Environ. Sci. Technol. 2010, 44, 4525–4530
Effects of Ethanol-Based Fuel Contamination: Microbial Community Changes, Production of Regulated Compounds, and Methane Generation DENICE K. NELSON,† TIMOTHY M. LAPARA, AND PAIGE J. NOVAK* Department of Civil Engineering, University of Minnesota, 500 Pillsbury Drive Southeast, Minneapolis, Minnesota, 55455
Received December 28, 2009. Revised manuscript received May 3, 2010. Accepted May 6, 2010.
Ethanol-based fuels are becoming more heavily used, increasing the likelihood of ethanol-based fuel spills during transportation and storage. Although ethanol is well-known to be readily biodegradable, very little is known about the effects that such a spill might have on an indigenous microbial community. Of particular concern is that ethanol contamination could stimulate the growth of organisms that can generate regulated compounds and/or produce explosive quantities of methane gas. A column-based study was performed to elucidate the potential impacts of ethanol-based fuel (E85) on the indigenous microbial community during a simulated fuel spill. A continuous dilute supply of E85 resulted in profound shifts in both the bacterial and archaeal communities. The shift was accompanied by the production of high concentrations of volatile fatty acids and butanol, a compound that is regulated in groundwater by some states. Results also indicated that a continuous feed of dilute E85 generated explosive levels of methane within one month of column operation. Quantitative PCR data showed a statistically significant increase in methanogenic populations when compared to a control column. The elevated population numbers correlated to areas of the column receiving a sustained carbon load. Toxicity data indicated that microbial growth was completely inhibited (as evidenced by absence of ethanol breakdown products) at ethanol levels above 6% (v/v). These data suggest that ethanol from ethanol-based fuel can be readily degraded, but can also produce metabolic products that are regulated as well as explosive levels of methane. The core of an E85 spill may serve as a longterm source of contamination as it cannot be degraded until significant dilution has occurred.
Introduction The Energy Independence and Security Act of 2007 requires the production of 36 billion gallons of renewable fuel, including ethanol-based fuels, by 2022 (1). This constitutes a 75% increase in renewable fuel production over the next 14 years. As a result, an increased availability of ethanol* Corresponding author phone: (612) 626-9846; fax: (612) 6267750; e-mail:
[email protected]. † Current address: ARCADIS U.S. Inc., 430 First Avenue North, Suite 720, Minneapolis, Minnesota, 55401. 10.1021/es903940q
2010 American Chemical Society
Published on Web 05/20/2010
based fuels will undoubtedly occur over the next decade, leading to an increased likelihood of ethanol-based fuel spills during transportation and storage. Ethanol-based fuel spills can result in numerous adverse environmental impacts. The biodegradation of ethanol initially yields several possible products, including propionate and butyrate (2, 3). Butyrate is produced via the butyryl-CoA pathway, which can also be used to produce butanol and acetone (2, 4). While butanol and acetone do not currently have an assigned federal Maximum Contaminant Level (MCL), they may be regulated by individual states. For example, the Minnesota Department of Health has set a Health Risk Limit (HRL) of 0.7 mg/L for n-butanol and a Health Based Value (HBV) of 4 mg/L for acetone in groundwater (5). These metabolic products are then typically further transformed to produce acetate and hydrogen, which are subsequently used by methanogenic organisms to generate methane, potentially at levels sufficient to pose an explosion hazard. A factor complicating the degradation of ethanol in fuel is that high levels of ethanol (6% by volume, or higher 6, 7) are toxic to microorganisms. The core of an ethanol fuel plume, therefore, may be recalcitrant and serve as a longterm source of carbon for the plume fringes. To our knowledge, the toxicity of ethanol on complex anaerobic communities, which drive the breakdown of ethanol and subsequent methane formation, has not been evaluated. Because a spill of ethanol-based fuel to an aquifer environment is a complex and unique problem, it is important to understand how ethanol contamination stimulates and alters the community structures of Bacteria and Archaea as well as the subsequent formation of anaerobic metabolic products. This study was designed to improve our understanding of these complex processes, which should facilitate the assessment and modeling of ethanol-based fuel spills, enabling one to predict the human health risk stemming from product formation and methane generation.
