Environ. Sci. Techno/. 1995, 29, 2778-2781
Rapid T h n e J(((ineralifation by Aquifer Micmrmanisms at Adak, Environments PAUL M. BRADLEY* AND F R A N C I S H . CHAPELLE U.S. Geological Survey- Water Resources Division, Stephenson Centei-Suite 129, 720 Gracern Road, Columbia, South Carolina 29210-7651
Sediments from a relatively cold (5 "C), petroleum hydrocarbon-contaminated aquifer in Adak, AK, mineralized [14C]tolueneat an aerobic rate (16.3% day-' at 5 "C) comparable to that (5.1% day-' at 20 "C) of sediments from a more temperate aquifer at Hanahan, SC. In addition, rates of overall microbial metabolism in sediments from the two aquifers, as estimated by [l-14C]acetate mineralization, were similar (-10.6% h - l ) at their respective in situtemperatures. These results are not consistent with the common assumption that biodegradation rates in cold groundwater systems are depressed relative to more temperate systems. Furthermore, these results suggest that intrinsic bioremediation of petroleum hydrocarbon contaminants in cold groundwater systems may be technically feasible, in some cases.
Introduction It has long been recognized that ambient temperature affects the activity of biological systems ( 1 ) . The activity of a specific enzyme typically is reduced by about 50% for every 10 "C reduction in temperature below that enzyme's characteristic temperature optimum ( 1 , 2). Because the activity of a defined microbial community often exhibits a similar response to a reduction in growth temperature, this model is applied routinely to predict the rates of metabolism and growth of the communitywhen exposed to suboptimal temperatures ( 1 , 2). When evaluating the alternatives that are available for remediation of petroleum-contaminated aquifers, the general decline in the activity of a defined microbial community exposed to suboptimal growth conditions has been used as the basis for assuming that intrinsic rates of contaminant biodegradation are depressed in cold-temperature aquifers compared to more temperate aquifers (2-6). For example, microbial activity in groundwater systems is generally expected to be optimal between 20 and 40 "C ( 2 , 3 ) ,and microbial activity in low-temperature * Corresponding author telephone: (803)750-6125;fax: (803)7506181.
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systems is assumed to be significantly reduced in comparison (3, 4). In the same manner, rates of petroleum hydrocarbon degradation are presumed low in groundwater with temperatures ca. 5 "C (5). Others have suggested that the rates of natural biodegradation occurring under low temperature can be estimated based on literature values (typically determined at temperatures of 20-25 "C) by assuming that the rate is decreased by one-half for each 10 "C deficit in temperature (6). However, such assumptions do not take into account the fact that microbial populations are capable of adaptation, such that high rates of growth and metabolism can be achieved under ambient temperature conditions. Microbial adaptation to low temperatures is particularlylikely in aquifers because seasonal variation in groundwater temperature is low (7, 8) and because competition for availableresources is intense (2). The purpose of the studies reported here is to reexamine the assumption that rates of microbial hydrocarbon degradation in low-temperature, groundwater systems are necessarily depressed relative to the activities observed in more temperate systems. To this end, we compared rates of aerobic toluene mineralization for two shallow water table aquifers, one with a mean groundwater temperature of 5 "C and the other with amean temperature of 20 "C. At their respective in situ temperatures, rates of toluene mineralization observed in microcosms containing low-temperature sediments were comparable to those observed in the microcosms containing sediment from the temperate groundwater system. These results are not consistent with the assumption that hydrocarbon degradation rates are depressed in cold groundwater systems and suggest that bioremediation of such aquifers may be technically feasible.
