioremediation of BTEX. To develop a strategy for aromatic hydrocarbon bioremediation and to understand the impacts of ethanol on BTEX biodegradation under strictly anaerobic conditions, a microcosm experiment was conducted using pristine aquifer sand and groundwater obtained from Canadian Forces Base Borden, Canada. The initial electron accepter pool included nitrate, sulfate and/or ferric iron. The microcosms typically contained 400 g of sediment, 600800 ml of groundwater, and with differing EAs added, and were run under anaerobic conditions. Ethanol was added to some at concentrations of 500 and 5000 mg/L. Trends for biodegradation of aromatic hydrocarbons for the Borden aquifer material were first developed in the absence of ethanol, The results showed that indigenous microorganisms could degrade all aromatic hydrocarbons (BTEX and trimethylbenzene isomers-TMB) under nitrate- and ferric iron-combined conditions, but not under sulfate-reducing conditions. Toluene, ethylbenzene and m/p-xylene were biodegraded under denitrifying conditions. However, the persistence of benzene indicated that enhancing denitrification alone was insufficient. Both benzene and o-xylene biodegraded significantly under ironreducing conditions, but only after denitrification had removed other aromatics. For the trimethylbenzene isomers, 1,3,5-TMB biodegradation was found under denitrifying and then iron-reducing conditions. Biodegradation of 1,2,3-TMB or 1,2,4-TMB was slower under iron-reducing conditions. This study suggests that addition of excess ferric iron combined with limited nitrate has promise for in situ bioremediation of BTEX and TMB in the Borden aquifer and possibly for other sites contaminated by hydrocarbons. This study is the first to report 1,2,3-TMB biodegradation under strictly anaerobic condition. With the addition of 500 mg/L ethanol but without EA addition, ethanol and its main intermediate, acetate, were quickly biodegraded within 41 d with methane as a major product. Ethanol initially present at 5000 mg/L without EA addition declined slowly with the persistence of unidentified volatile fatty acids, likely propionate and butyrate, but less methane. In contrast, all ethanol disappeared with repeated additions of either nitrate or ferric iron, but acetate and unidentified intermediates persisted under iron-enhanced conditions. With the addition of 500 mg/L ethanol and nitrate, only minor toluene biodegradation was observed under denitrifying conditions and only after ethanol and acetate were utilized. The higher ethanol concentration (5000 mg/L) essentially shut down BTEX biodegradation likely due to high EA demand provided by ethanol and its intermediates. The negative findings for anaerobic BTEX biodegradation in the presence of ethanol and/or its biodegradation products are in contrast to recent research reported by Da
Corresponding author. Fax: +86 773 5897019. E-mail address: cyd0056@vip.sina.com (Y.D. Chen). 0169-7722/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jconhyd.2007.09.006

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Silva et al. [Da Silva, M.L.B., Ruiz-Aguilar, G.M.L., Alvarez, P.J.J., 2005. Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate, chelated ferric iron or nitrate. Biodegradation. 16, 105114]. Our results suggest that the apparent conservation of high residual labile carbon as biodegradation products such as acetate makes natural attenuation of aromatics less effective, and makes subsequent addition of EAs to promote in situ BTEX biodegradation problematic. 2007 Elsevier B.V. All rights reserved.
Keywords: Ethanol; BTEX; Trimethylbenzene isomers; Acetate; Gasohol; Enhanced bioremediation; Anaerobic condition; Nitrate; Ferric iron

1. Introduction Aromatic hydrocarbons such as benzene, toluene, ethylbenzene and xylenes and trimethylbenzenes (often referred to as BTEX and TMB) are primary constituents of gasoline. For example, American Petroleum Institute (API. 9101 gasoline contains 27 wt.% of BTEX and TMB (Barbaro et al., 1999). Aromatic hydrocarbons are of particular concern in gasoline contaminated groundwater, given their high solubility, mobility and toxicity. Biodegradation of BTEX has been demonstrated under aerobic condition (Barker et al., 1987; Alvarez and Vogel, 1995), however, the activity of aerobic hydrocarbon-degrading microorganisms can quickly exhaust limited dissolved oxygen in groundwater, which has a low solubility in water. Therefore, anaerobic conditions will often form in a plume of petroleum hydrocarbons and anaerobic biodegradation usually plays an important role in removing hydrocarbon contaminants (Cozzarelli et al., 2001). Under anaerobic conditions, benzene biodegradation can be variable (Johnson et al., 2003). Although benzene has been shown to biodegrade under denitrifying, ironreducing, sulfate-reducing and methanogenic conditions (Lovley et al., 1994; Kazumi et al., 1997; Cozzarelli and Baehr, 2003), many field studies reported that benzene was recalcitrant under denitrifying (Hutchins, 1991), ironreducing (Phelps and Young, 1999), and sulfate-reducing (Edwards et al., 1992; Ball and Reinhard, 1996; Davis et al., 1999) conditions. At Canadian Forces Base (CFB) Borden, field and supporting laboratory studies have been conducted to understand the biodegradability of BTEX under aerobic and anaerobic conditions. The BTEX compounds biodegraded rapidly under aerobic conditions and oxygen availability was found to be the limiting process in plumes derived from controlled sources (Barker et al., 1987, Schirmer et al., 1999; King et al., 1999). Under denitrifying conditions, BTX were observed to degrade in microcosms (Major et al., 1988). However, in subsequent studies (Barbaro et al., 1992; Acton and Barker, 1992), only toluene was observed to degrade quickly in field experiments. Benzene was