Materials and Methods Microcosms. Toxicity experiments were conducted in 150mL bottles fitted with rubber septa sealed with aluminum crimp caps. Each bottle contained 1.5 g of the aquifer mixture (described in the Supporting Information) and 75 mL of minimal groundwater media (described in the Supporting Information) containing resazurin and varying percentages (0, 0.5, 1.0, 2.5, 5.0, 6.0, 10.0, 12.0% by volume) of 100% ethanol. Microcosms were initially set up with an aerobic headspace, and individual treatments were evaluated in triplicate. The pH was adjusted to 8 by adding 10% HCl prior to the addition of the aquifer mixture. Microcosms were run for 66 days at which time a liquid sample was collected from each microcosm, filtered through a 0.2-µm polyethersulfone syringe filter and analyzed for alcohols, ketones, volatile fatty acids, and methane. Microcosms appeared to become anaerobic (identified via the use of the redox color indicator resazurin) within one week of ethanol addition. The presence or absence of microbial metabolic products was used to evaluate whether the added ethanol inhibited microbial activity. Columns. Two soil columns were operated continuously for 145 days to investigate the effect of a dilute E85 feed on indigenous microbial communities and their subsequent generation of anaerobic metabolic products and methane. Both columns, initially aerobic, were packed with aquifer material from southwestern Minnesota, which is further described in the Supporting Information. One column was VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Schematic of the column system setup. supplied with a continuous dilute E85 feed (0.52% E85 by volume in a minimal groundwater media) and the other served as a control for microbiological measurements (continuous feed of minimal groundwater media only). The column feeds were maintained under aerobic conditions, but the addition of the high concentrations of ethanol (0.36% by volume or 2840 mg/L) to the E-85 fed column was expected to produce anaerobic conditions very quickly. Each column was constructed from a 1.5-m long, 0.1-m inner diameter polyvinyl chloride (PVC) plastic pipe. The ends of the columns were sealed with 0.1-m PVC end-caps that were fitted with influent and effluent connections. Six threaded sample ports were tapped along the column length and fitted with Mininert valves (Valco, 3-mm NPT female) to allow for sampling along the length of the columns. Stainless-steel screen (150 mesh) tubes were placed into each sample port to prevent aquifer material from clogging syringe needles during sampling. Columns were operated horizontally to mimic groundwater flow in an aquifer setting. In addition to sample ports, 11 vertical 6-mm diameter glass tubes were connected to the top of the column via compression fittings (Cole-Parmer Instrument Co., Vernon Hills, IL) to allow for off-gassing during column operation. This was important to prevent the column from clogging and also to prevent dangerous pressure build-up due to biogenic gas formation. Tubing ends were connected to a headspace trap via flexible PVC tubing (Fisher Scientific, Pittsburgh, PA) to inhibit diffusion of oxygen into the columns during the experiment. A schematic of the column setup is shown in Figure 1. The minimal groundwater media used for the influent of both columns contained trace nutrients and minerals consistent with natural groundwater, and is described in the Supporting Information. Column feed stocks were adjusted to a pH of 8 using either 10 mM NaOH (control column) or 10% HCl (E85-fed column). E85 fuel was obtained from a local filling station (Minneapolis, MN). The content of ethanol within E85 fuel varies with the season; the E85 used in this experiment had an actual denatured ethanol content of 74% (anhydrous ethanol content of 70.3%), as analyzed by the Minnesota Department of Commerce. Adjusting for the above, the anhydrous ethanol percentage in the feed to the E85 column was 0.36% by volume. The feed solution was pumped at a flow rate of 0.06 mL/ min into each column using a Masterflex cartridge pump (Cole Parmer, Vernon Hills, IL). The feed to the columns was replaced every 3-4 days. The influent lines were disinfected twice weekly with a 10% (by vol) bleach solution followed by a rinse of deionized water and final rinse of the respective feed solution. This removed biomass growth within the influent tubing and prevented clogging of the feed lines. Two tracer tests were performed on the columns, the first after construction but before E85 addition, and the second 123 days after E85 introduction. The tracer studies consisted of a step input of solution containing bromide (0.5 g/L) to each column over a 3-day period. Samples were collected from selected sample ports and analyzed for bromide using ion chromatography. The mean residence time of each 4526
