U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2010–1291

Prepared in cooperation with the College of Engineering, Technology, and Computer Science, 

Tennessee State University

Partnership of Environmental Education and Research:

A Compilation of Student Research, 1999–2008

“...the state is our laboratory.” Dean Decatur Rogers (ret.) Tennessee State University
Cover photographs. Collage: students involved in research at Tennessee State University; background photo: Tennessee State 

University campus (from Google Earth images).
Partnership of Environmental Education and Research: 

A Compilation of Student Research, 1999–2008

Edited by Michael W. Bradley, Patrice Armstrong, and Thomas D. Byl

Open-File Report 2010–1291

U.S. Department of the Interior

U.S. Geological Survey

Prepared in cooperation with the College of Engineering, Technology, and Computer 

Science, Tennessee State University
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2011

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reproduce any copyrighted materials contained within this report.

Suggested citation:

Bradley, M.W., Armstrong, P., and Byl, T.D., eds., 2011, Partnership of Environmental Education and Research: 

A compilation of student research, 1999–2008: U.S. Geological Survey Open-File Report 2010–1291, 67 p.

ISBN 978 1 4113 3082 5 
iii

Foreword

Tennessee State University College of Engineering and the U.S. Geological Survey developed a 

collaborative partnership to coordinate and develop a synergy between the university and USGS 

research in bioremediation, to provide cross-discipline research opportunities to the students, 

and to develop research programs in hydrology, water quality, and bioremediation. The collaboration was eventually titled PEER—Partnership for Environmental Education and Research. 

PEER made significant contributions to the Tennessee State University Environmental Program in 

three major areas: Learning, Inquiry, and Engagement.

1. PEER-directed Learning took place in STEM-based pre-college programs, Bachelor of 

 Science Civil and Environmental Engineering Program, and Master of Environmental 

 Engineering Program. PEER directed Learning:

• stimulated student interest in the field of environmental engineering and science,

• helped mentor undergraduate and graduate students through classes, internships, and 

directed research, and

• promoted student development through participation in regional and national 

professional conferences.

2. PEER established a focused, sustained, and quality-directed program of Inquiry, which 

 resulted in science fair projects for pre-college youth, award-winning capstone projects 

 for Bachelor of Science Engineering Seniors and regional and national award winning 

 Master’s Theses. PEER focused Inquiry:

• helped students make a connection between laboratory research and field trials,

• promoted scientific advances in quantifying biodegradation of contaminants in 

karst aquifers,

• added to our understanding of how to stimulate biodegradation of recalcitrant compounds in groundwater, and

• demonstrated wetland remediation of urban non-point source contamination.

3. PEER-directed Learning, focused Inquiry, and broad based engagement resulted in an 

 enhanced, enriched, multi-disciplined, team-engaged quality Environmental Engineering 

 Program. PEER broad based Engagement:

• improved the TSU Environmental Program by bringing in significant grants and contracts, 

• promoted cross-discipline research, uniting teams of engineers, biologists, chemists, 

mathematicians, and other earth scientists, and

• helped advance USGS research in natural remediation.

TSU and USGS have both benefited from the partnership. Significant advances have been made 

in fields of karst hydrology and contaminant remediation. More importantly, environmental 

engineering students have benefited the most. The students learn what it takes to get out of the 

class room, to get on the ground—in the mud and muck of wetlands and marshes—to use their 

education and the experiences from PEER to solve urgent real-world environmental problems.

As you read this document and marvel at the depth, breadth and quality of the students’ work, 

take particular note of the PEER research faculty, research scientists, research institutions and 

research organizations that contributed to the exciting achievements of the TSU students!

Dr. Decatur Rogers, Former Dean

College of Engineering, Technology, and Computer Science

Tennessee State University, Nashville, TN 37209
iv
v

Contents

Foreword ........................................................................................................................................................iii

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

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

PEER Research Areas...................................................................................................................................3

Karst—Bioremediation........................................................................................................................3

Microcosm Study to Assess the Potential for Intrinsic Bioremediation at 

a Karst Site ......................................................................................................................4

Biodegradation of Fuel in a Karst Aquifer ...............................................................................5

Comparison of Different Lactic Acid Formulations Used to Enhance 

Biodegradation of Perchloroethylene ........................................................................6

Microbial Strategies for Degradation of Organic Contaminants in Karst..........................7

Optimum pH for Biodegradation of Benzene and Toluene in a Karst Aquifer ...................9

Enhanced Biodegradation of TCE in a Karst Aquifer Using Lactic Acid, 

Molasses, and Soy Milk ..............................................................................................10

Evaluating Oxygen-Releasing Compounds to Enhance Fuel Biodegradation 

by Free-Living Bacteria ...............................................................................................11

Biorestoration of a TCE-Contaminated Karst Aquifer by Stimulating 

Sulfate–Reducing Bacteria ........................................................................................12

Supplements to Enhance Groundwater-Microbial Growth and 

Biodegradation Processes .........................................................................................13

The Role of Free-Living and Attached Bacteria in Processing 

Contamination in Karst Aquifers................................................................................14

Comparison of Redox Measurements and Geochemical Conditions in 

a Contaminated Karst Aquifer and Their Influence on 

Biodegradation .............................................................................................................15

Comparison of Water Geochemistry and Redox Electrodes to Identify 

Terminal Electron Acceptor Processes in an Aquifer............................................16

Free-Living Bacteria Versus Attached Bacteria: Which Contributes More to 

Bioremediation? ...........................................................................................................17

Quantifying Peroxide-Enhanced Toluene Biodegradation in a SingleWell Injection ................................................................................................................18

Evaluating Oxygen-Releasing Compounds to Enhance Toluene 

Biodegradation by Free-Living Bacteria ..................................................................19

Lactate Induction of the Ammonia Mono-Oxygenase Enzyme and 

PCE Cometabolism .......................................................................................................20

Biodegradation of Toluene as it Continuously Enters a 5-Liter 

Karst System .................................................................................................................21

Ammonia Oxidation by Bacteria Collected from a Karst-Bedrock Well (a).....................22

Ammonia Oxidation by Bacteria Collected from a Karst-Bedrock Well (b).....................23

Free-Living Bacteria or Attached Bacteria: Which Contributes More 

to Bioremediation?.......................................................................................................24

Bacteria Induced Dissolution of Limestone in Fuel-Contaminated 

Karst Wells ....................................................................................................................25

Geochemical and Microbial Evidence of Fuel Biodegradation in a 

Contaminated Karst Aquifer in Southern Kentucky, June 1999............................26
vi

Karst—Contaminant Degradation Models.....................................................................................27

Numerical Modeling of Flow Simulations and Rate of Biodegradation in 

a Karst Microcosm.......................................................................................................27

Numerical Simulation of Flow and Contaminant Transport in a Karst 

Aquifer Conduit.............................................................................................................28

Use of Residence-Time Distribution Coupled with a Biodegradation 

Rate to Predict Toluene Removal in an Artificial Karst System............................29

Development of a Computer Program that Uses Residence-Time 

Distribution and First-Order Biodegradation to Predict BTEX 

Removal in Karst Aquifers ..........................................................................................30

Development of a Protocol for Enhanced Bioremediation in Karst 

Using a Single Injection Well .....................................................................................31

Evaluating Peclet Values and the Role of Advection, Dispersion and 

Diffusion in Tracer Studies..........................................................................................32

Application of the RTD Model to Analyze the Fate and Transport of 

Ammonia in Laboratory Karst System ......................................................................33

Development and Verification of a Computer Program That Predicts 

Fuel Biodegradation in Karst Aquifers......................................................................34

Residence Time Distribution Derived from Independent Gamma 

Distributions of Tracer Travel Distance and Linear Velocity.................................35

Adaptation of the Residence Time Distribution (RTD)-Biodegradation 

Model to Quantify Peroxide-Enhanced Fuel Biodegradation in 

a Single Karst Well.......................................................................................................36

Karst—Hydrology ..............................................................................................................................37

Advection Versus Dispersion as Determined by Single-Well Tracer Studies ................37

Enhanced Groundwater Recharge Can Alleviate Storm-Water Flow

 and Dilute Groundwater Pollution in Karst Terrane...............................................38

Characterization of Bacteria and Geochemistry of Springs in 

Nashville, Tennessee...................................................................................................39

Land-Use Effects on Bacteria Loads and Water Quality in Small 

Karst Catchments of the Upper Duck River Watershed ........................................40

Methods in Contaminant Evaluation................................................................................................41

Use of Polymerase Chain Reaction and Oligonucleotide 

Hybridization Probes to Determine the Source of Fecal 

Contamination in Karst Terranes ...............................................................................41

Effectiveness of m-TEC Agar for Quantifying Escherichia coli in the

Duck River near Shelbyville, Tennessee ..................................................................42

Evaluation of Passive-Diffusion Samplers to Measure Dissolved Oxygen, 

Geochemistry, and Volatile Organic Compounds in Groundwater ......................43

The Value of Single-Well Tracer Studies for Characterizing Karst Sites .........................44

Contaminant Transport and Remediation in Wetlands and Streams ........................................45

Survival of Fecal Bacteria in Sediments and Development of a 

Numerical Model to Predict Storage and Transport in a River............................45

Modification of a Numerical Model to Predict Transport and Flux of 

Fecal Bacteria in a River.............................................................................................46

A Flux Term to Describe the Movement of Fecal Bacteria between the 

Sediment and Water Column in a Riverine System................................................47
vii

Effect of a Riverine Wetland on Parking Lot Runoff at Tennessee 

State University ............................................................................................................48

Use of Tanks-in-Series Method to Predict Nitrate Removal in Wetlands........................49

Wetland Removal of Nutrients and Pollution from a Mixed Sewer and

 Karst Spring System in Nashville, Tennessee........................................................50

Water-Quality Monitoring—Luminescence...................................................................................51

Evaluating a Bioluminescent Bacteria for Measuring Toxicity of Industrial 

Wastewaters.................................................................................................................51

Evaluation of Bioluminescent Bacteria as Indicators of Water Quality 

in Wastewater Treatment Plants ...............................................................................52

Comparison of Bioluminescent Bacteria and Oxygen Consumption

 as Indicators of Water Quality ..................................................................................53

Bioluminescent Bacteria as Indicators of Water Quality in a WastewaterTreatment Plant.............................................................................................................54

Sensitivity of the Oxydase-Enzyme Induced Chemiluminescent to Water 

Quality Parameters ......................................................................................................55

Development of an Aquatic Plant Chemiluminescent Bioassay to Assess 

Water Quality ................................................................................................................56

Other Research—Degradation and Transport...............................................................................57

Desorption Isotherms For Toluene And Karstic Materials And Implications

 For Transport in Karst Aquifers ................................................................................57

Reductive Dechlorination of Tetrachloroethylene by Vitamin B12 

and Electrokinesis........................................................................................................58

Are Aquifers at Greater Risk from Alternative Alcohol-Fuel Mixes Compared

 to Regular Gasoline?...................................................................................................59

The Interaction between Calcite Precipitation and Dissolution, Carbon 

Dioxide, and Perchloroethylene Sorption ...............................................................60

Reductive Dechlorination of Perchloroethylene with a Direct Current, 

Different Salts, and Vitamin B12 ..................................................................................61

Student Awards............................................................................................................................................62

Outreach........................................................................................................................................................63

Summary........................................................................................................................................................65

References....................................................................................................................................................66

Figure

1. Map showing karst areas in Tennessee and location of research sites for the 

U.S. Geological Survey/Tennessee State University Partnership in Environmental 

 Education and Research Program, 1998–2008 .................................................................................2

Table

1. Summary of student participation, research areas, and awards for students in the 

PEER program, 1998–2008 ....................................................................................................................3
viii

Multiply By To obtain

Length

inch (in.) 2.54 centimeter (cm)

foot (ft) 0.3048 meter (m)

mile (mi) 1.609 kilometer (km)

Area

square foot (ft2) 929.0 square centimeter (cm2)

square mile (mi2) 2.590 square kilometer (km2) 

Volume

gallon (gal) 3.785 liter (L) 

cubic foot (ft3) 0.02832 cubic meter (m3) 

Flow rate

acre-foot per day (acre-ft/d) 0.01427 cubic meter per second (m3/s)

foot per second (ft/s) 0.3048 meter per second (m/s)

gallon per minute (gal/min) 0.06309 liter per second (L/s)

gallon per day (gal/d) 0.003785 cubic meter per day (m3/d)

Conversion Factors

Inch/Pound to SI

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C=(°F-32)/1.8

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius 

(μS/cm at 25 °C).

Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L) or 

micrograms per liter (μg/L).

Note: Any use of trade, product, or firm names in this document is for descriptive purposes only 

and does not imply endorsement by the U.S. Government.

Photographs used in this report were by U.S. Geological Survey employees unless 

otherwise noted.
Partnership of Environmental Education and Research: 

A Compilation of Student Research, 1999–2008

Edited by Michael W. Bradley1, Patrice Armstrong2, and Thomas D. Byl1

Abstract

The U.S. Geological Survey (USGS) Tennessee Water 

Science Center and the College of Engineering and Technology at Tennessee State University developed a Partnership in 

Environmental Education and Research (PEER) to support 

environmental research at TSU and to expand the environmental research capabilities of the USGS in Tennessee. The 

PEER program is driven by the research needs to better define 

the occurrence, fate, and transport of contaminants in groundwater and surface water. Research in the PEER program has 

primarily focused on the transport and remediation of organic 

contamination in karst settings. Research conducted through 

the program has also expanded to a variety of media and 

settings. Research areas include contaminant occurrence and 

transport, natural and enhanced bioremediation, geochemical 

conditions in karst aquifers, mathematical modeling for contaminant transport and degradation, new methods to evaluate 

groundwater contamination, the resuspension of bacteria from 

sediment in streams, the use of bioluminescence and chemiluminescence to identify the presence of contaminants, and 

contaminant remediation in wetlands. 

The PEER program has increased research and education 

opportunities for students in the College of Engineering, Technology, and Computer Science and has provided students with 

experience in presenting the results of their research. Students 

in the program have participated in state, regional, national 

and international conferences with more than 140 presentations since 1998 and more than 40 student awards. The PEER 

program also supports TSU outreach activities and efforts to 

increase minority participation in environmental and earth 

science programs at the undergraduate and graduate levels. 

TSU students and USGS staff participate in the TSU summer 

programs for elementary and high school students to promote 

earth sciences. The 2007 summer camps included more than 

130 students from 20 different States and Washington DC. 

Introduction

The U.S. Geological Survey (USGS) Tennessee Water 

Science Center and the College of Engineering, Technology, 

and Computer Science at Tennessee State University (TSU) 

developed a partnership in environmental education and 

research (PEER). The partnership was initially developed in 

1998 between TSU and the USGS to support environmental 

research at TSU and to expand the environmental research 

capabilities of the USGS in Tennessee. The objectives of the 

partnership are to conduct environmental research and to promote environmental education through research and hands-on 

activities. Dean Decatur Rogers (TSU, retired) in describing 

one benefit, said of the PEER program, “The entire State is 

now our laboratory.” Research conducted by USGS scientists, 

TSU professors, and students in the PEER program has been 

presented in a wide range of local, State, national and international conferences. 

The PEER program is driven by research needs to better 

define the occurrence, fate, and transport of contaminants in 

groundwater and surface water. Contamination of surface 

water and groundwater affects the adequacy of water resources 

to support healthy ecosystems and to meet the ever-increasing 

demands for municipal, agricultural, and industrial use. 

Research conducted through the PEER program in the USGS 

Tennessee Water Science Center currently is focused on the 

occurrence, fate, and transport of contaminants in karst terrain, 

in unconsolidated aquifers, and in wetlands. 

The PEER program includes students in the engineering, 

environmental engineering, chemistry, and biology programs. USGS scientists assist and mentor students in defining 

research topics and mentor students in proper data collection, 

documentation, and quality assurance and quality control. Students in the PEER program have worked in USGS Water Science Centers in Nashville, Tennessee, Louisville, Kentucky, 

Indianapolis, Indiana, and Columbia, South Carolina and in 

the USGS Biological Science Center in Rolla, Missouri.

The PEER program also supports TSU outreach activities and efforts to increase minority participation in environmental and earth science programs at the undergraduate and 

graduate levels. The PEER program has increased research 

and education opportunities for the College of Engineering 

and Technology. Students in the program have participated in 

state, regional and nation al conferences with more than 140 

presentations since 1998 and more than 40 student awards. 

USGS scientists have developed new curriculum for the environmental engineering programs. PEER students and USGS 

staff participate in the TSU summer programs for elementary 

and high school students to promote earth sciences. The 2007 

1

 U.S. Geological Survey, Tennessee Water Science Center, Nashville, Tennessee

2

 Tennessee State University, College of Engineering, Technology, and Computer Science
2 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

summer camps included more than 130 students from 20 

States and from Washington DC.

The PEER program and the involvement of USGS were 

instrumental in assisting TSU in receiving a grant from the 

National Science Foundation (NSF) to increase the number of 

minority students in earth sciences (geology, hydrology, and 

environmental science and engineering). The NSF grant has 

allowed TSU, with assistance from the PEER program and the 

USGS, to develop programs at TSU with the National Park 

Service (Mammoth Cave) to encourage, develop, and mentor minority students in the earth sciences. TSU, through the 

PEER program, has been recognized by the American Society 

for Engineering Education (ASEE) for educating, on average, 

more than 10 percent of the Nation’s African-American students entering graduate programs in environmental engineering; this percentage increases to 30 percent during some years 

(Dr. Decatur Rogers, former Dean of Engineering ret, TSU, 

oral comm., 2006). Research conducted on natural attenuation 

by students in the PEER program was featured in an article 

on environmental research at historically black colleges and 

universities (Witherspoon, 2008).

The PEER program has included more than 70 students 

in studies in seven general research topics at 10 locations 

across Tennessee (fig. 1). The research has resulted in more 

than 50 abstract, articles, and presentations at professional 

conferences at the local, regional, national, and international 

level (table 1). The purpose of this report is to provide a 

description of the PEER program including descriptions of the 

research areas, a summary of the accomplishments in awards 

and outreach activities, and a compilation of abstracts and 

papers developed through the USGS and TSU research. 

Figure 1. Karst areas in Tennessee and location of research sites for the U.S. Geological Survey/Tennessee State University 

Partnership in Environmental Education and Research Program, 1998–2008.

0 50 100 MILES

0 50 100 KILOMETERS

Base from U.S. Geological Survey

digital data, 1: 100,000, 1983

StatePlane projection, datum NAD27

Zone 5301

EXPLANATION

Karst areas characterized by limestone or dolomite bedrock, sinkholes, caves, and

 disappearing streams 

Karst sites used in the research of karst hydrogeology, bacteria occurrence in

 karst aquifers and bioremediation in karst aquifers.

Research sites in the collection of soil and subsurface material to evaluate

 contaminant transport, bioflouresence, and crude oil contamination and remediation.

35°

36°

89°

88° 87° 86° 85° 84° 82° 83°
 PEER Research Areas 3

PEER Research Areas

More than two-thirds of the State of Tennessee is underlain by limestone and dolomite with karst features such as 

caves, sinkholes, and disappearing streams (figure 1). Contamination in karst areas can be difficult to fully characterize and 

remediate. Groundwater and surface water in karst settings 

are linked in an interactive system that can be complex and 

difficult to predict or model. Temporal variations in natural 

water quality and contaminant concentrations and highly variable geochemical, biological, and hydrogeological processes 

affect the fate and transport of contaminants in karst. Research 

conducted through the PEER program can provide additional 

information to better understand these processes and to help 

improve strategies for pollution prevention, mitigation, and 

remediation in karst.

Research in the PEER program has focused primarily 

on the transport and remediation of organic contamination in 

karst settings. Accordingly, most of the abstracts and student 

presentations have described contaminant occurrence and 

transport, natural and enhanced bioremediation, and geochemical conditions in karst aquifers. As the activities in karst areas 

expanded, PEER research included mathematical modeling for 

contaminant transport and degradation, the adaptation of residence-time-distribution (RTD) and Lattice-Boltzmann models 

to karst aquifers, the occurrence and role of bacteria in karst 

aquifers, and the occurrence and fate of different contaminants 

in karst aquifers.

Although the PEER activities have focused on karst settings, research has expanded to a variety of media and settings. 

Research has been conducted on new methods to evaluate 

groundwater contamination, the resuspension of bacteria from 

sediment in streams, the use of bioluminescence and chemiluminescence to identify the presence of contaminants, and 

contaminant remediation in wetlands. 

 The research compiled in this report is grouped by subject. The first group contains abstracts and papers that describe 

the research conducted in karst areas, including bioremediation, research on modeling contaminant degradation in karst 

aquifers, and other research on karst hydrology. Additional 

research conducted through PEER includes methods in contaminant evaluation, contaminant transport and remediation 

in wetland and streams, the use of bioluminescence to monitor water quality, and the degradation and transport of other 

contaminants in the environment. 

Karst—Bioremediation

Initial research conducted through the USGS and 

TSU partnership supported USGS investigations of contamination in karst areas of Tennessee in general (Wolfe and 

others, 1997). Research examining bioremediation in karst 

aquifers was generally lacking and many felt that karst aquifers were not suitable for bioremediation due to rapid flow 

conditions, a perceived relatively low surface to volume ratio 

and perceived absence of bacteria. Conceptual models developed by Wolfe and others (1997) indicated that contaminants 

in karst systems occurred not only in the active, large conduit, 

but also along small fractures, bedding planes and other isolated features. Groundwater and contaminants in the isolated 

areas could provide an environment for biodegradation if the 

bacterial and geochemical conditions were suitable. 