recalcitrant and ethylbenzene and xylenes were only incompletely biodegraded. Based on these subsequent studies, addition of nitrate was considered inefficient for BTEX bioremediation due to the recalcitrance of benzene. Under sulfate-reducing conditions, Acton and Barker (1992) found only toluene biodegraded. In addition to denitrification and sulfate reduction, little evidence of other anaerobic electron acceptor (EA) processes is available under Borden conditions. For example, natural gradient field experiments (e.g., King and Barker, 1999) and in situ column experiments (Acton and Barker, 1992) demonstrate little generation of Fe2+ and methane even when excess hydrocarbons were available. This is in contrast to aquifer systems studied by Cozzarelli et al. (2001) and Cozzarelli et al. (1999) where iron reduction was a very significant EA process. Therefore, while the potential for anaerobic biodegradation of benzene exists, this potential is not realized at most sites for reasons that remain unclear (Kazumi et al., 1997). In addition, a change of gasoline formulation may impact the fate of BTEX in contaminated groundwater. Ethanol is a likely replacement for methyl tertiary-butyl ether (MTBE) as a gasoline oxygenate. In fact, about half of gasoline is amended with 22% ethanol (by volume) in Brazil, and many US states use 10% ethanol blended gasoline (Powers et al., 2001). The ethanol-blended gasoline is called gasohol. A few provinces in Canada, China and Australia are or are encouraging blending 10% ethanol into gasoline. Increasingly, ethanol will be a new constituent along with BTEX in groundwater contaminated by gasohol. Ethanol itself will not pose a risk to drinking water sources. However, ethanol and its biotransformation intermediates such as acetate (Kim et al., 1994; Wu and Hickey, 1996) can rapidly consume existing EAs that could otherwise have been used to biodegrade aromatics. A relevant field study was conducted with methanol (Barker et al., 1992) which could cause similar effects as ethanol. Two mixtures of gasoline-contacted groundwater containing BTEX only and BTEX with methanol were injected into the research aquifer at CFB Borden. Here, BTEX biodegradation was dominantly

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aerobic, as little nitrate and sulfate were present and iron reduction did not appear significant (King et al., 1999; Schirmer et al., 2000). Results showed that BTX persisted longer in groundwater containing alcohol likely due to oxygen removal by methanol biodegradation. Molson et al. (2002) suggested that ethanol may make benzene more persistent in groundwater contaminated by gasohol as compared to normal gasoline. Ruiz-Aguilar et al. (2002a), however, found little evidence for longer benzene and toluene plumes in releases of ethanol-blended gasoline. Using microcosms or sand column in the laboratory, Corseuil et al. (1998) showed that toluene biodegradation was not significantly affected by 50 mg/L ethanol under denitrifying conditions but was not biodegraded under iron-reducing and methanogenic conditions. Ruiz-Aguilar et al. (2002b) showed that ethanol was often biodegraded before BTEX compounds and though that impacts on BTEX biodegradation were likely to be site-specific. Significantly, Da Silva et al. (2005) reported that additions of anaerobic EAs could help maintain BTEX biodegradation in the presence of about 100 mg/L ethanol. Recently, gasoline containing 10% ethanol (gasohol or E10), ethanol denatured with 5% gasoline (E95) and ethanol-free gasoline were released in a controlled manner into well-monitored segments of the shallow sandy aquifer at CFB Borden. To develop and demonstrate novel in situ remedial techniques, laboratory microcosms were established with Borden material for anaerobic bioremediation of aromatic hydrocarbons in this study. Also, this study described the anaerobic biotransformation of ethanol and the impacts of ethanol at target concentrations of 500 mg/L and 5000 mg/L (hereafter referred to as Low EtOH and High EtOH, respectively) on BTEX bioremediation under strictly anaerobic conditions. These concentrations of ethanol are similar to the maximum field concentrations of 690 and 5000 mg/L observed about 15 m downgradient of the E10 and E95 sources respectively. An initial electron acceptor pool was set in each microcosm, with nitrate and sulfate and/or ferric iron tested as added EAs to promote bioremediation. 2. Materials and methods

upgradient of all the experimental zones at the site. Nitrate was below detection, total dissolved iron was 0.4 mg/L, and sulfate was approximately 190 mg/L as it was apparently slightly affected by an underlying landfill plume (Nicholson et al., 1983). The materials were stored at 4 C until required. 2.2. Medium To provide nutrients, sterile Modified Bushnell-Haas (MBH) medium was prepared according to Mueller et al. (1991). It consisted of the following compounds at the concentrations (millimolar) indicated in parentheses: K2HPO4 (5.75), KH2PO4 (7.35), NH4NO3 (12.44), MgSO47H2O (0.81), CaC122H2O (0.136), FeCl36H2O (0.019); pH adjusted to 7.0. This medium also contributed limited nitrate, ferric iron and sulfate to the initial electron acceptor pool and they are included in Table 1.

Table 1 Microcosm groups and initial additions in the experiment Microcosm groups Microcosm MBH and initial Initial electron marks additions acceptor pools (mmol)
3+ SO2 NO 3 4 Fe

Sterile Control

M1

M2

BTEX Only

c a

M3 M4 M5 M6 M7 M8 M9 M10 M11

BTEX + Low EtOH

BTEX + High EtOH

2.1. Aquifer materials Pristine sediment was collected from just above the water table at the CFB Borden site. It consisted of fine sand with an average organic carbon content of 0.023%. Groundwater was collected from a shallow well located

150 ml MBH 5 ml NaNO3, 5 ml FeCl3 250 ml MBH, 5 ml NaNO3, 5 ml FeCl3 50 ml MBH 100 ml MBH, 5 ml NaNO3 100 ml MBH, 5 ml FeCl3 150 ml MBH 150 ml MBH, 5 ml NaNO3 150 ml MBH, 5 ml FeCl3 250 ml MBH 250 ml MBH, 5 ml NaNO3 250 ml MBH, 5 ml FeCl3

5.1

1.77

3.58

6.35 1.65

3.58

0.63 1.92 4.48 1.85 1.25 1.85 1.88 1.8 5.11 1.79 1.88 1.79 3.13 1.68 6.35 1.67 3.13 1.67

b 0.001 b 0.002 3.58 b 0.003 b 0.003 3.58 b 0.005 b 0.005 3.58

Notes: Low EtOH: target ethanol concentration of 500 mg/L; High EtOH: target ethanol concentration of 5000 mg/L; MBH: Sterile Modified Bushnell-Haas (Mueller et al., 1991); : sulfate was from groundwater. a: without further addition (non-enhanced); b: with further addition of nitrate (nitrate-enhanced); c: with further addition of ferric iron (iron-enhanced).

