Prepared in cooperation with the

TENNESSEE DEPARTMENT OF ENVIRONMENT AND CONSERVATION,

DIVISION OF SUPERFUND

Biodegradation of Chlorinated Ethenes at a

Karst Site in Middle Tennessee

Water-Resources Investigations Report 99-4285

U.S. Department of the Interior

U.S. Geological Survey
Biodegradation of Chlorinated Ethenes at a

Karst Site in Middle Tennessee

By Tom D. Byl and Shannon D. Williams

U.S. GEOLOGICAL SURVEY

Water-Resources Investigations Report 99-4285

Prepared in cooperation with the 

TENNESSEE DEPARTMENT OF ENVIRONMENT AND CONSERVATION, 

DIVISION OF SUPERFUND

Nashville, Tennessee

2000
U.S. DEPARTMENT OF THE INTERIOR

BRUCE BABBITT, Secretary

U.S. GEOLOGICAL SURVEY

Charles G. Groat, Director

Any use of trade, product, or firm names in this report is for identification 

purposes only and does not constitute endorsement by the 

U.S. Geological Survey.

For additional information write to: Copies of this report may be purchased from:

District Chief U.S. Geological Survey

U.S. Geological Survey Branch of Information Services

640 Grassmere Park, Suite 100 Box 25286

Nashville, Tennessee 37211 Federal Center

Denver, Colorado 80225
Contents iii

CONTENTS

Abstract.................................................................................................................................................................................. 1

Introduction ........................................................................................................................................................................... 1

Chlorinated-Ethene Biodegradation Processes...................................................................................................................... 2

Reductive Dechlorination ............................................................................................................................................ 3

Aerobic Cometabolism ................................................................................................................................................ 3

Direct Oxidation .......................................................................................................................................................... 5

Previous Studies Relevant to Biodegradation in Bedrock Aquifers...................................................................................... 5

Study Site............................................................................................................................................................................... 7

Methods and Procedures........................................................................................................................................................ 15

Water-Quality Data Collection .................................................................................................................................... 15

Geochemical Indicators ............................................................................................................................................... 16

Chlorinated-Ethene and Degradation Product Data .................................................................................................... 16

Microcosm Experiments.............................................................................................................................................. 17

Bacteria Identification and Enumeration..................................................................................................................... 18

Results and Interpretation...................................................................................................................................................... 20

Biodegradation of Chlorinated Ethenes in the Shallow Water-Bearing Zone ............................................................. 20

Biodegradation of Chlorinated Ethenes in the Karst Aquifer...................................................................................... 25

Multiple Lines of Evidence from Well 12D ...................................................................................................... 29

Multiple Lines of Evidence from Well 1D ........................................................................................................ 31

Multiple Lines of Evidence from Well 3D ........................................................................................................ 33

Multiple Lines of Evidence from Well 2D ........................................................................................................ 36

Data Collected from Other Deep Wells............................................................................................................. 39

Lessons Learned .................................................................................................................................................................... 39

Summary and Conclusions .................................................................................................................................................... 42

References ............................................................................................................................................................................. 56
iv Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

FIGURES

1-3. Diagrams showing:

1. Common biological degradation processes for chlorinated ethenes ............................................................. 3

2. Oxidation-reduction potentials for selected microbial processes.................................................................. 4

3. Examples of cometabolic processes that degrade chlorinated ethenes ......................................................... 5

4-7. Maps showing:

4. Location of ground-water monitoring and remediation activities at the study site in 

Marshall County, Tennessee.......................................................................................................................... 8

5. Typical potentiometric surface conditions for the shallow water-bearing zone............................................ 10

6. Bedrock surface at the study site................................................................................................................... 11

7. Spatial distribution of chlorinated ethenes in the shallow water-bearing zone............................................. 12

8. Diagram showing the generalized hydrogeology of the Middle Tennessee study site ......................................... 13

9. Map showing the effect of pump-and-treat wells on water levels in deep wells.................................................. 14

10. Radial diagrams used to illustrate electron acceptor processes ............................................................................ 17

11. Map showing geochemical conditions of water samples collected 

from selected shallow wells, August 21, 1997 ..................................................................................................... 21

12. Bar graph showing chlorinated-ethene data for water samples collected from well 33S..................................... 23

13. Map showing nitrate and ammonia concentrations in samples from selected shallow wells............................... 24

14-15. Bar graphs showing:

14. Geochemical data for water samples from well 7S....................................................................................... 24

15. Daily rainfall totals for a 1-month period before each ground-water sampling event .................................. 25

16. Radial diagram showing typical geochemical conditions for anaerobic water samples 

collected from deep wells ..................................................................................................................................... 28

17-27. Graphs showing:

17. Concentrations of chlorinated ethenes in water samples collected from selected 

deep wells on November 4 and 5, 1997 ........................................................................................................ 28

18. Temporal changes in chlorinated-ethene concentrations in water samples 

from wells 3D and 4D ................................................................................................................................... 30

19. Continuous ground-water monitoring data collected from well 12D for water level, 

specific conductance, dissolved oxygen, and oxidation-reduction potential, 

March 13 through May 19, 1998................................................................................................................... 31

20. Continuous ground-water monitoring data collected from well 12D showing rapid 

changes in water level, specific conductance, dissolved oxygen, and 

oxidation-reduction potential, March 31 through April 1, 1998 .................................................................. . 32

21. Chlorinated-ethene concentrations in water samples collected from well 12D ............................................ 33

22. Trichloroethylene concentrations during microcosm experiment 2.............................................................. 34

23. Relative amounts of trichloroethylene and reductive-dechlorination degradation 

products in experiment 1 microcosms after 10 months of incubation .......................................................... 34

24. Continuous ground-water monitoring data collected from well 1D for water level, 

specific conductance, dissolved oxygen, and oxidation-reduction potential, 

March 18 through May 20, 1998................................................................................................................... 35

25. Continuous ground-water monitoring data collected from well 3D for water level, 

specific conductance, dissolved oxygen, and oxidation-reduction potential, 

March 19 through May 20, 1998................................................................................................................... 36

26. Continuous ground-water monitoring data collected from well 2D for water level, 

specific conductance, dissolved oxygen, and oxidation-reduction potential, 

March 31 through April 5, 1998.................................................................................................................... 37

27. Continuous ground-water monitoring data collected from well 2D for water level, 

specific conductance, dissolved oxygen, and oxidation-reduction potential, 

March 13 through May 20, 1998................................................................................................................... 38

28-30. Diagrams showing:

28. Lines of evidence needed to evaluate biodegradation of chlorinated solvents in the 

shallow water-bearing zone........................................................................................................................... 41

29. Lines of evidence needed to evaluate biodegradation of chlorinated 

ethenes in the karst aquifer............................................................................................................................ 42

30. Hydrogeology determines residence time of ground water, which influences the biological community,

the geochemistry, and the biodegradation of chlorinated ethenes in karst aquifers ........................................ 42
Contents v

TABLES

1. Terminal electron acceptors and byproducts involved in microbial processes..................................................... 4

2. Well completion and hydrogeologic data for selected wells ................................................................................ 45

3. Geologic and hydrologic units at the study site ................................................................................................... 9

4. Geochemical indicators of terminal electron acceptor processes measured during this study ............................. 16

5. Organic constituents frequently measured at chlorinated-solvent contaminated sites ......................................... 18

6. Description of microcosms used to examine biodegradation of chlorinated ethenes........................................... 19

7. Water-quality data for samples collected from selected shallow wells ................................................................ 22

8. Chlorinated-ethene data for samples from selected wells..................................................................................... 46

9. Water-quality data for samples collected from selected deep wells ..................................................................... 26

10. Results from bacteria identification for samples collected on 

November 4 and 5, 1997 from selected wells....................................................................................................... 33

11. Microcosm data used to examine biodegradation of chlorinated ethenes ............................................................ 51

12. Aerobic and facultative anaerobic heterotrophic bacteria enumeration data........................................................ 55

13. Summary and interpretation of multiple lines of evidence using 

data collected from deep wells.............................................................................................................................. 40

CONVERSION FACTORS, VERTICAL DATUM, WATER-QUALITY UNITS, AND ACRONYMS

Temperature in degrees Celsius (°C) can be converted to degrees Fahrenheit (°F) as follows: °F = 1.8 x °C + 32

Concentrations in micromoles per liter (μM/L) and moles per liter (M/L) can be converted to micrograms per liter (μg/L) and 

grams per liter (g/L), respectively, by multiplying by the atomic or formula weight of the constituent.

Sea level: In this report “sea level” refers to the National Geodetic Vertical Datum of 1929—a geodetic datum derived from a 

general adjustment of the first-order level nets of the United States and Canada, formerly called Sea Level Datum of 1929.

Multiply By To obtain

micrometer (μm) 0.00003937 inch

centimeter (cm) 0.3937 inch

meter (m) 3.281 foot

cubic meter (m3

) 35.31 cubic foot

kilometer (km) 0.6214 mile

square meter (m2

) 10.6 square foot 

liter (L) 33.82 liquid ounce

microliter (μL) 0.00003381 liquid ounce

milliliter (ml) 0.03381 liquid ounce

hectare (ha) 2.471 acre

kilogram (kg) 2.205 pound avoirdupois

liters per minute (L/min) 0.2642 gallons per minute 
vi Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

WATER-QUALITY UNITS

CFU/100 mL colony forming units per 100 milliliters of sample

mg/L milligrams per liter

mV millivolts 

μg/L micrograms per liter

m/h meters per hour

cells/mL cells per milliliter

μm micrometer

ACRONYMS

AMO Ammonia monooxygenase

DCA 1,2-Dichloroethane

DCE Dichloroethylene

cDCE cis-1,2-Dichloroethylene

tDCE trans-1,2-Dichloroethylene

DO Dissolved oxygen

DNAPL Dense nonaqueous phase liquid

MMO Methane monooxygenase

NADH Nicotinamide adenine dinucleotide

ORP Oxidation-reduction potential

PB Phosphate buffer

PCE Tetrachloroethylene

RNA Ribonucleic acid

rRNA Ribosomal ribonucleic acid

TCE Trichloroethylene

TDEC-DSF Tennessee Department of Environment and Conservation, Division of Superfund

USGS U.S. Geological Survey

VC Vinyl chloride 
Introduction 1

Biodegradation of Chlorinated Ethenes at a 

Karst Site in Middle Tennessee 

By Tom D. Byl and Shannon D. Williams

Abstract

This report presents results of field and laboratory investigations examining the biodegradation of chlorinated ethenes in a karst aquifer 

contaminated with trichloroethylene (TCE). The 

study site, located in Middle Tennessee, was 

selected because of the presence of TCE degradation byproducts in the karst aquifer and available 

site hydrologic and chlorinated-ethene information. Additional chemical, biological, and hydrologic data were gathered to evaluate whether the 

occurrence of TCE degradation byproducts in the 

karst aquifer was the result of biodegradation 

within 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. 

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 ammoniaoxidizers, 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. These chemical and biological results provide circumstantial 

evidence that several biodegradation processes 

are active in the aquifer. Additional site hydrologic information was developed to determine if 

appropriate conditions persist 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 

indicated that pH, specific conductance, dissolved 

oxygen, and oxidation-reduction potentials 

changed very little in areas isolated from active 

ground-water flow paths. These stable areas in the 

karst aquifer had geochemical conditions and bacteria conducive to reductive dechlorination of 

chlorinated ethenes. Other areas of the karst aquifer were associated with active ground-water flow 

paths and 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 chemical, biological, and hydrologic data demonstrate that a variety of biodegradation processes are active in this karst aquifer. 

INTRODUCTION

The biodegradation of chlorinated solvents such 

as trichloroethylene (TCE) in aquifers consisting of 

unconsolidated materials has been well documented, 

and several guidelines have been developed from 

those studies (Remediation Technologies Development Forum, 1997; U.S. Environmental Protection 

Agency, Region 4, 1997; and Wiedemeier and others, 
2 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

1998). The guidelines for evaluating sites contaminated with chlorinated solvents incorporate hydrogeology, microbiology, and geochemistry, and groundwater modeling. A large component of those guidelines is for evaluating if significant biodegradation is 

taking place. One environment where chlorinatedsolvent biodegradation has not been adequately investigated is karst aquifers because of the complex 

hydrology associated with these aquifers. Although 

chlorinated-solvent biodegradation products such as 

cis-1,2-dichloroethylene (cDCE) have been detected 

in karst aquifers, little research has been done in examining if biodegradation occurred in the karst aquifers 

or if the biodegradation products originated elsewhere.

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 chlorinated solvents in karst areas are 

poorly understood because of the distinctive hydraulic 

characteristics of karst aquifers (Field, 1993). Groundwater 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 ground-water models 

are not applicable in fractured rock or karst aquifers 

(U.S. Environmental Protection Agency, 1997).

The U.S. Geological Survey (USGS), in cooperation with the Tennessee Department of Environment 

and Conservation, Division of Superfund (TDECDSF), is conducting a study of the occurrence, fate, 

and transport of chlorinated solvents in karst regions 

of Tennessee. One objective of this study was to examine the role of biodegradation in the fate and transport 

of chlorinated solvents in karst aquifers. This report 

presents results from field and laboratory data collected to examine chlorinated-ethene biodegradation at 

a TCE-contaminated karst site in Middle Tennessee. 

The study site was selected because of the presence of 

chlorinated-solvent degradation products in ground 

water and because of the availability of hydrologic and 

chlorinated-ethene data in TDEC-DSF files. The 

objectives of this report are to:

1. Summarize the current understanding of:

a. chlorinated-ethene biodegradation processes 

and

b. biodegradation of organic compounds in bedrock aquifers.

2. Characterize the microorganisms and geochemical 

conditions present in ground water at the study 

site.

3. Evaluate which chlorinated-ethene degradation processes may be occurring in ground water at the 

study site. 

4. Document lessons learned from this study that may 

assist future investigations of chlorinated-solvent 

biodegradation in karst areas.

The body of the report is divided into six main 

sections. The first section is a general overview of 

chlorinated-ethene degradation processes. The second 

section reviews previous studies relevant to the effects 

of hydrology, bacteria, and geochemical conditions on 

the biodegradation of organic compounds in bedrock 

aquifers. The third section describes the study site with 

emphasis on hydrogeology and monitoring and remedial activities. The fourth section describes methods 

and procedures used to collect and interpret geochemical, chlorinated ethene and degradation product, and 

microbiological data. The fifth section presents results 

and interpretation of the multiple lines of evidence 

used to examine chlorinated-ethene degradation at the 

study site. Finally, the sixth section describes lessons 

learned during this study.

CHLORINATED-ETHENE 

BIODEGRADATION PROCESSES

Microbial organisms use a wide variety of metabolic processes to generate energy and maintain cellular growth. These processes involve the transfer of 

electrons from an electron donor (food source) to an 

electron acceptor. Three categories of metabolic processes are involved in the biological degradation of 

chlorinated ethenes. Reductive dechlorination is an 

anaerobic process in which chlorinated ethenes are 

used as electron acceptors (McCarty, 1994; Montgomery and others, 1994). Cometabolism is an aerobic 

process in which chlorinated ethenes are degraded as a 

result of fortuitous biochemical interactions which 

yield no benefit to the bacteria (Alvarez-Cohen and 

McCarty, 1991; Hanson and Brusseau, 1994; Ely, Williamson, and others, 1995). Direct oxidation is an aerobic or mildly anaerobic (iron reducing) process in 
Chlorinated-Ethene Biodegradation Processes 3

EXPLANATION

AEROBIC COMETABOLISM

DIRECT OXIDATION

Intermediate steps in the 

aerobic cometabolism

and direct oxidation 

processes are not shown.

REDUCTIVE DECHLORINATION

Figure 1. Common biological degradation processes for 

chlorinated ethenes.

Carbon dioxide,

chloride,

and water

Tetrachloroethylene (C2Cl4)

Trichloroethylene (C2HCl3)

Dichloroethylene (C2H2Cl2)

Vinyl chloride (C2H3Cl)

Ethene (C2H4)

which sparsely chlorinated ethenes are used as electron donors (McCarty and Semprini, 1994; Bradley 

and Chapelle, 1996). One or all of these processes 

could be occurring at a given site, depending on 

environmental conditions. 

Reductive Dechlorination 

Generally, organic molecules with abundant 

carbon-hydrogen bonds are good food sources (electron donors) because they contain high-energy electrons. Highly chlorinated solvents such as 

tetrachloroethylene (PCE) and TCE, however, are 

electron poor because they have chlorine-carbon 

bonds. These compounds are likely to be used by bacteria as final electron acceptors instead of electron 

donors (Wiedemeier and others, 1998). During reductive dechlorination, chlorine atoms are replaced by 

electrons coupled to hydrogen atoms, resulting in 

sequential dechlorination from PCE to TCE to dichloroethylene (DCE) to vinyl chloride (VC) to ethene 

(fig. 1). During reductive dechlorination, cis-1,2-

dichloroethylene (cDCE) is the most commonly 

formed isomer of DCE. 

Soil and ground-water bacteria use a variety of 

natural electron acceptors. The use of these final electron acceptors is not arbitrary but is based on energytransfer efficiency and availability (Montgomery and 

others, 1994). The most common inorganic electron 

acceptor in ground water is oxygen. Once the oxygen 

has been depleted, bacteria will preferentially use the 

next most efficient electron acceptor; usually this is 

nitrate (NO3

-

) or insoluble manganese (Mn4+). After 

NO3

-

 and Mn4+ have been depleted, the bacteria will 

use insoluble ferric iron (Fe3+), followed by sulfate 

(SO4

2-), and carbon dioxide (CO2), respectively. The 

byproducts resulting from transfer of electrons to various electron acceptors are shown in table 1.

Dechlorination of PCE and TCE to DCE can 

occur under mildly reducing conditions such as NO3

-

or Fe3+ reduction (fig. 2); however, the dechlorination 

of DCE to VC, and VC to ethene requires the stronger 

reducing conditions of SO4

2--reduction or methanogensis (Vogel and others, 1987). The effectiveness of 

chlorinated solvents to serve as electron acceptors is 

proportional to the number of chlorine molecules 

attached (Vogel and McCarty, 1985). For example, 

PCE is more likely to serve as an electron acceptor and 

lose a chlorine atom through reductive dechlorination 

than is TCE, DCE, or VC.

Aerobic Cometabolism 

Wilson and Wilson (1985) first reported that 

TCE was degraded under aerobic conditions by methanotrophic bacteria in a soil enriched with CH4 and 

Figure 1. Common biological degradation processes for chlorinated ethenes.
4 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Figure 2. Oxidation-reduction potentials for selected microbial processes. 

(Modified from Stumm and Morgan, 1981.)

-1000 -500 0 500 1000

OXIDATION-REDUCTION POTENTIAL (Eh), IN MILLIVOLTS

Aerobic respiration

Nitrate reduction

Manganese reduction

Iron reduction

Sulfate reduction

Methanogenesis 

oxygen (O2). Further studies revealed that the methane 

monooxygenase (MMO) enzyme was responsible for 

catalyzing the oxidation of TCE (Alvarez-Cohen and 

McCarty, 1991; Henry and Grbic ́-Galic ́, 1994). Other 

oxygenase enzymes such as ammonia monooxygenase 

(AMO) (Arciero and others, 1989; Rasche and others, 

1991) and toluene dioxygenase (Nelson and others, 

1988; Hopkins and others, 1993; Heald and Jenkins, 

1994) also have been shown to oxidize certain chlorinated solvents. This oxidation reaction is called cometabolism because the reaction uses metabolic enzymes, 

but does not contribute any energy in return.

These oxidative enzymes catalyze a reaction 

that incorporates O2 into the target substrates (fig. 3). 

This oxidation reaction requires an energy molecule 

such as nicotinamide adenine dinucleotide (NADH) to 

incorporate the O2. The enzymes lack the ability to 

efficiently distinguish CH4, NH3, or toluene from certain chlorinated solvents. This lack of substrate 

specificity results in a chemical reaction in which oxygen is incorporated into the solvent molecule forming 

an unstable molecule such as TCE epoxide during 

MMO cometabolism (Alvarez-Cohen and McCarty, 

1991). The unstable molecule will spontaneously 

degrade to one of several chloroacetic acids, such as 

dichloroacetic acid. These chloroacetic acids are soluble in water and will slowly degrade to CO2, chloride, 

and water (fig. 3). 

Cometabolism is limited to chlorinated solvents 

that have at least one hydrogen atom attached to the 

carbon (fig. 1). No studies have found a cometabolic 

pathway capable of degrading PCE (Henry and Grbic ́-

Galic ́, 1994). Cometabolism tends to be an unsustainable process under stagnant conditions because of substrate competition and enzyme inhibition and 

inactivation (Ely and others, 1997). Competition 

occurs between the natural substrates, such as CH4, 

NH3, or toluene, and chlorinated solvents for binding 

Table 1. Terminal electron acceptors and byproducts involved in 

microbial processes 

Electron acceptor1

1

The electron acceptors are arranged from most energy efficient at the top to 

least efficient. 

Byproduct Microbial process

Oxygen (O2) CO2 Aerobic respiration

Nitrate (NO3

-

) Ammonia (NH3) NO3

- reduction

Manganese (Mn4+) Manganese (Mn2+) Mn4+ reduction

Iron (Fe3+) Iron (Fe2+) Fe3+ reduction

Sulfate (SO4

2-) Sulfide (S2-) SO42- reduction

Carbon dioxide (CO2) Methane (CH4) Methanogenesis

Figure 2. Oxidation-reduction potentials for selected microbial processes. (Modified from Stumm and 

Morgan, 1981.)
Previous Studies Relevant to Biodegradation in Bedrock Aquifers 5

Methane monooxygenase (MMO):

NADH

(methane) 

CH4

(methanol)

CH3OH

(carboxylate) MMO HCOOH

NADH, O2

(carbon dioxide)

CO2

Ammonia monooxygenase (AMO):

(ammonia)

NH3 AMO

NADH, O2

(ammonium

hydroxide)

NH2OH

NADH

(nitrite)

NO2

Trichloroethylene (TCE) cometabolism by MMO:

(carbon

dioxide)

CO2,

(TCE)

C2HCl3

MMO

NADH, O2

(TCE 

epoxide)

C2HCl3O

(dichloroacetic acid)

C2H2Cl2O2

(chloride)

Cl, (water)

H2O

Figure 3. Examples of cometabolic processes that degrade chlorinated ethenes

(information from Arciero and others, 1989; Hanson and Brusseau, 1994; 

Henry and Grbic-Galic, 1994; Ely, Williamson, and others, 1995). 