PEER students conducting research in this area documented the presence of bacteria in karst systems that were 

suitable for biodegradation, documented the occurrence of 

natural and enhanced bioremediation in karst systems, utilized 

laboratory microcosm studies to simulate degradation in karst 

aquifers, and defined geochemical conditions suitable for biodegradation. Research was conducted in Dickson, Marshall, 

and Montgomery Counties, Tennessee.

Table 1. Summary of student participation, research areas, and 

awards for students in the PEER program, 1998–2008.

Category Number of

abstracts

Number of

students

Number of

Awards*

Karst bioremediation 22 25 8

Karst modeling 10 8 5

Karst hydrology 5 7 1

Methods 5 6 —

Wetlands and streams 6 11 4

Luminescence 4 8 3

Other contaminants 5 11 1

* Does not include University awards
4 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

The potential for bioremediation of trichloroethylene 

(TCE) is being examined at a karst site in Middle Tennessee 

where “pump and treat” wells are used to slow the movement 

of ground water away from the site and to remove some of the 

TCE. Ground-water samples were collected from nearby wells 

and analyzed to identify geochemical indicators of bioremediation (electron donors and acceptors), and to monitor trends 

in the concen¬trations of TCE and intermediate degradation 

products. Bacteria associated with the degradation of TCE 

were identified in water samples using the RNA oligonucleotide hybridization technique. Water-quality data indicate that 

both aerobic and anaerobic conditions occur in the contaminated karst aquifer. Sulfate-reducing and methanogenic bacteria, 

both associated with the reductive dechlorination of TCE (an 

anaerobic process), were present in samples from anaerobic 

wells. Methanotrophs and ammonia-oxidizing bacteria, both 

associated with co-metabolic degradation of TCE (an aerobic process), were present in samples from aerobic wells. 

Aerobic and anaerobic bacteria were identified in samples 

from wells that fluctuated in dissolved-oxygen concentration. 

Microcosm studies of ground water collected from the karst 

aquifer suggest that both biodegrada¬tion pathways are active. 

The rate of biodegradation was rapid in some samples, with 

a TCE half- life of 35 days. Degradation products indicative 

of the reductive dechlorination process were detected in other 

samples at 10 months. Additional water-quality and hydrologic monitoring of the karst aquifer are being conducted to 

determine temporal and spatial changes in water quality and 

to evaluate how these changes may affect the biodegradation 

processes at the site.

Microcosm Study to Assess the Potential for Intrinsic Bioremediation at a Karst Site

P.R. Franklin1, S.D. Williams2, J.J. Farmer2, T.D. Byl2

Published as Franklin, P.R., Williams, S.D., Farmer, J.J., Byl, T.D., Microcosm study to assess the potential for intrinsic bioremediation at a karst site: in

Crabtree, L.R., Bradley, M.W., Blunt, Tiffany, and Pierre, Salnave, eds., 1999, Proceedings from the Ninth Annual Tennessee Water Resources Symposium, 

April 1999, Nashville, Tennessee: Tennessee AWRA, p. 1B-51.

1

 Tennessee State University, Department of Civil and Environmental Engineering

2

 U.S. Geological Survey
Karst—Bioremediation 5

Biodegradation of Fuel in a Karst Aquifer

Kamalah Minor1, Raushanah Muhammad1, Tavy Wade1, Allyn Allison2, and Tom D. Byl3

Complex hydrogeologic conditions coupled with poorly 

understood biodegradation processes in karst aquifers have led 

many to believe that the potential for natural attenuation of petroleum fuel hydrocarbons is limited. This research addressed 

the capacity for biodegradation processes in karst. Groundwater samples were collected for bacteria and geochemical analysis from two monitoring wells (MCI-1 and MCI-4) in a karst 

bedrock aquifer. Water from the MCI-1 well has consistently 

tested positive for fuel contamination during the past 3 years 

of semi-annual monitoring. Water from MCI-4 has been relatively uncontaminated during the same time period. Bacteria 

concentrations were 50% greater in ground-water samples 

from the fuel-contaminated well. Additionally, bacteria isolated from fuel-contaminated ground-water samples readily grew 

on Petri dishes with dissolved toluene and benzene as the only 

source of food. Water from the less contaminated MCI-4 well 

had a greater dissolved oxygen concentration (6.4 milligrams 

per liter) than the fuel-contaminated water (dissolved oxygen 

less than 0.1 milligram per liter). Also, where the oxygen concentrations were diminished, geochemical evidence indicated 

that anaerobic processes were active. This evidence includes 

elevated levels of ammonia, sulfide, and ferrous iron in the 

fuel-contaminated ground-water samples. Based on these 

results, biodegradation of fuel constituents in the karst aquifer 

is indicated, and therefore, natural attenuation should not be 

disregarded because of preconceptions about low microbial 

activity in karst aquifers.

Published as Minor, K., Muhammad, R., Wade T., Allison, A., and Byl, T., Biodegradation of fuel in a karst aquifer in Tennessee Section, American Water 

Resources Association, 2001, Proceedings from the Eleventh Annual Tennessee Water Resources Symposium, April 2001, Montgomery Bell State Park, Burns, 

Tennessee: Tennessee Section of the American Water Resources Association, p. P-6.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Army Corps of Engineers, 801 Broadway, Nashville, TN

3

 U.S. Geological Survey
6 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Comparison of Different Lactic Acid Formulations Used to Enhance Biodegradation 

of Perchloroethylene

Ahlam Ary1, LyTreese Hampton1, and Tom D. Byl2

A sanitary landfill situated on a karst terrain in northern 

Tennessee has leaked chlorinated solvents, such as perchloroethylene (PCE) and trichloroethylene (TCE), into a karst aquifer. Some biodegradation apparently has occurred based on the 

presence of breakdown products such as cis-dichloroethylene 

(cDCE) found in water from wells screened in the karstic 

bedrock. Sulfur-reducing bacteria, which are known to biodegrade chlorinated solvents, have been identified in water 

collected from these same bedrock wells. Previous studies 

have found that sulfur-reducing bacteria are stimulated by 

sodium lactate. A study was conducted to evaluate different formulations of lactic acid to determine which was the 

best for PCE biodegradation by indigenous karst bacteria. 

Water from the karst bedrock wells was collected and used 

to make 250-milliliter water microcosms. The microcosms 

were stocked with 2.5 milligrams per liter PCE and different 

formulations of lactic acid, including sodium lactate, lactic 

acid, magnesium lactate, iron lactate, crude lactate, methyllactate, potassium lactate, calcium lactate, ethyl-lactate, 

propyl-lactate, and ammonium lactate. The concentrations 

of the lactic acids were normalized so that each microcosm 

had the equivalent of 50 lactic-acid (reducing) electrons for 

each PCE molecule. Preliminary results indicate that all of the 

lactic acids stimulated the reductive dechlorination of PCE 

to TCE and cDCE. Trace amounts of trichloroethane (TCA) 

and 1,1-dichloroethylene also were detected in some of the 

microcosms. Within 4 weeks, a 40- to 60-percent decrease 

in PCE occurred in the treated microcosms compared to the 

sterile control microcosms. A concurrent rise in TCE also 

occurred during the same time period. The microcosms treated 

with ammonium lactate had PCE concentrations initially drop 

twice as fast compared to the other treatments during the first 

10 days. But after 21 days, no significant difference between 

the ammonium lactate treatment and the other treatments 

was observed. After 45 days incubation, the microcosms 

treated with iron-lactate and magnesium lactate resulted in 

a 90-percent reduction of the initial PCE. The other lactic 

acid treatments contributed to a 50-to 70-percent reduction of 

the initial PCE after a 45-day incubation period. The sterile 

controls had a 30-percent reduction in PCE caused by abiotic 

processes such as volatilization and sorption in the same time 

period. In conclusion, ammonium lactate initially appears 

to stimulate PCE biodegradation, but the effect diminishes 

within 21 days. Iron and magnesium lactate appear to maintain 

enhanced PCE biodegradation over a longer period of time 

than the other lactic acid formulations tested.

Published as Ary, A., Hampton, L., and Byl, T.D., Comparison of different lactic acid formulations used to enhance biodegradation of perchloroethylene in

Tennessee Section, American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources Symposium, April 9–11, 2003, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-2.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science 

2

 U.S. Geological Survey
Karst—Bioremediation 7

Microbial Strategies for Degradation of Organic Contaminants in Karst

Tom D. Byl1, Gregg E. Hileman1, Shannon D. Williams1, David W. Metge2, and Ron W. Harvey2

Approximately 40 percent of the United States east of 

the Mississippi River is underlain by various types of karst 

aquifers (Quinlan, 1989) and more than two-thirds of the 

State of Tennessee is underlain by carbonate rocks and can be 

classified as karst (Wolfe and others, 1997). Potential industrial sources of ground-water contamination are common in 

karst regions; however, the fate and transport of contaminants 

such as fuels in karst areas are poorly understood because 

of the distinctive hydraulic characteristics of karst aquifers 

(Field, 1993). Ground-water models that predict the fate and 

transport of contaminants in sandy aquifers have limited 

application to karst aquifers. Most natural attenuation and 

bioremediation guidelines specify that they are not applicable in fractured rock or karst aquifers (U.S. Environmental 

Protection Agency, 1997).

The lack of studies examining biodegradation in karst 

aquifers may be due to the widespread perception that contaminants are rapidly flushed out of karst aquifers. In highly 

developed and well-connected conduit systems, the rate of 

contaminant migration is expected to be much faster than the 

rate of biodegradation. Field (1993) states that remediation 

techniques such as ground-water extraction or bioremediation 

are impractical in karst aquifers dominated by conduit flow; 

however, he also states that the belief that contaminants are 

rapidly flushed out of karst aquifers is a popular misconception. Large volumes of water may be trapped in fractures along 

bedding planes and other features isolated from active groundwater flowpaths in karst aquifers (Wolfe and others, 1997). 

In areas isolated from the major ground-water flowpaths, 

contaminant migration may possibly be slow enough that 

biodegradation could reduce contaminant mass if favorable 

microorganisms, food sources, and geochemical conditions are 

present (Byl and Williams, 2000; Byl and others, 2001). The 

capacity for biodegradation processes in a karst setting was 

evaluated at sites in Tennessee and Kentucky.

The potential for biodegradation of trichloroethylene 

(TCE) was studied in a karst aquifer at Lewisburg, Tennessee. 

This site was selected because of the presence of TCE degradation by-products in the karst aquifer, available site hydrologic and chlorinated-ethene information. Additional chemical, 

biological and hydrological data were gathered to evaluate if 

the occurrence of TCE degradation by-products in the karst 

aquifer was the result of biodegradation in the aquifer or 

simply transport into the aquifer. Geochemical analysis established that sulfatereducing conditions, essential for reductive 

dechlorination of chlorinated solvents, existed in parts of the 

contaminated karst aquifer. Geochemical conditions in other 

areas of the aquifer fluctuated between anaerobic and aerobic 

conditions and contained compounds associated with cometabolism, such as ethane, methane, ammonia and dissolved 

oxygen. A large, diverse bacteria population inhabits the 

contaminated aquifer. Bacteria known to biodegrade TCE and 

other chlorinated solvents, such as sulfate-reducers, methanotrophs, and ammonia-oxidizers, were identified from karstaquifer water using the RNA-hybridization technique. Results 

from microcosms using raw karst-aquifer water found that 

aerobic cometabolism and anaerobic reductive dechlorination 

degradation processes were possible when appropriate conditions were established in the microcosms. The chemical and 

biological results provide circumstantial evidence that several 

biodegradation processes are potentially active in the karst 

aquifer. Additional site hydrologic information was developed 

to determine if appropriate conditions persisted long enough 

in the karst aquifer for these biodegradation processes to be 

significant. Continuous monitoring devices placed in four 

wells during the spring of 1998 documented a dual phase 

ground-water flow system within the karst aquifer. Dynamic 

areas were present within the karst aquifer where active flow 

occurred, as well as, stable areas in the karst aquifer that were 

isolated from active flow. The pH, specific conductance, low 

Published as Byl, T.D., Hileman, G.E, Williams, S.D., Metge, D.W. and Harvey, R.W. Microbial Strategies for Degradation of Organic Contaminants in 

Karst in Aiken, G.R., and Kuniansky, E.L., eds., 2002, U.S. Geological Survey Artificial Recharge Workshop proceedings, April 2–4, 2002, Sacramento, 

California: U.S. Geological Survey Open-File Report 02–89, p. 61–62.

1

 U.S. Geological Survey, Nashville, Tennessee

2

 U.S. Geological Survey, Boulder, Colorado
8 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

dissolved oxygen levels and low oxidation-reduction potentials changed very little in the stable areas isolated from active 

flow. The stable areas in the karst aquifer had geochemical 

conditions and bacteria conducive to reductive dechlorination 

of chlorinated ethenes. The dynamic areas of the karst aquifer 

associated with active flow fluctuated between anaerobic and 

aerobic conditions in response to rain events. Associated with 

this dynamic environment were bacteria and geochemical 

conditions conducive to cometabolism. In summary, multiple 

lines of evidence developed from biological, chemical and 

hydrological data demonstrate that a variety of biodegradation 

processes were active in this karst aquifer.

A second karst-aquifer site contaminated with jet fuel was 

also investigated. The site is located at an airfield in southern 

Kentucky. Ground-water samples were collected for bacteria 

and geochemical analysis from several contaminated monitoring wells in an unconsolidated regolith and karst aquifer that 

had varying concentrations of dissolved fuel. Bacteria counts 

ranged from approximately 700,000 bacteria per milliliter to 

1.2 million depending on the well and sample collection time. 

These bacteria counts were derived using two methods, direct 

counts and BART growth tests, and the results of the two 

tests were within 20 percent of each other. These numbers are 

much greater than previously reported when tryptic soy agar 

was used to quantify heterotrophic bacteria in the same wells 

(Byl and others, 2001). Bacteria from the fuel-contaminated 

part of the karst aquifer had a 5% lighter buoyant density and 

a wider range of sizes than the bacteria from the non-contaminated well. Additionally, bacteria isolated from fuel-contaminated ground-water samples readily grew with dissolved 

gasoline as the only source of food. Static microcosms (n=3) 

set up using aerated raw karst water spiked with benzene at 1 

mg/L established a biodegradation rate of 50% loss (T1/2) in 

3 days. Sterile control microcosms had less than 10% benzene 

loss over the same time period. Additional field evidence that 

biodegradation was taking place in the aquifer was established by measuring geochemical indicators. The wells with 

screens intersecting non-contaminated sections of the aquifer 

had greater dissolved oxygen concentrations (generally above 

2 milligrams per liter) than those intersecting more contaminated sections (dissolved oxygen less than 0.1 milligrams per 

liter). Also, where the oxygen concentrations were diminished, 

geochemical evidence indicated that anaerobic processes were 

active. This evidence includes elevated levels of ammonia, 

sulfide and ferrous iron in the fuel-contaminated ground-water 

samples. Based on these results, biodegradation of fuel constituents in the karst aquifer is indicated, and therefore, natural 

attenuation should not be disregarded because of preconceptions about low microbial activity in karst aquifers.

Microbial Strategies for Degradation of Organic Contaminants in Karst—Continued
Karst—Bioremediation 9

Optimum pH for Biodegradation of Benzene and Toluene in a Karst Aquifer

Natascha Morris1, Gregg Hileman2, and Tom D. Byl2

Jet fuel leaking from underground pipes contaminated the 

regolith and karst aquifer underlying a south-central Kentucky 

airfield. Benzene and toluene, components of jet fuel, were 

detected in seven fuel-contaminated wells at the airfield. 

Ground-water pH measured in these seven wells ranged from 

5 to 11. Some microorganisms are capable of degrading aromatic hydrocarbons such as benzene and toluene, but the rate 

of metabolism varies with pH. A study was conducted to determine the optimum pH for biodegradation of benzene and toluene by bacteria indigenous to the karst aquifer. Batch microcosms (three replicates per treatment and sampling time) were 

set up using karst bacteria enriched with benzene and toluene. 

The pH of the water in the microcosms was adjusted to pH 2, 

5, 7, 9, and 12. Little to no biodegradation occurred outside 

the pH range of 5 to 9; pH values higher than 9 or lower than 

5 caused the rate of biodegradation to decrease rapidly. Tests 

of biochemical oxygen demand verified that dissolved-oxygen 

consumption stopped when pH values were outside the range 

of 5 to 9. Microcosms with a pH of 5 showed the greatest decrease in benzene and toluene concentrations (approximately 

an 80-percent reduction in 6 days), followed by pH values of 7 

(70-percent reduction), 9 (65-percent reduction), 2 (25-percent 

reduction), and 12 (10-percent reduction). 

During a 3-month pump-and-treat remediation process, ground-water pH was monitored in the seven fuelcontaminated wells. The pH of the water in six of the seven 

wells ranged from 5.2 to 6.8. One well, which initially had 

a pH of 11 attributed to grout water from a newly installed 

well 9 feet away, retained a pH above 10 during the 3-month 

remediation. Increased biological activity was observed in 

most of the wells because of the remediation. However, bacterial growth and oxygen consumption of fuel biodegradation 

were not indicated in the well where the pH was greater than 

10. These results demonstrate that fuel biodegradation can be 

slowed by very high or low pH values.

Published as Morris, N., Hileman, G., and Byl, T.D., Optimum pH for biodegradation of benzene and toluene in a karst aquifer in Tennessee Section, American 

Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources Symposium, April 9–11, 2003, Montgomery Bell State Park, 

Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-20.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
10 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Enhanced Biodegradation of TCE in a Karst Aquifer Using Lactic Acid, Molasses, and Soy Milk

Koushik Chakraborti1, Gregg Hileman2, LyTreese Hampton1, Martin Greene1, and Tom D. Byl2

A sanitary landfill situated on a karst terrain in northern 

Tennessee has leaked chlorinated solvents, primarily trichloroethylene (TCE), into a karst aquifer. TCE has been found in 

water samples collected from eight wells screened in the karst 

bedrock aquifer. Some anaerobic biodegradation apparently 

has occurred in four of the eight wells tested based on the 

appearance of trace amounts of breakdown products. Sulfurreducing bacteria have been found in all eight wells. In July 

2002, a mixture of dye, lactic acid, molasses, and soymilk was 

injected into six of the eight wells to determine if the mixture would enhance reductive dechlorination of TCE. Water 

samples were collected and electronic monitoring devices 

were placed in selected wells over a period of 4 months following injection to monitor changes for geochemistry and 

concentrations of TCE and breakdown products, and dye. 

Prior to injection, TCE concentrations ranged from 1 part per 

billion (ppb) to 74 ppb. After approximately 4 weeks, there 

was a noticeable decrease in TCE and cis-dichloroethylene 

(cDCE) concentrations in all but one of the wells. After 

4 months, there was an 85- to 100-percent decrease in TCE 

concentrations in the eight wells, and a 65- to 100-percent 

decrease in cDCE concentrations in six of the eight wells. Two 

wells showed an increase in cDCE as TCE degraded to cDCE. 

Concurrent with decreases in TCE and cDCE concentrations 

were increases in sulfide concentrations. No dissolved oxygen 

was found in any of the wells 1 week after the mixture was 

injected. Dye concentrations decreased at varying rates during 

the 4-month sampling period, indicating that the injection 

mixture was being degraded or transported down-gradient. 

Trace amounts of 1,1-dichloroethylene (1,1-DCE) and trichloroethane (TCA) were found in some well-water samples, possibly due to secondary abiotic chemical reactions. These data 

indicate that a mixture of lactic acid, molasses, and soy milk 

enhanced biodegradation of TCE in a karst aquifer. However, 

further work is needed to determine how long the enhancement will last.

Published as Chakraborti, K., Hileman, G., Hampton, L., Greene, M., and Byl, T.D., Enhanced biodegradation of TCE in a karst aquifer using lactic acid, 

molasses, and soy milk, in Tennessee Section, American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources 

Symposium, April 9–11, 2003, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-5.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 11

Evaluating Oxygen-Releasing Compounds to Enhance Fuel Biodegradation by 

Free-Living Bacteria

Lashun King1, LyTreese Hampton1, and Tom D. Byl2

Anaerobic biodegradation of fuels is generally slower 

than aerobic biodegradation. During an anaerobic experiment, 

almost 100 days were required to biodegrade 2.5 milligrams 

per liter (mg/L) benzene and toluene in liquid-karst microcosms to less than 1 microgram per liter. However, it took less 

than 1 week to biodegrade the same amount of benzene and 

toluene under aerobic conditions. Considering that water in 

many karst aquifers moves at a relatively rapid pace, it follows 

that aerobic conditions are better whether efficient biodegradation processes are desired. The objective of this study was to 

evaluate if oxygen-release compounds (ORCs) enhance fuel 

biodegradation by free-living bacteria found in karst aquifers. 

One ORC that was evaluated is hydrogen peroxide (H2O2). 

