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Microbial Strategies for Degradation of Organic Contaminants in Karst

By Tom D. Byl1, Gregg E. Hileman1, Shannon D. Williams1, David W. Metge2, and Ron W. Harvey2
1U.S. Geological Survey 640 Grassmere Park, Suite 100, Nashville Tennessee 37211
2U.S. Geological Survey, Boulder, Colorado 80303

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Approximately 40 percent of the United States east of the Mississippi River is underlain by various types of karst aquifers (Quinlan, 1989) and more than two-thirds of the State of Tennessee is underlain by carbonate rocks and can be classified as karst (Wolfe and others, 1997). Potential industrial sources of ground-water contamination are common in karst regions; however, the fate and transport of contaminants such as fuels in karst areas are poorly understood because of the distinctive hydraulic characteristics of karst aquifers (Field, 1993). Ground-water models that predict the fate and transport of contaminants in sandy aquifers have limited application to karst aquifers.  Most natural attenuation and bioremediation guidelines specify that they are not applicable in fractured rock or karst aquifers (U.S. Environmental Protection Agency, 1997).

The lack of studies examining biodegradation in karst aquifers may be due to the widespread perception that contaminants are rapidly flushed out of karst aquifers. In highly developed and well-connected conduit systems, the rate of contaminant migration is expected to be much faster than the rate of biodegradation. Field (1993) states that remediation techniques such as ground-water extraction or bioremediation are impractical in karst aquifers dominated by conduit flow; however, he also states that the belief that contaminants are rapidly flushed out of karst aquifers is a popular misconception. Large volumes of water may be trapped in fractures along bedding planes and other features isolated from active ground-water flowpaths in karst aquifers (Wolfe and others, 1997). In areas isolated from the major ground-water flowpaths, contaminant migration may possibly be slow enough that biodegradation could reduce contaminant mass if favorable microorganisms, food sources, and geochemical conditions are present (Byl and Williams, 2000; Byl and others, 2001).   The capacity for biodegradation processes in a karst setting was evaluated at sites in Tennessee and Kentucky.

The potential for biodegradation of trichloroethylene (TCE) was studied in a karst aquifer at Lewisburg, Tennessee.  This site was selected because of the presence of TCE degradation by-products in the karst aquifer, available site hydrologic and chlorinated-ethene information.  Additional chemical, biological and hydrological data were gathered to evaluate if the occurrence of TCE degradation by-products in the karst aquifer was the result of biodegradation in the aquifer or simply transport into the aquifer.  Geochemical analysis established that sulfate-reducing conditions, essential for reductive dechlorination of chlorinated solvents, existed in parts of the contaminated karst aquifer.  Geochemical conditions in other areas of the aquifer fluctuated between anaerobic and aerobic conditions and contained compounds associated with cometabolism, such as ethane, methane, ammonia and dissolved oxygen.  A large, diverse bacteria population inhabits the contaminated aquifer.  Bacteria known to biodegrade TCE and other chlorinated solvents, such as sulfate-reducers, methanotrophs, and ammonia-oxidizers, were identified from karst-aquifer water using the RNA-hybridization technique.  Results from microcosms using raw karst-aquifer water found that aerobic cometabolism and anaerobic reductive dechlorination degradation processes were possible when appropriate conditions were established in the microcosms.  The chemical and biological results provide circumstantial evidence that several biodegradation processes are potentially active in the karst aquifer.  Additional site hydrologic information was developed to determine if appropriate conditions persisted long enough in the karst aquifer for these biodegradation processes to be significant.  Continuous monitoring devices placed in four wells during the spring of 1998 documented a dual phase ground-water flow system within the karst aquifer.  Dynamic areas were present within the karst aquifer where active flow occurred, as well as, stable areas in the karst aquifer that were isolated from active flow.  The pH, specific conductance, low dissolved oxygen levels and low oxidation-reduction potentials changed very little in the stable areas isolated from active flow.  The stable areas in the karst aquifer had geochemical conditions and bacteria conducive to reductive dechlorination of chlorinated ethenes.  The dynamic areas of the karst aquifer associated with active flow fluctuated between anaerobic and aerobic conditions in response to rain events.  Associated with this dynamic environment were bacteria and geochemical conditions conducive to cometabolism.  In summary, multiple lines of evidence developed from biological, chemical and hydrological data demonstrate that a variety of biodegradation processes were active in this karst aquifer.