(nicotinamide adenine

dinucleotide)

(oxygen)

Figure 3. Examples of cometabolic processes that degrade chlorinated ethenes. (Information from Arciero and others, 1989; Hanson and Brusseau, 1994; Henry and Grbic ́-Galic ́, 

1994; Ely, Williamson, and others, 1995.)

on the active site of the nonspecific oxygenase enzyme 

(Semprini and others, 1991; Broholm and others, 

1992). Competitive inhibition occurs when enzymes 

cometabolize chlorinated solvents to the exclusion of 

natural substrates, ultimately depleting the bacteria of 

energy molecules (Chang and Alvarez-Cohen, 1995) 

(NADH in fig. 3). The TCE oxidation byproducts such 

as TCE epoxide may result in the inactivation of the 

oxygenase activity caused by damage to the enzymes 

(Ely, Hyman, and others, 1995). Inhibition and inactivation may be overcome by additional natural substrates (Alvarez-Cohen and McCarty, 1991; Ely and 

others, 1997).

Direct Oxidation 

Recent studies have reported that chlorinated 

solvents with only one or two chlorine atoms (the least 

oxidized compounds) can serve as electron donors by 

bacteria. Several studies have shown that VC and 1,2-

dichloroethane (DCA) can serve as food under aerobic 

conditions (McCarty and Semprini, 1994). Ironreducing bacteria also can mineralize VC (Bradley and 

Chapelle, 1996) and DCE (Bradley and Chapelle, 

1997) as a food source under aerobic conditions. 

Direct oxidation is limited to degrading lightly chlorinated solvents such as DCA, DCE, and VC; however, 

direct oxidation may serve a vital role in the sequential 

steps of chlorinated-solvent biodegradation. Aerobic 

or iron-reducing zones are commonly found downgradient of methanogenic or sulfate-reducing zones. 

Thus, partially dechlorinated byproducts (DCE and 

VC) produced by reductive dechlorination in the 

methanogenic or sulfate-reducing zones may be consumed in the more oxidized zones downgradient.

PREVIOUS STUDIES RELEVANT TO 

BIODEGRADATION IN BEDROCK 

AQUIFERS 

Previous studies examining the biodegradation 

of organic compounds in fractured rock or karst settings generally have focused on biodegradation in the 

overburden or regolith above bedrock (Christensen 

and others, 1997). Wiedemeier and others (1998) 

described a case study in New Hampshire, where biodegradation of benzene, toluene, and TCE in overburden was documented by using microcosms and field 
6 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

data. Few studies have examined biodegradation in 

bedrock aquifers. Major and others (1995) determined 

that microorganisms in a fractured bedrock aquifer in 

New York dechlorinated TCE to ethene. Acetone and 

methanol, which were also present in the aquifer, 

served as electron donors, and SO4

2- and CO2 were the 

predominant electron acceptors. Chapelle and others 

(1995) examined the microbial degradation of organic 

carbon in the Floridan aquifer, which consists of Tertiary limestone characterized by locally extensive conduits and karst topography. These researchers 

experienced difficulty in using patterns of electron 

acceptor consumption as a means to identify discrete 

oxidation-reduction potential zones in the aquifer; 

however, they did identify sulfate-reducing and methanogenic conditions in the aquifer. 

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 flow paths 

in karst aquifers (Wolfe and others, 1997). In areas isolated from the major ground-water flow paths, contaminant migration may possibly be slow enough that 

biodegradation could reduce contaminant mass if 

favorable microorganisms, food sources, and 

geochemical conditions are present. 

Some researchers have implied that natural 

bioremediation in karst or fractured rock is unlikely to 

occur because of the microbiological characteristics of 

karst aquifers. Many microbes attach to the aquifer 

matrix and are not motile (swimmers); therefore, contact between those microbes and dissolved contaminants would be limited by the lack of surface area in 

fractures and conduits. However, adequate information does not exist to accept or reject the assumption 

that nonmotile bacteria dominate bacteria communities in karst conduits. Other researchers have implied 

that biodegradation in bedrock aquifers is limited by a 

lack of appropriate microorganisms. Vogel (1994), for 

example, states that hydrologic and geologic characteristics that would not benefit the natural bioremediation of chlorinated solvents include fractured rock 

systems where small microbial populations exist. 

A few reports in the literature dispute that statement. Typical microbial numbers in material from 

unconsolidated aquifers have been reported to range 

from 1 x 104 to 1 x 107 cells/mL (Ghiorse and Wilson, 

1988; Bedient and others, 1994). Studies have shown 

that water from bedrock (granite and karst) aquifers 

also may contain microbial populations within this 

range. For example, total microbial populations of 

approximately 1 x 105 cells/mL were detected in 

ground-water samples from a deep granite formation 

[800 meters (m) below land surface] in Sweden (Pedersen and Ekendahl, 1990). Total microbial populations of 9.7 x 105

 to 8.5 x 106 cells/mL and 

heterotrophic bacteria populations of 3.5 x 103 to 

5.0 x 105 cells/mL were detected in ground-water samples from a gasoline-contaminated karst aquifer in 

Missouri (O’Connor and Brazos, 1991). 

Ground-water studies have detected significant 

bacteria concentrations in water samples from karst 

aquifers in Tennessee. For example, fecal coliform and 

fecal streptococci bacteria were detected and cultured 

in ground-water samples from wells completed as 

deep as 90 m below land surface in Williamson 

County in Middle Tennessee (Hanchar, 1991). Fecal 

coliform and fecal streptococci bacteria also have been 

detected in several wells in Bedford and Coffee Counties in Middle Tennessee (Roman-Mas and others, 

1991) and in Grainger County in East Tennessee 

(Weaver and others, 1994). The detection of viable 

fecal bacteria suggests that a wide variety of other 

types of bacteria also may be present in karst aquifers 

in Tennessee. 

Microorganisms responsible for chlorinatedethene degradation have been detected in water samples from bedrock aquifers in other regions. Denitrifying and sulfate-reducing bacteria were identified in 

nutrient-poor, anoxic ground water from granite formations (240 m below land surface) in Manitoba, Canada, and lab studies indicated that the bacteria were 

able to mineralize a variety of organic substances (Jain 

and others, 1997). 

Aerobic and anaerobic bacteria have several 

characteristics that may improve their ability to 

degrade chlorinated ethenes in karst aquifers. These 

characteristics include diverse metabolisms and the 

ability to withstand fluctuating anaerobic and aerobic 

conditions. These bacteria can derive energy from a 
Study Site 7

wide variety of foods. Research has shown that methanotrophs are capable of using ammonia instead of 

methane (Bedard and Knowles, 1989; Dalton, 1977) 

and that ammonia-oxidizing bacteria are capable of 

using methane (Arciero and others, 1989; Bedard and 

Knowles, 1989). Research also indicates that 

ammonia-oxidizing bacteria and methanotrophs have 

slow death rates and can shift into a dormant stage for 

extended periods when growth substrates are absent. 

Methanotrophs also function at low oxygen concentrations and are not inhibited until oxygen is completely 

consumed (Henry and Grbic ́-Galic ́, 1994).

Many bacteria, particularly those adapted for 

aqueous environments, are capable of moving through 

the use of flagella. For example, the soil bacteria order 

Pseudomonales have flagella at one or both ends 

(Chapelle, 1993) allowing them to swim upgradient or 

downgradient. Many chemolithotrophic bacteria such 

as ammonia- and sulfur-oxidizers develop flagella to 

swim towards or away from chemical stimuli as the 

need arises. At other times they become nonmotile to 

conserve energy or metabolize material that flows past 

them (Atlas, 1987). 

Further evidence that bacteria are capable of 

degrading solvents in a conduit was revealed in a laboratory experiment that examined the aerobic degradation of TCE by methanotrophs in a 30-m by 5.0-cm 

stainless-steel pipe. TCE contaminated water (approximately 2 mg/L) was pumped into the pipe at a rate of 

1 liter per minute, and methanotrophs (32 milligrams 

dry weight per liter) were added to the pipe water at a 

rate of 0.1 liter per minute. Results indicated that the 

TCE was reduced by 88 percent as the contaminated 

water traveled the length of the pipe at a velocity of 

110 meters per hour (m/h) (residence time of 

0.91 hour) (U.S. Environmental Protection Agency, 

1993). 

The ground-water velocity used in the experiment was within the range reported for karst conduits 

(10 to 500 m/h) by Quinlan (1989). The initial TCE 

concentration in the flow reactor was also within 

ranges reported for chlorinated solvent contaminated 

aquifers; however, the concentration of methanotrophs 

in the flow reactor may have been higher than concentrations typically found in karst aquifers. Assuming 

that cell mass is equal to 10-12 grams/cell (Chapelle, 

1993), the concentration of methanotrophs in the flow 

reactor would be approximately 3 x 103 cell/mL. This 

concentration is within the range normally reported for 

total bacteria concentrations in water from karst aquifers, but the concentration does not account for species 

diversity. 

STUDY SITE

The site used in this study is a manufacturing 

facility located on 65 ha in Marshall County, in Middle 

Tennessee (fig. 4). The manufacturing facility has 

been in operation since 1937 and is currently used to 

assemble air conditioning and heating equipment. A 

degreaser was installed in 1973 near the south end of 

the main manufacturing building. The chlorinated solvent TCE was piped underground from an aboveground storage tank to the degreaser. In 1980, the 

underground pipe connecting the storage tank to the 

degreaser ruptured and released an estimated 

13,000 liters of TCE into a piping trench, allowing 

TCE to migrate into sanitary sewer pipes and the soil. 

The highest concentration of TCE (950,000 mg/L) was 

detected in 1987, when a 61-cm column of dense nonaqueous phase liquid (DNAPL) was measured in a 

shallow well screened near the top of the bedrock 

(Wolfe and others, 1997). In 1997, TCE was detected 

in a shallow well at a concentration (171 mg/L) indicating that DNAPL is still present beneath the manufacturing building. 

Approximately 50 ground-water monitoring 

wells have been completed at the site (fig. 4). Shallow 

wells are screened near the top of bedrock (as much as 

6 m into the bedrock) in a shallow water-bearing zone. 

Deeper wells range in depth from 18 to 67 m below 

land surface and are screened in a karst aquifer that 

consists of several water-bearing zones in the bedrock. 

Additional well completion data are given in table 2 

(at end of the report). Remedial activities at the site 

include construction of two ground-water collection 

trenches on the top of the bedrock in 1989, excavation 

and thermal treatment of 1,150 m3

 of contaminated 

soil near the above-ground storage tanks in 1995, 

installation of several ground-water pump-and-treat 

wells, and construction of an aeration weir in Snell 

Branch, a small stream that flows through the study 

site (fig. 4). Air strippers are used to remove chlorinated solvents from water collected from the recovery 

trenches and the pump-and-treat wells. 

The site is underlain by the Ordovician-age Lebanon, Ridley, and Pierce Limestones (Crawford, 1992; 

Crawford and Ulmer, 1994; Farmer and Hollyday, 

1999) (table 3). The Ridley Limestone is susceptible to 

dissolution, especially where exposed at land surface. 

Sinkholes are common, and streams flowing off the 

Lebanon Limestone lose water and sink into the Ridley Limestone. Caves and cave streams primarily 

develop at contacts between the aquifer and confining 
8 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

4D

21S

19S

34

33S 32S 7D

18S

5D 17S

15S 16S

31S

1D

7S

1S

3S 2S

40S

3D

5S 11S 12S

10S 4S

30S

9S

9D

6S 2D

13S

36S

35S

12D

11D

15D-A 15D-B

15D-C

10D-A

10D-B

10D-C

13D

14D

37S

BN2

PL1

BN1

W1

8D

25S

16D-B

42S

20S

Snell Branch

Manufacturing building

Wastewater ponds

N

EXCAVATED SOIL AREA

GROUND-WATER RECOVERY TRENCH

PUMP-AND-TREAT WELL AND NUMBER

MONITORING WELL AND NUMBER

EXPLANATION

32S

19S

Aeration

weir

Figure 4. Location of ground-water monitoring and remediation activities at the

study site in Marshall County, Tennessee.

Tennessee

Study area

100 METERS

300 FEET

0

0

MARSHALL

COUNTY

Figure 4. Location of ground-water monitoring and remediation activities at the study site in 

Marshall County, Tennessee.
Study Site 9

units (Crawford, 1992). The Ridley Limestone is 

exposed just north of the study site (Wilson and 

Luther, 1963), and Snell Branch sinks into the formation and flows to a spring approximately 2.7 km northeast of the site (Crawford, 1992). Water yields are low 

unless a well intersects a well-developed conduit or 

cave stream in the Ridley Limestone (Crawford and 

Ulmer, 1994).

The general flow direction of the shallow 

ground water is north-northwest toward Snell Branch 

creek as indicated by water levels in shallow wells 

(fig. 5). Water levels in the shallow water-bearing zone 

do not appear to be affected by the pump-and-treat 

wells. Along the western side of the manufacturing 

building, shallow ground water flows toward a trough 

in the bedrock surface (fig. 6). This trough is downgradient of the original TCE spill and appears to be the 

main route for horizontal transport of chlorinated 

ethenes in the shallow water-bearing zone (fig. 7). 

Well-driller logs and well-pressure test data 

from unpublished TDEC-DSF files indicate that the 

karst aquifer at the site consists of at least three 

distinctive water-bearing zones located along bedding 

planes in the upper part of the Ridley Limestone (fig. 8). 

The first water-bearing zone is located at an altitude of 

approximately 208 m above sea level and was detected 

during the construction of wells 3D and 12D and the 

10D wells. The second water-bearing zone is located 

at an altitude of approximately 200 m above sea level. 

The third zone is located just above the thin-bedded 

member of the Ridley Limestone at an altitude of 

190 m above sea level. The second and third zones 

were intersected during the construction of several of 

the deep wells. Water-bearing zones were not detected 

in the lower part of the Ridley or Pierce Limestones.

Well-driller logs for wells 12D, 10D-A, 10D-B, 

and 10D-C, which are located near Snell Branch, indicate the presence of several highly fractured zones 

between the altitudes of 190 and 217 m above sea 

level. Driller logs for the three 10D wells (fig. 4) 

describe a highly fractured zone at an altitude of 

216 m above sea level that is hydraulically connected 

to Snell Branch as well as several zones of fractures 

between the altitudes of 190 and 208 m above sea 

Table 3. Geologic and hydrologic units at the study site

1 From Wilson, 1949. 

2

 From J.J. Farmer and E.F. Hollyday, U.S. Geological Survey, written commun., 1999.

Stratigraphic unit Lithology1

Thickness at

study site2

(meters)

Hydrogeologic

characteristics at

study site2

Regolith Low permeability clay 1 to 6 Shallow water-bearing zone 

near top of bedrock.

Lebanon Limestone Relatively pure limestone with beds averaging 5.1 

to 7.6 cm in thickness. Limestone beds are separated by partings of calcareous shale ranging from 

0 to 1.3 cm in thickness.

6 to 24 Leaky confining unit

Upper Ridley Limestone Relatively pure, massively bedded limestone with 

little insoluble material. Beds ranging from 10 to 

120 cm in thickness.

31 Karst aquifer

Thin-Bedded Member Relatively pure limestone with beds 1.3 to 10 cm in 

thickness. Limestone beds are separated by thin 

partings of calcareous shale.

9 Confining unit

Lower Ridley Limestone Similar to upper part of the Ridley Limestone 6 No water-bearing zones

Pierce Limestone Thinly bedded limestone. Beds average 5.1 cm in 

thickness. One or more units of massively bedded 

limestone may be present. Limestone beds are 

separated by thin partings of calcareous shale as 

much as 1.3 cm in thickness.

9 Confining unit
10 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

220.7

222.2

224.2

224.9

224.4

222.9

223.6

223.4

224.5

226.9

226.3

225.8

225.4

226.1

226.2

227.3

227.3

227.3

228.2

231.5

229.7

228.0

229.6

228.9

230.4

229.6 229.3

228.1

227.6

223

224

225

226

227

228

229

230

Snell Branch

Figure 5. Typical potentiometric surface conditions for the shallow waterbearing zone.

Water levels are from unpublished Tennessee Division of 

Superfund site files.

Manufacturing building

Wastewater ponds

EXPLANATION

223 POTENTIOMETRIC SURFACE CONTOUR—Shows 

 altitude, in meters, at which water level would have

 stood in tightly cased wells, October 14, 1996. Contour 

 interval 1 meter. Datum is sea level. Dashed where

 approximate

PUMP-AND-TREAT WELL—Number indicates water 

 level on October 14, 1996 in meters above sea level

MONITORING WELL—Number indicates water level 

 on October 14, 1996 in meters above sea level

225.7

220.7

227.3

 100 METERS

300 FEET

0

0

N

225.8

225.7

226.4 225.0

228.3

Figure 5. Typical potentiometric surface conditions for the shallow water-bearing zone.
Study Site 11

EXPLANATION

BEDROCK SURFACE CONTOUR—Shows altitude, in meters, of top of

 Lebanon Limestone. Contour interval 1 meter. Datum is sea level.

 Dashed where approximate

PUMP-AND-TREAT WELL—Number indicates altitude of top of 

 Lebanon Limestone in meters above sea level 

MONITORING WELL—Number indicates altitude of top of 

 Lebanon Limestone in meters above sea level

Snell Branch

Manufacturing building

Wastewater ponds

221

222

223

224

225

226

227

228

229

230

220.1

219.4

Bedrock surface altitudes are from unpublished 

Tennessee Division of Superfund site files.

Figure 6. Bedrock surface at the study site.

224

100 METERS

300 FEET

0

0

N

231.1

230.9 229.3

229.5

229.8

228.9

227.3 227.6

227.0

227.1

225.8

226.2

226.9

226.8 224.8

229.4

227.8

225.0

223.0

223.1

220.1

220.1

219.4

223.1

222.5

224.8

231.2

226.1

224.7

Figure 6. Bedrock surface at the study site.
12 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Snell Branch

Manufacturing building

>10,000

>1,000

>100

>100

EXCAVATED SOIL AREA

GROUND-WATER RECOVERY TRENCH

PUMP-AND-TREAT WELL—Number represents 

 chlorinated-ethene concentration in micrograms 

 per liter, May 10, 1996

MONITORING WELL—Number represents 

 chlorinated-ethene concentration in micrograms 

 per liter, May 10, 1996 

EXPLANATION

1.1

120

Chlorinated-ethene data are from unpublished Tennessee

Division of Superfund files.

Figure 7. Spatial distribution of chlorinated ethenes in the shallow 

water-bearing zone.

Wastewater ponds

N

100 METERS

300 FEET

0

0

1.1

19

220

510

27

7.6

100

1.5

1.3

14,000

20,000

50,000

230,000

11 3,000

37

3,300

4,300

<5.0

<5.0

21,000

120

17,000

150,000

400,000

<5.0

<5.0

<5.0

<5.0

<5.0

<100

<50

Figure 7. Spatial distribution of chlorinated ethenes in the shallow water-bearing zone.
Study Site 13

Figure 8. Generalized hydrogeologic diagram of the Middle Tennessee

study site.

3D 1D

12D

Not to scale

11D

2D 9D

10D-B

10D-A

10D-C

13D

14D Snell Branch

Regolith

Shallow water-bearing

zone

Lebanon Limestone

Upper Ridley Limestone

Lower Ridley Limestone

Pierce Limestone

Thin-bedded Member

Water-bearing zones

Karst aquifer

Manufacturing building

Figure 8. Generalized hydrogeology of the Middle Tennessee study site.

level that are hydraulically connected. During the construction of well 12D, an uncompleted well boring 

intersected a mud-filled cavity 1.2 m in height at an 

altitude of 208 m above sea level that was hydraulically connected to Snell Branch. The extensive vertical fracturing beneath Snell Branch, which runs 

approximately north 40° east through the study site, 

may be a result of jointing. Geologic investigations 

described in unpublished Tennessee Division of 

Superfund site files indicate that the local joint orientation is north 40° east and north 50° west. 

Well-completion data indicate that the waterproducing zones are not continuous beneath the site, 

and in some areas fractures appear to be isolated from 

the major zones of ground-water flow. For example, 

well 11D intersects the three water-producing zones in 

the karst aquifer, yet the well produced less than 

3 L/min. Other wells intersected water-bearing zones 

and produced abundant water.

Because of the slight differences in water levels 

(fig. 9) when the pump-and-treat wells are operating 

and because the wells are screened in different waterbearing zones, determining the general direction of 

ground-water flow in the upper part of the Ridley 

Limestone is difficult. Since well 11D is screened in 

fractures isolated from the major zones of groundwater flow, changes in the water level of this well lag 

behind changes in other wells, and the water levels in 

11D may be significantly higher or lower than water 

levels in other deep wells (fig. 9). 