Hydrogen peroxide can be toxic to many bacteria in high concentrations (3 percent volume:volume), but is relatively nontoxic in low concentrations (less than 0.1 percent). Two H2

O2

will break down into oxygen (O2) and water (2 H2O). In a 

preliminary study, 250-milliliter liquid-karst microcosms were 

spiked with benzene and toluene. Half of the microcosms also 

were enriched with H2O2 to a final concentration of 30 milligrams per liter (mg/L), which would break down into 15 mg/L 

of dissolved O2. The microcosms enriched with H2O2 continued to biodegrade benzene and toluene at a fast rate, whereas 

biodegradation in the non-enriched microcosms slowed down 

after 2 days. After 7 days, the H2O2-enriched microcosms had 

biodegraded three times as much benzene and toluene as the 

non-enriched microcosms. These preliminary results indicate 

that biodegradation by free-living bacteria found in karst aquifers can be enhanced by H2

O2. Additional ORCs to be evaluated include magnesium peroxide and calcium peroxide. These 

ORCs differ from H2O2 by generating hydroxide ions, in addition to oxygen, which raise the pH significantly; they also tend 

to become solid and diffuse slowly into the water column.

Published as King, L., Hampton, L., and Byl, T.D. Evaluating oxygen-releasing compounds to enhance fuel biodegradation by free-living bacteria in Tennessee Section, American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources Symposium, 

April 9–11, 2003, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-18.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
12 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Biorestoration of a TCE-Contaminated Karst Aquifer by Stimulating Sulfate-Reducing Bacteria

Gregg Hileman2, Koushik Chakraborti1, and Tom D. Byl2

A sanitary landfill situated on a karst terrain in northern 

Tennessee has leaked chlorinated solvents, primarily trichloroethylene (TCE), into a karst aquifer. TCE has been found 

in water samples collected from eight wells screened in the 

karst bedrock aquifer. In July 2002, a mixture of dye, methyllactate, sodium lactate, molasses, and soymilk was initially 

injected into six of the eight wells to determine if the mixture 

would enhance biological reductive dechlorination of TCE. 

The mixture was later injected into a 7th well. Water samples 

were collected and electronic monitoring devices were placed 

in selected wells following injection to monitor changes in 

bacteria, geochemistry, TCE and breakdown products, and 

dye. After 8 months, there was an 85- to 100-percent decrease 

in TCE concentrations in the injection wells, and a 65- to 

100-percent decrease in cDCE concentrations in six of the 

eight wells. Two wells showed an increase in cDCE as TCE 

degraded to cDCE. Concurrent with decreases in TCE and 

cDCE, there were 10-fold increases in sulfur-reducing bacteria 

& sulfide. No dissolved oxygen was found in any of the wells 

1 week after the mixture was injected. Also, the lactic acid 

was metabolized into acetic acid after 4 months.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 13

Supplements to Enhance Groundwater-Microbial Growth and Biodegradation Processes

LeMiracle Hendking1, Patricia Burton1, and Tom D. Byl2

Recent research found that there is a large diverse bacteria population in karst aquifers. These bacteria have been 

shown to biodegrade fuels and chlorinated solvents in liquid 

microcosms. In one chlorinated solvent experiment, bacteria ceased biodegrading PCE. Biodegradation resumed only 

after vitamin B12 was added. Thus it appears that the karst 

conduit water is oligotrophic and requires additional vitamins 

to sustain a vigorous bioremediation. To test this hypothesis, 

microcosms were established using karst bacteria and toluene. 

The growth of bacteria was monitored over time. Then different vitamin supplements were provided and continued bacteria 

growth was evaluated. Bacteria supplied with a mixture of 

complete vitamin B (B1, B2, Niacin, B6, Folate, B12, Biotin, 

Pantothenic acid) grew 50% faster and 5-times more than 

control with no supplement. Vitamin B12 alone did not have a 

significant influence on growth or number. Bacteria supplied 

a complete vitamin supplement (Vitamins A, all the B’s, C, D, 

E, and various minerals) also grew faster than controls, but did 

not remain viable as long as the complete–B supplement. The 

optimum pH for biodegradation of toluene by karst bacteria 

was also found to be 6–8. Based on these results, future studies will evaluate different buffers to maintain optimum pH in 

conjunction with different concentrations of the B-vitamins.

Published as Hendking, L., Burton, P. and Byl, T.D., Supplements to Enhance Groundwater-Microbial Growth and Biodegradation Processes in Tennessee 

Section, American Water Resources Association, 2004, Proceedings from the Fourteenth Tennessee Water Resources Symposium, March 31–April 2, 2004, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-25.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
14 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

The Role of Free-Living and Attached Bacteria in Processing Contamination in Karst Aquifers

By Tom Byl2, Kelly Ray1, Chad Walden1, Valetta Watson1, and Roger Painter1

In karst aquifers, biodegradation can be accomplished 

both by surface-attached and free-living bacteria. Biodegradation by attached bacteria is dependent upon and limited by the 

relatively low surface area to volume ratio (SA/V) of karst 

aquifers. Biodegradation due to free-living bacteria, however, 

is not limited by SA/V, but by residence time. The objective 

of this research was to determine if free-living karst bacteria 

contributed as much to the removal of ammonia (NH3) as 

attached, indigenous karst bacteria. These results were compared with the results of a toluene biodegradation study conducted using the same set up (Painter and others, 2005 KIG). 

The experimental setup included flow-through karst microcosms with high and low SA/V ratios. The low SA/V ratio 

system consisted of three 1-L cylinders connected together 

with non-stick tubing. The high SA/V ratio karst system was 

similar except the cylinders were packed with glass beads to 

increase the SA/V ratio by approximately 500%. Microscopic 

examination confirmed that bacteria colonized the interior 

surfaces of the lab karst systems. Fresh spring water containing between 10,000 and 20,000 indigenous karst bacteria was 

continuously pumped through each 

system. A known quantity of NH3 

was added as a food source and 

measured at the exit port. Flow rates 

were similar and residence time 

differences were compensated for 

with the residence-time distribution 

(RTD) formula described by King 

and others (2005). First-order NH3-

biotransformation rate constants 

were 0.17 day-1 for the low SA/V system and 0.27 day-1 for 

the high SA/V system. In the previous toluene study, the firstorder rate constants were 0.014 hour-1 for the low SA/V system and 0.016 hour-1 for the high SA/V ratio system leading 

Painter (2005) to conclude that free-living bacteria contribute 

as much to toluene biodegradation processes as attached bacteria in karst aquifers. This study suggests that this is not the 

case with respect to NH3 biotransformation in karst systems.

Published as Byl, T., Ray, K., Walden, C., Watson, V., and Painter, R., The role of free-living and attached bacteria in processing contamination in karst 

aquifers, in Kuniansky, E.L., ed., 2008, U.S. Geological Survey Karst Interest Group Proceedings, Bowling Green, Kentucky, May 2008: U.S. Geological 

Survey Scientific Investigations Report 2008–5023, p. 68.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 15

Comparison of Redox Measurements and Geochemical Conditions in a Contaminated Karst 

Aquifer and Their Influence on Biodegradation

R. Darlington1, Gregg Hileman2, and Tom Byl2

Redox measurements measure the capacity of a system 

for oxidation or reduction. The capacity is determined by 

which inorganic ions are present and which redox processes 

are taking place. These redox processes identified by the redox 

measurements characterize the biodegradation process in a 

karst aquifer. Biodegradation is important because by this 

process bacteria break down organic contaminants in a contaminated karst aquifer. Many scientist and engineers hold the 

view that redox probes are incapable of producing an accurate 

determination of the redox processes taking place in an aquifer 

system and consequently whether biodegradation is taking 

place. Expensive geochemical analyses are considered more 

accurate. However by comparing redox measurements, using 

redox probes, with redox processes indicated by geochemical 

analyses, this study proves that redox probes can be used to 

identify the different biological redox processes. The inorganic ions considered in this study are O2

, H2S, NO3

–

, Fe3

+

, 

SO4

2– and CO2

 and these are called terminal electron acceptors (TEAs). TEA characterization is traditionally done using 

geochemical tests. Ground water samples were collected from 

clean and contaminated wells screened in the karst bedrock 

formation every three weeks using bailers. Field tests of temperature, pH, alkalinity, specific conductance and dissolved 

oxygen were conducted. In addition to the field tests, laboratory tests using a spectrophotometer were done to determine 

the concentration of sulfate, nitrate, soluble iron (2+), ammonia and sulfide in the water. Additional chemical information 

was collected using YSI datasonde units that recorded redox 

potential, DO, pH, specific conductance and temperature every 

15 minutes. The units were deployed for months in clean and 

contaminated wells. These data were used to observe trends in 

aquifer geochemistry and depth overtime. Comparison of the 

two data sets showed correlation’s between the concentration 

of TEAs and the datasonde redox measurements. However 

there was a lag time between when the units were deployed 

and when accurate readings were achieved. Once the probes 

reached a true equilibrium with the aquifer they provided an 

accurate assessment of aquifer redox conditions and thus an 

indication of biodegradation efficiency.

Presented as an unpublished poster session at the Tennessee Section, American Water Resources Association, 2002 conference. Tennessee Section, American 

Water Resources Association, 2002, Proceedings from the Twelfth Annual Tennessee Water Resources Symposium, April 2002, Montgomery Bell State Park, 

Burns, Tennessee: Tennessee Section of the American Water Resources Association [variously paged].

1

Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Comparison of Water Geochemistry and Redox Electrodes to Identify Terminal Electron Acceptor 

Processes in an Aquifer

Ramona Darlington1 and Tom D. Byl2

16 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Several metabolic pathways of fuel biodegradation exist 

that have different efficiencies based on the terminal electron 

acceptor process (TEAP) present in the aquifer. The TEAP 

is determined by the oxidation-reduction (redox) potential 

of the aquifer, which is a measure of the system’s capacity 

to give (oxidation) or receive (reduction) electrons. Because 

biodegradation can be an effective way to remove organic 

contaminants from ground water, depending on the TEAP, 

accurate redox measurements are essential. A study was initiated to determine whether a meter equipped with a platinum 

redox electrode could identify the TEAPs as effectively as 

geochemical analysis of the ground water. YSI* datasonde 

units equipped with a redox electrode, dissolved oxygen (DO), 

pH, specific conductance, temperature, and depth probes 

were deployed for 4 to 18 months in uncontaminated and 

fuel-contaminated wells screened in a karst bedrock formation. The datasonde units were serviced (probes re-calibrated, 

data downloaded, and batteries changed) every 2 to 3 weeks. 

Groundwater samples also were collected from the same wells 

during the datasonde servicing. The water samples were tested 

for temperature, pH, alkalinity, specific conductance, and DO 

within minutes of sample collection. Additional water samples 

were placed in clean glass amber bottles, packed on ice, and 

brought back to the laboratory for sufate, nitrate, soluble iron 

(Fe2+), ammonia, and sulfide analyses. These data were used 

evaluate trends in aquifer geochemistry, redox potential, and 

depth over time. An equilibrium time of 2 to 4 hours was 

needed for the YSI datasonde redox electrodes to equilibrate 

with their surroundings. Comparison of the geochemical 

and datasonde data showed a good correlation between the 

concentration of geochemical constituents and the datasonde 

redox measurements, indicating the potential to use a platinum 

redox electrode to identify TEAPs in a contaminated aquifer., 

ammonia and sulfide in the water. Additional chemical information was collected using YSI datasonde units that recorded 

redox potential, DO, pH, specific conductance and temperature every 15 minutes. The units were deployed for months 

in clean and contaminated wells. These data were used to 

observe trends in aquifer geochemistry and depth overtime. 

Comparison of the two data sets showed correlation’s between 

the concentration of TEAs and the datasonde redox measurements. However there was a lag time between when the units 

were deployed and when accurate readings were achieved. 

Once the probes reached a true equilibrium with the aquifer 

they provided an accurate assessment of aquifer redox conditions and thus an indication of biodegradation efficiency.

Published as Darlington, R., and Byl, T.D., Comparison of water geochemistry and redox electrodes to identify terminal electron acceptor processes in an 

aquifer, in Tennessee Section American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources Symposium, 

April 9–11, 2003, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-7.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

*

 Any use of trade, product, or firm name in this abstract is for identification purposes only and does not constitute endorsement by the U.S. Geological 

 Survey or Tennessee State University.
Karst—Bioremediation 17

Free-Living Bacteria Versus Attached Bacteria: Which Contributes More to Bioremediation?

Fuzail Faridi1, Tom Byl2, and Roger Painter1

Researchers have implied that natural bioremediation in karst or fractured rock is unlikely to occur because 

of the lack of bacteria biofilm in karst aquifers. It has been 

stated that hydrologic and geologic characteristics of fractured rock aquifers are not suited for natural bioremediation 

because small microbial populations exist. If bioremediation in bedrock aquifers is dependent upon contact between 

surface-attached bacteria and contaminants, then bioremediation would be limited by the low surface area to volume ratio 

(SA/V) of karst aquifers. However, a quantitative basis for 

accepting or rejecting the assumption that attached bacteria 

dominate bacteria the biodegradation process in karst conduits 

has not been shown. The objective of this research was to 

determine if free-living karst bacteria contributed as much to 

toluene biodegradation as attached bacteria. To accomplish 

this objective, two flow-through reactor systems were established. One reactor system consisted of three 1-liter cylinders 

connected together with Teflon® tubing for a total open 

volume of 3.3 liters. The second reactor system was similar 

to the open system except the cylinders were filled with flat, 

circular glass beads that increased surface area to volume ratio 

approximately 500 percent compared to the open system. Raw 

karst water containing live indigenous bacteria was pumped 

through each system. The flow rate was slightly less in the 

high SA/V system than in the open system to compensate for 

the reduced volume space. Additionally, the residence-time 

distribution of each system was established using rhodamine 

dye in order to calculate biodegradation rates as a function of 

residence-time distribution. Results from the conservative 

tracer studies and toluene degradation studies were used to 

mathematically determine first-order degradation-rate constants for both systems. The resulting first-order rate constants 

were 0.014 hour-1 for the open system and 0.016 hour-1 for 

the high SA/V ratio system, respectively. If surface-attached 

bacteria were the main contributors to the biodegradation process and the SA/V ratio was increased 500 percent, a significantly higher biodegradation rate should have occurred in the 

high SA/V reactor. The biodegradation process was predominantly a result of free-living bacteria in the open volume. The 

15 percent increase in the degradation rate for a 500 percent 

increase in SA/V indicates that attached bacteria may have 

contributed to the observed biodegradation rate.

Published as Faridi, F., Byl, T.D., and Painter, R., Free-living bacteria versus attached bacteria: which contributes more to bioremediation?, in Tennessee 

Section, American Water Resources Association, 2005, Proceedings from the Fifteenth Annual Tennessee Water Resources Symposium, April 2005, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-6.

1

 Tennessee State University, College of Engineering, Technology and Computer Science

2

 U.S. Geological Survey
18 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Quantifying Peroxide-Enhanced Toluene Biodegradation in a Single-Well Injection

Lashun King1, Kendra Smith1, and Tom D. Byl2

Microcosm studies indicate that anaerobic biodegradation of toluene is generally 50 times slower than aerobic 

biodegradation. Because of the potential for the rapid transport 

of dissolved contaminants in karst conduits, aerobic conditions are needed to enhance bioremediation. This study was 

divided into two phases. The objective of the first phase was 

to evaluate oxygenrelease compounds (ORCs) to enhance fuel 

biodegradation by free-living bacteria found in karst aquifers. 

The objective of the second phase was to develop a numerical 

method to quantify the rate of enhanced biodegradation using 

a single well for injection and monitoring. In the first phase 

of this study, the ORCs evaluated were hydrogen peroxide 

(H2O2), calcium peroxide (CaO2), and magnesium peroxide 

(MgO2). The H2O2 molecules break down into oxygen (O2) 

and water (H2O). The CaO2 and MgO2 break down in the 

presence of water into O2 and either CaOH or MgOH, respectively. In this study, 2.25-liter liquid-karst microcosms (for 

example, flasks containing water and free-living karst bacteria) were spiked to 100 micrograms per liter (μg/L) toluene, 

and different ORC concentrations were added. Sterile controls 

also were established with toluene and ORCs to verify that 

toluene removal resulted from biological processes. Additional controls with live bacteria, but no ORC supplements, 

also were established for comparison. Microcosms enriched 

with 3 milligrams per liter (mg/L) H2O2, CaO2, or MgO2 all 

showed greater than 95 percent toluene removal in 7 days, 

as compared to 45 percent removal in live microcosms with 

no ORCs. When the microcosms were enriched with 300 

mg/L H2O2, CaO2, or MgO2, only the H2O2 treatment elicited 

a reduction in toluene of greater than 99 percent in 7 days. 

The other peroxide treatments had slightly enhanced toluene 

removal compared to the live control, but generally were not 

effective at this higher concentration. The decline in MgO2

and CaO2 performance possibly was caused by the simultaneous release of hydroxide, which was found to inhibit biodegradation processes.

In the second phase of the project, a numerical method 

capable of quantifying biodegradation was developed by 

coupling the equation for residence-time distribution to a 

first-order rate of biodegradation. This numerical method was 

evaluated in a laboratory simulation. The simulation included 

a single-well injection of H2O2 and sodium chloride (conservative tracer) into a 5-gallon carboy containing karst water with 

100 micrograms per liter toluene. The carboy was connected 

to a pump that delivered a constant flow of fresh bacteriacontaining karst water (3 milliliters per minute) through the 

5-gallon carboy, thereby diluting and transporting the conservative tracer from the carboy. The toluene was also diluted 

and transported from the carboy, but was also subject to 

biodegradation processes since it is a non-conservative chemical. The rate of toluene removal predicted by the numerical 

model and the observed rate of removal in the experiment 

were within close agreement (18 percent), confirming the 

numerical approach.

Published as King, L., Smith, K., and Byl, T.D., Quantifying peroxide-enhanced toluene biodegradation in a single-well injection, in Tennessee Section, 

American Water Resources Association, 2005, Proceedings from the Fifteenth Annual Tennessee Water Resources Symposium, April 2005, Montgomery Bell 

State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-11.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 19

Evaluating Oxygen-Releasing Compounds to Enhance Toluene Biodegradation by 

Free-Living Bacteria

Lashun King1, Khalid Woods1, LyTreese Hampton1, and Tom D. Byl1,2

Microcosm studies done in our lab found that anaerobic 

biodegradation of toluene was generally 25 to 50 times slower 

than aerobic biodegradation. Considering the potential for 

rapid transport of dissolved contaminants in karst conduits, 

it follows that aerobic conditions are desired for enhancing 

bioremediation. The objective of this study was to evaluate the 

ability of three oxygen release compounds (ORCs) to enhance 

fuel biodegradation by free-living bacteria found in karst aquifers. The ORCs that were evaluated were hydrogen peroxide 

(H2O2), calcium peroxide (CaO2) and magnesium peroxide 

(MgO2). The H2O2 molecules 

will break down into oxygen (O2) 

and water (H2O). The CaO2 and 

MgO2 will break down in the 

presence of water into O2 and 

either CaOH or MgOH, respectively. In this study, 2.25-liter 

liquid-karst microcosms 

(i.e., flasks containing water 

and free-living karst bacteria) 

were spiked to 100 μg/L toluene and different ORC concentrations were added. Sterile controls were also established 

with toluene and ORCs to verify toluene removal was due to 

biological processes. Additional controls with live bacteria, 

but no ORC supplements were also established for comparison. Microcosms enriched with 3 mg/L H2

O2, CaO2, or MgO2

all showed >95% toluene removal in 7 days, as compared to, 

45% removal in live microcosms with no ORCs. When the 

microcosms were enriched with 300 mg/L H2O2, CaO2, or 

MgO2, only the H2O2 treatment elicited a >99% reduction in 

toluene in 7 days. The other peroxide treatments had slightly 

enhanced toluene removal compared to the live control, but 

were generally not effective at this higher concentration. The 

decline in MgO2 and CaO2 performance was possibly due to 

the simultaneous release of hydroxide, which was found to 

inhibit biodegradation processes. Other parameters that will 

be discussed in the poster are dissolved oxygen concentration, 

bacteria concentration, and pH. Based on these results H2O2

appears to be the best ORC candidate for enhancing biodegradation in karst aquifers.

Published as King, L., Woods, K., Hampton, L., and Byl, T.D., Evaluating Oxygen-Releasing Compounds to Enhance Toluene Biodegradation by Free-Living 

Bacteria, in Tennessee Section, American Water Resources Association, 2004, Proceedings from the Fourteenth Tennessee Water Resources Symposium, 

March 31–April 2, 2004, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-18.

1

 College of Engineering, Tennessee State University, Nashville, Tennessee

2

 United States Geological Survey, Nashville, Tennessee
20 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Lactate Induction of the Ammonia Mono-Oxygenase Enzyme and PCE Cometabolism

LyTreese Hampton1, Roneisha Graham1, and Tom D. Byl2

Water containing bacteria was collected from a 

PCE-contaminated karst aquifer in northern-central Tennessee to establish liquid, 1-liter microcosms. The microcosms 

were spiked with known concentrations of perchloroethylene (PCE) and 11 different formulations of lactic acid. The 

ammonia-lactate formulation caused a rapid removal of 

PCE and oxygen (O2). Similar results achieved using a 

second set of microcosms established with ammonia-lactate 

to re-test the removal rate of PCE and O2 indicated a possible co-metabolic PCE-removal process. Although only one 

report of PCE-cometabolism was found in the literature, we 

hypothesized that ammonia-oxidizing bacteria indigenous to 

the karst aquifer were capable of cometabolizing PCE with 

the ammonia mono-oxygenase (AMO) pathway. To test this 

hypothesis, microcosms were established using different forms 

of ammonia (ammonia-lactate, ammonia-chloride, ammonium 

plus sodium lactate), reference controls (sterile, live-no food, 

sodium lactate, sterile + ammonia lactate), and ammonia 

mono-oxygenase inhibitor (2-chloro-6-(trichloromethyl) pyridine). Microcosms treated with ammonia-lactate had the most 

rapid reduction of PCE and O2, followed by the ammonium + 

Na-lactate treatment. The other live microcosms treated with 

ammonia also experienced significant drops in PCE and O2

after 24 hours. The control (sterile and live-no food) microcosms did not experience a significant drop in PCE in the 

same time period. After 24 hours, the rapid PCE removal in 

all the ammonia-treated microcosms slacked off, due to the 

consumption of the oxygen. Tests with the AMO inhibitor did 

not prevent the PCE removal or O2 consumption, indicating 

the inhibitor did not work on this particular AMO enzyme 

or bacteria. It is possible that the lactate stimulates AMO or 

protects the enzyme from inhibition. Additional tests need to 

be conducted to characterize the optimum pH, stoichiometric 

balance, and different AMO inhibitors. Nonetheless, these 

preliminary results provide strong evidence that karst bacteria 

indigenous to this aquifer can cometabolize PCE.