A second karst-aquifer site contaminated with jet fuel was also investigated.  The site is located at an airfield in southern Kentucky.  Ground-water samples were collected for bacteria and geochemical analysis from several contaminated monitoring wells in an unconsolidated regolith and karst aquifer that had varying concentrations of dissolved fuel.  Bacteria counts ranged from approximately 700,000 bacteria per milliliter to 1.2 million depending on the well and sample collection time.  These bacteria counts were derived using two methods, direct counts and BART growth tests, and the results of the two tests were within 20 percent of each other.  These numbers are much greater than previously reported when tryptic soy agar was used to quantify heterotrophic bacteria in the same wells (Byl and others, 2001).  Bacteria from the fuel-contaminated part of the karst aquifer had a 5% lighter buoyant density and a wider range of sizes than the bacteria from the non-contaminated well.  Additionally, bacteria isolated from fuel-contaminated ground-water samples readily grew with dissolved gasoline as the only source of food.  Static microcosms (n=3) set up using aerated raw karst water spiked with benzene at 1 mg/L established a biodegradation rate of 50% loss (T1/2) in 3 days.  Sterile control microcosms had less than 10% benzene loss over the same time period.  Additional field evidence that biodegradation was taking place in the aquifer was established by measuring geochemical indicators.  The wells with screens intersecting non-contaminated sections of the aquifer had greater dissolved oxygen concentrations (generally above 2 milligrams per liter) than those intersecting more contaminated sections (dissolved oxygen less than 0.1 milligrams per liter).  Also, where the oxygen concentrations were diminished, geochemical evidence indicated that anaerobic processes were active.  This evidence includes elevated levels of ammonia, sulfide and ferrous iron in the fuel-contaminated ground-water samples. Based on these results, biodegradation of fuel constituents in the karst aquifer is indicated, and therefore, natural attenuation should not be disregarded because of preconceptions about low microbial activity in karst aquifers.


Byl, T.D., and Williams, S.D., 2000, Biodegradation of Chorinated ethenes at a karst site in Middle Tennessee.  U.S. Geological Survey Water-Resources Investigations Report 99-4285, 58 pages.  Also available at

Byl, T.D., Hileman, G.E., Williams, S.D., and Farmer, J.J., 2001,  Geochemical and microbial evidence of fuel biodegradation in a contaminated karst aquifer in southern Kentucky, June 1999, in U.S. Geological Survey Karst Interest Group Proceedings, St. Petersburg, Florida, February 13-16, 2001. E.L. Kuniansky, ed., WRIR 01-4011, pages 151-156.

Field, M.S., 1993, Karst hydrology and chemical contamination: Journal of Environmental Systems, v. 22, no.1, p. 1-26.

Quinlan, J.F., 1989, Ground-water monitoring in karst terranes: recommended protocols and implicit assumptions: Las Vegas, Nev., U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, EPA/600/X-89/050, 100 p.

U.S. Environmental Protection Agency, Region 4, 1997, Draft EPA Region 4 Suggested practices for evaluation of a site for natural attenuation (biological degradation) of chlorinated solvents, Version 3.0: Atlanta, Ga., U.S. Environmental Protection Agency, Region 4, 41 p.

Wolfe, W.J., Haugh, C.J., Webbers, Ank, and Diehl, T.H., 1997, Preliminary conceptual models of the occurrence, fate, and transport of chlorinated solvents in karst aquifers of Tennessee: U.S. Geological Survey Water-Resources Investigations Report 97-4097, 80 p.

In George R. Aiken and Eve L. Kuniansky, editors, 2002, U.S. Geological Survey Artificial Recharge Workshop Proceedings, Sacramento, California, April 2-4, 2002: USGS Open-File Report 02-89

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