Study Sites Low-temperature aquifer sediments were collected at the Naval Air Station, Adak, AK, located near the western end of the Aleutian Island chain. Groundwater chemistry data for the site are given in Table 1. Sediments at this site consisted ofwell-sorted, andesitic beach sand wind-blown fine sand; man-made fill; and layers of low-permeability peat. At the time of sample collection (August 1994), the depth to the water table was about 1 m. The annual range of groundwater temperature at the site was 4-6 "C. Contaminated sediments were collected at a depth of 1.5 m from the leading edge of a plume of contaminated groundwater that extended from a lens of JP-5jet fuel-free product. At this location, the dissolved concentration of BTX compounds was approximately 50 pglL. Uncontaminated sediments were collected upgradient of the contaminant plume at a depth of 1.5 m. Sediments collected from both locations were medium-grained, andesitic beach sands with total organic carbon contents (loss on ignition) of 0.3-0.8% dry weight. All sediment samples were collected with a hand auger that was rinsed in alcohol and flame sterilized. Samples were transported on ice and stored at 0 "C. Sediments from a temperate aquifer system were collected from a petroleum-contaminated, surficial aquifer underlying a fuel tank farm at Hanahan, SC. Groundwater chemistry data for the site are given in Table 1. The
This article not subject to U S . copyright. Published 1995 by the American Chemical Society.
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TABLE 1
Groundwater Chemistry Data for Two Sites at the Naval Air Station, Adak, AK, and two Sites at a Fuel Tank Farm in Hanahan, SC parameter
PH
temperature ("0 0 2 (mg/L)
NO3 (mg/L) PO4 (mg/L) BTX (pglL)
Hanahan-1
Hanahan-2
Adak-1
Adak-2
7.6 22.0 1.2 1.4 50.28 45
6.3 20.7 0.6
5.1 5.8 8.5 0.5 50.28 50
6.8 6.0 2.1 8.3 50.28 526
0.2
50.28 5 26
a Indicates dissolved concentration was less than detection limit of 0.2 mg/L. b Indicates dissolved concentration was less than detection limit of 2 pg/L.
sediments at the site consisted of medium-grained quartz sands underlain by predominantly clay material at a depth of 6-11 m. At the time of sample collection (August 19941, the depth to the water table was approximately 1.5 m, and the groundwater temperature was 20 "C. Groundwater temperatures at the site range from 19 to 22 "C annually. Contaminated sediment was collected at the water table contaminated groundwater. from within a plume of JP-4, The dissolved BTXconcentration at this location was about 45pglL. Uncontaminated sediments were collected at the water table from a point adjacent to but outside of the contaminant plume. Sediments collected from both locations were medium-grained quartz sands with total organic carbon content (loss on ignition) of 0.5-0.9% dry weight. Sediments were collected aseptically and stored as described above.
Methods Uniformly ring-labeled [ 14C]toluene(9.7 mcilmmol) was obtained from Sigma Chemical Co. (St. Louis, MO). The purity of the radiolabeled toluene was determined by flame ionization detection gas chromatographyto be greater than 98%. [ l-l4C1Acetate(53.7 mcilmmol) was obtained from Sigma Chemical Co. The purity of the radiolabeled acetate was determined by radiochemical detection high-pressure liquid chromatography to be greater than 98%. The effect of incubation temperature on aerobic, petroleum hydrocarbonmineralization was investigated using toluene as a model petroleum compound. Microcosms consisted of 10 g of moist aquifer material (8 g dry weight) and 0.5 mL of a sterile, aqueous solution of uniformly ringlabeled [14C]toluenein sterile 30-mL serum vials. The toluene solution was prepared using groundwater collected at the point of sediment sample collection. The concentration of non-radiolabeled toluene present in the microcosms was estimated (based on laboratory adsorption data and in situ concentrations of dissolved BTX) to be less than 10% of the final microcosm toluene concentration of approximately 10pmollkg dry weight. Because the groundwater at the sample collection points was aerobic, microcosms were prepared aerobically and sealed with butyl rubber stopper/base-trap assemblies as described previously (9). Abiological controls were prepared by adding HgC12 (5 mM) and autoclaving the microcosms (121 "C, 1 h). The microcosms were incubated statically, in the dark and at a temperature of 5, 20, or 35 "C. To quantify the fraction of toluene label recovered as l4CO2,microcosms were sacrificed immediatelyafter label addition (time zero) and at 5, 10, and 25 h after addition of label.