Published as Hampton, L., Graham, R., and Byl, T.D., Lactate induction of the ammonia mono-oxygenase enzyme and PCE cometabolism, in Tennessee 

Section, American Water Resources Association, 2005, Proceedings from the Fifteenth Annual Tennessee Water Resources Symposium, April 2005, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-9.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 21

Biodegradation of Toluene as it Continuously Enters a 5-Liter Karst System

The collaborative research on this project was presented at different conferences by the two 

student researchers.

a) Shawkat Kochary1, Roger Painter1, and Tom Byl2

b) Fuzail Faridi1, Roger Painter1, and Tom Byl2

Contamination releases can occur as slow, long-term 

spills rather than as instantaneous spills. These continuous 

releases can result in a steady state of contaminants that can 

last months to years. Predicting the fate and transport of 

these contaminants in a karst aquifer is especially challenging because of the complex hydrogeology and uncertainties 

in residence time. The objective of this research was to adapt 

the residence-time distribution (RTD) biodegradation model, 

which was developed to predict the biotransformation of a 

single spill in a karst aquifer, for a continuous input of contaminants. Theoretically, the RTD for a karst system calculated 

from either a pulse- or a continuous-input tracer study would 

be identical, but mathematical manipulation of the data for the 

two approaches is quite different. Determination of the RTD 

from a continuous input requires numerical differentiation of 

tracer response data as opposed to numerical integration for 

the pulse approach. Three experimental runs were conducted 

involving the application of a continuous input: (1) rhodamine 

dye alone to establish RTDs for 

the systems, (2) sterile toluene 

(25 micrograms per liter) to quantify abiotic sorption, and (3) toluene with karst bacteria to quantify biodegradation. The three 

replicate karst systems were each 

5 liters and had a continuous flow 

rate of 3.3 milliliters per minute. 

The difference between the RTD-based model prediction and 

the experimental toluene conversions was 17 percent. The 

continuous-input approach (numerical differentiation) had the 

tendency to magnify experimental and random errors in the 

tracer response data as compared to the pulse-input method 

(numerical integration).

This work was supported in part by the U.S. Army Corps of 

Engineers DACW62-00-H-0001 contract.

a) Published as Kochary, S., Painter, R. and Byl, T.D., Biodegradation of Toluene as it continuously enters a 5-liter karst system, in Tennessee Section, 

American Water Resources Association, 2005, Proceedings from the Fifteenth Annual Tennessee Water Resources Symposium, April 2005, Montgomery 

Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-12

b) Unpublished poster presented at Society of Environmental Toxicology and Chemistry (SETAC), 2005 conference

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
22 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Ammonia Oxidation by Bacteria Collected from a Karst-Bedrock Well

Kelly Ray1 and Tom Byl2

Elevated ammonia concentrations can pose an environmental and health problem in groundwater. Animal and human 

wastes, fertilizers and decaying plant material all contribute 

to ammonia-nitrogen into the 

subsurface. Ammonia issues can 

be exacerbated in karst systems 

where it can enter directly 

through sinkholes or disappearing streams without any filtration. 

The rate of ammonia oxidation 

and optimum conditions for 

autotrophic nitrifying bacteria 

in a karst system are not known. 

The objective of this study was to 

characterize the rate of ammonia oxidation using bacteria 

indigenous to a karst aquifer in 

middle Tennessee. Liquid microcosms (300 mL, n=3) were 

established with an initial ammonia concentration of 20 mg/L. 

The pH in one-third of the microcosms was adjusted to either 

pH 3 with HCl, pH 10 with NaOH, or 7 (no addition). The 

growth in bacteria was monitored using a spectrophotometer. 

Ammonia concentrations were also measured through time. 

The microcosms with the greatest rate of ammonia-oxidation 

were pH 7, followed by pH 10 and pH 3, respectively. The 

growth pattern also confirmed that bacteria incubated at pH 7 

had the greatest growth. Bacteria incubated at pH 3 or 10 did 

not demonstrate an appreciable increase in optical density, 

indicating they did not replicate to any significant degree. It 

is hypothesized that bacteria incubated at pH 3 were not able 

to take up ammonia as fast because the ammonia is ionized 

(NH4+) at this pH. It is possible that ionized ammonia less 

able to transfer across the cell membrane than the un-ionized 

form. Further research is needed to answer this hypothesis.

Published as Ray, K., and Byl, T.D., Ammonia oxidation by bacteria collected from a karst-bedrock well, in Tennessee Section, American Water 

Resources Association, 2006, Proceedings from the Sixteenth Tennessee Water Resources Symposium, April 19–21, 2006, Montgomery Bell State Park, 

Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-12.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 23

Ammonia Oxidation by Bacteria Collected from a Karst-Bedrock Well

By Kelly Ray1, Roger Painter1, and Tom Byl2

(NOTE – Although the abstract has the same title as previous abstract, this abstract represents a continuation of the research)

Elevated ammonia concentrations in groundwater pose 

health and environmental problems. In karst systems where 

water can enter directly through sinkholes or disappearing 

streams without any filtration, ammonia contamination can 

be exacerbated. The rate of ammonia oxidation by nitrifying bacteria in karst systems is not known. The objective of 

this study was to characterize the aerobic and anaerobic rate 

of NH3 oxidation using bacteria indigenous to a karst aquifer 

in Middle Tennessee. Static batch reactors using indigenous 

karst bacteria collected from a spring established a first-order 

rate of NH3-oxidation (k) = 0.0209 per day. Because this rate 

appeared to be slow, in a follow-up experiment, the effect of 

supplements and surface area were investigated. It was found 

that, in a flow-through karst system, a 500% increase in surface area to volume (SA/V) ratio increased the k value 54%. 

Addition of 1 g of lactate/L further increased the k value 

almost 10-fold. Because NH3-oxidizing bacteria are autotrophs 

(CO2 fixing), it is hypothesized the lactate stimulated the 

growth of symbiotic bacteria that significantly enhanced the 

activity of NH3-oxidizing bacteria. Anaerobic NH3 oxidation 

was also investigated using data collected in 2002 from an 

anaerobic karst site in northern Tennessee with high levels 

of NH3. The tracer and ammonia data were entered into the 

RTDB model and yielded a calculated ammonia oxidation k

of 0.0168/day. The observation of ammonia biotransformation 

at an anaerobic site is circumstantial evidence that the anaerobic ammonia oxidation pathway called anammox was active. 

Additional research, however, is needed to confirm if the 

anammox pathway was responsible for the observed anaerobic 

removal of NH3.

Published as Ray, K., and Byl, T.D., Ammonia oxidation by bacteria collected from a karst-bedrock well, in Tennessee Section, American Water 

Resources Association, 2006, Proceedings from the Sixteenth Tennessee Water Resources Symposium, April 19–21, 2006, Montgomery Bell State Park, 

Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-12.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
24 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Free-Living Bacteria or Attached Bacteria: Which Contributes More to Bioremediation?

Roger D. Painter1, Shawkat Kochary1, and Tom D. Byl2

Researchers have implied that natural bioremediation in 

karst or fractured rock is unlikely to occur because of the lack 

of bacteria biofilm in karst aquifers. Hydrologic and geologic 

characteristics of fractured rock aquifers have been described 

as not being suited for natural bioremediation because of small 

microbial populations. If bioremediation in bedrock aquifers 

is dependent upon contact between surface-attached bacteria 

and contaminants, then bioremediation would be limited by 

the low surface area to volume ratio (SA/V) of karst aquifers. 

A quantitative basis, however, for accepting or rejecting the 

assumption that attached bacteria dominate the biodegradation 

process in karst conduits has not been shown. The objective 

of this research was to determine if free-living karst bacteria 

contributed as much to toluene biodegradation as attached 

bacteria. Two flow-through reactor systems were established 

to test the different biodegradation rates. Each reactor system 

consisted of four 1.24-liter cylinders connected together with 

glass tubing for a total open volume of approximately 5 liters. 

The second reactor system was similar to the open system 

except the cylinders were filled with acid-washed, circular 

glass spheres that increased surface area to volume ratio 

approximately fivefold compared to the open system. Rhodamine dye was used to calculate the different residence-time 

distributions in each system. A sterile control study established 

that less than 3 percent of the toluene was lost to abiotic 

processes. Next, raw water from a karst aquifer containing 

live, indigenous bacteria was pumped through each system for 

5 days to establish a biofilm on the glass surfaces. Colonization of the surface was confirmed by microscope visualization before toluene was added to the systems. The resulting 

first-order rate constants were computed to be 0.014 per hour 

for the open system and 0.0155 per hour for the packed reactor 

system. If surface-attached bacteria were the main contributors to the biodegradation process and the SA/V ratio was 

increased fivefold, a significantly higher biodegradation rate 

should have occurred in the packed reactor. The results of 

this study indicate that the free-living bacteria indigenous to a 

karst aquifer contribute as much to the toluene biodegradation 

process as attached bacteria.

Published as Painter, R., Kochary, S., and Byl, T.D., Free-living bacteria or attached bacteria: which contributes more to bioremediation? in Kuniansky, 

E.L., ed., 2005, U.S. Geological Survey Karst Interest Group Proceedings, Rapid City, South Dakota, September 2005: U.S. Geological Survey Scientific 

Investigations Report 2005–5160, p. 180–187.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Bioremediation 25

Bacteria Induced Dissolution of Limestone in Fuel-Contaminated Karst Wells

Serge Mondesir1 and Tom D. Byl2

Karst landscapes are formed in water-soluble geologic 

formations, such as limestone, in which dissolution processes 

have enlarged water-transmitting openings. Approximately 

20 percent of the United States is underlain by carbonate rocks 

and is classified as karst, and 40 percent of the United States 

east of the Mississippi River is underlain by karst aquifers. 

Karst ground-water systems are extremely vulnerable to 

contamination. Many organic contaminants such as fuels 

can stimulate bacteria biodegradation and the production of 

carbon dioxide (CO2). The increased respiration by bacteria in 

contaminated karsts aquifers can lead to a significant increase 

in CO2 production and formation of carbonic acid. 

A quantitative study was conducted to determine the 

effect of elevated concentrations of carbonic acid due to 

bacteria action on limestone dissolution. Sealed flasks were set 

up that contained 250 milliliters of distilled water, limestone 

fragments of known size and weight, and varying concentrations of CO2

. The flasks with elevated CO2 concentration had 

a 3-fold increase in the rate of calcium carbonate dissolution. 

Water with elevated CO2 concentrations had a slightly lower 

pH than water with the lower CO2 concentrations, but the 

difference in pH was not statistically significant at the 0.05 

confidence level. Further tests were done to determine if these 

lab results applied to field conditions. Water samples were collected from wells completed in karst aquifers. The CO2

 concentrations in water samples collected from fuel-contaminated 

wells were higher than in samples collected from wells with 

no fuel contamination. Also, the dissolved calcium was usually two or three times greater in the fuel-contaminated wells. 

The results have implications for redesigning geochemical 

models that predict conduit enlargement when fuel contaminants are present in karst aquifers

This work was supported in part by the U.S. Army Corps of 

Engineers DACW62-00-H-0001 contract.

Published as Mondesir, S., and Byl, T.D., Bacteria Induced Dissolution of Limestone in Fuel-Contaminated Karst Wells, in, Kuniansky, E.L., ed., 2005, 

U.S. Geological Survey Karst Interest Group Proceedings, Rapid City, South Dakota, September 2005: U.S. Geological Survey Scientific Investigations 

Report 2005–5160, p. 192.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Geochemical and Microbial Evidence of Fuel Biodegradation in a Contaminated Karst Aquifer in 

Southern Kentucky, June 1999

Tom D. Byl1, Gregg E. Hileman1, Shannon D. Williams1, and James J. Farmer1,2

26 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Complex hydrogeologic conditions coupled with poorly 

understood biodegradation processes in karst aquifers have 

led many to believe that the potential for natural attenuation of petroleum fuel hydrocarbons is limited. This research 

addressed the capacity for biodegradation processes in karst. 

Ground-water samples were collected for bacteria and geochemical analysis from several contaminated monitoring wells 

in an unconsolidated regolith and karst aquifer that had varying concentrations of dissolved fuel. Bacteria concentrations 

were greatest in ground-water samples containing the greatest fuel contamination. Additionally, bacteria isolated from 

fuel-contaminated ground-water samples readily grew in Petri 

dishes with dissolved gasoline fuel as the only source of food. 

The wells with screens intersecting less contaminated sections 

of the aquifer had greater dissolved oxygen concentrations 

(6.3 milligrams per liter) than those intersecting more contaminated sections (dissolved oxygen less than 0.1 milligrams per 

liter). Also, where the oxygen concentrations were diminished, 

geochemical evidence indicated that anaerobic processes were 

active. This evidence includes elevated levels of ammonia and 

ferrous iron in the fuel-contaminated ground-water samples. 

Based on these results, biodegradation of fuel constituents in 

the karst aquifer is indicated, and therefore, natural attenuation 

should not be disregarded because of preconceptions about 

low microbial activity in karst aquifers.

Published as Byl, T.D., Hileman, G.E., Williams, S.D., and Farmer, J.J, Geochemical and microbial evidence of fuel biodegradation in a contaminated karst 

aquifer in southern Kentucky. June 1999, in Eve L. Kuniansky, editor, 2001, U.S. Geological Survey Karst Interest Group Proceedings, Water-Resources 

Investigations Report 01–4011, p. 151–156.

1

 U.S. Geological Survey

2

 U.S. Geological Survey and Tennessee State University
Karst—Contaminant Degradation Models 27

Karst—Contaminant Degradation Models

Karst aquifers are vulnerable to contamination and can 

have complex aquifer properties and flow characteristics. 

Ground-water models based on Darcy’s Law have limited application in karst aquifers, especially at a local scale 

typical of contamination sites. Research conducted by TSU 

professors, PEER students, and USGS scientists developed 

and applied non-standard modeling techniques to flow and 

contaminant degradation in karst aquifers. PEER students 

developed the non-standard models including a residiencetime-distribution (RTD) model and applied the Lattice 

Boltzmann method (LBM) to simulate flow rates in karst 

aquifers. The LBM is a simulation technique for complex fluid 

systems and was applied to PEER research to simulate flow 

in a laboratory-scale karst microcosm. The RTD model was 

coupled with a pseudo-first-order rate of biodegradation and 

then successfully modeled contaminant degradation through 

a laboratory-scale karst microcosm. The research continued 

with the evaluation of the model and the application of the 

RTD-biodegradation model to field sites. The results from 

the application of the LBM and RTD models indicate that 

these models can be applied to karst systems and can provide 

additional information to aid in characterizing and remediating 

contaminants in karst systems.

Numerical Modeling of Flow Simulations and Rate of Biodegradation in a Karst Microcosm

Valetta Watson1, Roger Painter1, Tom Byl2

The contamination of aquifers with petroleum hydrocarbons has lead to the development of several bioremediation strategies. Bioremediation has received special attention 

because it results in the mineralization of the petroleum 

hydrocarbon in the sub-surface. This eliminates the need for 

expensive excavation of the contaminated zone. In spite of 

its utility, bioremediation has not been aggressively pursued 

as a remediation option in karst aquifers. Skeptics theorize 

that bacteria concentrations in karst are negligible, and that 

contaminants are rapidly flushed out of a karst aquifer with 

little biodegradation. This research addresses the notion 

that insignificant biodegradation occurs in karst conduits. 

In this study, a dye tracer was used in a karst microcosm to 

gain information about its characteristics. We have utilized 

the Lattice Boltzmann method to simulate flow rates in 

karst aquifer microcosms. The Lattice-Boltzmann method 

(LBM) uses a statistical approach to solve the Navier-Stokes 

equations for fluid flow. The LBM uses the nearest neighbor 

relations on a micro scale to evolve the macroscopic dynamics 

of the system. Preliminary results show that the LBM can be 

used to model the complex interactions associated with karst 

aquifer flows.

Presented as an unpublished poster session during the 1999 Tennessee AWRA conference. 

Crabtree, L.R., Bradley, M.W., Blunt, Tiffany, and Pierre, Salnave, eds., 1999, Proceedings from the Ninth Annual Tennessee Water Resources 

Symposium, April 1999, Nashville, Tennessee: Tennessee AWRA, 271 p. 

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
28 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Numerical Simulation of Flow and Contaminant Transport in a Karst Aquifer Conduit

Valetta Watson1, Roger Painter1, and Tom D. Byl2

Computer models that simulate water flow or contaminant transport in unconsolidated aquifers have little transferability to karst aquifers because they are based on equations 

derived under the assumption of laminar flow. Flow through 

conduits developed in karst aquifers may be turbulent, and the 

locations of the conduits may not be well understood, limiting 

the usefulness of these models.

To address this issue, the Lattice-Boltzmann method 

(LBM), a powerful technique that can be used for computational modeling of complex fluid flow, was used to develop 

a numerical model to simulate water flow and contaminant 

movement in a theoretical karst conduit. The LBM is based 

on statistical physics and uses a numerical approach to solve 

the Navier-Stokes equations for fluid flow. In this study, twodimensional images were generated using object grids that 

delineated the structure and surface of the karst conduit within 

which the fluid flow was being modeled. Fluid movement 

was visualized by assembling simulation outputs as a series 

of time-step animations. For the volume simulation outputs, 

transparency was used to indicate fluid density and color 

intensity was used to indicate fluid velocity.

The numerical results obtained from the simulations were 

compared against those obtained from a laboratory dye-tracer 

experiment to validate the model. An artificial karst conduit 

of varying dimensions was constructed in the laboratory by 

connecting a series of glass tubes (≤ 2 inch diameter). Once 

a constant discharge was observed at the outlet, rhodamine 

dye was injected at the inlet of the conduit and measured at 

the outlet as a function of time. An analysis of the quantitative 

experiment indicated that distinct re-circulation zones existed 

within the artificial karst conduit. These same features were 

captured in the LBM simulation. The agreement between the 

LBM simulation and the laboratory study demonstrates the 

potential for using the LBM technique to model the transport 

characteristics and residence time of contaminants within a 

karst system.

Published as Watson, V., Painter, R. and Byl, T.D., Numerical simulation of flow and contaminant transport in a karst aquifer conduit in Tennessee 

Section, American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources Symposium, April 9–11, 2003, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-38.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Use of Residence-Time Distribution Coupled with a Biodegradation Rate to Predict Toluene 

Removal in an Artificial Karst System

R. Painter1, V. Watson1, and T. Byl2

Karst—Contaminant Degradation Models 29

Approximately 40 percent of the United States east 

of the Mississippi River is underlain by karst aquifers 

(Quinlan, 1989). Karst ground-water systems are extremely 

vulnerable to contamination; however, the fate and transport 

of contaminants in karst areas are poorly understood because 

of the complex hydraulic characteristics of karst aquifers. 

Ground-water models developed using Darcy’s Law coupled 

to rates of biodegradation are useful for predicting the fate of 

fuels in unconsolidated aquifers, but have little utility in karst 

conduits. Conceptual models developed for karst aquifers have 

a consistent theme of non-ideal flow, storage and active flow 

components. This research used a residence-time distribution 

(RTD) model approach that integrated residence times of contaminants isolated in storage areas with the residence time of 

contaminants moving through conduits coupled to a pseudofirst order rate of biodegradation. The microcosms consisted of 

4 1-liter chambers connected with small glass tubing. A peristaltic pump provided a consistent flow of karst water from a 

10-gallon reservoir. First, a quantitative dye study was done to 

establish the residence time distribution of the three systems. 

This was followed by a sterile toluene run to measure sorption 

of toluene to the microcosm systems. The third microcosm 

run incorporated karst bacteria and toluene. There was good 

agreement between the predicted toluene concentration by the 

RTD model and the experimental data. Eighty-seven micrograms of toluene was injected into each system. Recovery in 

the sterile systems ranged from 62.6 μg to 84.6 μg, indicating 

that sorption was minimal. Recovery of toluene in the biotic 

systems ranged from 36.5 to 41.6 μg indicating biodegradation occurred. The mean residence-time distributions for the 

3 systems were ranged from 40 to 43 hours. The RTD model 

predicted 44, 48 and 52 percent toluene biodegradation for the 

3 systems, respectively. The biodegradation calculated from 

the experiments was 44, 48 and 50 percent respectively. Thus, 

the RTD coupled to a pre-determined biodegradation rate 

appears to accurately predict the amount of toluene that the 

bacteria will degrade.