Pump-and-treat wells completed in the upper 

part of the Ridley Limestone (wells 9D, 13D, and 

14D) draw down water levels in many of the deep 

wells and affect local ground-water flow (fig. 9). The 

pumping of well 9D (approximately 100 L/min) 

affects water levels in all of the deep wells located 

south of Snell Branch. The pumping of wells 13D and 

14D (approximately 20 L/min at alternating intervals) 

affects a much smaller area north of Snell Branch 

(fig. 9). The pump-and-treat operations in the karst 

aquifer help to remove chlorinated solvent and contain 

the contaminant on site.

Ground-water tracing studies documented in 

unpublished TDEC-DSF site files verify that conduits 

between wells 3D and 9D and between wells 12D and 

9D are hydraulically connected. Sodium chloride was 
14 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Manufacturing building

Water level and drawdown data are from unpublished 

Tennessee Division of Superfund files.

PUMP-AND-TREAT WELL AND NUMBER

MONITORING WELL AND NUMBER

NUMBER INDICATES WATER LEVEL IN METERS ABOVE

 SEA LEVEL, OCTOBER 25, 1996, PUMP-AND TREAT

 WELLS NOT OPERATING

NUMBER INDICATES TYPICAL DRAWDOWN IN METERS 

 WITH PUMP-AND-TREAT WELLS OPERATING

APPROXIMATE AREA LIKELY AFFECTED BY

 PUMP-AND-TREAT WELLS

EXPLANATION

14D

11D

Figure 9. Effect of pump-and-treat wells on water levels in deep wells.

Snell Branch

220.24

 (11.0)

220.63

221.57

221.57

(12.8)

220.67

(12.1) 222.14

(13.0)

221.67

(13.4)

222.67

(13.5)

9D

222.77

(4.9)

207.20

11D

222.75 223.52

221.84 220.66

220.68

220.70

(0.6) 220.80

(4.7)

220.64

(2.3) 13D

14D

221.83

(2.3)

N

100 METERS

300 FEET

0

0

Wastewater ponds

Figure 9. Effect of pump-and-treat wells on water levels in deep wells.
Methods and Procedures 15

transported from well 3D to well 9D in 18 hours (average velocity 8 m/h) and rhodamine WT was transported from well 12D to well 9D in 28 hours (5 m/h). 

During these ground-water tracing studies, the pumping rate of well 9D was approximately 64 L/min. The 

reports were unclear on how much water was used to 

flush the tracers into the conduits. These tracer results 

indicate that ground water could flow relatively fast in 

the karst aquifer at this site but do not address the issue 

of ground-water storage.

Results from additional ground-water tracing 

studies demonstrate that some water-bearing zones are 

isolated from major zones of ground-water flow. 

These water-bearing zones may store water and contaminants for relatively long periods. Fluorescein 

(2.3 kg) was injected into well 8D on November 15, 

1991, and the well was flushed with water (750 L/min) 

for 26.5 hours. During the fluorescein study, the pumpand-treat wells were turned off and nearby springs 

were monitored for fluorescein. Fluorescein was 

detected in some nearby springs; however, the results 

were inconclusive because of the lack of background 

samples from the springs. The deep wells at the study 

site were not sampled for fluorescein until 1996, 

5 years after the dye injection. During those 5 years, 

pump-and-treat wells and other remediation efforts 

were active at the site. Unpublished TDEC-DSF files 

report that fluorescein was still present in water samples collected from deep wells during two separate 

sampling events in 1996. The dye was detected at significant concentrations in water samples from wells 

3D (11 to 18 μg/L), 4D (0.54 to 1.2 μg/L), 5D (0.07 to 

0.22 μg/L), and 8D (23 to 37 μg/L). 

METHODS AND PROCEDURES

Multiple lines of evidence are often needed to 

demonstrate biodegradation processes at contaminated 

sites (National Research Council, 1993; Wiedemeier 

and others, 1998). The lines of evidence used to examine biodegradation of chlorinated solvents include 

(1) geochemical data that indicate depletion of electron donors and acceptors and increasing concentrations of metabolic byproducts, (2) chemical data that 

indicate decreasing concentrations of chlorinated solvents and increasing concentrations of degradation 

byproducts, and (3) laboratory or field microbiological 

data that indicate the bacteria present at a site can 

degrade contaminants (Chapelle and others, 1996; 

U.S. Environmental Protection Agency, 1997a). This 

study used field and laboratory techniques to develop 

data and address the three lines of evidence. Geochemical evidence was collected from selected wells for 

over 2 years during this study; and organic chemical 

data collected during a 10-year period was compiled, 

tabulated, and reviewed for spatial and temporal 

trends. Microbiological evidence was developed using 

microcosms and identification techniques.

Water-Quality Data Collection

Much of the water-quality data was already 

available from TDEC-DSF files (site 59-502), including chlorinated-ethene, ethene, and ethane data. 

Chlorinated-ethene data (TCE and byproducts of TCE 

degradation) were available for water samples collected quarterly between 1985 and 1998 from shallow 

and deep monitoring wells. Ethene and ethane 

(byproducts of VC degradation) data were available 

for water samples collected from selected shallow and 

deep wells beginning in 1997. These data included 

analytical printouts, information on analytical methods, quality control, and quality assurance data. The 

data were tabulated and examined, and changes in the 

analytical laboratory methods used were noted. 

Additional data-collection activities conducted 

by the USGS included periodic water-quality sampling 

of selected shallow and deep monitoring wells, collection of water samples from selected deep wells for bacteria identification and enumeration, and continuous 

monitoring of water quality and water levels for 

selected deep wells. Water-quality samples were collected by the USGS during seven sampling events: 

November 1996; February, August, and November 

1997; and February, April, and May 1998. Field measurements of temperature, pH, specific conductance, 

alkalinity, and dissolved oxygen (DO) were conducted 

using methods described by Wood (1981) and Hach 

Company (1992). Nutrient, major anion and cation, and 

total organic carbon analyses were conducted at the 

USGS regional laboratory in Ocala, Fla., using methods 

described by Fishman and Friedman (1989). Nutrient 

and major anion and cation analyses for samples collected during 1998 were conducted using spectrophotometric methods described by Hach Company (1992). 

Continuous monitoring of water quality and 

water levels was conducted from March through May 

1998. Data were collected at 15-minute intervals from 

four wells (1D, 2D, 3D, and 12D). The water-quality 

monitors located in the screened intervals of wells 
16 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

near water-producing zones measured conductivity, 

temperature, pH, ORP, and DO. The various sensors in 

the monitors were calibrated prior to placement in the 

wells and included rapid-pulse DO sensors that do not 

require stirring of the water in a well. Water-quality 

and water-level monitors were checked and calibrated 

every 2 to 3 weeks. Precipitation data (15-minute 

intervals) were obtained from a USGS gaging station 

approximately 1.5-km southeast of the study site. Precipitation data (daily totals) collected by employees at 

the site were obtained to verify that the data collected 

from the USGS gaging station were consistent with 

data from the site.

Geochemical Indicators

Geochemical data were used as indicators of terminal electron acceptor processes occurring in ground 

water at the site (Bouwer, 1994; Chapelle and others, 

1995; Wiedemeier and others, 1998). The geochemical 

data used included DO, NO3

-

, NH3, Mn2+, Fe2+, SO4

2-, 

and S2- concentrations (table 4). Calcium carbonate 

saturation indexes were calculated using methods 

described by Eaton and others (1995). Field measurements such as pH, alkalinity, specific conductance, 

temperature, and ORP were used to provide additional 

information concerning biodegradation processes and 

changes in water chemistry associated with the site 

hydrology. 

Due to the complex ground-water flow paths in 

karst, geochemical isopleth maps may be difficult to 

construct in karst aquifers and the identification of discrete oxidation-reduction zones along a contaminantplume transect is impractical (Chapelle and others, 

1995). Thus, the geochemical data used to identify 

oxidation-reduction trends must be presented some 

other way. Radial diagrams can be used to identify 

spatial and temporal trends for geochemical indicators 

of biodegradation (Carey, 1998). Radial diagrams 

were used to illustrate oxidation-reduction conditions 

by arranging the axes of the diagrams in the sequential 

order of preferred electron acceptors (DO, NO3

-

, Mn, 

Fe, and SO4

2-). Axis ranges were based on concentrations considered indicative of specific geochemical 

conditions (Wiedemeier and others, 1998). The axes 

for Mn2+ and Fe2+ were oriented with concentrations 

increasing toward the origin since these compounds 

are byproducts of Mn4+ and Fe3+ reduction (fig. 10). 

Chlorinated-Ethene and Degradation Product 

Data

The chlorinated ethene released at this site was 

TCE. Degradation byproducts of TCE include DCE, 

VC, ethene, and ethane. Decreases in parent compounds and detection of chlorinated-ethene degradation byproducts may provide evidence that 

biodegradation has occurred (table 5). During reductive 

Table 4. Geochemical indicators of terminal electron acceptor processes measured during this study

Constituent Remarks concerning interpretation

Dissolved oxygen (DO) If DO is greater than 1 mg/L (aerobic conditions), reductive dechlorination is unlikely; however, cometabolism and direct oxidation of partially dechlorinated solvents are possible.

Nitrate (NO3

-

) Reductive dechlorination of tetrachloroethylene (PCE) and trichloroethylene (TCE) to cis-1,2-dichloroethylene (cDCE) can occur under these mildly reducing conditions. Direct oxidation of partially 

dechlorinated solvents also may occur under NO3

-

-reducing conditions.

Ammonia (NH3) Elevated NH3 concentrations may indicate NO3

-

-reduction if DO is less than 1 mg/L. If NH3 is present 

at 0.5 mg/L or greater and DO levels are greater than 1.0 mg/L, then partially dechlorinated solvents 

may be cometabolized through the NH3-monooxygenase pathway. 

Soluble manganese (Mn2+) 

and ferrous iron (Fe2+).

Increased concentrations of Mn2+ and Fe2+, coinciding with low DO concentrations, are indicators that 

insoluble manganese and iron (Mn4+ and Fe3+) are serving as electron acceptors. Reductive dechlorination of PCE and TCE to cDCE can occur under mildly reducing conditions. Mn4+- and Fe3+-

reducing bacteria are capable of consuming vinyl chloride (VC) and cDCE. 

Sulfate (SO4

2-) Reductive dechlorination of TCE to cDCE, then to VC and ethene can occur under SO42--reducing 

conditions. The chlorinated solvents compete directly with SO4

2- to serve as electron acceptors.

Sulfide (S2-) Elevated S2- concentrations may indicate SO4

2--reduction and conditions favorable for reductive 

dechlorination of chlorinated solvents.
Methods and Procedures 17

Figure 10. Radial diagrams used to illustrate electron acceptor 

processes. (Modified from Carey, 1998.)

All units are in milligrams per liter 100

10

1

0.1

10

1

0.1

0.01

Dissolved oxygen (02)

Nitrate (NO3-)

Manganese (Mn2+ Iron (Fe2+) )

Sulfate (SO42-)

>1.0

0.5 >1.0

0.5

 > 20

10

AEROBIC NITRATE REDUCING MANGANESE REDUCING

METHANOGENIC SULFATE REDUCING IRON REDUCING

Figure 10. Radial diagrams used to illustrate electron acceptor processes. (Modified from 

Carey, 1988.)

dechlorination, all three isomers of DCE can be produced; however, cDCE is the most commonly produced isomer and 1,1-DCE is the least commonly 

produced. Chlorinated-ethene, ethene, and ethane data 

for water samples from shallow and deep wells were 

converted to micromolar concentrations and examined 

for spatial and temporal trends that could indicate the 

occurrence of reductive dechlorination. Aerobic degradation of chlorinated ethenes often results in intermediate byproducts that spontaneously degrade into a 

variety of compounds. Intermediate byproducts of aerobic degradation, such as TCE-epoxide and chloroacetic acids, were not examined during this study.

Microcosm Experiments

In addition to the field evidence, laboratory 

microcosms were used to determine if microorganisms 

at the site had the potential to degrade contaminants. A 

standard microcosm method does not exist; however, a 

variety of methods have been reported. In many of the 

studies, solid and liquid aquifer materials were placed 

in the microcosms (Hopkins and others, 1993; Chapelle 

and others, 1996; Wilson and others, 1996). Other studies have used only ground water from contaminated 

sites in the microcosms (Nelson and others, 1988; 

Krumme and others, 1993; Moran and Hickey, 1997).

Batch-type microcosms containing only water 

were used during this study because they may be more 

representative of conditions in karst aquifers. Since 

preliminary field data indicated the occurrence of aerobic and anaerobic conditions at the site, microcosms 

were set up under aerobic conditions to monitor for the 

occurrence of cometabolic degradation pathways. 

After depletion of DO, the microcosms were 

monitored for anaerobic degradation (reductive 
18 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

dechlorination). Water samples collected from wells 

1D, 2D, 3D, and 12D on February 17, 1998 and 

May 21, 1998 were used to construct microcosms during two experiments. Four replicates were established 

for each treatment, and control procedures included 

sterilization of selected microcosms (table 6). During 

experiment 1, bacteria counts indicated that the sterilization procedure used for controls (experiment 1, 

treatment 9) were not successful. The control microcosms were cross-contaminated with bacteria from the 

other microcosms during the addition of TCE. The 

second microcosm experiment was set up to establish 

new sterile controls and define the loss of TCE due to 

abiotic factors.

Microcosms were set up in 40-mL glass vials 

equipped with Teflon-lined septum caps then stored 

inverted in the dark until the selected sampling dates. 

On the sampling dates, samples from the microcosms 

were analyzed for chlorinated ethenes using gas chromatography (GC) with hexane extractions and an electron capture detector. Additional chlorinated ethene 

analyses were performed using GC with headspace 

analysis and a photoionization detector. 

Bacteria Identification and Enumeration

The geochemical information described earlier 

in this report helped to determine if conditions were 

suitable for biodegradation of chlorinated ethenes and 

to tentatively identify microbial processes in the karst 

aquifer. Other lines of evidence such as laboratory 

microcosm data and bacteria identification and enumeration strengthen the interpretation of the geochemical information. Bacteria in water samples collected 

on August 20 and 21, 1998, from selected deep wells 

were identified by the use of the RNA-oligonucleotide 

hybridization method. Facultative and aerobic heterotrophic bacteria were enumerated from microcosm 

samples that contained water from various wells using 

tryptic soy agar plate counts. This information was 

used to provide further evidence that sufficient bacteria capable of solvent biodegradation were present in 

the karst aquifer.

The RNA-oligonucleotide hybridization method 

is a technique that takes advantage of unique nucleotide sequences in the ribosomal RNA (rRNA) to identify the bacteria (Amann and others, 1995). This 

method can be used to identify groups of bacteria such 

as sulfate-reducing bacteria or specific genera such as 

Table 5. Organic constituents frequently measured at chlorinated-solvent contaminated sites 

[Degradation byproducts are often indicative of biodegradation at the site]

Organic constituent Remarks concerning interpretation

Tetrachloroethylene (PCE) A commonly used chlorinated solvent that can be biodegraded only by reductive dechlorination. PCE was not used at this site and has not been detected in ground-water samples from 

the study site.

Trichloroethylene (TCE) A commonly used chlorinated solvent. The most efficient biodegradation mechanisms include 

reductive dechlorination and methane cometabolism. TCE was the chlorinated solvent 

spilled at the study site and has been detected in ground-water samples from the study site.

Dichloroethylene (DCE)

cis-1,2-DCE (cDCE) The most common byproduct of reductive dechlorination of TCE. If the concentration of 

cDCE is greater than 80 percent of total DCE concentration, then reductive dechlorination is 

likely to have occurred. Significant concentrations of cDCE have been detected in groundwater samples from the study site.

trans-1,2-DCE (tDCE) Trans-1,2-DCE is not produced significantly through any biologically mediated process. If 

tDCE is 50 percent or more of the total DCE, a good possibility exists that tDCE was 

released. Significant concentrations of tDCE have not been detected in ground-water samples from the study site.

1,1-DCE This is the least common byproduct of reductive dechlorination of TCE. 1,1-DCE is also produced when 1,1,1-trichloroethane (TCA) undergoes abiotic chloride elimination. Significant 

concentrations of 1,1-DCE have not been detected in ground-water samples from the study 

site. 

Vinyl chloride (VC) VC is a byproduct of cDCE reductive dechlorination. VC has been detected in ground-water 

samples from the study site.

Ethane and ethene These are strong indicators of complete reductive dechlorination. They have been detected in 

ground-water samples from the study site.
Methods and Procedures 19

Nitrosomonas sp. The identification level used in this 

study was general bacteria groups such as iron-oxidizers, 

methanotrophs, ammonia-oxidizers, and sulfate-reducers 

(Byl and others, 1997; Farmer and others, 1998).

Bacteria samples were collected with sterile disposable bailers. Each water sample was poured into a 

sterile 1-liter bottle, placed on ice, and brought back to 

the laboratory. Sterile polycarbonate filters with 

0.22-μm pores were used to concentrate the bacteria in 

the water samples. Bacteria were washed off the polycarbonate filters using a few drops of sterile phosphate 

buffer (PB) solution. The bacteria were preserved 

using a 4-percent paraformaldehyde-PB solution. The 

samples were stored in solution at 4 °C until they were 

subjected to the hybridization tests (Braun-Howland 

and others, 1992). 

The sequences for the RNA-oligonucleotide 

probes of the various bacteria groups were selected 

from previously published sequences (Tsien and others, 1990; Kane and others, 1993; Amann and others, 

1995). Bacteria have long been recognized to play an 

important role in oxidizing Fe2+ to Fe3+. The reverse 

process, bacteria mediated reduction of Fe3+ to Fe2+, 

has been studied as well (Chapelle, 1993). Field 

microbial investigations are indicating that bacteriacatalyzed Fe3+ reduction and Fe2+ oxidation are occurring within close proximity of each other (Lovley and 

Phillips, 1988; Lovley and others, 1990), which 

implies a synergistic relation between the organisms. 

This potential synergistic relation is one reason why 

these bacteria have been difficult to isolate and culture 

in the laboratory. To avoid this problem, Siering and 

Ghiorse (1997) have developed a rRNA-targeted 

hybridization probe to identify iron- and manganeseoxidizing bacteria in environmental samples. The 

RNA-hybridization probe was used to identify these 

potential synergistic bacteria in ground-water samples 

from this site.

The oligonucleotide probes were tagged with 

rhodamine, fluorescein, or acridine orange dye. The 

probes were stored at –20 °C until used. Bacteria were 

prepared for hybridization by taking a 100-μL sample 

from the preserved cells and washing the preservative 

out by using a series of washes and centrifugation 

steps (Brawn-Howland and others, 1992) and replacing the preservative with a hybridization buffer. The 

bacteria cells were incubated for 30 minutes at 90 °C 

to relax and open the RNA coils for hybridization with 

the oligonucleotide probes. The fluorescent-tagged 

probes were added to the hybridization mixture and 

incubated at 37 °C for 2 hours on a gentle shaker. 

Probes hybridized to complimentary sequences in the 

bacteria. The cells were spun down in a centrifuge and 

resuspended in clean PB solution. The un-hybridized 

probes were washed away in a series of PB-solution 

centrifugation washes. After two washes, the cells 

Table 6. Description of microcosms used to examine biodegradation of chlorinated ethenes

[TCE, trichloroethylene; mg/L, milligrams per liter; --, none; four replicates created for each treatment]

Source of water Microcosm treatments

Experiment Treatment Wells(s) Date TCE (mg/L) Additional

1 1 1D Feb. 17, 1998 0.17 --

1 2 1D Feb. 17, 1998 0.92 --

1 3 2D Feb. 17, 1998 0.57 --

1 4 2D Feb. 17, 1998 1.19 --

1 5 3D Feb. 17, 1998 0.30 --

1 6 3D Feb. 17, 1998 0.73 --

1 7 12D Feb. 17, 1998 0.08 --

1 8 12D Feb. 17, 1998 0.68 --

1 9 Multiple Feb. 17, 1998 0.94 Sterilized1

1

Bacteria data indicated sterilization not successful.

2 1 1D May 21, 1998 1.61 --

2 2 2D May 21, 1998 1.53 --

2 3 3D May 21, 1998 1.19 --

2 4 12D May 21, 1998 2.50 --

2 5 2D/3D May 21, 1998 0.73 Sterilized
20 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

were resuspended and filtered through a 0.2-μm, clear 

polycarbonate filter. The filter with the cells was 

placed directly on a slide and examined using an epifluorescent microscope (X 1,000 magnification) 

equipped with appropriate excitation and emission filters for the targeted dye. 

The tryptic soy agar and sterile dilution buffer 

used to count facultative anaerobic and aerobic heterotrophic bacteria were prepared as described by 

Eaton and others (1995). Autoclaved glass filtration 

devices were used to hold 0.45-μm pore-size sterile 

filters. A 0.01- or 1-mL aliquot of shaken well water 

was transferred to 20 mL of sterile dilution buffer and 

drawn onto the filter as described in the membranefiltration method (Britton and Greeson, 1989). The filters were placed on the agar plates by using sterile forceps and placed in an incubator at 35 °C. Bacteriacolony forming units were counted after 48 hours. 

Results were reported as colony forming units per 

100 mL of water.

RESULTS AND INTERPRETATION

TCE and TCE-degradation byproducts are 

present in both the shallow water-bearing zone near 

the top of bedrock and the deeper karst aquifer at the 

study site (fig. 8, table 8). The monitoring data was 

unclear whether the byproducts of TCE degradation 

were produced in the shallow water-bearing zone 

alone, or if conditions were suitable for additional biodegradation to occur in the karst aquifer. This investigation attempted to address the biodegradation issue 

by reviewing hydrogeologic and organic chemistry 

records, running geochemical analyses, identifying 

microbes, establishing microcosms, and integrating 

the results into multiple lines of evidence. The results 

for the shallow water-bearing zone are presented first, 

followed by results for the karst aquifer.