The effect of temperature on overall microbial activity was evaluated using [l-14C]acetateas a carbon substrate. The concentration of acetate in each microcosm was approximatelyl0pmollkg dry material. The in situ acetate concentration for the Ad& and Hanahan sediments was less than the analytical detection limit of 0.2 pmollkg dry material. Microcosms were prepared and incubated as described above. To quanufy the fraction of acetate label recovered as l4CO2,microcosms were sacrificed immediately after label addition (time zero) and at 0.5, 1, and 3 h after label addition. At each time point, triplicate experimental vials and a single control vial were acidified with 1000 pL of 13 N HJPO4. Evolved 14C02 was collected by placing 400 pL of 10 N KOH in the suspended base traps and shaking the acidified microcosms overnight. The 14Crecovered in the base solution was quantified by liquid scintillation counting. The recovery efficiency of 14C02in the sample material was determined using H14C03. Reportedvalueswere corrected for recovery efficiencies (57%for Ad&, 81% for Hanahan), the activity recovered at time zero (less than 0.2%),and the activity detected in sterilized control vials (less than 0.5%). The activity associated with radiolabeled volatile compounds other than COZwas estimated using toluene as a trapping agent and found to be less than 1%of the activity recovered in the base traps. During the incubations, the rates of l4COZproduction in the Ad& and Hanahan microcosmswere approximately first order. Thus, for each sediment and incubation temperature, rates of toluene and acetate mineralization were estimated by linearly regressing the percent recovery of radiolabel against time using the simple linear regression routine of Sigmastat (10). For a given sediment, statistically significant differences between treatment means were identified by analysis of variance and the StudentNewman-Keds multiple comparison test (10).
Results and Discussion Public concern over groundwatercontamination combined with an increasing emphasis on the costs of restoring contaminated aquifers has led environmental regulatory agencies to consider intrinsic bioremediation (natural attenuation) as an aquifer restoration technology (11).To identify the environmental conditions that favor natural biodegradation of contaminants and to avoid misapplication of this approach at sites or under circumstances that are inappropriate, several organizations have developed guidelines for site assessment to determine the suitability of intrinsic bioremediation as a remediation alternative (5, 6, 12, 13). Such guidelines often contain generalized assumptions about the circumstances under which microbial activity is either favored or inhibited. Because of the economic investment attached to the cleanup of contaminated sites and because of the environmental consequences inherent in any cleanup technology, some of these assumptions merit reexamination. Based primarily on the well-documented decrease in the activity of a defined microbial community exposed to suboptimal temperatures, it is widely assumed that rates of microbial hydrocarbon degradation in cold-temperature aquifers are low relative to rates in more temperate systems (5). In the present study, Hanahan microcosms exhibited maximum aerobic toluene mineralization rates at incubation temperatures of 20-35 "C with mean rates of 5.1 and 1.6% day-' for contaminated and uncontaminated sediVOL. 29, NO. 11, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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5 2
0.3
1
Ifl 0
0
5
HANAHAN-I HANAHAN-2
10
IS
20
30
25
35
. .
0
0
ADAK-I ADAK-2
R
C
e
-1
0.0
1 5
10
15
zn
l 25
TEMPERATURE ( 'C
30
35
)
FIGURE 2. Effect of incubation temperature ("C) on rates of toluene mineralization (Ohh)for sediments collected from a contaminated (Adak-1)and an uncontaminated (Adak9)site at the NavalAir Station, Adek, AK. Datum points are first-order rate constants. Error bars indicate the standard error of the estimate for the first-order rate constants. Rates with the same superscript are not statistically different according to analysis of variance and the StudentNewman-Keuls multiple comparisons test ( p < 0.05).