Published as Painter, R., Watson, V., and Byl, T.D., Use of Residence-Time Distribution Coupled with a Biodegradation Rate to Predict Toluene Removal 

in an Artificial Karst System, in Tennessee Section, American Water Resources Association, 2004, Proceedings from the Fourteenth Tennessee Water 

Resources Symposium, March 31–April 2, 2004, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p 2A-14.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Development of a Computer Program that Uses Residence-Time Distribution and First-Order 

Biodegradation to Predict BTEX Removal in Karst Aquifers

Ryan Fitzwater1, Roger Painter1, Valetta Watson1, and Tom D. Byl2

30 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Approximately 40 percent of the United States east of the 

Mississippi River is underlain by karst aquifers. Karst groundwater systems are extremely vulnerable to contamination; 

however, the fate and transport of contaminants in karst areas 

are poorly understood because of the complex hydraulic characteristics of karst aquifers. Ground-water models developed 

using Darcy’s Law coupled to rates of biodegradation are useful for predicting the fate of fuels in unconsolidated aquifers, 

but have little utility in karst conduits. Conceptual models 

developed for karst aquifers have a consistent theme of nonideal flow, storage and active flow components. This research 

used a residence-time distribution (RTD) model approach that 

integrated residence times of contaminants isolated in storage 

areas with the residence time of contaminants moving through 

conduits coupled to a pseudo-first order rate of biodegradation. 

The microcosms consisted of 4 1-liter chambers connected 

with small glass tubing. A peristaltic pump provided a consistent flow of karst water from a 10-gallon reservoir. First, 

a quantitative dye study was done to establish the residencetime distribution of the three systems. This was followed by 

a sterile toluene run to measure sorption of toluene to the 

microcosm systems. The third microcosm run incorporated 

karst bacteria and toluene. The removal of toluene predicted 

by the RTD-biodegradation model and the experiment were 

within 2 percent agreement (n=3). The RTD-biodegradation 

model was transformed into a user-friendly program that utilizes MS Excel® with Visual Basic interfaces. The input sheet 

of this prototype program requires site information, a biodegradation rate, and the results of a quantitative tracer study. The 

results, or output pages, provide residence-time distribution 

graphs and various statistical calculations. The output pages 

also report the calculated amount of BTEX removed during 

transport through the karst aquifer based on RTD and biodegradation. Additional work is needed to incorporate dilution 

into the model.

Note: Any use of trade, product or firm names in this document is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Published as Fitzwater, R., Painter, R., Watson, V., and Byl, T.D., Development of a computer program that uses residence-time distribution and first-order 

biodegradation to predict BTEX removal in karst aquifers, in Tennessee Section, American Water Resources Association, 2005, Proceedings from the 

Fifteenth Annual Tennessee Water Resources Symposium, April 2005, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American 

Water Resources Association, p. P-7.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Contaminant Degradation Models 31

Development of a Protocol for Enhanced Bioremediation in Karst Using a Single Injection Well

Tarra M. Beach1, Lashun K. King1, Roger Painter1,and Tom D. Byl2

Approximately two-thirds of Tennessee and Kentucky are 

underlain by karst terrain. The groundwater aquifers in karst 

terrains are particularly susceptible to contamination. Once a 

contaminant has entered a karst-bedrock aquifer it is difficult 

to determine its precise flow-path through the bedrock. The 

contaminant may reside for long periods in stagnant areas of 

the aquifer or it may be rapidly transported through tortuous 

conduits. All the while, the contaminants are susceptible to 

biodegradation processes in the aquifer. Recently, the RTDB 

model was developed to calculate the contaminant biotransformation as a function of residence time in the aquifer. There 

have been two field tests successfully applying RTDB to contaminated karst sites with enhanced biodegradation. However, 

there is currently no standard protocol for the application of 

RTDB on karst sites. The objective of this research was to 

develop a general protocol for enhanced bioremediation of 

organic contaminants in karst terrains and the application of 

the RTDB model. Furthermore, since the discharge point is 

often off-site, unknown, or diffused, a single-well method was 

developed for the protocol. This protocol is based on data and 

reports from field studies done at Ft. Campbell. The protocol 

is divided into six steps. The first step is to gather historical 

data about the spill and develop background information about 

the site (geochemical, biological, and hydrological information). Second, based on the information collected in step one, 

an enhancement mixture is decided upon and calculations are 

done to determine how much enhancement-mixture should 

be injected. Third, the enhancement mixture mixed with a 

conservative tracer is injected at a depth equal to the conduit opening(s). Fourth, the tracer concentration is measured 

through time along with the chemical, hydrological and biological measurements. Fifth, the tracer data is used to calibrate 

the residence-time distribution portion of the RTDB model. 

Sixth, once calibrated, the RTDB model is used to quantify the 

amount and rate of contaminant biodegradation in the aquifer 

around the well. The protocol is intended to be flexible so it 

can be applied to a variety of contaminated karst sites.

Published as Beach T.M., King, L.M., Painter, R., and Byl, T.D., Development of a protocol for enhanced bioremediation in karst using a 

single injection well, in Tennessee Section, American Water Resources Association, 2006, Proceedings from the Sixteenth Tennessee Water 

Resources Symposium, April 19–21, 2006, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water 

Resources Association, p. P-4.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
32 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Evaluating Peclet Values and the Role of Advection, Dispersion and Diffusion in Tracer Studies

Jameka Johnson1, Carlton Cobb1, Lonnie Sharpe1, and Tom Byl2

Karst aquifers have been recognized as one of the 

most challenging geological media in terms of groundwater 

modeling. Numerical models based on Darcy’s Law often are 

unable to accurately characterize contaminant flow through 

the heterogeneous fractures and dissolution features. Lab and 

field data were used to study dispersion, matrix diffusion and 

advection. The residence-time distribution (RTD) formula was 

used to calculate Peclet values, which are indicative of advection and dispersion properties. Based on field tracer studies 

published in the literature, it appears that when a tracer study 

was conducted with an artificial head (i.e., the dye is pushed 

or flushed through the system), the system was dominated by 

advection processes. When a natural water gradient was used, 

the system was dominated by dispersion processes and a longer tail is produced. However, it was not clear if the dispersion 

was due to turbulence or matrix dispersion. Lab experiments 

were conducted to try and differentiate between the hydrodynamic dispersion, heterogenous advection and matrix diffusion. The lab system, consisting of a plexiglass box holding 

limestone rocks with 

permeable fractures, 

was used to measure tracer diffusion under different 

hydrologic conditions. Preliminary 

results from those 

studies back the field 

observations, that 

pushing the system tends to result in plug-like flow (advective flow) and less matrix diffusion. Additional tracer studies 

are scheduled for a wetland to evaluate the Peclet values, 

dispersion and advection values for the system under stormand base-flow. These findings will have implications for the 

interpretation of tracer tests designed to measure advection, 

dispersion and diffusion of contaminants as they transport 

through complex hydrologic systems.

Published as Johnson, J., Cobb, C., Sharpe, L., and Byl, T.D., Evaluating peclet values and the role of advection, dispersion and diffusion in tracer 

studies, in Tennessee Section, American Water Resources Association, 2007, Proceedings from the Seventeenth Tennessee Water Resources 

Symposium, April 17–19, 2007, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p. P-10.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Contaminant Degradation Models 33

Application of the RTD Model to Analyze the Fate and Transport of Ammonia in Laboratory 

Karst System

Kelly Ray1, Roger Painter1, and Tom Byl2

Elevated ammonia concentrations in groundwater pose 

health and environmental problems. The impact of ground 

water contamination can be exacerbated in karst systems 

where water can enter directly through sinkholes or disappearing streams without any filtration. Karst aquifers are highly 

heterogeneous and cannot be adequately described by Darcian 

principles used to characterize flow in sandy aquifers. The 

objective of this research was to determine if a residence time 

distribution (RTD) based model in conjunction with the advection dispersion equation could adequately describe the fate and 

transport of ammonia in a karst aquifer. To accomplish this 

task, a laboratory karst system was constructed to simulate the 

non-ideal flow. Feeding ammonia solution to this simulated 

system in a controlled fashion allowed the model to be validated by comparing the actual concentration versus time data 

at the systems effluent to the model predicted values. Static 

batch reactors using indigenous karst bacteria established a 

first-order rate of NH3-oxidation with a k value of 0.0209 per 

day. These results suggest that ammonia degradation does not 

occur at significant rate in karst under ambient conditions. 

In a follow-up experiment, supplements and surface area 

were investigated. A flow-through karst system with a 400% 

increase in surface area to volume (SA/V) ratio increased the 

k value 54%. The addition of 1 g of lactate/L increased the 

k value almost 10-fold.

Published as Ray, K., Painter, R. and Byl , T.D., Application of the RTD model to analyze the fate and transport of ammonia in laboratory karst 

system, in Tennessee Section, American Water Resources Association, 2007, Proceedings from the Seventeenth Tennessee Water Resources 

Symposium, April 17–19, 2007, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p. P-14.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
34 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Development and Verification of a Computer Program That Predicts Fuel Biodegradation in 

Karst Aquifers

Ryan Fitzwater1, Patricia Burton1, Roger Painter1, and Tom Byl2

Approximately 40 percent of the United States east of 

the Mississippi River is underlain by karst aquifers. Karst 

ground-water systems are extremely vulnerable to contamination; however, the fate and transport of contaminants in karst 

areas are poorly understood because of the complex hydraulic 

characteristics of karst aquifers. Ground-water models developed using Darcy’s Law coupled to rates of biodegradation 

are useful for predicting the fate of fuels in unconsolidated 

aquifers, but have little utility in 

karst conduits. Conceptual models developed for karst aquifers 

have a consistent theme of nonideal flow, storage, and active 

flow components. This research 

used a residence-time distribution (RTD) model approach that 

integrated residence times of 

contaminants isolated in storage 

areas with the residence time of 

contaminants moving through 

conduits coupled to a pseudofirst order rate of biodegradation. 

This RTD-biodegradation model was adapted for large sites 

with a known source-to-discharge connection, and, for small, 

singlewell, injection-monitoring systems. The first method 

assumes second points are measured (a release point and 

discharge point) and RTD is calculated using numerical 

integration. The second method assumes a single measuring 

point (as it leaves the release area) and uses differential integration to solve for RTD. These two approaches were tested 

in laboratory karst systems and fuel-contaminated field sites. 

The results found that the 2-point strategy worked well in the 

lab, but failed to account for dilution in the large-scale field 

study. Dilution must be factored into large scale site evaluations. The single-point strategy worked well in the lab and 

field studies. Both methods show great promise. These two 

numerical equations were converted to computer algorithms. 

Next a Microsoft Excel® 2002 and the Visual Basic® programming language were utilized to create a user friendly interface 

for the computer program. The RTD method that utilizes 

2-monitoring points requires additional work on the dilution 

term before it is ready for use. The single point injectionmonitoring method appears to be ready for use at contaminated karst sites.

Published as Fitzwater, R., Burton, P., Painter, R., and Byl, T.D., Development and verification of a computer program that predicts fuel biodegradation in karst aquifers, in Tennessee Section, American Water Resources Association, 2006, Proceedings from the Sixteenth Tennessee Water 

Resources Symposium, April 19–21, 2006, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p. P-7.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Residence Time Distribution Derived from Independent Gamma Distributions of Tracer Travel 

Distance and Linear Velocity

By Roger Painter1, Tom Byl2, and Valetta Watson1

Karst—Contaminant Degradation Models 35

The advection dispersion equation (ADE) is widely used 

as a predictor of residence time distributions (RTDs) for tracer 

breakthrough curves for karst systems. Solutions of the ADE 

for tracer breakthrough studies with near plug flow behavior 

are characteristically Gaussian in appearance. However, very 

few, if any, quantitative tracer studies result in tracer concentrations that are normally distributed about the mean residence 

time. While the symmetry of Gaussian breakthrough curves 

often correctly predicts finite tracer concentrations at zero 

time, it generally does not accurately predict actual tracer 

breakthrough curves, which invariably are characterized by 

relatively long tails. This suggests that a different conceptual 

approach may be appropriate for describing these systems in 

easily visualized terms. The objective of this project was to 

develop a more descriptive approach of tracer break-through 

data based on the gamma probability density function. The 

tracer travel distance and tracer linear velocity were assumed 

to be randomly distributed variables with gamma distributions. 

The RTD for tracer breakthrough curves was derived from 

the individual distributions of tracer travel distance and linear 

velocity. This approach was compared and contrasted with the 

traditional approach based on the ADE for modeling tracer 

breakthrough data at a karst site, as well as, modeling the rate 

of biodegradation of toluene in laboratory karst aquifers.

Originally published as Painter, R., Byl, T., and Watson, V., Residence time distribution derived from independent gamma distributions of tracer travel 

distance, in Kuniansky, E.L., ed., 2008, U.S. Geological Survey Karst Interest Group Proceedings, Bowling Green, Kentucky, May 2008: U.S. Geological 

Survey Scientific Investigations Report 2008–5023, p. 75.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Adaptation of the Residence Time Distribution (RTD)-Biodegradation Model to Quantify

 Peroxide-Enhanced Fuel Biodegradation in a Single Karst Well

Lashun K. King1,3, Roger D. Painter1, and Tom D. Byl2

36 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

This field study was conducted to determine if a numerical model incorporating residence time distribution (RTD) 

coupled to a first-order rate of biodegradation (k’) could be 

used to quantify toluene and benzene removal in a single 

karst-well injection system. This study involved injecting 

sodium chloride (NaCl) as a conservative tracer, as well as 

hydrogen peroxide (H2O2), to enhance aerobic biodegradation 

of toluene and benzene. A 100-gallon volume of fuel-contaminated karst aquifer water was pumped into a container. NaCl 

(1.25 kilograms) and 33 percent H2O2 (4 liters) were mixed 

into the water and injected back into the bedrock aquifer. The 

NaCl, dissolved oxygen, benzene and toluene concentrations 

were monitored for several weeks. Results show that benzene 

and toluene concentrations declined approximately 10 times 

faster than the NaCl concentrations, indicating enhanced biodegradation. The RTD was calculated by using the declining 

NaCl-concentration curve through time. The biodegradation 

rate was derived from the benzene and toluene data. The RTDbiodegradation formula (described in this paper) was used to 

predict and quantify the enhanced biodegradation of benzene 

and toluene in the karst aquifer. The RTD-biodegradation 

formula predicted benzene and toluene concentrations in the 

well through time to within 1 microgram per liter (μg/L) of 

the actual concentration. This close agreement between the 

RTD-biodegradation model prediction and the measured concentration confirms that this method can be used to quantify 

enhanced biodegradation in a single karst injection well.

Published as King, L.K., Painter, R.D., and Byl, T.D., Adaptation of the Residence Time Distribution (RTD)-Biodegradation Model to Quantify PeroxideEnhanced Fuel Biodegradation in a Single Karst Well, in Kuniansky, E.L., ed., 2005, U.S. Geological Survey Karst Interest Group Proceedings, Rapid City, 

South Dakota, September 2005: U.S. Geological Survey Scientific Investigations Report 2005–5160, p. 174–179.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

3

 Currently with Virginia Polytechnical Institute
Karst–Hydrology 

The occurrence and movement of contaminants in a 

karst aquifer are controlled by the hydraulic properties of 

the aquifer and the volume and rate of ground-water flow 

through the aquifer. As part of the process to better understand ground-water contamintation, the PEER program has 

included research on karst hydrology and aquifer characteristics. Students in the PEER program have conducted studies on ground-water recharge in a karst system, evaluated 

land-use effects on water quality in small watersheds in 

Middle Tennessee, applied methods to evaluate advective 

versus dispersive contaminant transport, and characterized the 

geochemistry of a shallow, local aquifer discharging at springs 

near the TSU campus. The PEER students have conducted the 

field studies and measured water quality and springflow. The 

research was conducted at multiple site in Middle Tennessee 

and included the Mississippian limestone aquifers in Dickson 

and Montgomery Counties, and the Ordovician limestone 

aquifers in Davidson and Bedford Counties, Tennessee.

Karst—Hydrology 37

Advection Versus Dispersion as Determined by Single-Well Tracer Studies

Tarra M. Beach1, Michael Bradley2, Roger Painter1, and Tom D. Byl2

Approximately two-thirds of Tennessee and Kentucky 

are underlain by karst terrain. The groundwater aquifers in 

karst terrains are particularly susceptible to contamination; 

once a contaminant has entered a karst-bedrock aquifer it 

is difficult to determine its precise flow-path through the 

bedrock. The contaminant may reside for long periods in 

stagnant areas of the aquifer or it may be rapidly transported 

through tortuous conduits. All the while, the contaminants are 

susceptible to biodegradation processes in the aquifer. Traditional dye trace studies carried out in karst terrains required 

two or more points of assessment. A dye, such as rhodamine 

or sodium chloride, would be injected into a well, sinkhole, 

or stream; that dye’s length of time to resurface at a spring 

would be measured–this could lead to extremely long-term 

projects which could provide no conclusive results. Therefore, 

the objective of this research was to develop a tracer injection procedure that would involve one well. A quantity of 

sodium chloride was injected and its dissipation over time was 

monitored. The data collected yielded a response curve for the 

specific conductance of the sodium chloride injections, and in 

turn, permitted an examination of the properties of advection 

and dispersion in the area around the well. These properties 

indicated whether a site was suitable for remediation. Monitoring and production wells in Dickson, Tennessee and Fort 

Campbell, Kentucky were utilized to determine these hydraulic conductivity properties.

Published as Beach, T.M., Bradley, M.W. Painter, R. and Byl , T.D., Advection versus dispersion as determined by single-well tracer studies, in

Tennessee Section, American Water Resources Association, 2007, Proceedings from the Seventeenth Tennessee Water Resources Symposium, 

April 17–19, 2007, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-3.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Enhanced Groundwater Recharge Can Alleviate Storm-Water Flow and Dilute Groundwater 

Pollution in Karst Terrane

Derek Lovett1, Nkechi Chieke1, and Tom Byl2

38 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Storm-water runoff is becoming a major issue as flashflooding becomes a common theme in many low-lying areas. 

As water is diverted to streams, groundwater recharge is 

diminished. This project focused on the development of a 

numerical model to quantify enhanced groundwater recharge 

in karst terrains. A karst-lab model system was set up to 

analyze the effects of enhanced recharge on spring discharge 

and contaminant dilution. Recharge was quantified using 

a modified water-budget method by measuring discharge 

and water levels during a rain simulation. Placement of the 

infiltration-basin (up-gradient, down-gradient, dispersed) 

affected transport and dilution of contaminants. Placing the 

enhanced infiltration up-gradient of a spill resulted in rapid 

transport of the contaminant to the spring. Enhancing infiltration down-gradient of the groundwater spill delayed migration 

of the contaminant. When infiltration was distributed over the 

entire aquifer, dilution effects 

varied depending on the rate of 

discharge. Analyzing various 

infiltration scenarios increased 

understanding of transport 

and dilution effects, as well as 

assisted development of a numerical approach for quantifying 

recharge in karst aquifers. Applying this approach to the field 

required extensive knowledge 

of the basin hydrology and soil 

hydraulic conductivity properties. 

Management of groundwater resources can be better optimized by understanding the influence that infiltration basins 

have on an aquifer system.

Presented as an unpublished poster session at the 2007 Tennessee Section, AWRA conference.

Tennessee Section, American Water Resources Association, 2007, Proceedings from the Seventeenth Tennessee Water Resources Symposium, 

April 17–19, 2007, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association [variously paged].

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Karst—Hydrology 39

Characterization of Bacteria and Geochemistry of Springs in Nashville, Tennessee

Patrice Armstrong1, Carlton Cobb3, Brandon Cobb3, Jennifer Stewart-Wright4, Tom Byl2

The objective of the project was to evaluate the water 

quality of four limestone bedrock springs in an urban environment during a severe drought in the summer of 2007. Three 

of the springs were discovered on the Tennessee State University (TSU) campus in Nashville, TN in May, 2007. Two 

are located near a poultry research facility and a third near 

the TSU athletic center. An additional spring flowing from a 

cave in the Charlotte Park neighborhood of west Nashville 

(Carlos Cave) was also included in the study. The two TSU 

springs behind the 

poultry barns were 

sampled approximately every week 

from June through 

September, 2007. 

The cave and TSU 

athletic center 

springs were sampled less frequently. 

Water quality parameters included temperature, specific 

conductance, and dissolved oxygen, pH, sulfate, nitrogen, E. 

coli, and bacteria Biological Activity Reaction Tests (BART). 

Continuous water-quality monitoring devices were installed 

at two of the springs to measure changes associated with different weather patterns. Water temperatures were very stable, 

ranging from 16oC in June to 19oC in September. Sulfate concentrations were consistently higher in the spring water than 

the receiving surface waters. Conversely, nitrogen levels were 

lower in the spring water (< 10 mg/L) than the surface waters. 

Fecal bacteria levels fluctuated randomly with no discernable 

correlation to weather pattern. BART tests confirmed the presence of denitrifying, iron-reducing, sulfur-reducing, and slimeproducing bacteria at each of the springs. Spring discharges 

decreased at all sites as the drought continued but never 

decreased below 10 gallons per minute. The data showed that 

each spring had unique water quality characteristics reflective 

of the different hydrologic recharge areas that replenish them.