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column and travel time to the various ports was determined by temporal first moment analysis (8).The tracer tests are described in more detail in the Supporting Information. Sample Collection. Liquid samples were withdrawn from the columns using syringes equipped with a 5-cm long 22gauge needle. All samples were filtered through a 0.2-µm polyethersulfone syringe filter (Nalgene, Rochester, NY) prior to analysis. A total of 8 mL was collected from each port during each sampling event, equating to 1200 mg/L) of anaerobic metabolic products were detected in the microcosms to which e5% ethanol was added. The typical end products of anaerobic ethanol metabolism, butyrate, acetate, and butanol, were all present (Figure 2). In contrast, only low concentrations (55 mg/L) of acetate were detected in the microcosm containing 6% ethanol and no fermentation products were detected in microcosms containing >6% ethanol. Although the possibility exists that this inhibition was temporary and the organisms would have recovered after a longer period of incubation, these data indicate that the microbial population was strongly inhibited when g6% ethanol was present. These results agreed well with previous studies, where ethanol was seen to be inhibitory at levels greater than 7% for Pseudomonas putida (7), and 3.1% for E. coli (14). Butanol was detected at concentrations exceeding 600 mg/L in both the 0.5% and 1% ethanol treatments. This concentration is approximately 3 orders of magnitude above the Minnesota regulatory standard of 0.7 mg/L. Acetone was not detected in the microcosms, but the presence of butanol indicates the potential for acetone generation via the acetone-butanol fermentation pathway (4). Methane was not detected in the microcosms. Column Study. Tracer Study. Data from the pre- and post-ethanol addition tracer studies indicated that flow through the E85-fed column averaged 1.2 and 1.9 in/day in the control and E85-fed columns, respectively. The travel times associated with each port location are summarized in Table 1. Ethanol Biotransformation. Ethanol and its metabolic products are shown over time for Ports A, C, and F in Figure 3. Ethanol concentrations decreased over time in Port A, likely as a result of increasing biomass and metabolic activity at that location. The majority of ethanol was consumed prior to reaching Port C (11 days travel time), and was never detected in Port F, located at the end of the column (28 days travel time). These data indicate that ethanol is consumed rapidly when present at levels that are not inhibitory to microbial growth. The disappearance of the metabolites along the length of the column is attributed to microbial uptake
port name
distance from inlet (inches)
control column travel time (days)
E85 column travel time (days)
A B C D E F
7 14 21 32 43 54
6 11 17 26 35 44
4 7 11 17 23 28
and subsequent degradation of these products. Dilution along the column length is not anticipated to affect these concentrations given the nature of this column experiment (i.e., continuous input of ethanol). As was observed in the microcosm study, acetate, butyrate, and butanol were the detected products of ethanol biotransformation. Port A contained the highest levels of these metabolic products throughout the course of the experiment, indicating that the majority of the biological activity occurred in the area of initial exposure to ethanol. The primary metabolic product observed in both Ports A and C was acetate, detected consistently throughout the column experiment. Low levels of butanol (discussed below) and butyrate were detected intermittently throughout the experiment in both Ports A and C. Acetate was present in Port F at the beginning of the study, but was not detected after Day 30. Butyrate and butanol were not detected at Port F during this experiment.
FIGURE 3. Ethanol and metabolic products versus time detected in the E85-fed column in (a) Port A, (b) Port C, and (c) Port F. Time ) 0 denotes the start of the E85 feed. VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Dissolved methane concentrations versus time in Ports A, C, and F of the E85-fed column. The dashed line represents the calculated dissolved methane concentration (2 mg/L) at which the lower explosive limit is reached upon equilibrium partitioning to the gas phase. E85 introduction began at Time ) 0.