Biodegradation of Chlorinated Ethenes in the 

Shallow Water-Bearing Zone

Ground water in the shallow water-bearing zone 

flows along a trough in the bedrock surface, which 

serves as the main route for horizontal transport of 

chlorinated ethenes in the shallow zone. The shallow 

ground-water contamination plume moved laterally 

from well 33S to well 7S to well 2S to well 5S 

(fig. 11). Because the general direction of groundwater flow and the extent of the chlorinated-ethene 

plume in the shallow water-bearing zone were known, 

identifying discrete oxidation-reduction zones along 

the flow path in the shallow zone was possible. The 

delineation of oxidation-reduction zones, supplemented with organic chemistry data, was used to infer 

which chlorinated-ethene degradation processes prevailed along the plume.

Geochemical data for water samples collected 

from well 33S indicate that anaerobic conditions were 

present in shallow ground water beneath the manufacturing building (fig. 11). Compared to water samples 

from other shallow wells, samples collected from well 

33S on August 22, 1997, contained low DO and NO3

-

concentrations (<0.1 and <0.06 mg/L, respectively) 

and high NH3, Mn2+, and Fe2+ concentrations (1.6, 

2.6, and 18 mg/L, respectively). Anaerobic conditions 

were present in water samples from well 33S (table 7), 

and concentrations of electron acceptors were within 

ranges normally associated with iron or possibly 

sulfate-reducing conditions. Those oxidationreduction conditions are associated with reductivedechlorination processes.

Chlorinated-ethene and degradation-product 

data from shallow wells under the building also support the inference that reductive dechlorination was 

occurring in shallow ground water beneath the manufacturing building [table 8 (at end of the report)]. 

Water samples from well 33S contained lower molar 

concentrations of TCE than molar concentrations of 

cDCE and VC (reductive-dechlorination byproducts) 

and cDCE and VC concentrations generally increased 

in water samples collected between 1994 and 1997 

(fig. 12). Ethene and ethane (degradation products of 

VC) also were detected in water samples from shallow 

wells screened beneath the manufacturing building. 

Water samples collected from well 33S contained 

60 to 93 μM/L of ethene and 28 to 34 μM/L of ethane 

(table 7). Water samples from well 32S, also screened 

beneath the manufacturing building, contained 278 to 

379 μM of ethene. A low molar ratio of parent material to byproducts and increasing concentrations of 

byproducts are evidence of biologically mediated 

reductive dechlorination of TCE.

As the shallow ground water moved downgradient from the building, direct recharge resulted in higher 

DO concentrations in the ground water. Water samples 

from shallow wells not screened beneath the manufacturing building commonly contained DO concentrations greater than 1.0 mg/L (fig. 11). Conditions in 

wells away from the manufacturing building, near 
Results and Interpretation 21

Manufacturing building

Figure 11. Geochemical conditions of water samples collected from 

selected shallow wells, August 21, 1997. 

Iron 

reduction

Aerobic

Dissolved oxygen 

(O2)

Nitrate 

(NO3

-

)

Manganese (Mn2+) Iron (Fe2+)

Sulfate

(SO4

2-)

Snell Branch

33S

N

100 METERS

300 FEET

0

0

5S

EXPLANATION

2S

7S

7S WELL NUMBER

Concentrations are in milligrams per liter

100

10

1

0.1

10

1

0.1

0.01

>1.0

0.5 >1.0

0.5

 > 20

10

well 5S for example, were consistently aerobic 

(table 7), which would preclude reductive dechlorination from occurring. Aerobic degradation (cometabolism) of chlorinated ethenes is possible if sufficient 

food and oxygen are available. Ethene and ethane, 

which were found in shallow ground water under the 

building, are suitable food substrates for inducing 

cometabolism. However, concentrations in water samples from wells to the west of the manufacturing building (5S, 6S, and 35S) were usually below detection 

limits. 

A more consistent food supply would be 

required to sustain cometabolism in the aerobic zone. 

Ammonia is an alternative food substrate of the AMO 

cometabolic pathway. The anaerobic conditions underneath the manufacturing building resulted in elevated 

concentrations of NH3 (approximately 0.5 to 2.5 mg/L) 

and low NO3

- concentrations in water samples from 

wells near the manufacturing building (table 7). 

Farther downgradient, where conditions were consistently aerobic (near well 5S), NH3 concentrations were 

lower (less than 0.10 mg/L) and NO3

-

 concentrations 

increased (0.59 to 1.8 mg/L) indicating that NH3 was 

oxidized to NO3

-

 (fig. 13). Concurrent decreases in 

NH3 concentrations and increases in NO3

- concentrations along the delineated flow path are consistent 

with the AMO pathway and the cometabolic degradation of TCE, cDCE, and VC. These geochemical data 

indicate that cometabolism by the AMO pathway may 

have been occurring in shallow ground water to the 

west of the manufacturing building. Direct oxidation 

of VC and cDCE is another potential biodegradation 

pathway in the aerobic part of the shallow waterbearing zone, but this was not confirmed in this study.

Geochemical conditions of water samples from 

wells located near the western side of the manufacturing building (7S and 2S) changed in response to rain 

Figure 11. Geochemical conditions of water samples collected from selected shallow wells, 

August 21, 1997.
22 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

events. DO and NO3

- concentrations decreased and 

NH3 and Mn2+ concentrations increased significantly 

between November 12, 1996 and February 11, 1997 in 

water samples from well 7S (fig. 14), indicating a 

change from aerobic conditions to manganese-reducing 

conditions. The geochemical sampling technique used 

during the November 12, 1996 and February 11, 1997 

sampling events may have resulted in some aeration of 

samples and actual DO concentrations were probably 

lower than the measured concentrations. The sampling 

technique was modified after the February 11, 1997 

sampling event to minimize aeration of samples. The 

geochemical data indicate that at times the zone of 

anaerobic reductive dechlorination may have extended 

beyond the edge of the building; however, whether significant reductive dechlorination occurred in these fluctuating zones of transition is not clear. 

Geochemical conditions in this fluctuating transition zone were probably affected by both infiltration 

of water from the surface after rainfall events (fig. 15) 

and transport of shallow ground water from the upgradient anaerobic zone beneath the manufacturing building. As the shallow ground water moves laterally out 

from under the building, recharge from rain would 

supply DO and dilute the contaminated water. Thus, 

the anaerobic water containing ammonia, small aliphatic hydrocarbons, and chlorinated ethenes coming 

from under the building mixes with aerobic waters, 

making the conditions suitable for cometabolism. 

Methanotrophic and ammonia-oxidizing bacteria 

would consume methane and ammonia as well as oxygen in this transition zone. Cometabolic destruction of 

chlorinated ethenes would also occur in this transition 

zone. Undoubtedly some of the water carrying bacteria, contaminants, electron donors, and electron acceptors in the shallow water-bearing zone also migrates 

down into the karst aquifer. Evidence that aerobic and 

anaerobic biodegradation pathways remained active in 

the karst aquifer is examined in the next section.

Table 7. Water-quality data for samples collected from selected shallow wells

[μS/cm, microsiemens per centimeter; CaCO3, calcium carbonate; mg/L, milligrams per liter; μM/L, micromoles per liter; <, less than; --, no data; a, 

sample possibly aerated during collection; *, see table 8 for date samples were collected for trichloroethylene, cis-1,2,-dichloroethylene, and vinyl chloride]

NitroNitro- gen

Spe- gen am- Mangacific Alka- nitrate, monia, nese, Iron, Sulfate,

pH con- linity Dis- dis- dis- dis- dis- dis-

 (stand- duct- (mg/L solved solved solved solved solved solved

Well ard ance as oxygen (mg/L (mg/L (mg/L (mg/L (mg/L 

name Date units) (μS/cm) CaCO3) (mg/L) as N) as N) as Mn) as Fe) as SO4)

Well 33S 11/12/96* 6.2 1,276 413 <0.1 0.21 1.40 1.70 5.70 61

02/11/97 6.7 1,250 386 <0.1 0.05 1.40 2.00 6.00 75

08/22/97 6.8 1,585 480 <0.1 0.06 1.60 2.60 18.10 170

5/98 -- -- -- -- -- -- -- -- --

Well 7S 11/12/96* 6.4 1,118 434 2.4a 0.63 0.84 0.03 0.01 37

02/11/97 6.7 1,024 441 1.1a 0.22 2.50 4.40 <0.01 32

08/21/97* 7.1 1,080 387 3.5 1.90 0.98 1.90 <0.01 110

11/05/97 6.7 1,311 350 5.1 0.20 1.47 -- 0.01 78

5/98 -- -- -- -- -- -- -- -- --

Well 2S 11/12/96* 6.4 908 335 2.5a 0.25 0.48 2.20 0.30 72

02/11/97 7.0 886 359 0.8a <0.02 0.48 1.90 0.24 69

08/22/97 6.8 918 363 2.1 <0.02 0.47 2.10 0.28 67

5/98 -- -- -- -- -- -- -- -- --

Well 5S 11/12/96* 7.4 678 329 -- 0.91 0.02 <0.01 <0.01 75

02/11/97 7.6 645 265 7.5a 0.59 0.02 <0.01 <0.01 75

08/22/97 -- 581 340 5.6 1.80 0.10 1.40 <0.01 78

11/05/97 7.5 578 255 5.4 1.10 0.05 -- 0.01 122

5/98 -- -- -- -- -- -- -- -- --
Results and Interpretation 23

Table 7. Water-quality data for samples collected from selected shallow wells—Continued

Chlo- cisride, Tri- 1,2-DiSulfide, dis- Calcite chloro- chloro- Vinyl

dissolved solved satur- ethy- ethy- chloWell (mg/L (mg/L ation lene lene ride Ethene Ethane

name Date as S2-) as Cl) index (μM/L) (μM/L) (μM/L) (μM/L) (μM/L)

Well 33S 11/12/96* -- 150 -0.52 <0.76 182.6 78.4 -- --

02/11/97 -- 140 -0.10 0.30 147.5 47.7 -- --

08/22/97 0.056 190 0.14 <0.76 150.6 56.0 92.67 27.57

5/98 -- -- -- <6.85 312.9 36.0 60.24 33.59

Well 7S 11/12/96* -- 80 -0.28 63.55 90.3 1.02 -- --

02/11/97 -- 70 -0.01 6.15 11.4 0.08 -- --

08/21/97* 0.060 56 0.49 50.23 142.4 <0.02 <0.93 <0.86

11/05/97 0.015 14 -- 28.92 168.1 0.98 <0.93 <0.86

5/98 -- -- -- 59.25 280 <7.20 <0.93 <0.86

Well 2S 11/12/96* -- 45 -0.47 1,415 330.1 32.8 -- --

02/11/97 -- 47 0.15 610.4 466.2 29.0 -- --

08/22/97 0.068 46 0.00 1,302 355.9 <8.00 7.77 3.72

5/98 -- -- -- 702.1 279.4 17.3 10.37 2.39

Well 5S 11/12/96* -- 15 0.42 0.74 0.47 0.06 -- --

02/11/97 -- 18 0.48 2.09 0.44 <0.02 -- --

08/22/97 0.034 11 -- -- -- -- <0.93 <0.86

11/05/97 0.005 137 -- 3.73 3.77 <0.02 <0.93 <0.86

5/98 -- -- -- 10.96 5.20 <0.02 <0.93 <0.86 5/1/94 8/1/94 11/1/94 2/1/95 5/1/95 8/1/95 11/1/95 2/1/96 5/10/96 8/10/98 11/10/96 2/11/97 8/22/97

300

0

100

150

200

250

50

SAMPLING DATE

Figure 12. Chlorinated-ethene data for water samples collected from well 33S.

EXPLANATION

VINYL CHLORIDE

cis-1,2-DICHLOROETHYLENE

TRICHLOROETHYLENE

PER LITER

CONCENTRATION, IN MICROMOLES
24 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Manufacturing building

Figure 13. Nitrate and ammonia concentrations in samples from selected

shallow wells. 

Snell Branch

 3.4 μM/L NO3

- 

 82 μM/L NH3

 

 15 μM/L NO3

- 

 1.1 μM/L NH3

 

 10 μM/L NO3

- 

 49 μM/L NH3

 

 4.0 μM/L NO3

- 

 28 μM/L NH3

 

N

100 METERS

300 FEET

0

0

Elevated ammonia concentration

because of reducing conditions

Elevated nitrate concentrations

because of ammonia oxidation

EXPLANATION

 10 μM/L NO3

- 

 49 μM/L NH3

 

GENERAL DIRECTION OF GROUND-WATER 

 FLOW IN THE SHALLOW WATER-BEARING

 ZONE

SHALLOW WELL AND CONCENTRATIONS OF 

 NITRATE AND AMMONIA IN MICROMOLES PER

 LITER IN WATER SAMPLES COLLECTED ON 

 NOV. 12, 1996 

Figure 13. Nitrate and ammonia concentrations in samples from selected shallow wells.

Nov. 12, 1996 Feb. 11, 1997 Aug. 21, 1997

SAMPLING DATE

4.5

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

CONCENTRATION, IN MILLIGRAMS PER LITER

EXPLANATION

DISSOLVED OXYGEN

NITRATE

AMMONIA

DISSOLVED MANGANESE

DISSOLVED IRON

Figure 14. Geochemical data for water samples from well 7S.
Results and Interpretation 25

10/22/96 11/12/96

4/23/98 4/30/98 5/7/98 5/14/98 5/21/98

Sampling event 7

0

1

2

3

4

5

0

1

2

3

4

5

10/8/97 10/15/97 10/22/97 10/29/97 11/5/97

Sampling event 4

RAINFALL, IN CENTIMETERS

4/2/98 4/9/98 4/16/98 4/23/98 4/30/98

Sampling event 6

0

1

2

3

4

5

10/15/96 10/29/96 11/5/96

Sampling event 1

0

1

2

3

4

5

1/15/97 1/22/97 1/29/97 2/5/97 2/12/97

Sampling event 2

0

1

2

3

4

5

7/24/97 7/31/97 8/21/97 8/7/97 8/14/97

Sampling event 3

0

1

2

3

4

5

1/20/98 1/27/98 2/3/98 2/10/98 2/17/98

Sampling event 5

0

1

2

3

4

5

Figure 15. Daily rainfall totals for a 1-month period before each ground-water sampling event.

Figure 15. Daily rainfall totals for a 1-month period before each ground-water sampling event.

Biodegradation of Chlorinated Ethenes in the 

Karst Aquifer

Water-quality data collected during periodic 

sampling by the USGS indicate that conditions in 

water-producing zones of the upper part of the Ridley 

Limestone vary spatially and temporally at the study 

site. Conditions in some zones, such as those intersected by wells 1D and 3D, were consistently anaerobic, whereas conditions in other zones, such as those 

intersected by wells 2D and 12D, fluctuated between 

anaerobic and aerobic (table 9). When anaerobic conditions were present, Mn4+ or Fe3+ reduction may 

have been the dominant electron acceptor (fig. 16); 

however, sulfate reduction also may have been occurring. Sulfide concentrations ranging from 0.002 to 

3.46 mg/L were detected in water samples from deep 

wells (table 9). The sulfate and sulfide concentrations 

were generally higher in samples from the karst aquifer compared to the shallow wells (tables 7 and 9). 

This is probably due in part to the availability of sulfate from the gypsum nodules of the karst bedrock. 

Bacteria can accelerate the remobilization of sulfate 

and convert the sulfate to sulfide.

Significant concentrations of chlorinated ethenes 

(TCE, cDCE, and VC) were detected in water samples 

from several deep wells (table 9). Those wells containing chlorinated ethenes, except for well 12D, had 

higher molar concentrations of reductive-dechlorination 

byproducts (cDCE and VC) than of TCE (fig. 17). The 

concentration of chlorinated ethenes varied with 

hydrologic conditions and weather events. Periodic 

increases in VC and cDCE concentrations in water 

samples were detected. For example, between May 10 

and August 10, 1996, cDCE concentrations in water 

samples from wells 3D and 4D increased from 
26 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Table 9. Water-quality data for samples collected from selected deep wells

[μS/cm, microsiemens per centimeter; CaCO3, calcium carbonate; mg/L, milligrams per liter; μM/L, micromoles per liter; --, no data; <, less than; *, see 

table 8 for date samples were collected for trichloroethylene, cis-1,2,-dichloroethylene, and vinyl chloride]

NitroNitro- gen

Water Spe- gen am- Mangalevel cific Alka- nitrate, monia, nese, 

(meters pH con- linity Dis- Total dis- dis- dis-

 above (stand- duct- (mg/L solved organic solved solved solved

Well sea ard ance as oxygen carbon (mg/L (mg/L (mg/L

name Date level) units) (μS/cm) CaCO3) (mg/L) (mg/L) as N) as N) as Mn)

Well 4D 02/12/97 215.01 7.4 476 180 0.2 -- <0.02 0.28 0.04

08/21/97* 213.12 7.0 179 90 1.2 -- 0.06 0.02 0.04

5/98 -- -- -- -- -- -- -- -- --

Well 1D 11/12/96* 210.34 6.9 830 265 <0.1 -- 0.42 0.04 0.07

 02/11/97 214.71 6.7 995 335 <0.1 -- 0.03 0.30 0.11

08/21/97* 213.36 7.6 818 259 <0.1 2.5 0.12 0.32 <0.01

11/05/97 206.24 7.5 756 253 <0.1 1.3 <0.02 0.50 <0.01

02/17/98 7.2 634 230 0.3 1.4 0.30 0.50 <0.20

04/30/98 210.85 7.4 708 202 <0.1 7.5 0.30 0.09 <0.20

05/21/98 207.80 6.2 736 220 0.2 0.7 0.20 0.13 <0.20

Well 3D 11/13/96* 210.30 7.1 725 251 -- -- 0.14 0.47 0.01

02/12/97 214.52 7.3 2,342 450 -- -- <0.02 3.40 <0.01

08/21/97* 213.23 7.5 818 284 <0.1 -- 0.04 0.43 0.05

11/05/97 206.18 7.3 844 319 <0.1 -- <0.02 0.71 0.01

02/17/98 7.1 711 294 <0.1 4.0 0.10 0.77 0.10

04/30/98 210.08 7.3 781 240 0.2 8.4 0.20 0.85 0.90

05/21/98 207.11 6.7 688 263 <0.1 1.3 0.20 0.78 0.20

Well 2D 02/11/97 214.27 7.0 598 -- <0.1 -- 0.26 1.70 0.02

08/20/97* 213.37 7.3 427 156 3.4 3.8 0.11 0.02 0.02

11/04/97* 205.73 7.3 406 153 3.3 -- 0.10 0.41 --

02/17/98 6.9 340 135 2.0 4.0 0.60 0.14 <0.20

04/30/98 210.57 7.3 509 138 <0.1 7.7 0.20 0.24 <0.20

05/21/98 207.21 6.4 400 159 0.1 7.9 0.20 0.26 <0.20

Well 11D 11/13/96* 209.47 8.2 1,242 113 <0.1 -- 0.03 0.47 <0.01

02/11/97* 213.35 7.7 1,095 163 <0.1 -- 0.02 0.30 0.02

08/20/97* 216.23 8.2 776 137 <0.1 -- 0.16 0.14 0.02

Well 12D 08/20/97* 222.65 7.1 507 105 5.5 6.3 0.72 0.05 0.05

11/04/97 218.43 7.2 557 266 0.4 -- 0.10 0.13 --

02/17/98 6.9 389 135 5.8 4.6 0.20 0.02 <0.20

04/30/98 220.32 7.4 369 82 5.3 5.9 0.30 0.10 <0.20

05/21/98 217.22 6.3 590 210 0.2 2.4 0.40 0.32 <0.20

Well 14D 11/13/96* 220.54 6.8 738 333 -- -- 0.04 0.06 0.14

02/11/97* 219.30 7.2 750 330 -- -- <0.02 0.16 0.14

11/04/97* 220.03 7.3 789 338 1.0 1.8 0.19 0.09 0.28

Well 15D-A 11/13/96 223.35 6.9 713 277 -- -- 0.58 0.03 0.03

02/12/97 223.60 7.2 830 230 -- -- 0.53 0.02 0.03

Well 15D-C 11/13/96 221.67 9.4 329 128 -- -- 0.05 0.06 <0.01

02/12/97 221.88 9.6 346 155 <0.1 -- -- 0.17 <0.01

Well 16D-B 08/22/97 -- 9.6 627 140 1.8 38.0 0.15 2.30 0.02

11/04/97 -- 10.5 697 126 0.3 -- 0.20 4.08 --

Well 10D-B 08/22/97 220.52 8.2 310 138 <0.1 2.7 0.06 0.05 0.04

11/04/97 220.24 7.0 531 227 <0.1 -- <0.02 0.08 --
Results and Interpretation 27

Table 9. Water-quality data for samples collected from selected deep wells—Continued

Chlo- cis-

 Iron, Sulfate, ride, Tri- 1,2-Didis- dis- Sulfide, dis- Calcite chloro- chloro- Vinyl

solved solved dissolved solved satur- ethy- ethy- chloWell (mg/L (mg/L (mg/L (mg/L ation lene lene ride Ethene Ethane

name Date as Fe) as SO4) as S2-) as Cl) index (μM/L) (μM/L) (μM/L) (μM/L) (μM/L)