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2.3. Laboratory microcosms Duplicate microcosms were set up for four groups: sterile controls, BTEX Only, BTEX + Low EtOH and BTEX + High EtOH (Table 1). Microcosms were prepared in 1400-ml glass bottles capped with Teflon Mininert-valved screw caps in an oxygen-free atmosphere (90% N2, 5% H2, 5% CO2) in an anaerobic glove box. Groundwater was purged with nitrogen prior to use. Anaerobic autoclaved stock solutions of NaNO3 (645.15 mM) and FeCl3 (716.85 mM) were prepared to provide EA enhancements. For the sterile controls (M1, M2), bottles were prepared with 400 g sediment and 500 ml groundwater and then autoclaved for 60 min on three successive days, allowed to cool and moved into the glove box. An additional 5 ml 4% HgCl2 was spiked into controls for further sterilization. After 400 g sediment filled, groundwater, medium and stock solutions were added into microcosms as indicated in Table 1. Also, 0.633 ml and 6.337 ml pure ethanol were added into the microcosms where required to attain 500 mg/L (Low EtOH) and 5000 mg/L (High EtOH) target concentrations. Then 150 ml of gasoline saturated groundwater, made by equilibrating 200 ml API 91-01 gasoline and 4 L groundwater, was added to each microcosm for a 15 mg/L target concentration of total BTEX. The composition of API 91-01 gasoline is presented by Barbaro et al. (1999). Finally, groundwater was added to make 1000 ml total water and about 220 ml headspace (glove box gas) in each microcosm. All microcosms were incubated statically in the dark at 23 C in the glove box. Initial nitrate and sulfate and ferric iron concentrations from stock solution, groundwater and MBH were

shown in Table 1. During the incubation, nitrate stock solution was repeatedly injected into microcosms M7 and M10 and ferric iron stock solution into microcosms M8 and M11 to ensure continued supply of EAs for biodegradation of ethanol and aromatics. When necessary, the pH level was adjusted to the range of 6.08.0 by adding HCl or NaOH solution. 2.4. Analyses Aqueous samples withdrawn from microcosms using a sterilized glass syringe were transferred into 2 ml and 18 ml vials prepared with a drop and 0.2 ml 10% sodium azide for ethanol and aromatic hydrocarbon analyses, respectively, and immediately capped with a Teflon coated septum and aluminum crimp cap. Ethanol concentrations were analyzed on a Hewlett Packard 5890 gas chromatograph (GC) equipped with a flame ionization detector (FID). The column was 3.05 m long with a 0.318 cm inner diameter and packed with 3% SP1500 on Carbopack B (80/100 mesh). The analysis was run isothermally at 115 C and the injection temperature was 230 C. The detection limit was approximately 0.06 mg/L for ethanol. Aromatic hydrocarbons (BTEX, TMB) were extracted with pentane and analyzed by direct injection of pentane onto a Hewlett-Packard 5890 gas chromatograph, equipped with a splitless injection port, a 0.25 mm 30 m DB5 capillary column with a film thickness of 0.25 m, and a flame ionization detector. The chromatographic conditions were: injection port temperature = 275 C; initial column temperature = 35 C; initial time = 0.5 min.; heating rate = 15 C/min.; final temperature = 300 C; final time = 2 min.; detector temperature = 325 C;

Fig. 1. BTEX and TMB concentrations in the sterile control were normalized to the initial concentrations. The initial concentrations of BTEX and TMB isomers were: Benzene 3.8 mg/L, Toluene 5.9 mg/L, Ethylbenzene 0.80 mg/L, m/p-xylene 1.7 mg/L, o-xylene 0.77 mg/L; 1,3,5-TMB 93 g/L, 1,2,4-TMB 302 g/L, and 1,2,3-TMB 82 g/L.

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column flow rate = 5 ml/min helium. The m- and p-xylene isomers were not resolved in this GC analysis and so they are reported together as m/p-xylene. The method detection limit for these aromatics is 2.5 g/L. Nitrate, sulfate and acetate were analyzed with a Dionex ICS-2000 ion chromatograph equipped with an Ion-Eluent Generator, a Dionex IonPac AS18 column (4 250 mm) and conductivity detector. Total iron as an indicator of Fe2+ was analyzed with a Hach instrument (DR/2400). Under anoxic condition at near-neutral pH the dominant aqueous species of iron is Fe2+ (Langmuir, 1997) and so total iron is taken to be Fe2+. Detection limits were 0.5 mg/L for nitrate, 0.6 mg/L for sulfate, 1.0 mg/L for acetate and 0.02 mg/L for total iron. Methane analysis was not preformed until day 78 and was done with a Varian 3800 GC. Detection limits range from 0.2 to 1.6 g/L for methane. 3. Results and discussion Most duplicated microcosms behaved similarly and average values were used to plot figures and calculate parameters. Toluene concentrations were very different between duplicates of microcosm M10 and so these results were treated individually. The concentrations of BTEX and TMB were relatively stable in the sterile controls (Fig. 1). Approximately 15% loss of total BTEX and 14% loss of total TMB were observed during the experiment. Loss of ethanol was b 20% in both Low EtOH and High EtOH sterile controls (Fig. 2). The decrease of ethanol, BTEX and TMB concentrations was attributed to abiotic processes. In all active Low EtOH microcosms,