Originally published as Armstrong, P., Cobb, C., Cobb, B., Stewart-Wright, J., and Byl, T.D., Characterization of bacteria and geochemistry of 

springs in Nashville, Tennessee, in Kuniansky, E.L., ed., 2008, U.S. Geological Survey Karst Interest Group Proceedings, Bowling Green, Kentucky, 

May 2008: U.S. Geological Survey Scientific Investigations Report 2008—5023, p. 69.

1

 Tennessee State University, Department of Biological Science

2

 U.S. Geological Survey

3

 Tennessee State University, College of Engineering, Technology, and Computer Science 

4

 Tennessee State University, Office of Research and Sponsored Programs
Land-Use Effects on Bacteria Loads and Water Quality in Small Karst Catchments of the Upper 

Duck River Watershed

James J. Farmer1

40 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Several streams in the upper Duck River watershed 

are listed by the Tennessee Department of Environment and 

Conservation as impaired by elevated bacteria levels. Potential 

sources of bacteria in the watershed include grazing, confinedanimal operations, and domestic septic systems. The relative 

contributions of these sources to elevated bacteria levels are 

not known. The effects of land-use on stream water-quality 

in karst topography will be determined by studying four rural 

catchments in the upper Duck River watershed. Two of the 

study catchments will be small and in close proximity to each 

other to control for natural variability in water chemistry, 

geology, and physiography. One catchment will encompass 

the two small catchments, and the fourth will be located on the 

main stem of the Duck River. This study will be conducted in 

two phases. The first phase will characterize the water quality and land use across the study to identify spatial patterns. 

The second phase will investigate temporal variability of 

bacteria counts and the use of surrogates, such as turbidity, for 

predicting bacteria loads.

Published as Farmer, J.J., Land-use effects on bacteria loads and water quality in small karst catchments of the Upper Duck River watershed, in Tennessee 

Section, American Water Resources Association, 2008, Proceedings from the Eighteenth Tennessee Water Resources Symposium, April 2008, Montgomery 

Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. 2C-23.

1

 Tennessee State University and U.S Geological Survey
Use of Polymerase Chain Reaction and Oligonucleotide Hybridization Probes to Determine the 

Source of Fecal Contamination in Karst Terranes

Tom D. Byl1 and James J. Farmer2

Methods in Contaminant Evaluation 41

Fecal contamination of surface and ground water remains 

a serious health problem in the U.S. and the world. This 

problem cannot be adequately addressed until the source of the 

contamination is known and remediated. At present, no standard monitoring technique exists that can identify the source 

of the bacteria. We have been working to modify and apply 

the molecular technique known as “oligonucleotide hybridization probes” to identify bacteria sources in the karst terrane of 

Middle Tennessee. The oligonucleotide hybridization probes 

can be used to target ribosomal RNA and DNA. Fecal bacteria 

unique to a host species can be identified using this molecular 

technique. Tests have been conducted using RNA hybridization probes that target universal sequences (all bacteria), 

E. faecalis (warm blooded animals), Lachnospira multiparus

(ruminants), Fibrobacter succinogenes (ruminants), Fibrobacter succinogenes (ruminants), Fibrobacter intestinales

(ruminants), Bacteroides distasonis (humans), Bacteroides 

vulgates (primarily human), Bacteroides fragilis (human) and 

Salmonella sp. (poultry & human pathogen). We were able 

to differentiate between these organisms in blind studies, but 

the concentration of bacteria had to be equal to or greater than 

1,000 bacteria per liter before the cells were visually detected. 

The sensitivity was increased by using polymerase chain reaction (PCR) to amplify nucleic acid sequences on DNA (instead 

of RNA). PCR increased the sensitivity so that only 5 cells per 

liter were required for positive detection. Using PCR, we identified Bacteroides fragilis, a bacterium unique to humans, and 

E. coli in a water and biofilm sample collected from a karst 

spring-fed stream in Middle Tennessee. These preliminary 

results indicate this technique has potential to identify sources 

of fecal bacteria in hydrologically complicated karst terranes.

Originally published as Byl, T.D., and Farmer, J.J., Use of polymerase chain reaction and oligonucleotide hybridization probes to determine the source 

of fecal contamination in karst terranes, in Tennessee Section, American Water Resources Association, 2001, Proceedings from the Eleventh Annual 

Tennessee Water Resources Symposium, April 2001, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p. 2B-41.

1

 U.S. Geological Survey

2

 Tennessee State University and U.S. Geological Survey

Methods in Contaminant Evaluation

Researchers in the PEER program were often faced with 

inadequate or an absence of established methods to address 

research questions about contaminants in karst aquifers. Karst 

aquifers are complex, and resource managers are often faced 

with trying to identify the best way to determine the sources 

and extent of contamination and to characterize ground-water 

flow conditions. Student researchers in the PEER program 

have adapted and pioneered methods to better characterize 

karst sites and to identify specific sources of bacterial contamination in karst areas. The students in the PEER program have adapted new methods and technology to evaluate 

ground-water quality in karst aquifers. The students have constructed and deployed diffusion bag samplers to monitor water 

quality and evaluate vertical movement in karst aquifers. The 

student retrieved the samplers from field sites, analyzed the 

water samples and evaluated the changes in water quality due 

to water movement in the aquifer. Student researchers also 

applied mathematic models to evaluate water movement and 

contaminant transport using water-level and water-quality data 

collected from single-well injection and withdrawal tests. The 

research conducted in the PEER program has provided new 

tools that can be used to better understand karst hydrology and 

contaminant transport.
Effectiveness of m-TEC Agar for Quantifying Escherichia coli in the Duck River near 

Shelbyville, Tennessee

Allyson M. Morgan1, James J. Farmer2, Anthony O. Ejiofor1, and Terrance L. Johnson1

42 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

The filter membrane method using m-TEC agar* for 

quantifying Eshcerichia coli (E. coli) concentration is based 

on the assumption that most of the lactose-fermenting, urease 

negative (LFUN) bacteria that grow on the agar are E. coli. 

Standard Methods (1995) published by the American Public 

Health Association recommends specific identification of 

LFUNs to determine the percentage of the bacteria that are 

E. coli; however, this procedure rarely is performed. A study 

was conducted to test the hypothesis that LFUN species can 

be highly variable in a specific watershed. Four water grab 

samples were collected from two sites on the Duck River 

near Shelbyville, Tennessee. Site 1 is upstream, and site 2 

is downstream of Shelbyville. The samples were collected 

once during base-flow conditions and once during storm-flow 

conditions at each site. Water samples were filtered through 

0.45-micrometer filters and placed onto m-TEC agar. Plates 

were incubated at 44.5oC for 24 hours. Filters were transferred 

to a pad saturated with urea containing phenol red. Isolates 

that remained yellow or brown after 15 minutes were considered to be urease negative. LFUN colonies were streaked 

onto tryptic soy agar for isolation. A total of 124 isolates were 

identified using the Biolog®* system. At site 1, E. coli composed 75 percent and 88 percent of the LFUN isolates from 

the base-flow and storm-flow samples, respectively. At site 2, 

60 percent and 40 percent of the LFUN isolates were identified as E. coli for base-flow and storm-flow samples, respectively. The LFUN isolates that were not E. coli were identified 

as other enteric bacteria. These results indicate that for the 

samples from site 2, colony counts of LFUN bacteria would 

result in a poor quantification of E. coli.

Published as Morgan, A.M., Farmer, J.J., Ejiofor, A.O., and Johnson, T.L., Effectiveness of m-TEC agar for quantifying Escherichia coli in the Duck River 

near Shelbyville, Tennessee, in Tennessee Section, American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee 

Water Resources Symposium, April 9–11, 2003, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p. P-19.

*Any use of trade, product, or firm name in this abstract is for identification purposes only and does not constitute endorsement by the U.S. Geological 

Survey.

1

 Tennessee State University, Department of Biological Sciences

2

 U.S. Geological Survey
Evaluation of Passive-Diffusion Samplers to Measure Dissolved Oxygen, Geochemistry, and 

Volatile Organic Compounds in Ground Water

Sumedha Namuduri1, Gregg Hileman2, Koushik Chakraborti1, and Tom Byl2

Methods in Contaminant Evaluation 43

The efficiency of fuel or solvent biodegradation is 

dependent upon the amount of dissolved oxygen and other 

geochemical electron acceptors present in the water. Accurate 

measurements of ground-water geochemistry are essential 

when evaluating the potential for biodegradation at a contaminated site. Collecting ground-water samples with a bailer 

or pump can disrupt the concentration of constituents in the 

water and provide misleading information about the aquifer. 

The objective of this study was to determine if geochemical 

constituents and volatile organic compounds (VOCs) could 

be monitored more effectively using passive-diffusion (PD) 

samplers as compared to disposable bailers. Initially, laboratory experiments were conducted to determine whether polyethylene or cellulose-dialysis tubing should be used to make 

the PD samplers. PD samplers were constructed by filling 

polyethylene or cellulose-dialysis tubing with ultra-pure water, 

sealing the ends, and placing the samplers in 4-liter bottles of 

water with a known geochemistry and VOC concentration. 

The samplers were removed from the water at specific time 

intervals, and the contents of the samplers and bottled water 

were analyzed to determine the VOC concentration, DO, dissolved iron (Fe2+), nitrate (NO3

2–), sulfate (SO42–), sulfide (S2–), 

pH, alkalinity, and specific conductance. Results of laboratory 

studies indicate that the concentration of VOCs and DO in the 

polyethylene bags were at equilibrium with the bottled water 

by 48 hours. The polyethylene bags were not suitable for measuring pH, specific conductance, or any inorganic constituents. 

The dialysis tubing was better suited for measuring pH, specific conductance, and dissolved inorganic compounds such 

as Fe2+, NO3

2–, SO42–, and S2–. The geochemistry of the bottled 

water and the cellulose-dialysis sampler came into equilibrium 

within 4 to 10 hours in the laboratory experiments. In field 

trials where the results of PD samplers were compared with 

bailers, DO concentrations obtained using PD samplers were 

consistently lower by 0.1 to 0.5 milligrams per liter as compared to water collected with disposable bailers. Concentrations of Fe2+, NO3

2–, SO42–, and S2– were similar between water 

collected with dialysis tubing and water collected with bailers 

in field trials. Geochemical and VOC samples collected with 

PD samplers did not require filtering before analysis, which 

was beneficial where geochemical and VOC monitoring was 

done in wells enriched with molasses, lactic acid and soy milk. 

One drawback of the cellulose-dialysis PD sampler was that 

ground-water microbes often generated holes in the membrane 

when the samplers were placed in wells with aerobic conditions for 4 weeks or longer. However, the cellulose-dialysis 

samplers maintained integrity in anaerobic conditions over 

the same time period. There was no observable disintegration in the polyethylene samplers when they were placed in 

aerobic or anaerobic conditions over a similar time period. In 

summary, the cellulose-dialysis PD samplers were useful for 

obtaining accurate geochemical concentrations, and the polyethylene PD samplers were useful for obtaining accurate VOC 

and DO concentrations.

Published as Namuduri, S., Hileman, G., Chakraborti, K., and Byl, T.D., Evaluation of passive-diffusion samplers to measure dissolved oxygen, geochemistry, and volatile organic compounds in ground water, in Tennessee Section, American Water Resources Association, 2003, Proceedings from the Thirteenth 

Tennessee Water Resources Symposium, April 9–11, 2003, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water 

Resources Association, p. P-21.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
44 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

The Value of Single-Well Tracer Studies for Characterizing Karst Sites

Tarra M. Beach1, Michael W. Bradley2, Roger Painter1, and Thomas D. Byl2

Water movement, contaminant migration and energy 

transfer in heterogeneous karst aquifers is challenging to 

describe quantitatively. Karst ground-water aquifers may be 

characterized by high velocity, turbulent flow within solutionenhanced fractures, conduits, or stratiform passageways. 

In other parts of the same aquifer, ground-water flow may be 

stagnant or very slow. Once a contaminant has entered a karst 

aquifer, it is often difficult to determine its precise flow-path 

or residence time in the bedrock. Attempts to characterize a 

karst site with traditional tracer tests may yield limited information, especially if the tracer cannot be detected at downgradient monitoring sites. This project applied an integrated 

approach using local geology, data from fracture mapping, 

borehole geophysics, and hydraulic testing, as well as geochemical and single-well tracer tests to characterize aquifer 

hydraulic properties at several karst sites in Middle Tennessee. 

Two types of wells; existing wells with the characteristics of 

typical domestic-water wells, and wells constructed to meet 

project specifications were also evaluated. Single well tracer 

studies were conducted by injecting a conservative salt tracer 

of known concentrations into several wells at known depths 

and measuring the decrease in tracer concentration with time. 

Changes in tracer concentration were then analyzed to provide 

information about aquifer advection and dispersion properties 

in the immediate proximity of the wells. Unfortunately, some 

of the wells used in this study were drilled approximately 

25 years ago and proved to be less than ideal for single-well 

injection studies. The most difficult problem encountered 

involved wells characterized by long vertical sections with 

no bedrock openings for the tracer to enter the aquifer freely. 

These wells yielded very little useful information. For example, the mean residence time in the water column of bedrock 

wells with few openings was 65 hours or greater. Conversely, 

tracer injections in wells with good hydraulic communication 

with the bedrock aquifer provided useful data with mean residence times ranging from 14 to 45 hours. Single-well tracer 

studies conducted in properly designed wells provided valuable hydrologic information on the residence-time distribution 

and dispersion of the tracer in the vicinity of the injection 

wells. The information collected from single-well injection 

tests when combined with water chemistry and water level 

data can be useful for the design of remediation strategies at 

contaminated karst sites.

Originally published as Beach, T.M., Bradley, M.W., Painter, R.,and Byl, T.D., The value of single-well tracer studies for characterizing karst sites, in

Kuniansky, E.L., ed., 2008, U.S. Geological Survey Karst Interest Group Proceedings, Bowling Green, Kentucky, May 2008: U.S. Geological Survey 

Scientific Investigations Report 2008–5023, p. 99.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Survival of Fecal Bacteria in Sediments and Development of a Numerical Model to Predict 

Storage and Transport in a River

J. Finke1, R. Graham1, J. Carpenter1, T. Rashid1, L. Sharpe1, J. Farmer2 , and T. Byl2

Contaminant Transport and Remediation in Wetlands and Streams 45

Various models, such as CE-QUAL-W2, predict the rate 

of bacteria removal from the water column based on density, 

settling rates and water velocity. Such models, however, do not 

consider survival and reproduction of bacteria in sediments, 

or re-suspension. Flume experiments are being conducted to 

measure the survival, reproduction and resuspension of fecal 

bacteria in sediments. These results will be used to improve 

numerical models by incorporating survival of bacteria in bed 

sediments and re-suspension into the water column, in addition to other parameters such as water velocity, initial bacteria 

concentration, and settling rate. The flume is 10 feet long and 

6 inches wide with 2 inches of pre-sterilized sediment (gravel, 

sand and organic matter) spread on the bottom and 12 inches 

of water over the sediments. Water was circulated at a velocity of 0.1 meters per second using 2 small pond pumps. Two 

strains of Escherichia coli (E. coli) and two strains of Klebsiella were introduced into the circulating water at known 

concentrations and monitored as they settled or remained suspended. Bacteria concentrations were measured in the water 

column and the sediment along the flume to determine bacterial fate and transport. The model accurately predicted bacteria 

settling from the water column. Once the bacteria settled onto 

the sediments, the population declined at an exponential rate 

over several weeks. The bacteria decomposition in the sediments was described by the equation: 

Bt = Bie–μt

where

Bt = bacteria concentration in the sediment at 

 time t,

Bi = initial bacteria concentration in sediments,

–μ = exponential decomposition rate 

 (experimental value = 0.2735), and

t = residence time, in days.

This decomposition rate will be coupled to the numerical 

model after additional tests are done on bacteria re-suspension 

associated with different velocities to improve the bacterial 

storage and transport model.

Contaminant Transport and Remediation in 

Wetlands and Streams 

TSU is located in an urban area, and several nearby 

streams have been adversely affected by sewer overflows and 

other sources of bacterial contamination. Students in the PEER 

program have conducted research on the occurrence and fate 

of bacteria in streams and sediment. The research included 

studies and model development on the settling, reproduction, and resuspension of fecal bacteria in stream sediment. 

The PEER program also has a growing program in wetlands 

research to determine the effect of wetlands on attenuating 

urban runoff. The research is facilitated by the location of a 

wetland on the TSU campus. The wetland, adjacent to the 

main TSU campus, receives urban runoff from adjoining 

streets, nearby parking lots, and residential and commercial 

neighborhoods. Students in the PEER program have conducted 

research at the wetlands and documented the reduction of volatile organic compounds and nutrients as water moved through 

the wetland. The students deployed water-quality monitors 

at key points in the wetland and monitored the water-quality 

changes following storm events and collected and analyzed 

samples to evaluate the changes due to remediation in the wetland. The results on the research were also utilized within the 

engineering program to evaluate engineering practices that can 

be used to reduce the effects of non-point source contaminants 

in an urban setting.

Published as Finke, J.,Graham, R., Carpenter, J., Rashid, T., Sharpe, L., Farmer, J., and Byl,T.D., .Survival of Fecal Bacteria in Sediments and 

Development of a Numerical Model to Predict Storage and Transport in a River, in Tennessee Section, American Water Resources Association, 2004, 

Proceedings from the Fourteenth Tennessee Water Resources Symposium, March 31–April 2, 2004, Montgomery Bell State Park, Burns, Tennessee: 

Tennessee Section of the American Water Resources Association, p. P-26.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
46 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Modification of a Numerical Model to Predict Transport and Flux of Fecal Bacteria in a River

James Davis, III1, Tiffany Hines, John Brew1, John Finke1, Lonnie Sharpe1, and Tom D. Byl2

Fecal pollution in surface waters is a serious waterquality problem. As a result, scientists have developed a number of models in an attempt to predict the fate and transport 

of fecal pollution in riverine systems. Various models predict 

the rate of bacteria removal from the water column based 

on density, settling rates and water velocity. Such models, 

however, do not consider survival and reproduction of bacteria 

in sediments, or re-suspension. Flume and stream experiments 

were conducted to measure the survival, reproduction and 

resuspension of fecal bacteria in sediments. These results will 

be used to modify a numerical model by incorporating survival of bacteria in bed sediments and re-suspension into the 

water column, in addition to other parameters such as water 

velocity, initial bacteria concentration, and settling rate. E. coli

and Klebsiella were introduced into the circulating-water 

flume at known concentrations and monitored as they settled 

or remained suspended. Bacteria concentrations were measured in the water column and the sediment along the flume to 

determine bacterial fate and transport. The model accurately 

predicted bacteria settling from the water column. The sediment fecal bacteria population declined at an exponential rate 

over several weeks (experimental decay value = –0.2735). 

This decomposition rate was coupled to the numerical model 

and additional tests were done in a small stream contaminated 

with fecal coliform. Comparison of the model and stream data 

were mixed due to irregular resuspension of bacteria-contaminated sediments. Additional work is needed on factors that 

control resuspension of sediments.

Published as Davis III, J., Hines, T., Brew, J., Finke, J., Sharpe, L.,and Byl, T.D., Modification of a numerical model to predict transport and flux 

of fecal bacteria in a river, in Tennessee Section, American Water Resources Association, 2005, Proceedings from the Fifteenth Annual Tennessee 

Water Resources Symposium, April 2005, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources 

Association, p. P-5.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
A Flux Term to Describe the Movement of Fecal Bacteria between the Sediment and Water 

Column in a Riverine System

Tiffany Hines1, James Davis1, Lonnie Sharpe1, and Tom Byl2

Contaminant Transport and Remediation in Wetlands and Streams 47

Fecal pollution in surface waters is a serious water-quality problem. As a result, scientists have developed a number 

of models in an attempt to predict the fate and transport of 

fecal pollution in riverine systems. Various models predict 

the rate of bacteria removal from the water column based 

on density, settling rates and water velocity. Such models, 

however, do not consider survival and reproduction of bacteria 

in sediments, or re-suspension. Flume and stream experiments 

were conducted to measure the survival, 

reproduction, and resuspension of fecal 

bacteria in sediments. These results can 

be used to modify a numerical model 

by incorporating survival of bacteria in 

bed sediments and re-suspension into 

the water column, in addition to other 

parameters such as water velocity, initial 

bacteria concentration, and settling rate. 

Fecal coliform bacteria were introduced 

into the circulating-water flume at known concentrations and 

monitored as they settled or remained suspended. Bacteria 

concentrations were measured in the water column and the 

sediment along the flume to determine bacterial fate and transport. The model accurately predicted bacteria settling from the 

water column. The sediment fecal bacteria population declined 

at an exponential rate over several weeks (experimental decay 

value = –0.2735). This decomposition rate was coupled to the 

numerical model, and additional tests 

were done in a small stream contaminated with fecal coliform. Comparisons 

of the model and stream data were mixed 

due to irregular resuspension of bacteriacontaminated sediments. Additional work 

was done to incorporate resuspension as 

a function of water velocity, particle size 

and density into the formula.

Published as Hines, T., Davis, J., Sharpe, L., and Byl, T.D., A flux term to describe the movement of fecal bacteria between the sediment and water column in a riverine system, in Tennessee Section, American Water Resources Association, 2006, Proceedings from the Sixteenth Tennessee Water Resources 

Symposium, April 19–21, 2006, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-8.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
48 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Effect of a Riverine Wetland on Parking Lot Runoff at Tennessee State University

Carlton Cobb1, Jameka Johnson1, Tom Byl2

A major contributor to non-point source pollution is parking lot runoff during rain storms. Wetlands have been shown 

to attenuate suspended sediments, bacteria and agricultural 

pollution. The objective of this project was to determine if 

a natural riverine wetland located down gradient of a TSU 

parking lot helped to mitigate the NPS for the runoff. The first 

phase of the project required walking through the wetlands 

and the parking lot during a rain storm to observe where the 

water would flow. This was done to establish sampling points. 