Well 4D 02/12/97 0.27 50 -- 7 -0.15 0.01 0.06 <0.02 -- --

08/21/97* 0.72 2 0.038 3 -- <0.01 <0.01 <0.02 -- --

5/98 -- -- -- -- -- <0.01 <0.01 <0.02 <0.04 <0.03

Well 1D 11/12/96* 0.41 22 -- 91 -0.39 0.42 0.13 0.74 -- --

 02/11/97 0.13 52 -- 150 -0.44 <0.01 0.06 0.09 -- --

08/21/97* 0.17 36 0.024 86 0.45 0.11 0.10 0.05 -- --

11/05/97 0.01 34 0.002 66 0.25 0.02 0.36 1.90 3.39 67.18

02/17/98 0.04 72 0.009 45 -- -- -- -- -- --

04/30/98 0.05 50 0.003 47 -- -- -- -- -- --

05/21/98 0.02 50 0.028 77 -- 0.06 0.12 0.08 <0.04 <0.03

Well 3D 11/13/96* 0.06 93 -- 23 -0.16 0.02 1.89 1.17 -- --

02/12/97 <0.01 110 -- 440 0.27 0.02 0.62 0.31 -- --

08/21/97* 0.03 67 1.570 53 0.41 0.01 0.06 <0.02 -- --

11/05/97 0.11 71 2.340 47 0.19 <0.01 0.20 <0.02 <0.93 126.71

02/17/98 0.13 115 0.130 38 -- -- -- -- -- --

04/30/98 0.08 88 0.013 12 -- -- -- -- -- --

05/21/98 0.04 90 0.014 23 -- <0.01 2.37 2.24 <0.04 21.22

Well 2D 02/11/97 0.02 48 -- 26 0.18 6.96 14.34 3.92

08/20/97* 0.01 35 0.028 10 -0.04 1.28 4.82 4.10

11/04/97* 0.01 46 0.011 8 -- 6.70 58.80 12.16 6.10 40.48

02/17/98 0.02 49 0.031 9 -- 4.54 11.04 2.50 <0.07 4.42

04/30/98 0.08 36 0.006 >10 -- -- -- -- -- --

05/21/98 0.02 45 0.019 11 -- 0.88 5.27 1.17 <0.04 <0.03

Well 11D 11/13/96* 0.01 590 -- 9 0.71 0.52 1.76 <0.02 -- --

02/11/97* 0.03 450 -- 6 0.35 0.02 1.46 <0.02 -- --

08/20/97* <0.01 260 3.460 3 0.66 <0.01 0.73 <0.02 -- --

Well 12D 08/20/97* 0.03 100 0.030 8 -0.35 3.04 0.98 <0.02 -- --

11/04/97 0.03 72 0.014 8 -- 8.56 3.88 <0.02 <0.93 <0.87

02/17/98 0.01 75 0.023 37 -- 0.14 0.04 <0.03 <0.07 <0.07

04/30/98 0.06 74 0.012 >10 -- -- -- -- -- --

05/21/98 <0.01 90 0.010 18 -- 1.29 0.78 0.06 <0.04 <0.03

Well 14D 11/13/96* 0.03 65 -- 9 -0.24 2.21 2.39 0.10 -- --

02/11/97* 0.23 63 -- 10 0.16 1.51 3.94 0.44 -- --

11/04/97* 0.01 120 0.015 11 0.36 2.32 5.21 0.93 <0.93 <0.87

Well 15D-A 11/13/96 0.02 130 -- <1 -0.21 <0.02 -- -- -- --

02/12/97 0.13 130 -- 5 -- <0.01 -- -- -- --

Well 15D-C 11/13/96 <0.01 26 -- 6 0.52 -- -- -- -- --

02/12/97 <0.01 26 -- 4 -- -- -- -- -- --

Well 16D-B 08/22/97 <0.01 160 0.034 15 1.33 <0.01 0.05 <0.02 -- --

11/04/97 <0.01 150 0.014 17 -- <0.01 0.03 <0.02 <0.93 4.19

Well 10D-B 08/22/97 0.01 19 0.504 3 0.43 <0.01 0.06 0.09 -- --

11/04/97 <0.01 49 0.124 4 -- 0.07 0.15 0.12 <0.93 <0.87
28 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Figure 16. Typical geochemical conditions for anaerobic water 

samples collected from deep wells.

 WELL 1D (February 11, 1997)

WELL 3D (August 21, 1997)

EXPLANATION

Concentrations are in milligrams per liter

100

10

1

0.1

10

1

0.1

0.01

1.0

>1.0

0.5

 > 20

10

0.5

Dissolved oxygen 

(O2)

Nitrate 

(NO3

-

)

Manganese (Mn2+) Iron (Fe2+)

Sulfate

(SO4

2-)

Figure 16. Typical geochemical conditions for anaerobic water samples collected from deep wells.

16D-B 1D 3D 2D 12D 14D 10D-B

WELL NUMBER

0

80

10

20

30

40

50

60

70

CONCENTRATION, IN MICROMOLES PER LITER

EXPLANATION

VINYL CHLORIDE

cis-1,2-DICHLOROETHYLENE

TRICHLOROETHYLENE

Figure 17. Concentrations of chlorinated ethenes in water samples collected from selected deep 

wells on November 4 and 5, 1997.
Results and Interpretation 29

0.01 to 7.48 μM/L and from 0.04 to 5.41 μM/L, 

respectively (fig. 18). Ethene and ethane also were 

detected in water samples from wells 1D, 2D, and 3D 

(table 9). The ethene and ethane are final products of 

reductive dechlorination of VC and are readily consumed by bacteria. 

The high ratio of degradation byproducts to 

TCE implies that reductive dechlorination is occurring 

at the site. The location of the reductive-dechlorination 

process is not clear from the chlorinated-ethene data. 

The degradation byproducts may have been formed in 

the shallow water-bearing zone and transported down 

into the upper part of the Ridley Limestone without 

any additional dechlorination occurring in the karst 

aquifer. Traditionally, sequential patterns of oxidationreduction zones along a contaminant plume transect 

are used to identify where reductive dechlorination is 

active in the aquifer. A simple contaminant flow path 

and sequential oxidation-reduction zones could not be 

identified for the karst aquifer because of the complex 

hydrogeology at the study site. The geochemical and 

biological information derived from sampling karstaquifer wells were considered individually instead of 

as a section of a continuum along a flow path. The data 

for an individual well represents geochemical and biological conditions in that part of the karst aquifer, 

whether the aquifer is in an active or less active flow 

zone. Multiple lines of biodegradation evidence are 

presented for karst wells 12D, 1D, 3D, and 2D in the 

following text.

Multiple Lines of Evidence from Well 12D 

Water-quality conditions of samples from well 

12D, located near Snell Branch and screened in the 

upper water-producing zone of the Ridley Limestone, 

changed significantly after rainfall events. Continuous 

data collected from well 12D indicated that water levels rose (sometimes to land surface), specific conductance decreased due to dilution by rainwater, and DO 

concentrations and ORP (expressed as Eh) increased 

(fig. 19). These changes typically occurred within 

hours of rainfall events (fig. 20) indicating that water 

was quickly transported from land surface to the upper 

water-producing zone of the Ridley Limestone intersected by well 12D. These changes in water chemistry 

are consistent with the detection of a mud-filled cavity 

that was hydraulically connected to Snell Branch during the construction of well 12D. After rainfall events, 

the DO would start to decrease and anaerobic conditions would develop until the next rainfall (fig. 19).

During dry periods such as late May 1998, 

anaerobic conditions persisted and oxidation-reduction 

potentials decreased (fig. 19D) to levels associated 

with iron-reducing conditions (fig. 2). However, during the 3 months of continuous monitoring, anaerobic 

conditions did not persist long enough for reductive 

dechlorination to occur. The lack of VC (fig. 21), 

ethene, and ethane (table 9) in water samples from 

well 12D also suggests that little reductive dechlorination had occurred. Some periodic increases in cDCE 

concentrations were detected in water samples collected from well 12D; however, these increases were 

often accompanied by increases in TCE (fig. 21) and 

are most likely the result of transport processes, not 

reductive dechlorination in the water-producing zone 

intersected by well 12D.

These results indicate that natural attenuation of 

chlorinated ethenes in the part of the karst aquifer 

intersected by well 12D is probably limited to aerobic 

degradation or mechanisms such as dilution. 

Ammonia-oxidizing bacteria and methanotrophs 

(table 10) were detected in water samples from well 

12D; however, microcosms containing water from 

well 12D (experiment 2, treatment 4) did not exhibit 

significant aerobic degradation of TCE during a 17-

week period (fig. 22). Microcosm data are given in 

table 11 (at end of the report). The water samples used 

to construct these microcosms were collected during a 

dry period (May 21, 1998) and contained unusually 

low DO concentrations (table 9) and ORP (fig. 19D), 

which limited aerobic degradation. Between weeks 17 

and 23, some reduction in TCE concentrations 

occurred in excess of the reductions in control samples 

(fig. 22) which indicate that reductive dechlorination 

was beginning to occur. 

Data from microcosm experiment 1 indicate the 

occurrence of reductive dechlorination in microcosms 

containing water from well 12D. During microcosm 

experiment 1, the discovery was made that control 

microcosms (experiment 1, treatment 9) were not sterile. Plate counts for facultative and aerobic heterotrophic bacteria indicated greater than 2 x 104

bacteria colony-forming units per 100 milliliters 

(CFU/100mL) of water sample in the control microcosms after a 1-week incubation period. Bacteria enumeration data are given in table 12 (at end of the 

report). 

Without adequate controls, determining to what 

degree the TCE decreases in the microcosms was 

caused by biodegradation was impossible. Significant 
30 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

VINYL CHLORIDE

cis-1,2-DICHLOROETHYLENE

TRICHLOROETHYLENE

EXPLANATION

VINYL CHLORIDE

cis-1,2-DICHLOROETHYLENE

TRICHLOROETHYLENE

EXPLANATION

Figure 18. Temporal changes in chlorinated-ethene concentrations in water samples

from wells 3D and 4D.

Figure 18. Temporal changes in chlorinated-ethene concentrations in water samples from wells 

3D and 4D.
Results and Interpretation 31

0

1

2

215

220

225

A. Water level

0

1

2

0

200

400

600

800 B. Specific conductance

0

1

2

0

2

4

6

8

10 C. Dissolved oxygen

3/13/983/17/983/22/983/26/983/30/98

4/3/984/7/98

4/11/984/16/984/20/984/24/984/28/98

5/2/985/6/98

5/11/985/15/985/19/98

0

1

2

-200

0

200

400

600 D. Oxidation-reduction potential (Eh)

Figure 19. Continuous ground-water monitoring data collected from well 12D

for (A) water level, (B) specific conductance, (C) dissolved oxygen, and

(D) oxidation-reduction potential, March 13 through May 19, 1998. (Line gaps

indicate missing data.) 

Figure 19. Continuous ground-water monitoring data collected from well 12D for (A) water level, 

(B) specific conductance, (C) dissolved oxygen, and (D) oxidation-reduction potential, March 13 

through May 19, 1998. (Line gaps indicate missing data.)

cDCE and VC concentrations, however, were detected 

in several of the treatments after a 10-month incubation period, indicating that some reductive dechlorination had occurred in the microcosms. VC and cDCE 

were not present in the microcosms when they were 

first constructed, and the detection of the compounds 

could only be due to reductive dechlorination of TCE.

After a 10-month incubation period, cDCE represented 98 and 67 percent of the total chlorinatedethene concentration remaining in microcosm treatments containing water from well 12D (experiment 1, 

replicates 7 and 8, respectively) (fig. 23). Microorganisms responsible for reductive dechlorination (sulfate 

reducers) were detected in water samples from well 

12D (table 10), and the microcosms from experiment 1 

indicate that the microorganisms were able to dechlorinate TCE after oxygen and other electron acceptors 

were depleted during the 10-month incubation period. 

In spite of microcosm evidence that reductive dechlorination can occur in water from 12D, it is unlikely 

that reductive dechlorination is a significant biological 

process in that part of the karst aquifer. Field 

geochemical conditions indicate that the reducing conditions in 12D are disrupted with each rainfall event. 

Multiple Lines of Evidence from Well 1D

Well 1D is screened in the lower water-producing 

zone of the karst aquifer and is located close to the 

manufacturing building. Water-quality conditions in 

the part of the karst aquifer intersected by well 1D 

exhibited little change in response to storms during the 

2 months (March 1998 to May 1998) of continuous 
32 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

TIME, IN 2-HOUR INCREMENTS

0

0.5

1

215

220

225

A. Water level

0

0.5

1

0

200

400

600

800 B. Specific conductance

0

0.5

1

0

2

4

6

8

10 C. Dissolved oxygen

00:0002:0004:0006:0008:0010:0012:0014:0016:0018:0020:0022:0000:0002:0004:0006:0008:0010:0012:0014:0016:0018:0020:0022:00

0

0.5

1

-200

0

200

400

600 D. Oxidation-reduction potential (Eh)

Figure 20. Continuous ground-water monitoring data collected from well 12D showing

rapid changes in (A) water level, (B) specific conductance, (C) dissolved oxygen,

and (D) oxidation-reduction potential, March 31 through April 1, 1998. (Line gaps

indicate missing data.)

Figure 20. Continuous ground-water monitoring data collected from well 12D showing rapid changes 

in (A) water level, (B) specific conductance, (C) dissolved oxygen, and (D) oxidation-reduction 

potential, March 31 through April 1, 1998. (Line gaps indicate missing data.)

monitoring. Anaerobic conditions (less than 0.3 mg/L 

DO) were consistently detected in water samples collected from well 1D during quarterly sampling events 

(table 9), and geochemical data indicate manganesereducing conditions (fig. 16). Continuous water-quality 

monitoring indicated that although water levels 

increased after precipitation events, specific conductance, DO, and ORP (expressed as Eh) changed little 

(fig. 24). Equilibrium of the ORP electrode took 

approximately 4 days to come to equilibrium with the 

aquifer water. Once equilibrium was reached, ORP 

measured during continuous monitoring was normally 

between –150 and –200 millivolts (mV), which is normally associated with nitrate-, iron-, and sulfatereducing conditions (figs. 2 and 24D). 

During a 23-week period, significant aerobic degradation of TCE was not detected in microcosms using 

water collected from well 1D on May 1, 1998 (experiment 2, treatment 1) (fig. 22). Ammonia oxidizers were 

not detected in water samples collected from well 1D; 

however, methanotrophs were detected (table 10). 

Based on the geochemical data and the microcosm 

results, biodegradation of chlorinated ethenes in the 

water-producing zone intersected by well 1D would be 

limited to reductive dechlorination. Between 17 and 

23 weeks, TCE concentrations in the microcosms using 

well 1D water decreased faster than in the control samples (fig. 22) which could indicate that reductive 

dechlorination was beginning to occur. During the 10-

month incubation period of microcosm experiment 1, 

TCE degradation byproducts (VC and cDCE) increased 

from zero to approximately 80 and 50 percent of the 

average total chlorinated-ethene concentration remaining in microcosms containing water from well 1D 
Results and Interpretation 33

Table 10. Results from bacteria identification for samples collected on November 4 and 5, 1997 from selected wells

[N, not detected; Y, detected; --, data not available] 

Wells

Type of bacteria 1D 2D 3D 12D 16D-B 10D-B

Ammonia oxidizers N N YYYY

Methanotrophs Y Y Y Y Y Y

Manganese/iron oxidizers -- Y Y Y N Y

Sulfate reducing -- Y Y Y Y Y

E. coli Y -- Y Y Y N

VINYL CHLORIDE

cis-1,2-DICHLOROETHYLENE

TRICHLOROETHYLENE

EXPLANATION

Figure 21. Chlorinated-ethene concentrations in water samples collected from

well 12D.

Figure 21. Chlorinated-ethene concentrations in water samples collected from well 12D.

(experiment 1, treatments 1 and 2, respectively) 

(fig. 23). Results from microcosm experiment 1 demonstrate that reductive dechlorination of TCE is possible in the water from well 1D. 

The lag time for reductive dechlorination to 

occur in the microcosms was probably due to aeration 

of the experimental water during set up. No lag time 

would be expected in the aquifer around well 1D since 

the conditions are consistently anaerobic. Further evidence of reductive dechlorination in well 1D is found 

in the chlorinated-ethene data (table 8). Byproducts of 

TCE reductive dechlorination, DCE and VC, were 

found in every sample reported. Thus, the multiple 

lines of evidence indicate reductive dechlorination 

occurs in the part of the karst aquifer intersected by 

well 1D.

Multiple Lines of Evidence from Well 3D 

Well 3D is located near the center of the manufacturing building and is screened in the lower waterproducing zone of the upper part of the Ridley Limestone. Continuous water-quality data indicate that 

rainfall events and changes in the pumping rate of well 

9D affect water-quality conditions in the part of the 

karst aquifer intersected by 3D. When pump-and-treat 

well 9D was operating (March 19, 1998 to April 8, 

1998) the water moving through the screened interval 

of well 3D appeared to have been stored in the karst 

aquifer longer than the water moving through the 

screened interval of well 12D. Evidence for this inference includes increased specific conductance after precipitation events (fig. 25B). The higher specific 

conductance is indicative of increased dissolved solids 
34 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

0

0

5 10 15 20 25

Figure 22. Trichloroethylene (TCE) concentrations during microcosm experiment 2.

TIME, IN WEEKS

Treatment 2 (well 2D)

Treatment 3 (well 3D)

Treatment 1 (well 1D) Treatment 4 (well 12D)

TRICHLOROETHYLENE REMAINING,

IN PERCENT

10

20

30

40

50

60

70

80

90

100

Treatment 5 (control samples)

Figure 22. Trichloroethylene (TCE) concentrations during microcosm experiment 2.

Figure 23. Relative amounts of trichloroethylene and reductive-dechlorination 

degradation products in experiment 1 microcosms after 10 months of incubation.

1(1D) 2(1D) 3(2D) 4(2D) 5(3D) 6(3D) 7(12D) 8(12D)

TREATMENT NUMBER (WELL)

0

100

0

10

20

30

40

50

60

70

80

90

MOLAR PERCENTAGES OF TOTAL

CHLORINATED-ETHENE CONCENTRATION

EXPLANATION

VINYL CHLORIDE

cis-1,2-DICHLOROETHYLENE

TRICHLOROETHYLENE
Results and Interpretation 35

Figure 24. Continuous ground-water monitoring data collected from well 1D for

(A) water level, (B) specific conductance, (C) dissolved oxygen, and (D) oxidationreduction potential, March 18 through May 20, 1998. (Line gaps indicate missing

data.)

Figure 24. Continuous ground-water monitoring data collected from well 1D for (A) water level, 

(B) specific conductance, (C) dissolved oxygen, and (D) oxidation-reduction potential, March 18 

through May 20, 1998. (Line gaps indicate missing data.)

normally associated with limestone dissolution over 

time. Also, dye was detected in water samples collected from well 3D in 1996, 5 years after the dye was 

injected into well 8D which was screened in the same 

lower water-producing zone of the karst aquifer. Water 

samples collected from well 3D during quarterly sampling normally had low DO concentrations (less than 

0.2 mg/L) (table 9). During the continuous monitoring 

period, DO concentrations in the well water were less 

than 0.5 mg/L and ORP was less than –100 mV 

(fig. 25D) when the pump-and-treat well was operating. 

The ORP (fig. 25D) was within ranges normally 

associated with nitrate-, iron-, and sulfate-reducing 

conditions (Stumm and Morgan, 1981). Sulfide concentrations detected in some water samples from 

well 3D (>1 mg/L) were significantly higher than 

sulfide concentrations detected in water samples from 

shallow wells (tables 7 and 9), which indicate sulfatereduction occurred in the aquifer near well 3D. 

Sulfate-reducing bacteria were identified in water 

samples collected from well 3D (table 10). Results 

from microcosm experiment 1 indicate that bacteria 

from well 3D can reductively dechlorinate TCE. After 

a 10-month incubation period, cDCE represented 98 

and 24 percent of the total chlorinated ethene remaining in microcosms using well 3D water (experiment 1, 

treatments 5 and 6, respectively) (fig. 23). 

The specific conductance of water in well 3D 

decreased, and DO increased, when pump-and-treat 

well 9D was not operating (various times between 

April 8, 1998 and May 20, 1998) (fig. 25). The slight 

decrease in specific conductance and increased DO 
36 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Figure 25. Continuous ground-water monitoring data collected from well 3D for

(A) water level, (B) specific conductance, (C) dissolved oxygen, and (D) oxidationreduction potential, March 19 through May 20, 1998. (Line gaps indicate missing

data.)

Figure 25. Continuous ground-water monitoring data collected from well 3D for (A) water level, 

(B) specific conductance, (C) dissolved oxygen, and (D) oxidation-reduction potential, March 19 

through May 20, 1998. (Line gaps indicate missing data.)

associated with pump-and-treat downtime indicate 

recharge of the karst aquifer near well 3D by fresh 

rainwater. The resulting change would tend to benefit 

the aerobic bacteria, ammonia oxidizers and methanotrophs, detected in water samples from well 3D. 

Results from the second microcosm experiment using 

water collected on May 21, 1998, suggest that significant cometabolism occurred when aerobic conditions 

prevailed. During a 3-week incubation period, TCE in 

the microcosms containing well 3D water (experiment 

2, treatment 3) was completely degraded (fig. 22). 

The multiple lines of evidence combined indicate 

that aerobic and anaerobic degradation occurred in the 

water-producing zone of the karst aquifer intersected 

by well 3D. Anaerobic conditions were present with the 

pump-and-treat well operating (the normal condition 

during this study) and reductive dechlorination was 

likely. When pump-and-treat well 9D was turned off, 

aerobic conditions suitable for aerobic cometabolic 

degradation of chlorinated ethenes prevailed. Bacteria 

suitable for both anaerobic and aerobic degradation 

were present in well 3D, and evidence of both degradation pathways was found in the microcosm studies.

Multiple Lines of Evidence from Well 2D

Well 2D is located to the west of the manufacturing building near pump-and-treat well 9D and is 

screened in the lower water-producing zone of the 

karst aquifer, as is well 9D. Because well 2D is located 

very close to pump-and-treat well 9D, water passing 

through the screened interval of well 2D probably 

flows from several parts of the karst aquifer in 

response to the gradient imposed by pumping well 9D. 