analysis of ethanol ceased on day 55 when it was below the detection limit. 3.1. Biodegradation of BTEX 3.1.1. Denitrification Nitrate was amended into microcosms M3, M4 and M5 to produce initial concentrations of 0.63 mM, 4.48 mM and 1.25 mM, respectively (Table 1). During incubation, concentrations of nitrate decreased significantly in all active microcosms (Fig. 3-b). Nitrate was below detection on day 13 and day 111 in the nitratelimited microcosms M3 and M5 respectively, whereas about 56% of the initial nitrate mass was consumed in the excess-nitrate microcosm M4 by day 204. Previous research with Borden material confirmed such nitrate loss was due to denitrification (Major et al., 1988; Barbaro et al., 1992), therefore, the significant utilization of nitrate indicated that denitrification was a viable bioprocess in all nitrate-amended microcosms. In the absence of ethanol, the typical BTEX biodegradation trend under denitrifying conditions is seen in the excess-nitrate situation (Fig. 3, M4-a). Toluene was most readily biodegraded, followed by ethylbenzene and then by o-xylene. Degradation of these three aromatics was concurrent and initiated within 7 d. Toluene biodegraded to 0.34 mg/L (i.e. C/C0 = 0.07) and ethylbenzene biodegraded to 0.40 mg/L (C/C0 = 0.02), whereas o-xylene persisted at C/C0 = 0.62 over 204 d. First-order biodegradation rate constants fitted for toluene, ethylbenzene and o-xylene were 0.170 d 1, 0.030 d 1 and 0.013 d 1 over 17, 118 and 20 d respectively. Unlike these three aromatics, significant biodegradation of m/p-xylene occurred only after a lag period of 55 d, and it biodegraded to 26% of the initial mass at a first-order rate of 0.018 d 1 over 118 d. Benzene did not degrade significantly in comparison to the controls and by day 204 benzene only reached a C/C0 of 0.66 in M4 compared to 0.83 in the control. Biodegradation of benzene under denitrifying condition in Borden material exposed to BTX contamination has been reported (Major et al., 1988), but generally field and laboratory experiments have found no significant benzene degradation under denitrifying conditions (Barbaro et al., 1992, 1999). The experiments described here indicate enhancing denitrification alone has little potential for efficient benzene bioremediation in the Borden aquifer. 3.1.2. Iron reduction Microcosm M5 was amended with 5 ml ferric iron stock solution and 100 ml MBH medium, giving an

Fig. 2. Normalized concentrations ethanol in sterile controls. Concentrations of Low EtOH (M1) and High EtOH (M2) were 500 mg/L (10.8 mM) and 5000 mg/L (108.1 mM), respectively.

22 Y.D. Chen et al. / Journal of Contaminant Hydrology 96 (2008) 1731 Fig. 3. Inorganic and normalized BTEX concentrations in active microcosms. Initial concentrations of BTEX and TMB: benzene 3.13.5 mg/L, toluene 5.15.8 mg/L, ethylbenzene 0.700.79 mg/L, m/p-xylene 1.51.7 mg/L, o-xylene 0.680.76 mg/L; 1,3,5-TMB 94103 g/L, 1,2,4-TMB 274301 g/L, 1,2,3-TMB 7680 g/L.

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initial electron acceptor pool including 1.25 mM nitrate, 3.58 mM ferric iron, and 1.85 mM sulfate (Table 1). Denitrification was certainly active until day 69 and possibly to day 111 (Fig. 3, M5-b). Iron reduction was evident after day 69 with the rising total iron (as an indicator of Fe2+) concentration starting between day 69 and 90. Toluene, ethylbenzene, o-xylene and m/p-xylene were biodegraded to 3%, 6%, 68% and 61% of their initial concentrations, respectively, by day 69 under denitrifying conditions. During the transition from denitrifying to ironreducing conditions (day 69 to day 111), ethylbenzene and m/p-xylene continued to degrade. More importantly, significant biodegradation of both benzene and o-xylene was now observed (Fig. 3 M5-a). By day 204, benzene and o-xylene biodegraded to 13% and 1% of their initial concentrations and degradation after day 69 was fitted by the first-order rate constants of 0.014 d 1 and 0.018 d 1 respectively. Most of the benzene loss occurred under iron-reducing conditions and after most of ethylbenzene had been biodegraded. Previous studies suggested that benzene was biodegraded under iron-reducing condition in the field (Anderson et al., 1998) and in the laboratory with aquifer sediments (Lovley et al., 1994). In our experiment, the onset of benzene biodegradation was during the transition from nitrate to Fe3+ as the dominant EA and was coincident with Fe2+ generation. Perhaps denitrifying microbes played an important role in prompting this process by degrading other aromatic hydrocarbons (perhaps ethylbenzene) whose presences may have inhibited benzene biodegradation (Cunningham et al., 2001, Da Silva and Alvarez, 2004). Unfortunately, the role of denitrifyers in facilitating benzene degradation under iron-reducing conditions could not be directly demonstrated since no microcosm was prepared with Fe3+ as the sole EA. However, 5 ml of ferric iron stock solution was injected into the microcosm M3 on day 154, in which BTEX were persisting in the presence of sulfate (discussed below). Interestingly, concentrations of total iron increased by day 174 and quickly rose to concentrations much higher than in microcosm M5. Significant decreases in ethylbenzene and slight decreases in m/p-xylene were noted only after day 174 (Fig. 3, M3-a). In this case, no benzene or oxylene degradation was found under iron-reducing conditions, perhaps due to the persistence of ethylbenzene. Undoubtedly, ferric iron was used to degrade other organic compounds within the API 91-01 gasoline. No apparent delay in ethylbenzene biodegradation was found under denitrifying conditions. Therefore, denitrification seemed to have a higher potential than