ments, respectively (Figure 1). The contaminated and the uncontaminated sediments from Adak exhibited rates of toluene mineralization at the in situ temperature of 5 "C that were approximatelytwice those observed for Hanahan sediments at their in situ temperature of 20 "C (Figures2). Adak microcosms exhibited daily toluene mineralization rates for contaminated and uncontaminated sediments of 16.3 and 17% day-' at 5 "C, respectively. These results should not be construed as evidence that hydrocarbon compounds are degraded more readily in cold-temperature groundwater systems than in temperate systems. The authors have measured hydrocarbon biodegradation in several JP-4 or JP-5 contaminated, shallow aquifers in South Carolina using similar radiolabeled techniques and have observed aerobic toluene mineralization rates varying from 2.4 to 29% day-' (unpublished results). The rates of aerobic toluene mineralization observed in Adak microcosms in this study are within the range observed for hydrocarboncontaminated, shallow aquifers in South Carolina. These results indicate that hydrocarbon biodegradation rates in 2780
l6
0
0
ADAK-I HANAHAN-1
15
20
25
30
3s
TEMPERATURE ( "C )
FIGURE 1. Effect of incubation temperature ("C) on rates of toluene mineralization (%h) for sediments collected from a Contaminated (Henahan-1)end an uncontaminated(Hanahan-2)site at a tank farm near Charleston, SC. Datum points are first-order rate constants. Error bars indicate the standard error of the estimate for the firstorder rate constants. Rates with the same superscript are not statistically different according to analysis of variance and the Student-Newman-Keuls multiple comparisons test ( p < 0.05). 2.0
18
2
10
TEMPERATURE ( "C )
y
w
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29. NO. 11, 1995
FIGURE 3. Effect of incubation temperature ("C) on rates of acetate mineralization (%h)forsediments collected from contaminated sites at Adak Island, AK (Adak-1) and a tank farm near Charleston, South Carolina (Hanahan-1). Datum points are first-order rate constants. Error bars indicate the standard arror of the estimate for the firstorder rate constants. The observed rates of acetate mineralization were wt statistically different according to analysis of variance and the Student-Newman-Keuls multiple comparisons test (p < 0.05).
cold-temperature environments are not necessarily lower than in temperate systems. This conclusion is supported by the results of the acetate mineralization study (Figure 3). Although estimates of microbial biomass can provide useful information about the productivity of the microbial populations that exist under varying environmental conditions, these estimates do not necessarily indicate the level of microbial activity occurring under in situ conditions. In this study, the rate of acetate mineralization in sediment microcosms was used to evaluate the effects of low-temperature conditions on rates of overall microbial metabolism in these sediments. The results indicate that rates of aerobic microbial metabolism occurring at the Adak and Hanahan sites under in situ conditions were quite similar (mean rate of 10.6% h-l). The lack of significantvariation in the rates of aerobic metabolism observed at different incubation temperatures indicated that the microorganisms involved in acetate mineralization were less sensitive to short-term changes in incubation temperature than those microorganisms involved in toluene mineralization. It is important to note that the results of the toluene mineralization studies are consistent with the general model of decreasing activity with temperature for a defined microbial community. For example, both sediments from the Hanahan site demonstrated a significant decrease in the rate of toluene mineralization with decreasing temperatures (Figure 1). Although the toluene mineralization rate of the uncontaminated Adak sediment did not vary significantly at 5 and 20 "C incubation temperatures, the rate of toluene mineralization by the contaminatedsediment microbial community was approximately twice as high at 20 as at 5 "C (Figure 2). Nevertheless, the microbial communities indigenous to the aquifer at Adak readily mineralized toluene even at 5 "C. This rapid mineralization of toluene by Adak microorganisms at 5 "C illustrates that it is not appropriate to estimate rates of hydrocarbon degradation by a cold-temperature acclimated community based on the temperature response of a microbial community that is indigenous to and adapted to a more temperate environment.