Water in well 2D represents a composite of waters 
Results and Interpretation 37

Figure 26. Continuous ground-water monitoring data collected from well 2D for

(A) water level, (B) specific conductance, (C) dissolved oxygen, and (D) oxidationreduction potential, March 31 through April 5, 1998. (Line gaps indicate missing

data.)

from parts of the karst aquifer affected by the pumping. The ORP and DO conditions in well 2D tended to 

be moderate whereas the bacteria composition ranged 

from aerobic to anaerobic bacteria types.

Water-quality conditions in the part of the karst 

aquifer intersected by well 2D changed in response to 

rainfall events, although not as quickly as conditions 

in well 12D. Specific conductance decreased after 

rainfall events due to dilution by rainwater (fig. 26B). 

DO concentrations initially increased after rainfall 

events then rapidly decreased as anaerobic aquifer 

water with a high chemical oxygen demand mixed 

with the rainwater (fig. 26C). In a pattern similar to the 

DO concentrations, ORP decreased after rainfall 

events (fig. 26D). These data indicate that freshwater 

was initially transported toward the pump-and-treat 

well after rainfall events (fig. 26). However, this fresh 

rainwater was soon followed by a mixture of anaerobic aquifer water and rainwater as indicated by the 

lower specific conductance, DO, and ORP concentrations. Presumably, old aquifer water displaced by the 

rainwater contained chemicals such as sulfide and 

reduced-metals (Fe2+ and Mn2+) that would scavenge 

DO carried in by the rainwater, also driving the ORP 

down. When the rain ceased, the pre-rain event flow 

pattern would return to the karst aquifer within days. 

The implication is that the hydrology in the karst aquifer was stable during dry periods punctuated by 

recharge events that carried in water, DO, food, and 

other compounds. Bacteria as diverse as those found in 

the karst aquifer would take advantage of these changing environments, switching between anaerobic and 

aerobic conditions.

The quarterly geochemical data (table 9) and 

continuous ORP monitoring (approximately +200 mV) 

(fig. 27D) indicate that baseline conditions ranged 

from slightly aerobic to Mn4+ and NO3

-

 reducing. Aerobic bacteria and NO3

-

-reducing bacteria were identified in these water samples. During the anaerobic 

periods after a rain event, ORP commonly decreased 

to sulfate-reducing conditions (fig. 27D), and 

Figure 26. Continuous ground-water monitoring data collected from well 2D for (A) water level, 

(B) specific conductance, (C) dissolved oxygen, and (D) oxidation-reduction potential, March 31 

through April 5, 1998. (Line gaps indicate missing data.)
38 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Figure 27. Continuous ground-water monitoring data collected from well 2D for

(A) water level, (B) specific conductance, (C) dissolved oxygen, and (D) oxidationreduction potential, March 13 through May 20, 1998. (Line gaps indicate missing

data.)

Figure 27. Continuous ground-water monitoring data collected from well 2D for (A) water level, 

(B) specific conductance, (C) dissolved oxygen, and (D) oxidation-reduction potential, March 13 

through May 20, 1998. (Line gaps indicate missing data.)

SO4-reducing bacteria were detected in water samples 

from well 2D (table 10). However, the sulfatereducing conditions lasted only a few days in well 2D, 

which would be insufficient time for significant reductive dechlorination to take place. The ORP conditions 

and bacteria types found in well 2D represent the wide 

range of conditions present in the karst aquifer. The 

pattern of ORP and DO with regard to baseline and 

post-rain conditions provides insight into the displacement of older ground water by recharge from 

rainwater. 

Cometabolism appeared to be a more significant 

biological degradation process than reductive dechlorination in microcosms set up with water from well 

2D. Experiment 2 microcosms using water from well 

2D exhibited significant aerobic degradation. Within 

3 weeks, TCE in the microcosms was completely 

degraded (fig. 22, treatment 2). Methanotrophs were 

identified in well 2D water samples; however, ammonia oxidizers were not detected. Bacteria identification 

and microcosm results in conjunction with geochemical monitoring indicate that environmental conditions 

suitable for cometabolism occurred more consistently 

in well 2D than did conditions for reductive dechlorination. However, results from microcosms in experiment 1 indicate that native bacteria from well 2D were 

capable of reductively dechlorinating TCE. Evidence 

of reductive dechlorination was found in microcosms 

after a 10-month incubation period. The TCE degradation byproduct cDCE was present in microcosms as a 

result of reductive dechlorination (experiment 1, treatments 3 and 4) (fig. 23). 
Lessons Learned 39

Data Collected from Other Deep Wells 

Geochemical data from other deep wells at the 

site indicated a similar pattern of anaerobic zones in 

the aquifer or zones fluctuating between aerobic and 

anaerobic conditions. The DO concentration in water 

samples from well 4D ranged from 0.2 to 1.2 mg/L 

and the DO concentration in water samples from well 

16D-B ranged from 0.3 to 1.8 mg/L (table 9). Both 

aerobic (ammonia oxidizers and methanotrophs) and 

anaerobic (sulfate reducers) bacteria were detected in 

water samples from well 16D-B (table 10).

Water samples from monitoring wells 10D-B 

and 11D contained less than 0.1 mg/L of DO in three 

or fewer sampling events. Well 11D is screened in a 

bedding plane that appeared to be isolated from the 

active ground-water-flow system. Water from well 

11D had such high sulfide concentrations that it had a 

strong odor and would start to form a black precipitate 

within minutes after being brought to the surface. 

These observations suggest that DO concentrations in 

well 11D were consistently less than 0.1 mg/L. Unlike 

well 11D, well 10D-B was screened in a highly fractured area of the karst aquifer. Limited geochemical 

data (table 9) indicate anaerobic conditions occurred 

in well 10D-B; however, both aerobic and anaerobic 

bacteria were present in water samples (table 10), 

implying a close association with aerobic conditions. 

More data are required to determine whether conditions in 10D-B are consistently anaerobic.

Geochemical data from 11 deep wells indicate 

that geochemical conditions varied greatly throughout 

the karst aquifer at the site (table 9). For example, during the August 20-22, 1997 sampling event, DO 

ranged from less than 0.1 to 5.5 mg/L, ammonia 

ranged from 0.02 to 2.30 mg/L, and sulfide ranged 

from 0.02 to 3.46 mg/L.

A large diverse microbial population was also 

present throughout the karst aquifer (table 10). The 

concentration of viable bacteria present in water samples from the deep wells was higher than expected. For 

example, aerobic heterotrophic bacteria concentrations 

in water samples from the karst aquifer were greater 

than 6 x 104

 colony forming units per 100 mL of sample (table 12). This range of bacteria does not account 

for strict anaerobic bacteria or others that would not 

grow on the tryptic soy agar plates. Still, these unenriched bacteria concentrations occurring in the karst 

aquifer were equivalent to culture concentrations used 

in laboratory biodegradation studies (Dalton, 1977; 

Nelson and others, 1988). This suggests enough bacteria are present in the karst aquifer to support TCE 

biodegradation. 

The hydrologic, biological, and chemical data 

demonstrate the large range of hydrologic conditions 

in the karst aquifer and provide insight into its complexity. Some areas in the karst aquifer responded rapidly to rainfall events whereas other areas were more 

stable and changed little regardless of rainfall, probably reflecting longer ground-water residence times. 

This range of hydrologic conditions provides an 

opportunity for bacteria to enter, reproduce, and 

spread throughout the aquifer system. The bacteria 

appear to have flourished wherever conditions were 

right. As the bacteria multiplied, they influenced the 

geochemistry of the system and degraded chlorinated 

solvents. Despite the complexity of the karst system, 

the same biological degradation processes are active in 

this karst system as in any unconsolidated aquifer. The 

hydrologic, biological, and chemical data provide 

multiple lines of evidence, supported by laboratory 

microcosm results, that a variety of TCE biodegradation pathways are active in the karst aquifer (table 13). 

The central issue for bioremediation is not whether 

biodegradation is occurring in the karst aquifer, but 

whether water is retained long enough in the karst 

aquifer to degrade a meaningful mass of contaminants. 

The observation of dyes in various deep wells 5 years 

after injection and the stable anaerobic conditions in 

parts of the aquifer indicate long local residence times. 

This study, however, does not determine what volume 

of the aquifer is represented by such areas and how 

much contamination they contain. 

LESSONS LEARNED

This project examined the potential for biological degradation of TCE in karst aquifers. Biodegradation is often presumed to be an irrelevant attenuation 

process in karst aquifers because of short ground-water 

residence times, lack of bacteria, and unsuitable environmental conditions. To address these issues, hydrologic, biological, and geochemical information was 

gathered. The greatest challenge of this investigation 

was interpreting the results within the framework of 

the complex karst hydrology. Examining biodegradation in the shallow water-bearing zone near the top of 

the limestone bedrock was relatively straightforward. 

Identification was possible of a chlorinated-solvent 

plume, a general ground-water flow path, and a 

sequential pattern of oxidation-reduction zones in the 
40 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

shallow water-bearing zone. Two lines of evidence, 

geochemical and chlorinated-ethene data, sufficed to 

demonstrate the occurrence of TCE biodegradation in 

the shallow anaerobic zone (fig. 28). Since the aim of 

this study was to determine if biological degradation of 

chlorinated solvents was occurring in the karst aquifer, 

further examination of the shallow water-bearing zone 

was not done.

Byproducts of TCE reductive dechlorination 

were detected in water samples from the deeper karst 

aquifer; however, additional data was required to 

determine if the degradation byproducts were simply 

transported from the shallow water-bearing zone or if 

they were the result of biodegradation processes 

within the karst aquifer. Also, the complexity of the 

karst aquifer system made computerized fate-andtransport models useless for this site. In the karst aquifer, ground-water zones in close proximity to each 

other often varied in hydrology, biology, and 

geochemistry.

Geochemical parameters and chlorinated solvents measured quarterly in deep wells provided some 

understanding of spatial and temporal patterns in the 

karst aquifer. These data demonstrated that conditions 

were favorable for reductive dechlorination or cometabolic degradation pathways in various parts of the 

karst aquifer. After four sampling events, evidence 

showed that temporal patterns were not adequately 

characterized by the quarterly sampling schedule. 

Continuous monitoring devices were placed in four 

karst aquifer wells to gather information on temperature, pH, DO, ORP, specific conductance, and water 

levels to monitor aquifer stability. These continuous 

monitoring devices provided some of the most important information for identifying areas capable of sustaining biological degradation processes. The 

continuous monitoring devices confirmed water storage areas and active flow zones in the karst aquifer by 

linking specific conductance, ORP, and DO with 

hydrogeologic and weather information. This continuous monitoring information was used to interpret the 

bacteria and microcosms results (fig. 29).

Biological information similar to that described 

in other studies (Wilson and others, 1996) was gathered during the investigation. Additional biological 

data were needed because of the widespread perception that karst aquifers are not able to sustain large 

microbial populations. Determining if a sufficiently 

large and diverse microbial population capable of biodegrading the chlorinated solvents existed in the karst 

aquifer was critical for this investigation (fig. 30). This 

line of evidence was established by identifying bacteria known to degrade chlorinated solvents using 

molecular methods and traditional plate counts. 

Microcosms prepared from non-enriched well water 

demonstrated cometabolism or reductive dechlorination of TCE. Data resulting from these activities 

provided biological evidence that sufficient microbes 

were present in the karst aquifer and were capable of 

degrading TCE under expected site conditions. 

Table 13. Summary and interpretation of multiple lines of evidence using data collected from deep wells

Well Geochemical indicators

(quarterly data)

Geochemical indicators

(continuous monitoring data) Chlorinated-ethene data

1D Anaerobic, nitrate-reducing 

conditions. 

Anaerobic, sulfate-reducing or methanogenic conditions.cis-1,2-Dichloroethylene, vinyl chloride, ethene, and ethane detected. 

3D Anaerobic, nitrate-reducing conditions. Possible sulfate-reducing 

conditions at times (high sulfide 

concentrations detected).

Anaerobic, sulfate-reducing or methanogenic conditions. Aerobic conditions 

when the 9D pump-and-treat well is 

not operating.

cis-1,2-Dichloroethylene, vinyl chloride, and ethane detected. 

2D Fluctuates between aerobic and 

anaerobic conditions. Possible 

ammonia oxidization and nitrate 

reduction during aerobic and 

anaerobic conditions, respectively. 

Rapid fluctuations between aerobic and 

anaerobic conditions. Oxidationreduction potentials indicate manganese or iron-reducing levels.

cis-1,2-Dichloroethylene, vinyl chloride, ethene, and ethane detected.

12D Fluctuates between aerobic and 

anaerobic conditions. Possible 

ammonia oxidization and nitrate 

reduction during aerobic and 

anaerobic conditions, respectively. 

Anaerobic conditions only present for a 

few days after rainfall events. During 

dry periods, reducing conditions may 

be present for a few weeks.

cis-1,2-Dichloroethylene, vinyl chloride, ethene, and ethane detected. 

Higher molar concentrations of 

trichloroethylene than degradation 

products detected.

Other 

wells.

Aerobic and anaerobic conditions Data not available Reductive-dechlorination degradation 

products detected.
Lessons Learned 41

Figure 28. Lines of evidence needed to evaluate biodegradation

of chlorinated solvents in the shallow water-bearing zone. 

Concluded that reductive

dechlorination was occurring 

in anaerobic zones.

Chlorinated-ethene data used 

to identify decreasing

concentrations of TCE and 

increasing concentrations of 

VC and cDCE.

Geochemical data used to identify 

various oxidation-reduction zones

ANAEROBIC

ZONES

Chlorinated-ethene data used 

to identify decreasing

concentrations of TCE, 

cDCE, and VC. 

Conditions were determined 

appropriate for aerobic 

degradation such as 

cometabolism.

Additional data (bacteria

identification and microcosm) 

needed to verify aerobic 

biodegradation was occurring.

AEROBIC

ZONES

Table 13. Summary and interpretation of multiple lines of evidence using data collected from deep wells—Continued

Well Bacteria-identification data Microcosms Conclusions

1D Ammonia oxidizers not detected. 

Methanotrophs detected. Data for 

other types of bacteria not available.

No significant aerobic degradation. Reductive-dechlorination degradation products 

present after a 10-month incubation.

Reductive dechlorination possible. No 

aerobic degradation because of the continuously anaerobic conditions.

3D Ammonia oxidizers, methanotrophs, 

manganese and iron oxidizers, and 

sulfate reducers detected.

Significant aerobic degradation during a 

3-week incubation. Reductive-dechlorination degradation products present 

after a 10-month incubation.

Reductive dechlorination possible. Aerobic degradation may occur when the 9D 

pump-and-treat well is not operating.

2D Methanotrophs, manganese and iron 

oxidizers, and sulfate reducers 

detected.

Significant aerobic degradation during a 

3-week incubation. Reductive-dechlorination degradation products present 

after a 10-month incubation.

Rapid fluctuations between aerobic and 

anaerobic conditions may prevent 

reductive dechlorination from occurring. Some aerobic degradation is 

possible.

12D Ammonia oxidizers, methanotrophs, 

manganese and iron oxidizers, and 

sulfate reducers detected.

No significant aerobic degradation. 

Reductive-dechlorination degradation 

products present after a 10-month incubation.

Geochemical conditions would limit 

reductive dechlorination. Aerobic bacteria were present, but no aerobic 

degradation in microcosms.

Other 

wells.

Aerobic and anaerobic bacteria detected 

in samples from 10D-B and 16D-B.

Microcosm data not available Potential aerobic and anaerobic degradation. Additional data needed.

Figure 28. Lines of evidence needed to evaluate biodegradation of chlorinated solvents 

in the shallow water-bearing zone.
42 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Figure 29. Lines of evidence needed to evaluate biodegradation of chlorinated ethenes in 

the karst aquifer.

Geochemical conditions identified

(quarterly sampling results)

Used microcosms to determine 

if bacteria can degrade

chlorinated ethenes

Examined chlorinated-ethene

data for byproducts

Identified and enumerated 

bacteria present in aquifer

Temporal changes in

geochemistry of karst aquifer 

used to interpret results and 

assign significance of various 

evidence.

Hydrogeology

(residence time)

Geochemistry Biological community

BIODEGRADATION OF

CHLORINATED ETHENES

IN KARST AQUIFERS

Figure 30. Hydrogeology determines residence time of ground water, 

which influences the biological community, the geochemistry, and the 

biodegration of chlorinated ethenes in karst aquifers.

Hydrogeology was the principal factor influencing the biological and geochemical conditions in the 

karst aquifers (fig. 30). Therefore, the chemical and 

biological evidence had to be considered in the context 

of the hydrology. For example, temporal and spatial 

continuity of the aquifer geochemistry and ORP had to 

be taken into account before applying the results from 

the microcosm studies to the karst aquifer. Results 

from the microcosm studies indicated that anaerobic 

reductive dechlorination was possible in each well 

given strong anaerobic conditions. However, 

geochemical conditions in some parts of the aquifer 

fluctuate with the weather, preventing reductive 

dechlorination from occurring to the same extent as in 

the microcosms. 

Although hydrology had the principal influence 

on the karst system, the appropriate geochemical conditions and biological assemblages also had to be 

present for TCE biodegradation to occur. Individually, 

hydrologic, biological, or chemical observations were 

insufficient to prove that TCE biodegradation was 

occurring in the karst aquifer. Together they provided 

convincing evidence that anaerobic and aerobic biological degradation processes were active in distinct 

areas of the karst aquifer.

SUMMARY AND CONCLUSIONS

The biodegradation of chlorinated ethenes in 

aquifers consisting of unconsolidated material has 

been well documented; however, chlorinated-ethene 

biodegradation has not been adequately investigated in 

karst areas even though chlorinated-solvent biodegradation products have been detected in karst aquifers. 

Factors that affect the biodegradation of chlorinated 

Figure 29. Lines of evidence needed to evaluate biodegradation of chlorinated ethenes in the karst aquifer.

Figure 30. Hydrogeology determines residence time of ground water, which 

influences the biological community, the geochemistry, and the biodegradation of 

chlorinated ethenes in karst aquifers.
Summary and Conclusions 43

solvents include hydrology, microbiology, and 

geochemistry.

Three categories of metabolic processes are 

involved in the biological degradation of chlorinated 

ethenes. Reductive dechlorination is an anaerobic process in which chlorinated ethenes are used as electron 

acceptors and results in a sequential dechlorination 

from PCE to TCE to DCE to VC to ethene. Cometabolism is an aerobic process in which chlorinated ethenes 

are degraded as a result of oxygenase enzymes, such 

as methane and ammonia monooxygenase, inserting 

oxygen into TCE, DCE, or VC molecules. Cometabolism needs additional substrates like methane or 

ammonia, as well as dissolved oxygen, to sustain the 

process. Direct oxidation is an aerobic or mildly anaerobic process in which lightly chlorinated ethenes 

(DCE and VC) are used as electron donors. At a given 

site, one or all of these processes could be occurring 

depending on environmental conditions. 

Most studies of contaminant biodegradation in 

fractured rock or karst settings examined biodegradation in the overburden or saprolite above bedrock. The 

lack of studies examining biodegradation in karst 

aquifers may be due to the widespread perception that 

the hydrologic and microbiologic characteristics of 

karst aquifers prevent biodegradation of contaminants 

in karst aquifers. Previous research, however, has indicated that large volumes of water may be isolated from 

active ground-water flow paths in karst aquifers. Other 

studies have shown that water from bedrock aquifers 

may contain large and diverse microbial populations, 

which include bacteria responsible for the degradation 

of chlorinated ethenes. 

The biodegradation of chlorinated ethenes was 

examined at a TCE contaminated karst site in Middle 

Tennessee. A shallow water-bearing zone is present at 

the site and chlorinated-ethenes are transported along 

a trough in the bedrock surface. Some deep wells 

intersect fractures that are part of the active groundwater flow system of the karst aquifer, whereas other 

deep wells intersect fractures isolated from the active 

ground-water flow system. Pump-and-treat wells completed in the upper part of the Ridley Limestone draw 

down water levels in many of the deep wells and affect 

local ground-water flow.

Multiple lines of evidence usually are needed to 

evaluate potential biodegradation processes. These 

lines of evidence normally include: (1) geochemical 

data that indicate depletion of electron donors and 

acceptors and increasing concentrations of metabolic 

byproducts, (2) chemical data that indicate decreasing 

concentrations of chlorinated solvents and increasing 

concentrations of degradation products, and (3) laboratory or field microbiological data that indicate the 

bacteria present at a site can degrade contaminants. 

Chlorinated-ethene, ethene, and ethane data for water 

samples from shallow and deep wells were obtained 

from TDEC-DSF files. Additional data-collection 

activities conducted by the USGS included periodic 

water-quality sampling of selected shallow and deep 

monitoring wells, collection of water samples from 

selected deep wells for bacteria identification and enumeration, continuous monitoring of water quality and 

water levels for selected deep wells, and microcosm 

studies. 

Multiple lines of evidence indicate that reductive dechlorination was the dominant biodegradation 

process occurring in the anaerobic shallow waterbearing zone underneath the manufacturing building. 

Ground water in the shallow water-bearing zone was 

influenced by precipitation recharge as the ground 

water moved away from the manufacturing building. 

The recharge water supplied DO and diluted the contaminated water in this transition zone between the 

anaerobic zone upgradient and the aerobic zone downgradient. Geochemical data indicate aerobic bacteria 

capable of cometabolizing or directly oxidizing the 

chlorinated ethenes were active in the aerobic zone 

downgradient, further decreasing the concentration of 

chlorinated ethenes in the shallow water-bearing zone. 

Undoubtedly some of the water, carrying with it bacteria, contaminants, electron donors, and electron acceptors, in the shallow water-bearing zone also migrates 

down into the karst aquifer.