iron reduction in degrading some organics that can inhibit biodegradation of benzene or o-xylene. This hypothesis may also explain why initial denitrification can stimulate benzene biodegradation under ironreducing conditions as observed in the microcosm M5. It may be that all BTEX compounds can eventually be biodegraded under iron-only addition to the Borden aquifer, but only slowly. This study confirms that additions of ferric iron combined with some nitrate should promote biodegradation of all BTEX compounds in the Borden aquifer and so has significant potential for BTEX bioremediation at Borden. 3.1.3. Sulfate reduction Sulfate was present in groundwater added to all microcosms although not as a sole EA. Sulfate reduction was expected to occur after the complete consumption of nitrate in microcosm M3. However, no sulfate utilization was evident and BTEX persisted without significant loss up to day 146 (Fig. 3. M3). This study suggests that addition of sulfate would be of limited use in promoting BTEX biodegradation at the Borden Site. 3.2. Biodegradation of trimethylbenzene Isomers Trimethylbenzene isomers (1,2,3-TMB, 1,2,4-TMB, and 1,3,5-TMB) are often used as conservative tracers for assessing BTEX biodegradation at field sites since TMB isomers have Henry's Law constants and soil sorption coefficients similar to BTEX and, critically, are considered recalcitrant to biodegradation under anaerobic conditions (Wiedemeier et al., 1999; Kao and Wang, 2001). Some studies have reported biodegradation of 1,2,4-TMB under anaerobic conditions (Hutchins et al., 1991). Barker et al. (1986) suggested that 1,2,4-TMB was biodegraded in a landfill leachate plume. Acton and Barker (1992) reported that 1,2,4-TMB was biodegraded with sulfate addition but not with nitrate addition in microcosms derived from that plume. To date, no information on the biodegradability of other TMB isomers has been reported. In this study, control microcosms lost 14% of total TMBs (Fig. 1). Under denitrifying conditions (Fig. 3. M4), losses of 1,3,5-TMB exceeded that in the controls by day 17. After day 146, losses of all TMB isomers in M4 only slightly exceeded those in the controls. Overall, TMB isomers in M4 only declined to about 62% of the initial concentrations, compared to declines to 86% in controls. These differences are not large and thus the TMB isomers are not considered to degrade significantly in the presence of excess nitrate.

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Fig. 4. Concentrations of ethanol in both Low EtOH (M6, M7, M8) and High EtOH (M9, M10, M11) microcosms.

ethanol mass was lost in non-enhanced microcosms M6 by day 6, at the same time, 31% and 39% was lost in nitrate-enhanced microcosms M7 and in iron-enhanced microcosms M8, respectively (Fig. 4). Initial ethanol concentrations (9.810.7 mM) declined to below 0.02 mM (0.1 mg/L) within 13 to 34 d with first-order rate constants of 0.37, 0.26 and 0.21 d 1 in microcosms M6, M7 and M8, respectively. Though microcosms M7 and M8 were enhanced by additions of nitrate and ferric iron respectively, the enhancement did not increase the rates of ethanol biodegradation as compared with the non-enhanced case (M6). In the High EtOH microcosms (96.897.4 mM ethanol), in contrast, essentially complete removal of ethanol required longer periods and more EA addition. A 1428 day lag was apparent (Fig. 4). Without repeated nitrate or iron addition (microcosm M9), ethanol remained at 27% of the initial mass after 146 d with a first-order rate constant of 0.01 d 1 (coefficient of determination R-squared = 0.93). With repeated iron addition (M11), ethanol was detected below 1.5 mM with a first-order rate constant of 0.03 d 1 over the experiment (coefficient of determination R-squared = 0.96).These rates were slower than those observed in the Low EtOH microcosms (M6 or M8). Compared to

However, 1,2,4-TMB and 1,2,3-TMB (and to a lesser extent, 1,3,5-TMB) underwent more significant losses under iron-reducing condition (M5 after day 90). The fitted first order degradation rate constants (starting from day 90) for 1,2,4-TMB and 1,2,3-TMB were 0.006 d 1 and 0.013 d 1, respectively. The results provide clear evidence that 1,2,4-TMB and 1,2,3-TMB are at least slowly degradable under iron-reducing condition. TMB as conservative tracers for monitoring anaerobic BTEX biodegradation should be employed cautiously. With excess sulfate (Fig. 3. M3) and initial nitrate, some minor TMB loss may have occurred with denitrification, but losses were not sustained even with iron addition. As with BTEX, provision of nitrate and Fe3+ appear to be the better strategy to promote in situ bioremediation of the TMB isomers. However, TMB isomer degradation may still be too slow for practical needs. 3.3. Biotransformation of ethanol 3.3.1. Anaerobic ethanol biodegradation rate Ethanol was quickly depleted with b 7 day lag period in all Low EtOH microcosms. About 31% of the initial

Fig. 5. Concentrations of acetate in both Low EtOH (M6, M7, M8) and High EtOH (M9, M10, M11) microcosms.

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previous studies with even lower initial ethanol concentrations, results suggest that the rate of ethanol biodegradation is inversely related to its concentration. For example, by amending with BTEX and lower ethanol concentrations at 55 to 75 mg/L (1.2 to 1.6 mM), RuizAguilar et al. (2002b) reported similar or somewhat faster first-order rate constants for ethanol biodegradation, ranging from 0.8 to 2.0 d 1 under denitrifying conditions and 0.1 to 0.8 d 1 under sulfate-reducing conditions in microcosms prepared with materials from four sites. The presence of ethanol at high concentration, the absence or discontinuous addition of EA is likely to inhibit ethanol degradation. More interestingly, with repeated nitrate additions, ethanol depleted below detection limit (after day 118) under denitrifying conditions. The biodegradation rate of ethanol was more appropriately fitted by zeroorder kinetics with a constant of 0.80 mMd 1 (before 118 day, coefficient of determination R-squared = 0.97). These results indicate that nitrate addition has a high potential for bioremediation of ethanol, particularly ethanol at high concentration.