The second phase was to collect water at the sampling sites 

during a rainstorm that produced sufficient runoff after a minimum of 3 dry days. These samples were taken back to the lab 

for analysis. The samples were evaluated for turbidity, specific 

conductance, pH, and volatile organic compounds (VOC). The 

most dramatic change in water quality was associated with the 

VOCs. The water coming off the 

parking lot–driveway had 

62 μg/L benzene, 132 μg/L 

toluene and 106 μg/L xylenes, as 

well as, 4 unidentified peaks. As 

the water moved through the wetland and into a stream, the VOC 

concentration became undetectable. Additional work is needed 

to determine if the VOC removal 

was due to sorption, dilution or 

biotransformation. However, 

these preliminary results prove that wetlands are valuable for 

purifying contaminated runoff from parking lots.

Published as Cobb, C., Johnson, J., and Byl, T.D., Effect of a riverine wetland on parking lot runoff at Tennessee State University, in Tennessee 

Section, American Water Resources Association, 2007, Proceedings from the Seventeenth Tennessee Water Resources Symposium, April 17–19, 2007, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-5.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Contaminant Transport and Remediation in Wetlands and Streams 49

Use of Tanks-in-Series Method to Predict Nitrate Removal in Wetlands

Jameka Johnson1, Carlton Cobb1, Roger Painter1, Lonnie Sharpe1, and Tom Byl2

Quantitative tracer studies are 

frequently conducted to characterize 

hydrology through non-ideal flow 

systems and can provide very useful 

information, such as time of travel, 

discharge, residence-time distribution, 

advection and dispersion properties. 

The objective of this research was to 

determine if a tanks-in-series numerical model, incorporating residencetime distribution (RTD) coupled to a 

first-order rate of biodegradation (k’), 

could be used to predict contaminant 

removal in a series of small, natural, urban wetlands. The 

study site-wetland system consisted of an upper wetland 

(200 meters in length) and a lower wetland (400 meters in 

length) located on the campus of Tennessee State University 

and was modeled as two non-ideal flows, variable volume 

tanks-in-series. Flow characteristics used as input to the model 

were determined by quantitative tracer tests during base-flow 

and storm-flow conditions. Tracer 

data established there was an increase 

in mean residence time during stormflow conditions due to rising water 

being diverted through cattails and 

other vegetation. Dispersion values 

were also observed to increase during storm-flow. A first-order nitrate 

removal rate (k’) of 0.1748 per hour 

was derived from a static mesocosm 

test. The tanks-in-series model 

using storm-flow conditions (mean 

residence time of 45 hours) and the 

k’ value predicted 93% nitrate removal. Field data to test this 

model occurred when a leaking sewer provided nitrate-rich 

inflow to the wetland system during storm-flow conditions. 

Wetland discharge collected after 45 hours indicated an 

83% reduction in nitrate. There was a 10% difference between 

the measured nitrate concentration and the nitrate concentration predicted using the non-ideal flow tanks-in-series model.

Published as Johnson, J., Cobb, C., Painter, R., Sharpe, L., and Byl, T.D.., Use of tanks-in-series method to predict nitrate removal in wetlands in Tennessee 

Section, American Water Resources Association, 2008, Proceedings from the Eighteenth Tennessee Water Resources Symposium, April 2008, Montgomery 

Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-16.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

The wetland research site adjacent to the TSU campus. 

The wetland can be separated into two tanks to model 

contaminant removal.
Wetland Removal of Nutrients and Pollution from a Mixed Sewer and Karst Spring System in 

Nashville, Tennessee

Carlton Cobb1, Brandon Cobb1, Patrice Armstrong2, Jameka Johnson1, Lonnie Sharpe1, and Tom Byl3

50 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Wetlands have been shown to attenuate suspended sediments and agricultural pollution in rural areas but little work 

has been conducted regarding the benefits of the wetlands 

in mitigating urban non-point source pollution (NPS). The 

objective of this project was to determine if an 80 acre natural 

wetland located down gradient of bedrock springs, parking 

lots, city streets and leaky sewer systems in Nashville, Tennessee helped to mitigate urban NPS runoff. Sampling points 

were selected by reconnaissance during rainfall events to 

determine general flow paths. Water samples were collected at 

these sampling points during base-flow and rain runoff events. 

Water-quality monitors were also placed in the springs and 

along the flow path during the 12 month period of study. Water 

samples were analyzed within 48 hours for turbidity, specific 

conductance, pH, and volatile organic compounds (VOC). 

Additional analyses were performed for sulfate (SO4), nitrate 

(NO3) ammonia (NH3) and chemical oxygen demand (COD). 

It was found that runoff from parking lots and roads during winter storms had relatively high VOC levels (62 μg/L 

benzene, 132 μg/L toluene, 106 μg/L xylenes, and a number 

of unidentified compounds). Water samples collected downstream of the wetland, however, had VOC concentrations 

below detection levels. Water samples collected at the most 

downstream site also had significantly lower levels of turbidity (90 % lower), NH3

 (99% lower), COD (95% lower), 

NO3, (90% lower), and SO4 (63% lower) on average for the 

year. The results indicated that routing water through the 

urban wetland resulted in significant water-quality improvements during the study period.

Published as Hines, T., Davis, J., Sharpe, L., and Byl, T.D., A flux term to describe the movement of fecal bacteria between the sediment and water column 

in a riverine system, in Tennessee Section, American Water Resources Association, 2006, Proceedings from the Sixteenth Tennessee Water Resources 

Symposium, April 19–21, 2006, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-8.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 Tennessee State University, Department of Biological Sciences

3 U.S. Geological Survey
Water-Quality Monitoring—Luminescence 51

Evaluating a Bioluminescent Bacteria for Measuring Toxicity of Industrial Wastewaters

Robert Sarfo1, Tom D. Byl2 , and Paul Frymier3

Toxic compounds can have a negative effect on the performance of activated sludge systems in wastewater treatment 

systems. Monitoring the influent wastewaters by chemical 

analysis and periodic bioassays is too slow to avoid problems. 

The bioluminescent response of bacteria with the luciferase 

enzyme can provide quick and early toxicity information. 

Bacteria containing luciferase will bioluminesce under ideal 

conditions, but bioluminescence decreases as conditions 

deteriorate. The objective of this project was to evaluate the 

bioluminescent reporter in the bacterium Pseudomonas fluorescens to determine its suitability as an indicator of industrial 

toxicants. Bacteria containing the luceferase enzyme and a 

tetracycline-resistance gene were grown in a nutrient broth 

amended with 10 parts per million of tetracycline in batch 

cultures at 25 degrees Celsius. After 48 hours, the bacteria 

were tested for bioluminescence using a modified fluorometer. 

The bioluminescent cultures were standardized to 900 fluorescence standard units and used for dose-response bioassays. 

Toxins were added to the cultures in known concentrations 

and changes in bioluminescence were measured. Preliminary 

results found that a 0.02-percent sodium hypochlorite solution 

elicited an immediate decrease in bioluminescence. Solutions 

containing less than 1 milligram per liter chlorinated solvents, 

nickel (2+), and lead (2+), also elicited an immediate decrease 

in bioluminescence. However, other toxicants such as copper 

(2+), zinc (2+) and sodium thiosulfate enhanced bioluminescence at low concentrations (0.1 to 1.0 parts per million), but 

decreased initial bioluminescence by 50 percent at 100 parts 

per million. Petroleum compounds like toluene and benzene 

elicited an initial decrease in bioluminescence. However, incubating the bacteria with the toluene for 20 minutes increased 

the bioluminescence compared to control bacteria, possibly 

due to induction of toluene di-oxygenase. These preliminary 

results indicate bioluminescent bacteria might be useful as an 

early warning indicator of select toxicants.

Water-Quality Monitoring—Luminescence

Operators at water and waste-water treatment plants often 

need to monitor the incoming water stream for the efficient 

operation of the plant, to prevent contaminants from moving 

into the drinking-water system or to prevent contaminants 

from affecting wastewater treatment operations. Traditional 

methods for real-time or near real-time monitoring of water 

quality usually rely on easily measured surrogates for contaminants in water, such as turbidity for suspended solids 

or specific conductance for the concentrations of inorganic 

compounds or trace metals. The surrogates usually are only 

applicable for specific contaminants and do not provide broadspectrum protection. Monitoring water quality by chemical 

analysis or by periodic bioassays often is too slow in response 

to avoid problems.

Research into the use of a luminescent response from 

bioluminescent bacteria or plant oxidase enzyemes has been 

conducted by TSU students in the PEER program. The luminescent response of an organism or enzyme and the change in 

the luminescence can represent a direct response to toxicity. 

The PEER research was conducted to determine if the change 

in luminescence from biomarkers or bacteria could be used 

to monitor water quality. The research included two bioluminescent bacteria, Shk1 and PM6, which are gut bacteria from 

a Tennessee glow worm, Orfelia fultoni, and plant oxidase 

enzymes extracted from watercress and potatoes. Students 

in the program worked with USGS staff and staff from the 

Tennessee Wildlife Resources Agency on the field identification and collection of the Tennessee glow worm. Students 

conducted the laboratory work required to isolate the bioluminescent bacteria from the glow worm, from watercress, and 

from potatoes.

Published as Sarfo, R., Byl, T. D, and Frymier, P, Evaluating a bioluminescent bacteria for measuring toxicity of industrial wastewaters, in Tennessee 

Section, American Water Resources Association, 2001, Proceedings from the Eleventh Annual Tennessee Water Resources Symposium, April 2001, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association p. P-12.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

3

 University of Tennessee, College of Engineering
52 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Evaluation of Bioluminescent Bacteria as Indicators of Water Quality in Wastewater 

Treatment Plants

Martin Greene1, Janique Suber1, Tom D. Byl2, and Paul Frymier3

Toxic compounds in incoming wastewater can have a 

negative effect on the performance of activated sludge systems. Monitoring incoming wastewater by chemical analysis 

and periodic bioassays is often too slow to avoid problems. 

A more rapid response can be measured by respiration or by 

using bioluminescent bacteria with the luciferase enzyme. 

The objective of this study was to compare bioluminescence 

response with respiration response. The bioluminescence 

is correlated to metabolic activity and general health of the 

bacterium. Bacteria containing the luciferase enzyme will 

bioluminate under ideal conditions, and bioluminescence 

will decrease as conditions decline. Likewise, healthy sludge 

bacteria will respire (consume oxygen) at an even rate. During 

a toxic response, however, the bacteria will rapidly change 

their rate of respiration and reduce oxygen consumption. In 

this study, a bioluminescent bacterium, Pseudomonas sp.

(developed at the University of Tennessee, Knoxville), was 

compared to the oxygen consumption of sludge bacteria after 

exposure to varying concentrations of suspected wastewater 

toxins. Bacteria containing the luciferase enzyme and a tetracycline resistance gene were grown in nutrient broth amended 

with 10 parts per million of tetracycline in batch cultures at 

25oC. After 48 hours the bacteria cultures were tested for bioluminescence using a modified fluorometer. The bioluminescent cultures were standardized to 900 fluorescence standard 

units, and were used for dose-response bioassays. Toxins, such 

as sodium hypochlorite or heavy metals, were added to the 

cultures in known concentrations. Changes in bioluminescence 

were measured as a response to the toxins. Preliminary results 

indicate that a 0.02-percent sodium hypochlorite solution 

elicited an immediate decrease in bioluminescence and oxygen consumption. Nickel (Ni2+) and lead (Pb2+) also elicited 

rapid decreases in bioluminescence and oxygen consumption. 

However, sodium thiosulfate enhanced bioluminescence at 

low concentrations (0.1 to 1.0 parts per million), and increased 

oxygen consumption at all concentrations. These preliminary 

results indicate that bioluminescence may be a sensitive and 

rapid indicator of water quality.

Published as Greene, M., Suber, J., Byl, T. D., and Frymier, P., Evaluation of bioluminescent bacteria as indicators of water quality in wastewater treatment 

plants, in Tennessee Section, American Water Resources Association, 2003, Proceedings from the Thirteenth Tennessee Water Resources Symposium, 

April 9–11, 2003, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-16.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

3

 University of Tennessee, College of Engineering
Water-Quality Monitoring—Luminescence 53

Comparison of Bioluminescent Bacteria and Oxygen Consumption as Indicators of Water Quality

Dominic Anako1, Janique Suber1, Paul Frymier2, and Tom D. Byl3

Toxic compounds in influent wastewater can have a negative effect on the quality and performance of activated sludge 

systems. Monitoring the influent wastewater by chemical 

analysis and periodic bioassays is often too slow in response 

to avoid problems. Biolumincent in certain bacteria with a 

luciferase enzyme can provide quick and early toxicity information. Two bioluminescent bacteria, Shk1 and PM6, were 

exposed to increasing concentrations of selected chemicals 

and their bioluminescense monitored to develop dose-response 

toxicity curves. Likewise, activated sludge bacteria were 

exposed to increasing concentrations of the same chemicals 

and their oxygen consumption measured. The bioluminescense 

and oxygen consumption responses are both indicators of 

bacteria activity and health. These responses were compared 

to determine if the bioluminescent responses were comparable 

to the oxygen consumption response of activated sludge. A 

control test done with sodium chloride salt did not cause a 

noticeable change in bioluminescense or oxygen consumption at any of the test concentrations (10 to 100,000 μg/L). 

Chemicals that elicited a toxic response were zinc (Zn2+), 

nickel (Ni2+), silver (Ag2+), quaternary ammonia compounds, 

toluene and sodium hypochlorite. The responses of the two 

bioluminescent bacteria and the oxygen uptake were found to 

be similar in most cases with PM6 being the most sensitive 

and Shk1 being the least. These results indicate that water 

quality can be monitored using the bioluminescent response in 

a way that is protective of the activated sludge chamber of the 

wastewater treatment plant.

Published as Anako, D., Suber, J., Frymier, P., and Byl, T. D., Comparison of Bioluminescent Bacteria and Oxygen Consumption as Indicators of Water 

Qualityin Tennessee Section, American Water Resources Association, 2004, Proceedings from the Fourteenth Tennessee Water Resources Symposium, 

March 31–April 2, 2004, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-8.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 University of Tennessee, College of Engineering 

3

 U.S. Geological Survey
54 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Bioluminescent Bacteria as Indicators of Water Quality in a Wastewater-Treatment Plant

Dominic Anako1, Janique Suber1, Maurico Ricks1, Paul Frymier2, and Tom Byl3

Toxic compounds in influent wastewater can have a negative effect on the quality and performance of activated sludge 

systems. Monitoring the influent wastewater by chemical 

analysis and periodic bioassays is often too slow in response 

to avoid problems. Bioluminescence in certain bacteria with a 

luciferase enzyme can provide quick and early toxicity information. The two objectives of this project were to determine 

if a bioluminescent bacterium could serve as a onitor of water 

quality for incoming water, and, to design a way to apply 

this technology to provide a continuous toxicity measure. 

Two bioluminescent bacteria, Shk1 and PM6, were exposed 

to increasing concentrations of selected chemicals and their 

bioluminescence monitored to develop dose-response toxicity 

curves. Likewise, activated sludge bacteria were exposed to 

increasing concentrations of the same chemicals and their 

oxygen consumption measured. The bioluminescence and 

oxygen consumption responses are both indicators of bacteria 

activity and health. These responses were compared to determine if the bioluminescent responses were comparable to the 

oxygen consumption response of activated sludge. A control 

test done with sodium chloride salt did not cause a noticeable 

change in bioluminescence or oxygen consumption at any of 

the test concentrations (10 to 100,000 micrograms per liter). 

Chemicals that elicited a toxic response were zinc (Zn2+), 

nickel (Ni2+), silver (Ag2+), quaternary ammonia compounds, 

toluene, and sodium hypochlorite. The responses of the two 

bioluminescent bacteria and the oxygen uptake were found 

to be similar in most cases, with PM6 being the most sensitive and Shk1 being the least sensitive. These results indicate 

that water quality may be monitored using the bioluminescent 

response in a way that is protective of the activated sludge 

chamber of the wastewater-treatment plant. Implementing a 

continuous water-quality monitoring system in a wastewatertreatment plant using bioluminescent bacteria must include 

consideration of the volume entering the facility, equipment 

and placement of the monitoring system in-line, potential 

toxins, and the system’s response to a toxin.

Published as Anako, D., Suber, J., Ricks, M., Frymier, P., and Byl, T.D., Bioluminescent bacteria as indicators of water quality in a wastewater-treatment 

plant, in Tennessee Section, American Water Resources Association, 2005, Proceedings from the Fifteenth Annual Tennessee Water Resources Symposium, 

April 2005, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-3.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 University of Tennessee, College of Engineering 

3

 U.S. Geological Survey
Water-Quality Monitoring—Luminescence 55

Sensitivity of the Oxydase-Enzyme Induced Chemiluminescent to Water Quality Parameters

Farida Forouzon1, Lonnie Sharpe1, and Tom Byl2

Biomarkers or enzyme activity are often used to assess 

the quality of an environment. These indicators are useful 

because they represent a biological endpoint to toxicity. The 

objective of this research was to determine if the chemiluminescence from the catalase reaction could be used as a 

monitor of environmental quality. The primary task to achieve 

this objective was to evaluate the cause of the chemiluminescence when hydrogen peroxide is added. The second task was 

to determine whether microbial or plant sources of oxidase 

enzymes was better. And, the 

third task was to run assays to 

determine how water quality 

parameters affect the chemiluminescence response. This 

would include tests for optimum 

pH, optimum temperature, and 

dose-response tests for various 

environmental toxins. First a 

native organism of Tennessee, the 

glow warm named Orfelia fultoni

was collected and the gut bacteria 

were isolated. It was determined 

that luciferase was not present in the isolated bacteria and 

that an oxidase reaction in the presence of hydrogen peroxide 

catalyzed a chemiluminescent response. Additional work is 

needed to determine which oxidase enzyme is responsible. 

Since we achieved a chemiluminescent response with the 

bacteria oxidase enzymes, we tested other sources of oxidase, 

such as potato, and determined they also gave a chemilumescent response. The experiment shifted to a more available 

source of catalase, a plant such as potato. A simple experiment 

using different metals (Pb2+, Ag2+, Ni2+) found that the reaction 

was microbial and plant oxidase activity was sensitive to the 

metals at 500 mg/L concentrations. These preliminary tests 

indicate that plant and microbial sources of oxidase enzymes 

were capable of producing light. And, that these reactions 

were sensitive to dissolved metals. Additional work will be 

done to determine the optimum conditions for the reaction 

and to describe the dose-response to metals and some organic 

contaminants. It could be beneficial if this reaction is found to 

be suitable for monitoring environmental conditions such as 

water quality and air pollution.

Published as Forouzon, F., Sharpe, L., and Byl , T.D., Sensitivity of the oxydase-enzyme induced chemiluminescent to water quality parameters, 

in Tennessee Section, American Water Resources Association, 2007, Proceedings from the Seventeenth Tennessee Water Resources Symposium, 

April 17–19, 2007, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-9.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
56 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Development of an Aquatic Plant Chemiluminescent Bioassay to Assess Water Quality

Chris Beals1, Farida Forouzon2, and Tom Byl3

Biomarkers such as enzyme activity from fauna exposed 

to chemicals in the water column and sediments have been 

widely used by environmental toxicologists to assess the 

quality of an environment. Biomarkers are especially useful 

indicators because they represent a direct biological response 

to toxicity. The objective of this research was to determine 

if chemiluminescence from selected plant oxidase enzymes 

could be used as a biomarker of water quality in aquatic 

systems. The initial phase of this study included lab determination of optimum pH followed by dose-response assays of 

various environmental toxins with oxidase enzymes extracted 

from potato. The optimum pH for the potato oxidase chemiluminescence reaction ranged from 5 to 7. Initial experiments 

using dissolved metals (Pb2+, Ag2+, Ni2+) found that potato 

oxidase chemiluminescence was dose sensitive to metal 

concentrations above 500 mg/L and decreased proportionally 

with increasing metal concentrations. The chemiluminescent 

response of watercress collected near a relatively clean spring 

on Tennessee State University’s campus was also investigated. 

Watercress stems and leaves were macerated with a mortar 

and pestle and the oxidase enzymes were extracted from the 

plant material. The crude enzyme extract did provide a chemiluminescent response upon addition of hydrogen peroxide to 

the assay mixture. The optimum pH for running the watercress 

chemiluminescence assay was pH of 4. Additional doseresponse assays with whole-plant exposure will be needed 

before this bioassay can be used in water-quality assessments.

Published as Beals, C., Forouzon, F., and Byl, T.D., Development of an aquatic plant chemiluminescent bioassay to assess water quality, in

Tennessee Section, American Water Resources Association, 2008, Proceedings from the Eighteenth Tennessee Water Resources Symposium, 

April 2008, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-8.