Water-quality conditions in the karst aquifer 

varied both spatially and temporally. Significant concentrations of chlorinated-ethene biodegradation products were detected in the karst aquifer; however, the 

biodegradation products could have been transported 

from the shallow water-bearing zone. Because of the 

complex hydrology at the site, it was not possible to 

identify a chlorinated-ethene plume or discrete 

oxidation-reduction zones. The deep wells were 

treated as individual areas instead of as a direct continuum along a flow path. For example, well 12D is 

thought to intersect an active flow conduit in the aquifer. Wells 1D and 3D intersect water-bearing zones in 

the aquifer with less active flow. The data indicated 

that samples from wells which intersect the active 

ground-water flow system of the karst aquifer 
44 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

(well 12D) contained bacteria that dechlorinated TCE; 

however, the geochemical conditions present would 

limit the occurrence of reductive dechlorination. Samples from wells which intersected fractures isolated 

from the active ground-water flow system (well 1D) 

contained bacteria which could dechlorinate TCE. 

Anaerobic conditions persisted in these zones and the 

geochemical conditions in these fractures were suitable for reductive dechlorination. 

A vast consortia of bacteria capable of degrading chlorinated ethenes were detected in the water 

samples from the karst aquifer. Bacteria capable of 

cometabolism and direct oxidation of chlorinated 

ethenes were identified in water samples from wells 

that fluctuated between aerobic and anaerobic conditions (wells 2D and 3D). During periods of anaerobic 

conditions, constituents essential to cometabolism 

such as methane, ethane, and ammonia could be produced. Microcosm results indicated that the aerobic 

bacteria in samples from the karst aquifer could 

quickly (within less than 3 weeks) degrade TCE. 

The greatest challenge to this investigation was 

interpreting the results within the framework of the 

complex karst hydrology. Data such as continuous 

water-quality monitoring and microbiological data 

were necessary to compose sufficient evidence that 

significant biodegradation occurred in the karst aquifer. Continuous monitoring provided some of the most 

useful information about the geochemical conditions 

and variability in the karst aquifer. Together, the multiple lines of evidence helped to identify the relation 

between hydrology, geochemistry, and biology and the 

biodegradation of chlorinated ethenes in the karst 

aquifer.
Table 2 45

Table 2. Well completion and hydrogeologic data for selected wells

[--, data not available; altitudes are in meters above sea level; data from Tennessee Division of Superfund files]

Well

Top of

casing

Top of

Lebanon 

Limestone

Screen

interval

Bottom of 

benonite seal Notes

1D 232.48 227.0 171-192 194

2D 226.18 224.7 170-191 193

3D 231.31 229.4 175-193 194

4D 231.84 231.1 186-189 190

5D 232.14 229.8 188-191 191

7D 231.96 230.2 169-203 204 Well abandoned

8D 233.77 231.2 166-206 206 Well abandoned

9D 226.86 224.8 183-203 203 Pump-and-treat well

10D-A 221.58 219.3 203-206 207

10D-B 221.55 -- 185-188 188

10D-C 221.67 -- 197-200 201

11D 227.17 223.0 184-187 191

12D 223.81 222.5 203-206 206

13D 222.49 220.1 183-186 204 Pump-and-treat well

14D 222.57 220.1 180-183 205 Pump-and-treat well

15D-A 230.36 -- 209-212 210

15D-B 230.12 -- 186-189 190

15D-C 230.50 -- 168-174 176

16D -- -- -- --

1S 231.65 225.8 223- --

2S 231.65 225.8 223- -- Pump-and-treat well

3S 229.77 226.1 224- --

4S 228.40 226.1 224- --

5S 227.92 224.8 223- --

7S 230.64 227.1 225- --

9S 231.65 229.4 223- --

10S 227.71 226.9 223- --

11S 230.25 226.8 224- --

12S 231.86 227.8 226- --

13S 226.46 225.0 221- --

15S 230.93 228.9 226- --

16S 231.37 227.6 225- --

17S 231.36 228.9 226- --

18S 232.36 229.5 227- --

19S 232.36 230.9 228- --

20S 231.65 227.3 225- --

21S 231.49 229.3 226- --

25S 233.61 230.1 228- --

30S 231.33 -- 229- --

31S 231.58 -- 227- -- Pump-and-treat well

32S 231.55 -- 228- -- Pump-and-treat well

33S 231.52 -- 229- --

34S 230.75 -- 230- --

35S 224.12 -- 222- --

36S 224.70 -- 223- --

37S 222.76 -- 220- --

40S 230.83 -- 226- --

42S 230.55 -- 226- --

BN-1 227.09 -- 223- --

BN-2 225.61 -- 221- --

W-1 225.35 223.1 223- --

PL-1 225.52 223.1 223- --
46 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Table 8. Chlorinated-ethene data for samples from selected wells

[Data from Tennessee Division of Superfund files; TCE, trichloroethylene; cDCE, cis-1,2-dichloroethylene; VC, vinyl chloride; μg/L, micrograms per 

liter; --, no data; <, less than]

Date TCE cDCE VC Date TCE cDCE VC

Well sampled (μg/L) (μg/L) (μg/L) Well sampled (μg/L) (μg/L) (μg/L)

2S 10/31/85 950,000 -- -- 5S 08/10/96 137 89 <1

2S 11/05/85 750,000 -- -- 5S 11/10/96 97 46 4

2S 11/12/85 1,080,000 -- -- 5S 02/11/97 274 119 <1

2S 01/03/86 920,000 -- -- 5S 08/22/97 744 43 <1

2S 01/09/87 520,000 -- 5,300 5S 11/05/97 490 365 <1

2S 02/05/87 800,000 -- 88,000 5S 5/21/98 1,440 504 <1

2S 03/13/87 36,000 -- <10 6S 10/31/85 270 -- --

2S 11/13/88 730,000 -- -- 6S 11/05/85 490 -- --

2S 05/16/91 <10 -- <10 6S 11/12/85 720 -- --

2S 10/25/91 68,000 -- <5,000 6S 01/03/86 250 -- --

2S 02/10/92 80,000 -- 4,500 6S 01/09/87 32 -- <10

2S 04/17/92 170,000 -- <5,000 6S 02/05/87 120 -- 1,300

2S 07/31/92 1,800,000 -- <200,000 6S 03/13/87 <10 -- <10

2S 02/24/93 48,000 -- 1,900 6S 11/13/88 62 -- --

2S 05/14/93 52,000 -- 2,200 6S 05/16/91 2,100 -- <10

2S 05/01/94 300,000 100,000 3,000 6S 10/25/91 22 -- <10

2S 08/01/94 350,000 <20 2,900 6S 02/10/92 15 -- <10

2S 11/01/94 190,000 54,000 <500 6S 04/17/92 3 -- 23

2S 02/01/95 140,000 53,000 3,400 6S 07/31/92 5 -- <10

2S 05/01/95 170,000 46,000 <500 6S 10/30/92 8 -- <10

2S 08/01/95 270,000 35,000 <5,000 6S 02/24/93 <5 -- <10

2S 11/01/95 129,600 36,900 2,160 6S 05/14/93 3 -- <10

2S 02/01/96 151,200 36,450 2,300 6S 10/22/93 <5 -- 75

2S 05/10/96 352,000 48,000 3,300 6S 02/09/94 4 -- <10

2S 08/10/96 287,000 43,000 4,000 6S 05/01/94 95 1,300 <25

2S 11/10/96 186,000 32,000 2,050 6S 08/01/94 <25 340 <25

2S 02/11/97 80,200 45,200 1,810 6S 11/01/94 1 32 12

2S 08/22/97 171,000 34,500 <500 6S 02/01/95 <1 170 1

2S 5/21/98 92,250 27,090 1,080 6S 05/01/95 <1 86 21

5S 10/31/85 5,600 -- -- 6S 08/01/95 <3 39 <3

5S 11/05/85 9,800 -- -- 6S 11/01/95 <2 98 14

5S 11/12/85 14,800 -- -- 6S 02/01/96 <1 136 40

5S 01/03/86 16,600 -- -- 6S 05/10/96 <1 170 49

5S 01/09/87 110,000 -- 900 6S 08/10/96 <1 516 106

5S 02/05/87 280,000 -- 21,000 6S 11/10/96 12 81 14

5S 03/13/87 130,000 -- <10 6S 02/14/97 3 114 22

5S 11/13/88 68,100 -- -- 6S 08/27/97 <1 80 42

5S 05/16/91 320,000 -- 4,300 6S 5/21/98 <1 86 27

5S 10/25/91 3,000 -- <100 7S 10/31/85 48,000 -- --

5S 02/10/92 910 -- 30 7S 11/05/85 31,000 -- --

5S 04/17/92 470 -- 10 7S 11/12/85 71,000 -- --

5S 07/31/92 5,400 -- <500 7S 01/03/86 31,000 -- --

5S 10/30/92 4,400 -- <500 7S 01/09/87 430,000 -- 56

5S 02/24/93 2,700 -- <250 7S 02/05/87 120,000 -- <10

5S 05/14/93 160,000 -- <500 7S 03/13/87 150,000 -- <10

5S 02/09/94 980 -- <100 7S 11/13/88 18,900 -- --

5S 05/01/94 650 1,700 <25 7S 05/16/91 95,000 -- 400

5S 08/01/94 2,400 960 <25 7S 10/25/91 59,000 -- 290

5S 11/01/94 1,300 170 <25 7S 02/10/92 75,000 -- <5,000

5S 02/01/95 1,100 300 <50 7S 04/17/92 200,000 -- <5,000

5S 05/01/95 160 170 <50 7S 07/31/92 160,000 -- <10,000

5S 08/01/95 200 110 <5 7S 10/30/92 92,000 -- <10,000

5S 11/01/95 148 108 12 7S 02/24/93 93,000 -- <5,000

5S 02/01/96 71 110 3 7S 05/14/93 140,000 -- <10,000

5S 05/10/96 221 285 <1 7S 10/22/93 82,000 -- <10,000
Table 8 47

Table 8. Chlorinated-ethene data for samples from selected wells—Continued

Date TCE cDCE VC Date TCE cDCE VC

Well sampled (μg/L) (μg/L) (μg/L) Well sampled (μg/L) (μg/L) (μg/L)

7S 02/09/94 87 -- <10 20S 02/01/95 49,000 110,000 5,100 

7S 05/01/94 140,000 120,000 <500 20S 05/01/95 52,000 64,000 2,400 

7S 08/01/94 16,000 22,000 500 20S 08/01/95 120,000 79,000 4,800 

7S 11/01/94 17,000 28,000 <500 20S 11/01/95 6,300 1,200 36

7S 02/01/95 6,400 23,000 <500 20S 02/01/96 60,750 28,350 4,100 

7S 05/01/95 18,000 28,000 <500 20S 05/10/96 92,000 124,000 8,500 

7S 08/01/95 47,000 52,000 <500 20S 08/10/96 80,000 115,000 10,000 

7S 11/01/95 12,800 24,920 146 20S 11/10/96 78,000 102,000 8,500 

7S 02/01/96 810 1,163 21 20S 02/19/97 31,200 99,200 10,200 

7S 05/10/96 10,930 10,100 70 20S 08/27/97 40,000 77,000 3,000 

7S 08/10/96 5,660 7,850 85 20S 5/21/98 18,000 76,950 9,180

7S 11/10/96 8,350 8,750 64 33S 10/22/93 <5 -- 2,000

7S 02/11/97 808 1,110 5 33S 02/09/94 <100 -- 480

7S 08/22/97 6,600 13,800 <1 33S 05/01/94 2 110 140

7S 11/05/97 3,800 16,300 61 33S 08/01/94 1 1 5

7S 5/21/98 7,785 27,140 <450 33S 11/01/94 1 2,400 4,100

11S 01/09/87 35 -- <10 33S 02/01/95 3 2,400 2,900

11S 02/05/87 <10 -- 11 33S 05/01/95 300 3,400 1,500

11S 03/13/87 <10 -- <10 33S 08/01/95 140 4,800 1,600

11S 11/13/88 36 -- -- 33S 11/01/95 12 3,500 1,900

11S 02/10/92 14 -- <10 33S 02/01/96 <1 7,200 2,500

11S 04/17/92 <5 -- <10 33S 05/10/96 <100 10,700 3,100

11S 07/31/92 13 -- <10 33S 08/10/96 <100 11,200 4,600

11S 10/30/92 33 -- <10 33S 11/10/96 <100 17,700 4,900

11S 02/24/93 17 -- <10 33S 02/11/97 39 14,300 2,980

11S 05/14/93 20 -- <10 33S 08/22/97 <100 14,600 3,500

11S 10/22/93 31 -- <10 33S 5/21/98 <900 30,330 2,250

11S 02/09/94 18 -- <10 34S 10/22/93 <500 -- 770

11S 05/01/94 18 58 <1 34S 02/09/94 25,000 -- <5,000

11S 08/01/94 18 50 <1 34S 05/01/94 55 400 720

11S 11/01/94 15 41 <1 34S 08/01/94 140 1,800 2,400

11S 02/01/95 14 33 <1 34S 11/01/94 8,400 8,200 <100

11S 05/01/95 7 25 <1 34S 02/01/95 810 2,900 1,300

11S 08/01/95 11 26 <1 34S 05/01/95 300 2,500 130

11S 11/01/95 8 21 <1 34S 08/01/95 180 5,700 670

11S 02/01/96 9 33 <1 34S 11/01/95 675 5,490 900

11S 05/10/96 7 20 <1 34S 02/01/96 1,140 3,290 710

11S 08/10/96 6 38 <1 34S 05/10/96 460 15,620 3,940

11S 11/10/96 9 26 <1 34S 08/10/96 368 3,200 1,400

11S 02/18/97 7 40 <1 34S 11/10/96 115 1,850 709

11S 08/27/97 6 32 <1 34S 02/18/97 1,200 3,800 1,130

11S 5/21/98 <1 2 <1 34S 08/26/97 2 820 350

20S 01/09/87 270,000 -- <2,000 34S 5/21/98 212 1,449 338

20S 02/05/87 530,000 -- <10 35S 10/22/93 <5 -- <10

20S 03/13/87 500,000 -- <10 35S 02/09/94 6 -- <10

20S 11/13/88 79,000 -- -- 35S 05/01/94 280 110 2

20S 05/16/91 61,000 -- <10 35S 08/01/94 2 11 1

20S 10/25/91 54,000 -- <5,000 35S 11/01/94 5 38 <1

20S 02/10/92 290,000 -- <10,000 35S 02/01/95 <3 16 <3

20S 04/17/92 13,000 -- 11,000 35S 05/01/95 <1 7 <1

20S 10/30/92 460,000 -- <10,000 35S 08/01/95 3 27 <1

20S 02/24/93 360,000 -- <10,000 35S 11/01/95 6 38 8

20S 05/14/93 960,000 -- 4,700 35S 02/01/96 4 24 <1

20S 10/22/93 380,000 -- <25,000 35S 05/10/96 3 16 <1

20S 02/09/94 558,000 -- <50,000 35S 08/10/96 <1 20 3

20S 05/01/94 49,000 36,000 1,700 35S 11/10/96 1 18 2

20S 08/01/94 13,000 150,000 3,000 35S 02/13/97 4 8 <1

20S 11/01/94 180,000 90,000 <500 35S 08/27/97 2 29 5
48 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Table 8. Chlorinated-ethene data for samples from selected wells—Continued

Date TCE cDCE VC Date TCE cDCE VC

Well sampled (μg/L) (μg/L) (μg/L) Well sampled (μg/L) (μg/L) (μg/L)

35S 5/21/98 2 17 1 1D 11/05/97 3 35 119

37S 10/22/93 <5 -- <10 1D 5/21/98 8 12 5

37S 02/09/94 <5 -- <10 2D 02/14/89 <10 -- --

37S 05/01/94 <5 <5 <5 2D 04/20/89 <5 -- --

37S 08/01/94 <1 <5 3 2D 02/28/91 3,900 -- --

37S 11/01/94 <1 <1 <1 2D 04/11/91 5,200 -- 16

37S 02/01/95 <1 <1 <1 2D 07/25/91 16,000 -- 13

37S 05/01/95 <1 <1 <1 2D 10/18/91 90,000 -- 440

37S 08/01/95 <1 <1 <1 2D 02/19/92 130,000 -- 1,400 

37S 11/01/95 <1 <1 <1 2D 04/15/92 68,000 -- <5,000

37S 02/01/96 <1 <1 <1 2D 07/31/92 59,000 -- <5,000

37S 05/10/96 <1 1 <1 2D 10/30/92 130,000 -- <10

37S 08/10/96 <1 <1 <1 2D 02/26/93 51,000 -- <10,000

37S 11/10/96 <1 <1 <1 2D 05/19/93 17,000 -- <1,000

37S 02/18/97 <1 <1 <1 2D 10/22/93 27,000 -- 6,800 

37S 08/27/97 <1 <1 <1 2D 02/10/94 8,000 -- 1,000 

37S 5/21/98 <1 <1 <1 2D 05/01/94 14,000 16,000 <25

42S 08/01/94 1 10 1 2D 08/01/94 11,000 3,900 <250

42S 11/01/94 250 <50 <50 2D 11/01/94 38,000 12,000 <500

42S 02/01/95 <1 <1 <1 2D 02/01/95 1,700 540 <1

42S 05/01/95 <1 <1 <1 2D 05/01/95 510 8,300 <500

42S 08/01/95 <1 <1 <1 2D 08/01/95 8,300 2,000 <100

42S 11/01/95 <1 <1 <1 2D 11/01/95 101,700 106,200 4,500

42S 02/01/96 <1 <1 <1 2D 02/01/96 2,990 6,400 1,005

42S 05/10/96 <1 1 <1 2D 05/10/96 1,650 5,100 1,165

42S 08/10/96 <1 <1 <1 2D 08/10/96 1,500 2,300 50

42S 11/10/96 <1 <1 <1 2D 11/10/96 1,590 2,680 <10

42S 08/18/97 <1 <1 <1 2D 02/11/97 915 1,390 245

42S 08/27/97 <1 <1 <1 2D 05/20/97 5,290 520 38

42S 5/21/98 <1 4 <1 2D 08/22/97 168 467 256

1D 02/14/89 3,998,000 -- -- 2D 11/05/97 880 5,700 760

1D 04/20/89 260,000 -- -- 2D 2/17/98 597 1,070 156

1D 02/28/91 180 -- -- 2D 5/21/98 116 511 73

1D 04/11/91 180 -- 160 3D 01/25/90 26,000 -- --

1D 07/25/91 <10 -- 400 3D 02/28/91 2,900 -- --

1D 10/18/91 96 -- 17 3D 04/11/91 2,200 -- 120

1D 02/06/92 12 -- 2,600 3D 07/25/91 240 -- 13

1D 04/15/92 54 -- 8 3D 10/18/91 <5 -- 390

1D 07/31/92 48 -- <10 3D 02/06/92 130 -- 170

1D 10/30/92 <5 -- <10 3D 04/15/92 34 -- <10

1D 02/26/93 32 -- 450 3D 07/31/92 1,100 -- <50

1D 05/19/93 32 -- 9 3D 10/30/92 30 -- <10

1D 10/22/93 18 -- 1,000 3D 02/26/93 11 -- <10

1D 02/10/94 7 -- 3,500 3D 05/19/93 14 -- <10

1D 05/01/94 29 14 19 3D 10/22/93 <5 -- <10

1D 08/01/94 140 24 <3 3D 02/10/94 12 -- 16

1D 11/01/94 270 300 46 3D 05/01/94 39 25 19

1D 02/01/95 9 120 110 3D 08/01/94 3 48 12

1D 05/01/95 3 130 220 3D 11/01/94 43 47 <1

1D 08/01/95 5 18 25 3D 02/01/95 <1 2 <1

1D 11/01/95 28 47 65 3D 05/01/95 4 7 <1

1D 02/01/96 8 119 740 3D 08/01/95 5 7 <1

1D 05/10/96 10 81 940 3D 11/01/95 6 5 10

1D 08/10/96 89 20 24 3D 02/01/96 4 7 2

1D 11/10/96 55 13 46 3D 05/10/96 <1 1 <1

1D 02/11/97 <1 6 5 3D 08/10/96 1 725 176

1D 08/22/97 14 10 3 3D 11/10/96 3 183 73
Table 8 49

Table 8. Chlorinated-ethene data for samples from selected wells—Continued

Date TCE cDCE VC Date TCE cDCE VC

Well sampled (μg/L) (μg/L) (μg/L) Well sampled (μg/L) (μg/L) (μg/L)