3.3.2. Biotransformation products of ethanol Anaerobic biotransformation of ethanol can produce acetate, propionate or butyrate (Kim et al., 1994; Schink et al., 1987). However, fermentation of ethanol to acetate can only occur when methanogens are present to remove excess hydrogen (Hwang et al., 2004). For example, Pelobacter propionicus can produce both propionate and acetate from ethanol. If sulfate is available ethanol can be oxidized to acetate, otherwise ethanol may be combined with hydrogen and bicarbonate to form propionate (Wu and Hickey, 1996). Acetate (and propionate) from ethanol may accumulate and thus inhibit ethanol fermentation unless hydrogen consumers such as methanogens convert the associated hydrogen to methane (Schink et al., 1987). Fig. 5 shows formation of acetate in the microcosms containing ethanol. The accumulation and subsequent disappearance of acetate seems to depend on the concentration of ethanol and the type of additional EA. In Low EtOH microcosms, acetate accumulated quickly up to peak concentrations of 5.012.6 mM at

Fig. 6. Concentrations of initial EAs (nitrate and sulfate), acetate and methane in unenhanced microcosms M6 and M9. Note: methane analyses only began on day 78.

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rates of 0.390.97 mMd 1. Acetate apparently generated with initial nitrate- and sulfate-reduction and subsequent fermentation in microcosm M6 (see Fig. 6) was converted to methane by methanogens. In the nitrate-enhanced microcosm M7, acetate accumulated the least and disappeared the most rapidly with little methane remaining after 78 d (Figs. 5 and 7). Between repeated nitrate additions, fermentation of ethanol to acetate may have developed. However, while oxidizing both ethanol and acetate, denitrifiers apparently inhibited methane production. In contrast, much more acetate accumulated in the iron amended microcosms M8 and remained at about 2.7 mM (i.e. 23% of maximum concentration) over the 174 d of incubation (Fig. 5). Repeated iron addition also inhibited methane generation (Fig. 7). In the High EtOH microcosm M9, ethanol removal was incomplete. The presence of ethanol at high concentration may have inhibited methanogens and so limited fermentation. The lack of significant methane, at least since day 78 when it was first analyzed, further suggests that methanogens was inhibited. While only minor acetate and methane were formed, large peaks of unknown chemicals (propionate and butyrate, perhaps) were observed on the GC/IC chromatograms. This suggests that once the limited nitrate and sulfate were used, acetate was no longer the dominant product.

In the nitrate-enhanced microcosms (M10), acetate accumulated initially and very low concentration of methane was present (Figs. 5 and 7). With repeated nitrate addition, acetate appeared to deplete after most of the ethanol was biodegraded, suggesting denitrifiers were using ethanol and then acetate. In the iron-amended microcosms (M11), similar to the high EtOH microcosms (M9) but not the nitrate-amended microcosm (M10), large peaks from unknown chemicals (propionate and butyrate, perhaps) were also found. It appeared that iron addition permitted ethanol utilization with propionate or butyrate as the main products rather than acetate, and that iron addition limited methanogenesis. Overall, while nitrate enhanced ethanol biotransformation under both Low EtOH and High EtOH amended conditions, it led to accumulation and then only later biodegradation of acetate. However, when either no EA or ferric iron was added under High EtOH amended conditions, biotransformation of ethanol led to accumulation of intermediates other than acetate, and methanogenesis was also limited. 3.3.3. Utilization of electron acceptors and fermentation In all ethanol-amended microcosms, the initial electron acceptor pool, nitrate and sulfate (Table 1), was sequentially and quickly consumed. Ethanol can also be

Fig. 7. Concentrations of aqueous nitrate, total iron and headspace methane in enhanced microcosms. The symbol t represents the periods for EA balance calculation. Methane concentrations were percentages of headspace gas volumes.

Y.D. Chen et al. / Journal of Contaminant Hydrology 96 (2008) 1731 Table 2 The estimation of EAs required and actually consumed for selected periods Microcosms (period, d) M7 (041) M10 (6990) M8 (090) M11 (3469) Ethanol biodegraded 9.8 mM 30.1 mM 10.7 mM 54.2 mM Acetate accumulated 0 14.1 mM 6.1 mM 2.0 mM EAs required via Eqs. (1) (2) (3) (4) (5) (6) 20.6 mM nitrate, 1.79 mM sulfate 49.7 mM nitrate 1.79 mM nitrate, 1.88 mM sulfate, 60.8 mM ferric iron 739 mM ferric iron EAs consumed

27

19.7 mM nitrate, 1.79 mM sulfate 33.5 mM nitrate 1.79 mM nitrate, 1.88 mM sulfate, 49.3 mM ferric iron 31.5 mM ferric iron

Comment: Bolded microcosm periods appear to have undergone significant fermentation.

transformed by fermenters in the absence of sulfate (Wu and Hickey, 1996). This typical fermentation was observed in microcosms M6 and M9, after nitrate and sulfate were rapidly consumed (Fig. 6). Additions of nitrate (645 mM) and ferric iron (717 mM) were separately used to enhance the biodegradation of ethanol. During the incubation and upon depletion of nitrate, a total of 24.5 mmol and 132 mmol of nitrate were periodically injected into microcosms M7 and M10, respectively. Virtually all nitrate was utilized (Fig. 7), unidentified intermediates were not found and methanogenesis was effectively inhibited. All ethanol and acetate were removed in nitrate-enhanced Low EtOH microcosms, but some acetate remained in High EtOH microcosms. These transformations of ethanol and acetate occurred under mainly denitrifying conditions, even though there were times of nitrate depletion especially in M10. Similarly, a total of 80.6 mmol and 73.45 mmol of ferric iron were added to microcosm M8 and M11, respectively (Fig. 7). This produced the characteristic sulfide smell and a black precipitate (likely iron sulfide, FeS2, Edwards et al., 1992). Fig. 7 illustrated that concentrations of total aqueous iron always increased with additions of ferric iron, but the initial increases were likely muted somewhat by the FeS2 precipitation. Significantly, ethanol also biodegraded quickly under iron-enhanced conditions. Minor acetate was generated and persisted in both Low EtOH microcosm M8 and High EtOH microcosm M11. Some methane was produced, but probably only when ferric iron became depleted prior to the next cycle of iron addition. To estimate the relative significance of fermentation versus anaerobic respiration with external EAs, calculations of the amount of ethanol that could be potentially mineralized by EA-reactions were attempted. This approach was selected since not all fermentation products were identified and quantified and thus it was not possible to calculate the proportion of ethanol fermented. The calculations were based on the following hypothesis: (a) ethanol was oxidized to acetate and then