These results have significant implications for in situ bioremediation of petroleum-contaminated aquifer systems. First, the results demonstrate that the microorganisms indigenous to the surficial aquifer at the Naval Air Station,Adak, are capable of rapid, aerobic degradation of toluene at the in situ temperature of 5 "C. This response indicates that intrinsic biodegradation of petroleum hydrocarbon contamination can occur in cold-temperature aquifers at environmentally significant rates. Thus, the results suggest that intrinsic bioremediation may be a viable alternativefor cleanup of petroleum-contaminatedaquifers at NAS Ad& and at other contaminated, cold-temperature sites. Second, the results indicate that aprioriassumptions about the effect of cold climates on rates of microbial metabolic activity, in general, and microbial hydrocarbon degradation, in specific, are problematic at best. Thus, any effort to realistically estimate the rate of naturally occurringhydrocarbon biodegradation in cold-temperature aquifer systems should include a direct measurement of microbial degradation activity under the in situ temperature conditions.
Acknowledgments The authors thank C. F. Waythomas and G. L. Nelson of the Alaska District of the U.S. Geological Survey for their assistance in the collection of samples from Ad& Island. This work was supported by the U.S. Department of the Navy under an agreement with the U.S. GeologicalSurvey. The use of trade and firm names in this article is for the purpose of identification only and does not imply endorsement by the US.Geological Survey.
Literature Cited (1) Atlas, R. M.; Bartha, R. Microbial Ecology: Fundamentals and
Applications, 3rd ed.; The BenjamidCummings Publishing Company, Inc.: New York, 1993; pp 215-219.
(2) Chapelle, F. H. Groundwater Microbiology and Geochemistry; John Wdey and Sons: New York, 1993; pp 52-54. (3) Borden, R. C. In Handbook of Bioremediation; Matthews, J. E., Ed.; Lewis Publishers: Boca Raton, 1994; pp 182-183. (4) King, R. B.; Long, G. M.; Sheldon, J. K. Practicul Environmental Bioremediation; Lewis Publishers: BocaRaton, 1994 pp 34-36. (5) Wiedemeier, T. H.; Downey, D. C.; Wilson, J. T.; Kampbell, D. H.; Miller,R. N.; Hansen, J. E. TechnicalProtocolforImplernenting
the Intrinsic Remediation with Long-Term Monitoring Option for Natural Attenuation of Dissolved-Phase Fuel Contamination in Groundwater; United States Air Force Center for Environmental Excellence; Brooks Air Force Base: San Antonio, TX, 1994; p 129. (6) Naturally OccurringBiodegradutionas a RemedialAction Option for Soil Contamination:1994. Wisconsin Department of Natural Resources Emergency and Remedial Response Program: Madison, 1994; p 16. (7) Bouwer, H. Groundwater Hydrology; McGraw-Hill Book Company: New York, 1978; pp 378-380. (8) Matthess, G. The Properties of Groundwater; John Wiley and Sons: New York, 1982; pp 197-210. (9) Bradley, P. M.; Chapelle, F. H.; Landmeyer, J. E.; Schumacher, J. G. Appl. Environ. Microbiol. 1994, 60, 2170-2175. (10) SigmaStut Statistical Software User's Manual; Jandel Scientific: San Rafael, CA,1994. (1 1) National Research Council. In Situ Bioremediation, When Does It Work?;National Academy Press: Washington, DC, 1993. (12) US. Environmental Protection Agency. Chapter E,Natural attenuation of petroleum hydrocarbons. How to Evaluate Alternative Cleanup Technologiesfor UndergroundStorage Tank Sites: A Guide for Corrective Action Plan Reviewers: 1994; US. Government Printing Office: Washington, DC, 1994; EPA 510B-94-003. pp IX-17. (13) Groundwater MiringZone Guidance Document. South Carolina Department of Health and Environmental Control: Columbia, SC, 1994; 4 pp.
Received for review February 22, 1995. Revised manuscript received June 5, 1995. AcceptedJune 12, 1995.@ ES9501124 @Abstractpublished inAduanceACSAbstracts, September 1,1995.
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