FIGURE 4. Butanol and pH versus time in the E85-fed column in (a) Port A, (b) Port C, and (c) Port F. The dashed line represents pH ) 7. Introduction of E85 began on day 0. Butyrate and acetate are the predominant products of anaerobic biotransformation of ethanol at a near-neutral pH. In contrast, the generation of butanol and acetone is expected when the pH becomes acidic (2, 4). In this study, butanol was consistently detected when the pH was less than 7 (Figure 4), which was the case at Port A for most of the study. Port F, located at the end of the column, consistently had a pH g 7 throughout the experiment with no concomitant detection of butanol. Anaerobic metabolic products, such as acetate and hydrogen, can be directly used by methanogenic organisms to produce methane (15). Methane formation was observed throughout the E85-fed column approximately 30 days after E85 introduction (Figure 5). Dissolved methane was detected up to concentrations of 9 mg/L in both Ports C and F throughout the column experiment. This is similar to results observed in whole-aquifer experiments in which 500 mg/L ethanol was continuously injected (16). In those experiments dissolved methane concentrations reached 16-37.8 mg/L; the lower concentrations observed in our experiments, approximately half of solubility, were likely a result of the experimental setup in which methane could off-gas and be readily stripped with other biologically produced gases (i.e., carbon dioxide and hydrogen). Off-gassing was observed during this column study, suggesting this could occur even at the low dissolved phase methane concentrations shown in this experiment. Off-gassing of methane or partitioning of methane into the vapor phase is of concern as an explosion hazard can result when methane is present at approximately 4528
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5-20% of the gaseous environment (17). Indeed, the stripping of methane from groundwater in conjunction with the offgassing of other biogenic gases (carbon dioxide and hydrogen) can result in the release of a gas mixture containing a high percent composition of methane. Additionally, using an average Henry’s law constant for methane (18), a dissolved methane concentration of 2 mg/L can result in an explosive gaseous environment immediately above the air-water interface. In certain confined conditions (i.e., under concrete slabs, buildings, etc.) this could pose an explosion hazard. No ethanol, metabolic products of ethanol biotransformation, or methane were detected in the control column during the experiment. Microbial Community Structure. Automated ribosomal intergenic space analysis (ARISA) was used to track the community structures of the domains Bacteria and Archaea. ARISA data were analyzed using nMDS, a robust ordination method commonly used for ecological community data, with the distance between points representing dissimilarity (19, 20). Resulting ordination plots of the microbial community fingerprints reveal that both the Bacteria and Archaea clustered together in the control column, independent of the port from which a sample was collected (Figure 6). In contrast, the microbial communities detected along the length of the E85-fed column are spaced far from the control column for both the Bacteria and the Archaea. This indicates that the addition of E85 substantially changed the indigenous community. The community of Bacteria present at Port A was the most dissimilar, as indicated by the large distance between the data from Port A and that from the other sampling locations on the ordination plot (Figure 6a). This difference also corresponded to the elevated concentrations of anaerobic metabolic products detected at this port (Figures 3 and 4). In contrast, the community of Bacteria from Ports B and C in the E85-fed column appeared more similar to one another when compared to Ports D-F (Figure 6a). Port B (data not shown) and Port C (Figures 3 and 4) also contained similar concentrations of anaerobic metabolic products, which supports the presence of similar microbial communities in this portion of the column. Ports D-F contained sequentially lower concentrations of anaerobic metabolic products, with Port F containing nondetectable concentrations after Day 30 of the study (Figure 3). The fingerprints of the domain of Bacteria from these ports (D-F) are located sequentially closer to the community fingerprints from the control column (Figure 6a), indicating that these portions of the column, particularly the portion closest to Port F, were most similar to the indigenous soil community, as represented by the control column results. These data indicate
FIGURE 6. Results of nonmetric multidimensional scaling analysis for communities of (a) Bacteria (Stress 9.46), and (b) Archaea (Stress 11.63) using Bray-Curtis distance measure. Sample port labels are depicted by: column ID (C for control, E for E85-fed), and port location (A-F). Ellipses represent 95% confidence interval for triplicate soil sample analysis.