to carbon dioxide; (b) nitrate was reduced to nitrogen gas; (c) sulfate was reduced to sulfide; and (d) no cell growth occurred. Thus, the stoichiometric relationships for these reactions are: Under nitrate reduction:
CH3 CH2 OH 0:8NO 3 CH3 COO 0:4N2 0:2H 1:4H2 O

1
CH3 COO 1:6NO 3 2:6H 0:8N2 2CO2 2:8H2 O

2 Under iron reduction:

CH3 CH2 OH 4FeOH3 7H 4Fe2 CH3 COO 11H2 O

3
CH3 COO 17H 8FeOH3 8Fe
2

2CO2 22H2 O

4 Under sulfate reduction:

CH3 CH2 OH 0:5SO2 4 CH3 COO 0:5H2 S H2 O

5 6

CH3 COO SO2 4 3H H2 S 2CO2 2H2 O

From the above reactions, the complete mineralization of 1 mol ethanol to acetate and then to CO2 will require 2 2.4 mol NO3 , or 12 mol Fe3+ or 1.5 mol SO4 . Similarly, the accumulation of 1 mol acetate will consume 0.8 mol 2 NO3 , or 4 mol Fe3+ or 0.5 mol SO4 . Based on the net loss of ethanol and the net accumulation of acetate during specific periods (marked as t in Fig. 7), requirements of EAs were estimated (Table 2). The cases of greater requirements of EAs relative to EAs available (initial plus additions) or apparently consumed imply that some ethanol fermentation occurred. Some fermentation was inferred in M8, M10, and particularly in M11. Where denitrification was the dominant EA process, fermentation was generally less significant.

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Y.D. Chen et al. / Journal of Contaminant Hydrology 96 (2008) 1731

Fig. 8. Concentrations of BTEX in nitrate-amended microcosms with the presence of ethanol. M10-1 and M10-2 are duplicate nitrate enriched High EtOH microcosms.

3.4. Impacts of ethanol on biodegradation of BTEX Only toluene was clearly biodegraded in nitrateenhanced microcosms (Fig. 8) in the presence of ethanol. In the Low EtOH (10.8 mM) microcosm M7, even though approximately 30% of the toluene was

biodegraded by day 41, its biodegradation rate increased significantly after the disappearance of ethanol and acetate (Figs. 4 and 5). Curiously, in the presence of High EtOH (108.1 mM), toluene biodegradation was observed in one of the duplicate microcosms (M10-1) and only over the first 13 d (Fig. 8). Corseuil et al. (1998) reported that toluene biodegradation was not significantly affected by ethanol (b 300 mg/L or 6.3 mM) under denitrifying condition with excess nitrate. However, in our 10.8 mM initial ethanol experiment the presence of ethanol and/or acetate limited toluene biodegradation, and at higher ethanol and acetate concentrations, toluene biodegradation was significantly impaired. Apparently, toluene had a lower competitive ability for denitrification than ethanol or acetate. Edwards and Grbic-Galic (1994) and Da Silva and Alvarez (2004) also noted an inhibitory effect of acetate on toluene biodegradation under methanogenic conditions. Compared to sterile controls (Fig. 1), concentration of ethylbenzene in microcosm M7 was also significantly decreased (Fig. 8). In other cases where no EAs were added (i.e., microcosms M6 and M9) or only iron was repeatedly added (i.e., microcosms M8 and M11), however, BTEX concentrations had no significant decrease (Fig. 9). Apparent first-order biodegradation rate constants for BTEX compounds with or without the initial presence of ethanol are summarized in Table 3. BTEX biodegradation was dramatically affected by the presence of ethanol. For comparison, Ruiz-Aguilar et al. (2002b) reported similar or faster first-order rate constants for TEX biodegradation (benzene was recalcitrant) of 0.1 to 0.2 d 1 and 0 (ethylbenzene) to 0.3 d 1 under denitrifying and sulfate-

Fig. 9. Normalized BTEX concentrations in the microcosms (M6, M9 and M8, M11).

Y.D. Chen et al. / Journal of Contaminant Hydrology 96 (2008) 1731 Table 3 Summary of first order rates of BTEX biodegradation Chemicals Benzene Toluene Ethylbenzene m,p-Xylene o-Xylene BTEX Only Iron-reducing: 0.012 d Denitrifying: 0.17 d 1 Denitrifying: 0.025 d 1 Denitrifying: 0.018 d 1 Iron-reducing: 0.05 d 1
1

29

BTEX + Low EtOH NB Denitrifying: 0.1 d 1 Denitrifying: 0.006 d 1 NB NB

BTEX + High EtOH NB Denitrifying: 0.13 d 1 NB NB NB

Notes: = observed in only one duplicate microcosm; NB = no biodegradation observed.