3D 02/12/97 2 60 19 10D-A 02/06/92 3,600 -- <100

3D 08/22/97 2 6 <1 10D-A 02/12/92 3,100 -- <100

3D 11/05/97 <1 19 <1 10D-A 04/15/92 1,800 -- <250

3D 5/21/98 <1 230 140 10D-A 07/31/92 340 -- <100

4D 01/25/90 2,300 -- -- 10D-A 10/30/92 900 -- <10

4D 02/28/91 210 -- -- 10D-A 02/26/93 190 -- <10

4D 04/11/91 180 -- 150 10D-A 05/19/93 280 -- <100

4D 07/25/91 48 -- <10 10D-A 10/22/93 780 -- <100

4D 10/18/91 <5 -- <10 10D-A 02/10/94 160 -- <20

4D 02/06/92 31 -- 12 10D-A 05/01/94 110 33 <5

4D 04/15/92 1,100 -- <10 10D-A 08/01/94 70 71 <1

4D 07/31/92 250 -- <100 10D-A 11/01/94 120 79 <1

4D 10/30/92 <5 -- <10 10D-A 02/01/95 5 34 <1

4D 02/26/93 2 -- <10 10D-A 05/01/95 73 42 <1

4D 05/19/93 190 -- <10 10D-A 08/01/95 29 15 <1

4D 10/22/93 <5 -- <10 10D-A 11/01/95 86 39 <2

4D 02/10/94 <5 -- <10 10D-A 02/01/96 46 24 <1

4D 05/01/94 43 7 <1 10D-A 05/10/96 39 15 <1

4D 08/01/94 6 1 <1 10D-A 08/10/96 10 8 <1

4D 11/01/94 82 27 <1 10D-A 11/10/96 30 14 <1

4D 02/01/95 <1 3 <1 10D-A 02/21/97 35 18 <1

4D 05/01/95 9 3 <1 10D-A 08/26/97 1 <1 <1

4D 08/01/95 <1 <1 <1 10D-A 5/21/98 7 16 4

4D 11/01/95 2 <1 <1 10D-B 02/06/92 280 -- <10

4D 02/01/96 1 2 <1 10D-B 02/19/92 150 -- <10

4D 05/10/96 7 4 <1 10D-B 04/15/92 160 -- <10

4D 08/10/96 98 524 2 10D-B 07/31/92 140 -- <10

4D 11/10/96 8 60 7 10D-B 10/30/92 120 -- <10

4D 02/12/97 2 6 <1 10D-B 02/26/93 120 -- 15

4D 08/22/97 <1 <1 <1 10D-B 05/19/93 81 -- 32

4D 5/21/98 <1 <1 <1 10D-B 10/22/93 150 -- <10

5D 01/25/90 7 -- -- 10D-B 02/10/94 75 -- <4

5D 02/28/91 <5 -- -- 10D-B 05/01/94 51 45 6

5D 04/11/91 <5 -- <5 10D-B 08/01/94 59 91 6

5D 07/25/91 <10 -- <10 10D-B 11/01/94 32 130 33

5D 10/18/91 93 -- <5 10D-B 02/01/95 19 30 3

5D 02/06/92 20 -- <10 10D-B 05/01/95 33 45 <1

5D 04/15/92 1,300 -- <10 10D-B 08/01/95 37 38 <1

5D 07/31/92 49 -- <10 10D-B 11/01/95 60 46 10

5D 10/30/92 360 -- <100 10D-B 02/01/96 35 37 4

5D 02/26/93 <5 -- <10 10D-B 05/10/96 50 47 1

5D 05/19/93 170 -- <10 10D-B 08/10/96 11 42 14

5D 10/22/93 45 -- <10 10D-B 11/10/96 50 44 <1

5D 02/10/94 <5 -- <10 10D-B 02/21/97 24 38 1

5D 05/01/94 15 25 <1 10D-B 05/22/97 46 35 1

5D 08/01/94 31 5 <1 10D-B 08/22/97 <1 6 6

5D 11/01/94 20 7 <1 10D-B 11/04/97 9 15 8

5D 02/01/95 6 11 <1 10D-B 2/17/98 <2 <2 <2

5D 05/01/95 <1 <1 <1 10D-B 5/21/98 <1 <1 <1

5D 08/01/95 4 7 <1 10D-C 02/01/96 86 45 <1

5D 11/01/95 8 1 <2 10D-C 05/10/96 76 38 1

5D 02/01/96 <1 <1 <1 10D-C 08/10/96 36 33 <1

5D 05/10/96 <1 <1 <1 10D-C 11/10/96 35 25 <1

5D 08/10/96 <1 2 4 10D-C 02/21/97 21 20 <1

5D 11/10/96 2 <1 2 10D-C 08/26/97 7 4 <1

5D 02/13/97 <1 <1 <1 10D-C 5/21/98 <1 <1 <1

5D 08/26/97 2 <1 <1 11D 02/06/92 5,900 -- <100

5D 5/21/98 <1 <1 <1 11D 02/19/92 700 -- 13
50 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Table 8. Chlorinated-ethene data for samples from selected wells—Continued

Date TCE cDCE VC Date TCE cDCE VC

Well sampled (μg/L) (μg/L) (μg/L) Well sampled (μg/L) (μg/L) (μg/L)

11D 04/15/92 720 -- <50 12D 11/10/96 1,700 180 <10

11D 07/31/92 390 -- <100 12D 02/13/97 <1 <1 <1

11D 10/30/92 1,000 -- <50 12D 05/22/97 1,240 220 <10

11D 02/26/93 500 -- <50 12D 08/22/97 400 95 <1

11D 05/19/93 500 -- <100 12D 11/04/97 1,125 376 <1

11D 10/22/93 200 -- <100 12D 2/17/98 18 4 <2

11D 02/10/94 910 -- <50 12D 5/21/98 170 8 4

11D 05/01/94 1,400 1,400 10 13D 10/22/93 4,600 -- <10

11D 08/01/94 650 750 <25 13D 02/10/94 1,400 -- <100

11D 11/01/94 970 800 <50 13D 05/01/94 180 23 <5

11D 02/01/95 540 450 4 13D 08/26/97 255 203 3

11D 05/01/95 650 350 <25 13D 10/14/97 71 102 2

11D 08/01/95 540 420 <25 13D 11/05/97 62 72 <1

11D 11/01/95 420 335 9 14D 10/22/93 2,800 -- <10

11D 02/01/96 416 263 1 14D 02/10/94 610 -- 17

11D 05/10/96 140 410 <5 14D 05/01/94 470 140 12

11D 08/10/96 42 274 1 14D 08/01/94 210 130 <3

11D 11/10/96 68 171 <1 14D 11/01/94 130 150 <5

11D 02/12/97 3 142 <1 14D 02/01/95 150 170 2

11D 08/22/97 <1 71 <1 14D 05/01/95 180 84 <5

11D 5/21/98 <1 27 4 14D 08/01/95 120 92 <1

12D 02/06/92 200,000 -- <480 14D 11/01/95 198 138 16

12D 02/12/92 230,000 -- <1,000 14D 02/01/96 252 234 21

12D 04/15/92 14,000 -- <1,000 14D 05/10/96 428 230 10

12D 07/31/92 10,000 -- <1,000 14D 08/10/96 320 310 8

12D 10/30/92 2,300 -- <10 14D 11/10/96 290 232 6

12D 02/26/93 170 -- 15 14D 02/12/97 198 382 28

12D 05/19/93 2,100 -- 7 14D 05/22/97 520 310 <10

12D 10/22/93 2,200 -- <20 14D 08/26/97 270 330 24

12D 02/10/94 7 -- <10 14D 11/05/97 305 505 58

12D 05/01/94 1,400 550 5 16D-A 05/28/97 <1 2 <1

12D 08/01/94 1,800 1,100 <50 16D-A 08/26/97 <1 3 <1

12D 11/01/94 950 410 <5 16D-A 11/04/97 <1 2 <1

12D 02/01/95 620 670 3 16D-A 2/17/98 <2 <2 <2

12D 05/01/95 730 90 <50 16D-A 5/21/98 <1 <1 <1

12D 08/01/95 690 270 <25 16D-B 05/28/97 <1 2 <1

12D 11/01/95 920 240 11 16D-B 08/22/97 <1 5 <1

12D 02/01/96 1,500 300 6 16D-B 11/04/97 <1 3 <1

12D 05/10/96 1,250 250 <5 16D-B 2/17/98 <2 7 <2

12D 08/10/96 1,850 650 21 16D-B 5/21/98 <1 <1 <1
Table 11 51

Table 11. Microcosm data used to examine biodegradation of chlorinated ethenes

[Concentrations are in milligrams per liter; GC, gas chromatography; ECD, electron capture detector; PID, photoionization detector; cDCE, 

cis-1,2-dichloroethylene; ND, not detected; <, less than; --, no data] 

Microcosm identification

Experiment Treatment Treatment Time Replicate Trichloroethylene cDCE Vinyl chloride

number number description (weeks) number GC/ECD GC/PID GC/PID GC/PID

1 1 1D live 0 1 0.15 -- -- --

1 1 1D live 0 2 0.15 -- -- --

1 1 1D live 0 3 0.17 -- -- --

1 1 1D live 0 4 0.20 -- -- --

1 2 1D live 0 1 0.92 -- -- --

1 2 1D live 0 2 0.98 -- -- --

1 2 1D live 0 3 0.97 -- -- --

1 2 1D live 0 4 0.79 -- -- --

1 3 2D live 0 1 0.61 -- -- --

1 3 2D live 0 2 0.56 -- -- --

1 3 2D live 0 3 0.62 -- -- --

1 3 2D live 0 4 0.50 -- -- --

1 4 2D live 0 1 1.41 -- -- --

1 4 2D live 0 2 1.09 -- -- --

1 4 2D live 0 3 1.10 -- -- --

1 4 2D live 0 4 1.15 -- -- --

1 5 3D live 0 1 0.07 -- -- --

1 5 3D live 0 2 0.07 -- -- --

1 5 3D live 0 3 0.08 -- -- --

1 5 3D live 0 4 0.08 -- -- --

1 6 3D live 0 1 0.81 -- -- --

1 6 3D live 0 2 0.71 -- -- --

1 6 3D live 0 3 0.74 -- -- --

1 6 3D live 0 4 0.65 -- -- --

1 7 12D live 0 1 0.09 -- -- --

1 7 12D live 0 2 0.08 -- -- --

1 7 12D live 0 3 0.08 -- -- --

1 7 12D live 0 4 0.07 -- -- --

1 8 12D live 0 1 0.74 -- -- --

1 8 12D live 0 2 0.71 -- -- --

1 8 12D live 0 3 0.67 -- -- --

1 8 12D live 0 4 0.62 -- -- --

1 9 Unsterile control 0 1 -- -- -- --

1 9 Unsterile control 0 2 1.05 -- -- --

1 9 Unsterile control 0 3 0.87 -- -- --

1 9 Unsterile control 0 4 0.91 -- -- --

1 1 1D live 2 1 0.17 -- -- --

1 1 1D live 2 2 0.12 -- -- --

1 1 1D live 2 3 -- -- -- --

1 1 1D live 2 4 0.18 -- -- --

1 2 1D live 2 1 0.58 -- -- --

1 2 1D live 2 2 0.28 -- -- --

1 2 1D live 2 3 0.47 -- -- --

1 2 1D live 2 4 0.53 -- -- --

1 3 2D live 2 1 0.33 -- -- --

1 3 2D live 2 2 0.30 -- -- --

1 3 2D live 2 3 0.33 -- -- --

1 3 2D live 2 4 0.32 -- -- --

1 4 2D live 2 1 0.57 -- -- --
52 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Table 11. Microcosm data used to examine biodegradation of chlorinated ethenes—Continued

Microcosm identification

Experiment Treatment Treatment Time Replicate Trichloroethylene cDCE Vinyl chloride

number number description (weeks) number GC/ECD GC/PID GC/PID GC/PID

1 4 2D live 2 2 0.63 -- -- --

1 4 2D live 2 3 0.78 -- -- --

1 4 2D live 2 4 0.73 -- -- --

1 5 3D live 2 1 0.05 -- -- --

1 5 3D live 2 2 0.05 -- -- --

1 5 3D live 2 3 0.04 -- -- --

1 5 3D live 2 4 0.03 -- -- --

1 6 3D live 2 1 0.34 -- -- --

1 6 3D live 2 2 0.32 -- -- --

1 6 3D live 2 3 0.31 -- -- --

1 6 3D live 2 4 0.30 -- -- --

1 7 12D live 2 1 0.07 -- -- --

1 7 12D live 2 2 0.06 -- -- --

1 7 12D live 2 3 0.05 -- -- --

1 7 12D live 2 4 0.07 -- -- --

1 8 12D live 2 1 0.30 -- -- --

1 8 12D live 2 2 0.24 -- -- --

1 8 12D live 2 3 0.21 -- -- --

1 8 12D live 2 4 0.28 -- -- --

1 9 Unsterile control 2 1 0.30 -- -- --

1 9 Unsterile control 2 2 0.17 -- -- --

1 9 Unsterile control 2 3 0.13 -- -- --

1 9 Unsterile control 2 4 0.18 -- -- --

1 1 1D live 39 1 0.08 0.018 0.008 0.001

1 1 1D live 39 2 0.10 0.035 0.004 0.013

1 1 1D live 39 3 0.10 0.037 0.005 0.016

1 1 1D live 39 4 0.12 0.031 0.040 0.141

1 2 1D live 39 1 0.37 1.038 0.037 <0.002

1 2 1D live 39 2 0.56 0.824 0.106 <0.001

1 2 1D live 39 3 0.59 1.282 0.035 <0.001

1 2 1D live 39 4 0.66 0.963 2.878 <0.001

1 3 2D live 39 1 0.34 0.312 0.291 <0.001

1 3 2D live 39 2 0.32 0.331 0.096 <0.001

1 3 2D live 39 3 0.32 0.243 0.918 <0.001

1 3 2D live 39 4 0.42 0.458 0.118 <0.001

1 4 2D live 39 1 0.59 0.963 0.291 <0.002

1 4 2D live 39 2 0.88 1.038 0.162 <0.002

1 4 2D live 39 3 0.57 1.116 0.162 <0.002

1 4 2D live 39 4 0.75 1.197 0.111 <0.002

1 5 3D live 39 1 0.04 0.009 0.013 <0.001

1 5 3D live 39 2 0.03 0.007 0.179 0.001

1 5 3D live 39 3 0.03 0.007 0.495 <0.001

1 5 3D live 39 4 0.04 0.007 0.683 <0.001

1 6 3D live 39 1 0.51 0.759 0.131 <0.002

1 6 3D live 39 2 0.50 0.531 0.110 <0.002

1 6 3D live 39 3 0.48 0.584 0.131 <0.002

1 6 3D live 39 4 0.45 0.435 0.162 <0.002

1 7 12D live 39 1 0.09 0.027 0.855 <0.001

1 7 12D live 39 2 0.09 0.027 1.375 <0.001

1 7 12D live 39 3 0.07 0.023 0.179 <0.001
Table 11 53

Table 11. Microcosm data used to examine biodegradation of chlorinated ethenes—Continued

Microcosm identification

Experiment Treatment Treatment Time Replicate Trichloroethylene cDCE Vinyl chloride

number number description (weeks) number GC/ECD GC/PID GC/PID GC/PID

1 7 12D live 39 4 0.07 0.012 2.332 <0.001

1 8 12D live 39 1 0.46 0.391 1.056 <0.001

1 8 12D live 39 2 0.44 0.413 6.968 <0.001

1 8 12D live 39 3 0.43 0.331 0.320 <0.001

1 8 12D live 39 4 0.37 0.371 0.350 <0.001

1 9 Unsterile control 39 1 -- -- -- --

1 9 Unsterile control 39 2 -- -- -- --

1 9 Unsterile control 39 3 -- -- -- --

1 9 Unsterile control 39 4 -- -- -- --

2 1 1D live 0 1 1.74 -- -- --

2 1 1D live 0 2 1.90 -- -- --

2 1 1D live 0 3 1.69 -- -- --

2 1 1D live 0 4 1.11 -- -- --

2 2 2D live 0 1 1.60 -- -- --

2 2 2D live 0 2 1.38 -- -- --

2 2 2D live 0 3 1.75 -- -- --

2 2 2D live 0 4 1.37 -- -- --

2 3 3D live 0 1 1.03 -- -- --

2 3 3D live 0 2 1.25 -- -- --

2 3 3D live 0 3 1.19 -- -- --

2 3 3D live 0 4 1.27 -- -- --

2 4 12D live 0 1 2.14 -- -- --

2 4 12D live 0 2 3.10 -- -- --

2 4 12D live 0 3 2.19 -- -- --

2 4 12D live 0 4 2.57 -- -- --

2 5 2D/3D sterile 0 1 0.70 -- -- --

2 5 2D/3D sterile 0 2 0.83 -- -- --

2 5 2D/3D sterile 0 3 0.71 -- -- --

2 5 2D/3D sterile 0 4 0.66 -- -- --

2 1 1D live 3 1 0.70 -- -- --

2 1 1D live 3 2 1.05 -- -- --

2 1 1D live 3 3 0.92 -- -- --

2 1 1D live 3 4 1.59 -- -- --

2 2 2D live 3 1 ND -- -- --

2 2 2D live 3 2 ND -- -- --

2 2 2D live 3 3 <0.01 -- -- --

2 2 2D live 3 4 <0.01 -- -- --

2 3 3D live 3 1 <0.01 -- -- --

2 3 3D live 3 2 ND -- -- --

2 3 3D live 3 3 ND -- -- --

2 3 3D live 3 4 ND -- -- --

2 4 12D live 3 1 1.36 -- -- --

2 4 12D live 3 2 1.68 -- -- --

2 4 12D live 3 3 1.46 -- -- --

2 4 12D live 3 4 -- -- -- --

2 5 2D/3D sterile 3 1 0.43 -- -- --

2 5 2D/3D sterile 3 2 0.47 -- -- --

2 5 2D/3D sterile 3 3 0.34 -- -- --

2 5 2D/3D sterile 3 4 0.39 -- -- --

2 1 1D live 17 1 1.46 -- -- --
54 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

Table 11. Microcosm data used to examine biodegradation of chlorinated ethenes—Continued

Microcosm identification

Experiment Treatment Treatment Time Replicate Trichloroethylene cDCE Vinyl chloride

number number description (weeks) number GC/ECD GC/PID GC/PID GC/PID

2 1 1D live 17 2 1.21 -- -- --

2 1 1D live 17 3 0.72 -- -- --

2 1 1D live 17 4 1.04 -- -- --

2 2 2D live 17 1 ND -- -- --

2 2 2D live 17 2 ND -- -- --

2 2 2D live 17 3 <0.01 -- -- --

2 2 2D live 17 4 <0.01 -- -- --

2 3 3D live 17 1 ND -- -- --

2 3 3D live 17 2 <0.01 -- -- --

2 3 3D live 17 3 ND -- -- --

2 3 3D live 17 4 ND -- -- --

2 4 12D live 17 1 2.15 -- -- --

2 4 12D live 17 2 2.21 -- -- --

2 4 12D live 17 3 2.50 -- -- --

2 4 12D live 17 4 <0.01 -- -- --

2 5 2D/3D sterile 17 1 0.64 -- -- --

2 5 2D/3D sterile 17 2 0.51 -- -- --

2 5 2D/3D sterile 17 3 0.54 -- -- --

2 5 2D/3D sterile 17 4 0.27 -- -- --

2 1 1D live 22 1 0.39 -- -- --

2 1 1D live 22 2 0.44 -- -- --

2 1 1D live 22 3 0.26 -- -- --

2 1 1D live 22 4 <0.01 -- -- --

2 2 2D live 22 1 ND -- -- --

2 2 2D live 22 2 <0.01 -- -- --

2 2 2D live 22 3 <0.01 -- -- --

2 2 2D live 22 4 <0.01 -- -- --

2 3 3D live 22 1 <0.01 -- -- --

2 3 3D live 22 2 <0.01 -- -- --

2 3 3D live 22 3 <0.01 -- -- --

2 3 3D live 22 4 <0.01 -- -- --

2 4 12D live 22 1 0.94 -- -- --

2 4 12D live 22 2 0.05 -- -- --

2 4 12D live 22 3 <0.01 -- -- --

2 4 12D live 22 4 <0.01 -- -- --

2 5 2D/3D sterile 22 1 0.26 -- -- --

2 5 2D/3D sterile 22 2 0.35 -- -- --

2 5 2D/3D sterile 22 3 -- -- -- --

2 5 2D/3D sterile 22 4 0.19 -- -- --
Table 12 55

Table 12. Aerobic and facultative anaerobic heterotrophic bacteria enumeration data

[mL, milliliters; CFU, colony-forming units; CFU/100mL, colony-forming units per 100 milliliters; TNTC, too numerous to count; >, greater than] 

Replicate Incubation Source of water Enumeration results

Microcosm description number (weeks) (date collected) mL of sample CFU CFU/100 mL

Experiment 1, treatment 9 1 1 2D/12D (2/17/98) 1.00 TNTC > 2 x 104

Experiment 1, treatment 9 2 1 2D/12D (2/17/98) 1.00 TNTC > 2 x 104

Experiment 1, treatment 9 3 1 2D/12D (2/17/98) 1.00 TNTC > 2 x 104

Experiment 1, treatment 9 4 1 2D/12D (2/17/98) 1.00 TNTC > 2 x 104

Experiment 2, treatment 1 2 3 1D (5/21/98) 0.01 6 6 x 104

Experiment 2, treatment 1 4 3 1D (5/21/98) 0.01 89 8.9 x 105

Experiment 2, treatment 2 3 3 2D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 2 4 3 2D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 3 3 3 3D (5/21/98) 0.01 134 1.3 x 106

Experiment 2, treatment 3 4 3 3D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 4 3 3 12D (5/21/98) 0.01 121 1.2 x 106

Experiment 2, treatment 4 4 3 12D (5/21/98) 0.01 86 8.6 x 105

Experiment 2, treatment 5 (control) 1 3 2D/3D (5/21/98) 1.00 0 0

Experiment 2, treatment 5 (control) 2 3 2D/3D (5/21/98) 1.00 0 0

Experiment 2, treatment 5 (control) 3 3 2D/3D (5/21/98) 1.00 0 0

Experiment 2, treatment 5 (control) 4 3 2D/3D (5/21/98) 1.00 0 0

Experiment 2, treatment 1 1 17 1D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 1 4 17 1D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 2 1 17 2D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 2 4 17 2D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 3 1 17 3D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 3 1 17 3D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 4 4 17 12D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 4 4 17 12D (5/21/98) 0.01 TNTC > 2 x 106

Experiment 2, treatment 5 (control) 1 17 2D/3D (5/21/98) 0.01 0 0

Experiment 2, treatment 5 (control) 2 17 2D/3D (5/21/98) 0.01 0 0

Experiment 2, treatment 5 (control) 3 17 2D/3D (5/21/98) 0.01 0 0

Experiment 2, treatment 5 (control) 4 17 2D/3D (5/21/98) 0.01 0 0
56 Biodegradation of Chlorinated Ethenes at a Karst Site in Middle Tennessee

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