FIGURE 7. Number of methanogen gene copies per gram of soil (dry weight) along E85-fed and Control column lengths on Day 145 of experiment. Error bars represent the standard deviation of triplicate soil sample analysis. that a large shift in the community of Bacteria occurred in response to the addition of ethanol, with the magnitude of the shift corresponding to the presence and concentration of anaerobic metabolic products. The ARISA results for the Archaea (Figure 6b) parallel those for the community of Bacteria. The communities in the E85fed column and the control column were most dissimilar toward the point of E85 introduction (Port A). The data, however, show less of a difference in the community structure of Archaea along the length of the E85-fed column than that observed for the Bacteria. Shifts in the structure of Archaea in anaerobic environments have been attributed to methanogenic growth (16, 21); accordingly, the shift observed within the Archaea in the E85-fed column is likely a result of the growth of methanogens. This was further investigated by quantifying the 16S rRNA genes from methanogens in the soil at each sample port at the end of the experiment (Figure 7). A statistically significant increase (p < 0.05) in methanogen abundance in the E85-fed column was observed at Ports A, B, D, E, and F. The number of methanogens present along the length of the column also strongly correlated (r2 ) 0.995) to acetate concentration (Figure S2 in Supporting Information), indicating that the biotransformation of ethanol to acetate stimulated methanogenesis. The number of methanogens at Port A in this study (1.35 × 108 gene copies/g dry soil) are within the same order of magnitude as those detected in
other anaerobic systems containing high concentrations of carbon, such as in wetland soils (3.3 × 108 gene copies/g dry weight; 22), or cow rumen (2.85 × 108 gene copies/g wet weight; 11). When compared to a similar system in which ethanol was added to soil, however, the methanogen numbers measured in this study were 2 orders of magnitude higher than the Archaea measured by Ca´piro et al. (21) (1 × 106 gene copies/g dry weight). This difference is likely a result of the style of ethanol application, as Ca´piro et al. (21) added a one-time pulse of 100% neat ethanol, likely causing some toxicity, as opposed to the continuous low dose of E85 that was delivered over 145 days in this study. Engineering Significance. A simulated spill of ethanolbased fuel caused a number of significant microbial changes that led to the generation of a variety of products. The generation of anaerobic metabolic products such as acetate, butyrate, and butanol was observed; the composition of which was strongly affected by pH. Acetate appeared to drive the production of methane, which was present at levels that would be considered explosive in an aquifer where methane off-gassing, partitioning, and accumulation could occur. Indeed, the acetate concentration was statistically correlated to the number of methanogens present, which increased upon the addition of ethanol. A shift in the communities of both Bacteria and Archaea also occurred, which was thought to drive all of these changes via metabolism and fermentation. This study provides novel and important information regarding the potential impact of and remediation of ethanol-based fuel spills. Previous research has focused on the impact of comparatively small quantities of ethanol (used as fuel additive) on the behavior and remediation of gasoline spills (16, 23, 24); these studies, however, primarily focused on the adverse effect of ethanol on the remediation of aromatic hydrocarbons (e.g., benzene, toluene, xylenes, etc.) rather than on direct effects of ethanol contamination. The results presented herein are useful for assessing the potential risks posed by ethanolbased fuels. Our results can be used to more effectively model the fate and transport of an E85 spill. Anaerobic metabolic processes can be included to assess the potential generation of regulated compounds, such as butanol and acetone. Additionally, the generation of acetate can be used to predict the production of methane. Finally, care can be taken to avoid the generation of an explosion hazard VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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in areas in which methane could accumulate, such as under concrete slabs, or in confined underground spaces, following plume dilution and ethanol degradation.
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Acknowledgments This research was funded in part by the Minnesota Pollution Control Agency (MPCA) through the American Petroleum Institute (API), and a University of Minnesota Graduate School Thesis Research Grant. We extend a special thank you to Mark Toso, Tom Higgins, and Adam Sekely of the MPCA, and Bruce Bauman from the API for their interest and support of this research.
Supporting Information Available Additional information on the column setup, experimental methods, tracer study and molecular correlation. This information is available free of charge via the Internet at http://pubs.acs.org/.
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ES903940Q