1

 Tennessee State University, Department of Biological Sciences

2

 Tennessee State University, College of Engineering, Technology, and Computer Science

3

 U.S. Geological Survey
Other Research—Degradation and Transport 57

Desorption Isotherms For Toluene And Karstic Materials And Implications

 For Transport in Karst Aquifers

Mario Beddingfield1, Khalid Ahmed1, Roger Painter1, and Tom D. Byl2

Karst aquifers dominated by conduit flow are extremely 

vulnerable to fuel contamination such as from leaky underground storage tanks or spills. Direct flow paths through 

fractures and sinkholes often allow contaminants to move 

rapidly into the conduit system. Not much is known about how 

the fuel will interact with the carbonate rock in the conduit 

system. The objective of this research was to bridge this 

information gap by measuring sorption and desorption of fuels 

to karst materials. The first phase of this study involved the 

dissolution and desorption processes. Initial experiments (n=5) 

used karst bedrock fragments of known size soaked in toluene 

for 24 hours. Then the sterile toluene-soaked rocks were 

placed in sterile distilled water. The concentration of toluene 

dissolved in the water was measured over increasing time 

periods. These data were used to derive a first-order exponential rate of desorption [Cw(t)=Ciekt]. The empirical value for 

k was 0.8958. The toluene concentration in the water reached 

a maximum carrying capacity in approximately 3 weeks. The 

second phase of this project involved sorption studies using 

limestone fragments of known size and water containing a 

known concentration of dissolved toluene. The empirical 

value for the sorption k was 1.006. These results show that 

sorption is faster than desorption and have implications for 

designing a model that predicts the fate and transport of fuels 

in karst aquifers.

This work was supported in part by the U.S. Army Corps of 

Engineers DACW62-00-H-0001 contract

Published as Beddingfield, M., Ahmed, K., Painter, R., and Byl, T. D., Desorption isotherms for toluene and karstic materials and implications for 

transport in karst aquifers, in Kuniansky, E.L., ed., 2005, U.S. Geological Survey Karst Interest Group Proceedings, Rapid City, South Dakota, 

September 2005: U.S. Geological Survey Scientific Investigations Report 2005–5160, p. 188.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

Other Research—Degradation and Transport

Research conducted through the PEER program 

has, at times, included topics that were of specific interest 

to a student or researcher or were opportune for societal and 

environmental issues at the time. During a time of high fuel 

prices and increasing use of ethanol, students in the PEER 

program conducted research on the rate of contaminant transport for E-85 gasoline. Other research included the application 

of vitamin B12 to enhance contaminant degradation and 

measuring the sorption and desorption of fuel to carbonate 

rocks in karst aquifers. The research was conducted in the 

TSU environmental laboratory using carbonate rock, soil, and 

water samples collected from sites in Middle Tennessee and 

soil samples collected from West Tennessee. The students in 

the program utilized different methods to collect soil and rock 

samples, conducted laboratory tests, conducted appropriate 

QA/QC tests, and documented the results of the experiments. 
58 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Reductive Dechlorination of Tetrachloroethylene by Vitamin B12 and Electrokinesis

Tom D. Byl1 , Tracy Hughes2, Alishia Miller2, Pam Franklin2, and Shannon D. Williams1

The reductive dechlorination of aqueous tetrachloroethylene (PCE) by vitamin B12 and a small direct current 

(DC) was examined in saturated-soil columns and in water 

columns. A column (24 by 2 inches) filled with saturated soil 

and subjected to 200 millivolts (mV) DC and 1 micromolar 

(μM) vitamin B12 lost 10 μM (approximately 10 percent) 

of the initial PCE volume. There was a concurrent 16 μM 

increase in trichloroethylene (TCE) in the column. The other 

columns, lacking the vitamin B12, DC, or both, did not 

demonstrate a significant loss of PCE or gain of TCE over the 

7-day study. In water columns (12 by 0.2 inches) there was a 

38 percent decrease of PCE in control columns subjected to no 

DC or B12. The PCE decreased 55 percent in water columns 

subjected to DC (250 mV) without B12, and decreased 

75 percent in columns subjected to B12 (1 μM) and DC. There 

was a lack of PCE degradation products in these columns indicating electrokinesis played a major role in removing PCE. 

In a 4-day experiment, water columns with 0.1 molar (M) 

sodium chloride, vitamin B12 and 600 mV DC demonstrated 

a 44 percent loss of PCE (44 μM) and a concurrent gain of 6 

μM TCE. There was only a 20 percent loss of PCE in columns 

without vitamin B12 or DC and no TCE produced. The greater 

loss of PCE and the subsequent appearance of TCE support 

the hypothesis that vitamin B12 and a small DC can catalyze 

the reductive dechlorination of PCE.

Published as Byl, T.D., Hughes, T. H., Miller, A., Franklin, P., and Williams, S.D., Reductive dechlorination of tetrachloroethylene by vitamin 

B12 and electrokinesis, in Crabtree, L.R., Bradley, M.W., Blunt, Tiffany, and Pierre, Salnave, eds., 1999, Proceedings from the Ninth Annual 

Tennessee Water Resources Symposium, April 1999, Nashville, Tennessee: Tennessee AWRA 1B-43 to 1B-48.

1

 U.S. Geological Survey

2

 Tennessee State University, College of Engineering, Technology, and Computer Science
Other Research—Degradation and Transport 59

Are Aquifers at Greater Risk from Alternative Alcohol-Fuel Mixes Compared to Regular Gasoline?

Carlton Cobb1, Loreal Spear1, Keyshon Bachus1, Baibai Kamara1, Roger Painter1, Lonnie Sharpe1, Tom Byl2

The United States government is promoting alternative 

fuels that reduce our dependency on foreign oil. Tennessee is 

promoting E-85, a fuel that consists of 85 percent ethanol and 

15 percent gasoline. The environmental fate of gas-alcohol 

mixtures, however, has not been investigated. The consequences of an uncontrolled spill of E-85 or a related mixture 

would, therefore, be very difficult to predict. The objective 

of this research was to determine if a commercial grade E-85 

mixture would dissolve more readily in water and move faster 

through water-saturated soil than regular gasoline. A better 

understanding of E-85 mobility in the subsurface is of practical importance if E-85 is to become widely used and stored in 

underground storage tanks like conventional fuels. Solubilityin-water studies comparing gasoline with E-85 found that the 

ethanol component in E-85 acted as a co-solvent and enabled 

aromatic compounds to dissolve five times more rapidly in 

water than regular gasoline. These enhanced solubility characteristics may allow the aromatic rings to move faster and 

further through water-saturated soils and karst conduits than 

regular gasoline. Additional experiments were conducted to 

determine if regolith soils would affect the dispersal rate of 

E-85 fuel compounds. 

Sterile soil-column 

studies using soils collected from karst regions 

of Middle Tennessee 

demonstrated that aromatic compounds, such 

as benzene, toluene or 

xylene (BTX), from the 

E-85 moved 3 to 4 times 

faster than BTX compounds in regular gasoline when transported by water through 

the soil. Additional work compared the biodegradation of E-85 

with regular gasoline. Using static reactors with karst bacteria, E-85 biodegradation rates were almost 5 times greater 

than regular gasoline. This is in agreement with previous 

reports finding that dissolved-phase fuels were more bioavailable and degraded faster. Additional studies are needed to 

more thoroughly address issues concerning E-85 solubility 

and biodegradation.

Published as Cobb, C., Spear, L., Bachus, K., Kamara, B., Painter, R., Sharpe, L., and Byl, T. D.., Are aquifers at greater risk from alternative alcohol-fuel mixes compared to regular gasoline?, in Tennessee Section, American Water Resources Association, 2008, Proceedings from 

the Eighteenth Tennessee Water Resources Symposium, April 2008, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the 

American Water Resources Association, p. P-10.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey

Equipment used to collect soil samples.
60 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

The Interaction between Calcite Precipitation and Dissolution, Carbon Dioxide, and 

Perchloroethylene Sorption

Lipi Saha1 and Tom D. Byl2

Calcite (CaCO3) precipitation and dissolution is strongly 

affected by carbon dioxide (CO2) concentrations in water. As 

CO2 concentrations increase in water, a weak carbonic acid 

(H2CO3) is formed, thereby enhancing dissolution of calcite 

and, subsequently increasing the concentration of calcium 

(Ca2+) in solution. Conversely, as CO2 concentrations decrease, 

calcite precipitates from solution. Previous studies have shown 

that metals co-precipitate and dissolve with the calcite. Other 

studies have shown that nonpolar organic compounds can 

bind to calcite, but the influence of H2CO3 on co-precipitation 

during calcite precipitation was not studied. The objective of 

this study was to determine if carbonate-induced calcite dissolution and precipitation influenced perchloroethylene (PCE) 

sorption to CaCO3. The experiments were conducted using 

12-milliliter screw-top test tubes containing 2 grams of calcite 

(grain size = 0.062 to 0.500 millimeters) and distilled water, 

along with varying amounts of PCE and H2CO3. We used a 

minimum of three replicates per treatment. The first set of 

experiments established that CaCO3 dissolved and precipitated 

as expected under different concentrations of H2CO3. The second set of experiments established a linear sorption isotherm 

for PCE to calcite without the addition of H2CO3 (Kd = 1.35). 

The third set of experiments used calcite that was precontaminated with a known amount of PCE. Clean water containing 

no PCE and 0.00, 0.01, or 0.10 molar H2CO3 was added to 

the precontaminated calcite and desorption was allowed to 

occur for 4 hours. Desorption of PCE into solution occurred 

in all treatments. Approximately 12 percent of the sorbed PCE 

desorbed into the distilled water. Approximately 28 percent 

of the PCE desorbed from the calcite when it was placed in 

the 0.01 and 0.1 molar H2CO3 solutions. Thus, H2CO3 appears 

to enhance desorption of PCE 16 percent as it dissolves the 

calcite. The Ca2+, CO2, and pH were also measured to establish 

geochemical conditions in each test.

Published as Saha, L., and Byl, T.D., The Interaction between Calcite Precipitation and Dissolution, Carbon Dioxide, and Perchloroethylene 

Sorption in Tennessee Section, American Water Resources Association, 2001, Proceedings from the Eleventh Annual Tennessee Water Resources 

Symposium, April 2001, Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-11.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
Other Research—Degradation and Transport 61

Reductive Dechlorination of Perchloroethylene with a Direct Current, Different Salts, and 

Vitamin B12

Majharul Alam1 and Tom D. Byl2

Initial tests to reductively dechlorinate perchloroethylene 

(PCE) by using vitamin B12 dissolved in water and a small 

direct current were successful; however, the transformation 

process was inefficient (less than 30 percent transformation). 

If the process is to be utilized in the field, the efficiency must 

be improved. The objective of this study was to identify 

environmental conditions that increase PCE transformation 

into trichloroethylene (TCE) by electrokinesis and vitamin 

B12. Tests were conducted using 12-inch glass tubes filled 

with 3.5 milliliters of aqueous solutions containing 10 to 

50 milligrams per liter (mg/L) vitamin B12, 0.1 to 25.0 mg/L 

PCE, and a variety of salts. The ends of the tubes were sealed 

with silicone gel with a platinum electrode extending through 

the silicone. A direct current was applied to each test chamber 

using the platinum electrodes and a constant power supply. 

Preliminary results show that the salt type and concentration 

had an effect on PCE-transformation efficiency. For example, 

test solutions containing 25 mg/L vitamin B12 and 100 mg/L 

of CaCl2 had an 85-percent greater transformation of PCE to 

TCE than distilled water only containing vitamin B12. Solutions containing NaCl and CaCl2

 were twice as effective as 

solutions with FeCl3 and Fe(NH4)2(SO4)2 at enhancing transformation of PCE to TCE. Addition of the salts NH4

Cl and 

FeSO4 produced the lowest remaining concentrations of PCE 

but produced no significant amount of TCE in the test chamber. Whether PCE volatilized or was completely dechlorinated 

in those tests is not known. Also, a steady low voltage (200 

mV) was more effective than higher voltages (greater than 400 

mV) at inducing reductive dechlorination. The efficiency of 

the dechlorination reaction appeared to be unaffected by PCE 

concentration when PCE concentrations ranged from 0.1 to 

25.0 mg/L. This information will be useful for adapting the 

method from the laboratory to the field.

Published as Alam, M., and Byl, T.D, Reductive Dechlorination of Perchloroethylene with a Direct Current, Different Salts, and Vitamin B12, in Tennessee 

Section, American Water Resources Association, 2001, Proceedings from the Eleventh Annual Tennessee Water Resources Symposium, April 2001, 

Montgomery Bell State Park, Burns, Tennessee: Tennessee Section of the American Water Resources Association, p. P-1.

1

 Tennessee State University, College of Engineering, Technology, and Computer Science

2

 U.S. Geological Survey
62 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

Five PEER presentations by TSU students were made at the international 

Society of Environmental Toxicology and Chemistry (SETAC) conference 

in Montreal, November, 2006. (Left to right: Kelly Ray, won 1st place in 

the Elsevier Poster contest; Tarra Beach, won 2nd place in the student 

platform; Tom Byl and Tiffany Hines)

National Awards

 1. Mr. Doug Warden, 2000

 2. Ms. Sumedha Namuduri, 2002

 3. Ms. Valetta Watson, 2002

 4. Mr. Koushik Chakraborti, 2003

 5. Ms Tarra Beach, May 2006

 6. Ms Tarra Beach, Nov 2006 

 7. Ms Kelly Ray, Nov. 2006

Regional Awards

 1. Mr. Detoy James, 2000 

 2. Mr. James Murithi, 2000 

 3. Ms. Shimere Williams, 2001

 4. Ms. Kamalah Minor, 2002

 5. Ms. Rashaunna Mohmmad, 2002 

 6. Ms. Sumedha Namuduri, 2002 

 7. Ms. Tavy Wade, 2002 

 8. Ms. Valetta Watson, 2002

 9. Mr. Koushik Chakraborti, 2003 

10. Mr. Khalid Wood. 2003

11. Ms Lashun King, 2004 (twice)

12. Mr. Mario Beddingfield, 2005 (twice)

13. Mr.Carlton Cobb, 2006 

14. Ms. LyTreese Hampton, 2006 

15. Ms Jameka Johnson, 2006 

16. Ms. Lashun King , 2006

17. Mr. Carlton Cobb, 2007

18. Mr. Farida Forouzon, 2007 

19. Ms Jameka Johnson, 2007 

20. Mr. Derek Lovett, 2007

University Awards

 1. Ms. Christa Cruikshank, 1999

 2. Mr. Gopu Apo Rao, 1999

 3. Mr. Robert Sarfo, 2000

 4. Ms. Laltiha Akula, 2001

 5. Mr. Majhar Alam, 2001

 6. Mr. Allyn Allison, 2001

 7. Ms. Amini Boston, 2001

 8. Ms. Lipi Saha, 2001

 9. Mr. Daniel Agyemang, 2002

10. Ms. Ramona Darlington, 2002 

11. Ms. Pam Franklin, 2002 

12. Ms. Shaunna Haynes, 2002 

13. Ms. Natascha Morris, 2002 

14. Ms. Sumedha Namuduri, 2002 

15. Ms. Sudeshna Roy, 2002 

16. Ms. Valetta Watson, 2002 

17. Mr. Koushik Chakraborti, 2003 

18. Ms. LeMiracle Hendking, 2004 

19. Ms. Lashun King. 2004 

20. Mr. Dominic Anako, 2005 

21. Mr. John Brew, 2005 

22. Mr. James Davis, 2005 

23. Ms. Isabelle Ford, 2005 

24. Mr. Omar Hamawandi, 2005 

25. Ms. Tiffany Hines, 2005 

26. Mr. Serge Mondesir, 2005 

27. Mr. Carlton Cobb, 2006

28. Mr. Ryan Fitzwater, 2006 

29. Ms Jameka Johnson, 2006

30. Ms Charner Rogers, 2006

31. Ms Farida Fourizan, 2007

32. Ms Jameka Johnson, 2007

33. Mr Derek Lovett, 2007

Student Awards

Students in the PEER program are encouraged to actively 

participate in scientific conferences and to present the results 

of their investigations in proceedings, oral presentations, and 

poster sessions. PEER students have presented their research 

at the following conferences and meetings; American Water

Resources Association in Tennessee and Alabama, the USGS 

Karst Interest Group, the Society of Environmental Toxicology and Chemistry, the Tennessee Louis Stokes Alliance for 

Minority Participation and the TSU Campus-Wide Research 

Symposium and Department of Engineering Industrial Cluster. 

The quality of the research conducted by the PEER students is 

reflected in the awards they have received.
Outreach 63

Outreach

The PEER program has included a significant outreach 

componant that includes pre-college students, undergraduate 

students, and graduate students. As part of the PEER program, 

ground-water hydrologists with the USGS Tennessee 

Water Science Center organized educational field trips to 

hydrogeologic sites in Tennessee. A special field trip to 

Mammoth Cave National Park for engineering and biology 

students and faculty was conducted in cooperation with the 

U.S. National Park Service. A new graduate-level course in 

ground-water hydrology was developed and taught by Michael 

Bradley of the USGS Tennessee WSC. The students in the 

PEER program also conducted outreach activities for local 

schools. Several classes in earth sciences were taught by 

students in the PEER program each day at the TSU Summer 

Camps for pre-college students.

Carlton Cobb (second from left) uses his 

experiences from the PEER program to teach 

incoming engineering freshmen about nonpointsource pollution and water quality.

Brandon Cobb demonstrates contaminant 

movement in groundwater at the University of 

Tennessee Agricultural Extension Service SmallFarms Field Day in August, 2008.

Dr. Tom Byl (left photo) and TSU students (Ms. Tarra Beach shown in right photo) visit elementary 

schools in Nashville to teach them about science and math. The 4th graders learned about 

earthquakes and then designed houses to withstand a simulated earthquake.
64 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

The PEER program helped to sponsor the summer science and technology camps for more than 100 

pre-college teens from across the Nation. Small groups of students (four to eight) were instructed in 

lab safety, environmental engineering, groundwater, and water contamination. These small groups 

allowed more effective interaction between the students and instructors. TSU provided the camp 

space, laboratory facilities, and student instructors, and the USGS provided instructors, lessons and 

experiments, and materials.

TSU students interning at the USGS instructed more 

than 100 pre-college minority students about lab safety, 

hydrology, chemistry, stream biomonitoring, nonpointsource pollution, geology, microbiology, water-quality 

testing, and fossils at the 2006 summer engineering camps.

Michael Bradley, USGS Tennessee WSC, developed a new 

graduate course in groundwater hydrology for the College 

of Engineering. Here Mike discusses well construction and 

field techniques for the groundwater hydrology class.
Summary 65

Summary

The Partnership for Environmental Education and 

Research (PEER) is a joint partnership between the Tennessee State University, College of Engineering and Technology 

(TSU) and the U.S. Geological Survey (USGS) Tennessee 

Water Science Center (WSC). The partnership was developed to support environmental research at TSU, to expand 

the environmental research capabilities of the USGS in 

Tennessee, to conduct environmental research, and to promote 

environmental education. 

Student and faculty research in the PEER program is 

being conducted to better define the occurrence, fate, and 

transport of contaminants in groundwater and surface water. 

Contamination of surface water and groundwater affects the 

adequacy of water resources to meet ever-increasing demands 

for municipal, agricultural, and industrial use. Variations 

in natural water quality, contaminant concentrations, and 

geochemical, biological, and hydrogeological processes affect 

the fate and transport of contaminants in surface water and 

groundwater. Research conducted through the PEER program 

provides additional information to better understand these 

processes and help improve strategies for pollution prevention, 

mitigation, and remediation in karst.

Research in the PEER program has primarily focused 

on the transport and remediation of organic contamination in 

karst settings. As the activities in karst areas expanded, PEER 

research included mathematical modeling for contaminant 

transport and degradation, the adaptation of residence-time 

distribution and Lattice-Boltzmann models to karst aquifers, 

the occurrence and role of bacteria in karst aquifers, and the 

occurrence and fate of different contaminants in karst aquifers. The PEER program has expanded to apply research to a 

variety of media and settings. Research has been conducted 

on new methods to evaluate ground-water contamination, the 

resuspension of bacteria from sediment in streams, the use 

of bioluminescence and chemiluminescence to identify the 

presence of contaminants, and contaminant remediation in 

wetlands. 

The PEER program also supports TSU outreach activities 

and their efforts to increase minority participation in environmental and earth science programs at the undergraduate and 

graduate levels. PEER has increased research and education 

opportunities for the College of Engineering and Technology and provided students with experience in presenting the 

results of the research. Students in the program have participated in state, regional and national conferences, giving more 

than 140 presentations since 1998 and have won more than 

40 student awards.

Dr. Bill Wolfe, USGS Tennessee WSC, 

(center) shows TSU students Nkechi Chieko 

(left) and Derek Lovett (right) how to use the 

Guelph permeameter on TSU’s campus.

The TSU environmental engineering program was recognized 

at the Southeast U.S. Graduate School Dean’s conference in 

March 2007. (pictured, left to right, Dr. Farouk Mishu, head, Civil & 

Environmental Engineering; Professor Valetta Watson, Dr. Roger 

Painter, Dr. Melvin Johnson, President TSU; Ms. Tiffany Hines, 

(PEER student), and Dr. Tom Byl, USGS & PEER faculty.
66 Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008

References

Aiken, G.R., and Kuniansky, E.L., eds., 2002, U.S. Geological Survey Artificial Recharge Workshop proceedings, April 

2-4, 2002, Sacramento, California: U.S. Geological Survey 

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Bradley, Armstrong, and Byl—Partnership of Environmental Education and Research: A Compilation of Student Research, 1999–2008—Open-File Report 2010–1291

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