reducing conditions, respectively, in microcosms from four sites amended with BTEX and 55 to 75 mg/L ethanol. Impacts of ethanol on BTEX biodegradation can be mainly attributed to preferred utilization of not only ethanol (Corseuil et al., 1998) but also its biodegradation products such as acetate. The intrusion of ethanol leads to rapid consumption of EAs such as nitrate, ferric iron and sulfate in groundwater. In addition, biotransformation of ethanol may increase concentrations of readily biodegradable intermediates (acetate) also preferred over BTEX by EA processes involving nitrate and ferric iron. Though Reinhard et al. (2005) confirms that BTEX can degrade under methanogenic conditions, high ethanol concentration may have inhibited the activity of methanogens in our study. Da Silva et al. (2005) also noted slow BTEX biodegradation under methanogenic conditions in the presence of low ethanol concentrations. The recalcitrant behavior of BTEX in our microcosms initially containing ca. 500 or 5000 mg/L ethanol is in contrast to the findings reported recently by Da Silva et al. (2005). They separately amended columns contained BTEX-contaminated aquifer material with sulfate (160 mg/L), nitrate (120 mg/L) and Fe(III)-EDTA (4.2 g/L) in a basal mineral medium for one year. With ethanol concentrations of 100 mg/L and most oxygen purged from the influent water, 26 to 77% of the influent BTEX was biodegraded within about 1 day residence time after about one year of column operation. Similar to this study, Da Silva et al. (2005) noted the generation of acetate then methane in the column fed with ethanol and BTEX. However, their study found that BTEX biodegradation was enhanced through the addition of each EA, while our study just found some significant toluene biodegradation after complete removal of ethanol (500 mg/L) and its products under denitrifying conditions. Potential reasons for the differences between the two experiments were: the source of aquifer material and its pre-exposure to BTEX in the column experiments, dynamic versus static conditions, longer duration column experiments (1 versus 0.4 years) and higher levels of ethanol added in our microcosms. Perhaps their

findings are more applicable to a well-established, recurring contamination scenario with ethanol concentrations b 100 mg/L, and our microcosms are more indicative of near-source response in a previously pristine aquifer recently impacted with ethanol concentrations N 500 mg/L. While ethanol could be completely biodegraded anaerobically through provision of sufficient EAs in both studies, intermediates such as acetate still have a competitive advantage over BTEX for the available EAs. 4. Summary and conclusions This study indicates that indigenous microorganisms at the Borden Site have significant potential to degrade aromatic hydrocarbons under anaerobic conditions by utilizing nitrate and ferric iron as EAs. Benzene biodegradation was most rapid under ironreducing conditions, but only after denitrification had removed hydrocarbons such as ethylbenzene which may have inhibited benzene utilization. Toluene, ethylbenzene and m/p-xylene could be readily biodegraded under denitrifying conditions and o-xylene was biodegraded most effectively under iron-reducing conditions. For the TMB isomers, biodegradation was always slow. 1,2,3-TMB biodegraded clearly under iron-reducing conditions, with 1,2,4-TMB and 1,3,5-TMB showing some removal under denitrifying followed by ironreducing conditions. Overall, microcosm amended with both nitrate and ferric iron showed the greatest level of biodegradation of the BTEX and TMB compounds. After 204 d essentially complete removal of toluene, ethylbenzene and o-xylene occurred. Less than 15% of the benzene and m/p-xylene remained, less than 25% of the 1,2,3-TMB and less than 50% of the 1,3,5-TMB and 1,2,4-TMB remained. These residual concentrations could be readily biodegraded or polished under natural aerobic aquifer conditions (Schirmer et al., 2000). Therefore, addition of limited nitrate and excess ferric iron has a significant potential for in situ enhanced bioremediation of aromatic hydrocarbons at the Borden Site.

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Y.D. Chen et al. / Journal of Contaminant Hydrology 96 (2008) 1731

In non-enhanced microcosms, low ethanol concentrations (ca. 500 mg/L) depleted quickly, and acetate as a main intermediate of ethanol mineralization was also quickly biodegraded with methane as a major product. However, fermentation of higher concentrations of ethanol (ca. 5000 mg/L) without additional EAs resulted in significant ethanol and/or its intermediates such as acetate (perhaps propionate and butyrate) remaining for the 145 day incubation. These results suggest that bioremediation of ethanol at high concentration in the core of an ethanol plume is unlikely to meet the goals of monitored natural attenuation. Addition of either nitrate or ferric iron has significant potential for enhancing biodegradation of ethanol at both the low and high concentrations. Nitrate seemed to be the more advantageous EA for removing ethanol and its biotransformation products. However, even complete removal of ethanol still produced electron donors in the form of fatty acids (acetate and/or propionate/butyrate). They remain to compete for any EAs added to promote in situ bioremediation of the BTEX components of gasohol plumes. Therefore, the disappearance of ethanol does not end the likely impacts on BTEX biodegradation of acetate, etc., particularly with ethanol at initially high concentration. When 500 mg/L ethanol was present initially, only minor toluene biodegradation was observed under denitrifying conditions and only after ethanol and acetate were utilized. The higher ethanol concentration (5000 mg/L) essentially shut down BTEX biodegradation due to high EA demand provided by ethanol and its intermediates. The findings are in sharp contrast to the more positive findings of Da Silva et al. (2005). Based on our work, nitrate appears to have more potential for removing the impacts of ethanol on BTEX bioremediation. Also, this study suggests that further study is necessary to generalise the findings related to the fate of BTEX plumes in groundwater impacted by gasohol or denatured ethanol. Acknowledgements We especially acknowledge the assistance of Ms. Marianne Vandergriendt in preparing the microcosms and conducting the BTEX and TMB analyses. Ms. Shirley Chatten analyzed ethanol and Mr. Wayne Noble performed the nitrate and sulfate analyses. This study was funded by a research contract from the American Petroleum Institute and by an NSERC Collaborative Research & Development Grant (Barker, PI), and by the National Natural Science Foundation of China (40672200) and Natural Science Foundation of

Guangxi of China (0731022). The Soil and Groundwater Technical Task Force of API and in particular Mr. Bruce Bauman are thanked for their support and comments. Comments by anonymous reviewers were most helpful in developing this manuscript. References
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