1

United States

Environmental Protection

Agency

Office of Solid Waste

and Emergency

Response

Office of

Research and

Development

Ground Water Issue

In-Situ Bioremediation of

Contaminated Ground Water

Robert S. Kerr Environmental

Research Laboratory

Ada, Oklahoma

Superfund Technology Support Center for

Ground Water

J.L. Simsa, J.M. Suflitab, and H.H. Russellc

Technology Innovation Office

Office of Solid Waste and Emergency

Response, US EPA, Washington, DC

Walter W. Kovalick, Jr., Ph.D.

Director

An emerging technology for the remediation of ground water is

the use of microorganisms to degrade contaminants which are

present in aquifer materials. Understanding the processes

which drive in-situ bioremediation, as well as the effectiveness

and efficiency of the utilization of these systems, are issues

which have been identified by the Regional Superfund Ground

Water Forum as concerns of Superfund decision makers. The

Forum is a group of ground-water scientists and engineers,

representing EPA’s Regional Superfund Offices, organized to

exchange up-to-date information related to ground-water

remediation at Superfund sites.

Although in-situ bioremediation has been used for a number of

years in the restoration of ground water contaminated by

petroleum hydrocarbons, it has only been in recent years that

this technology has been directed toward other classes of

contaminants. Research has contributed greatly to

understanding the biotic, chemical, and hydrologic parameters

which contribute to or restrict the application of in-situ

bioremediation, and has been successful at a number of

locations in demonstrating its effectiveness at field scale.

This document is one in a series of Ground Water Issue

papers which have been prepared in response to needs

expressed by the Ground Water Forum. It is based on

findings from the research community in concert with

experience gained at sites undergoing remediation. The intent

of the document is to provide an overview of the factors

involved in in-situ bioremediation, outline the types of

information required in the application of such systems, and

point out the advantages and limitations of this technology.

For further information contact Dr. Hugh Russell, RSKERL,

FTS 743-2444, commercial number (405) 332-8800.

Summary

In-situ bioremediation, where applicable, appears to be a

potential cost-effective and environmentally acceptable

remediation technology. Suflita (1989) identified

characteristics of the ideal candidate site for successful

implementation of in-situ bioremediation. These characteristics

included: (1) a homogeneous and permeable aquifer; (2) a

contaminant originating from a single source; (3) a low

ground-water gradient; (4) no free product; (5) no soil

contamination; and (6) an easily degraded, extracted, or

immobilized contaminant. Obviously, few sites meet these

characteristics. However, development of information

concerning site specific geological and microbiological

characteristics of the aquifer, combined with knowledge

concerning potential chemical, physical, and biochemical fate

of the wastes present, can be used to develop a

bioremediation strategy for a less-than-ideal site.

Introduction

In-situ bioremediation is a technology to restore aquifers

contaminated with organic compounds. Organic contaminants

found in aquifers can be dissolved in water, attached to the

aquifer material, or as freephase or residual phase liquids

referred to as NAPLs which are liquids that do not readily

dissolve in water (Palmer and Johnson, 1989c). Generally,

a Soil Scientist, Utah Water Research Laboratory,

Utah State University b Professor, Dept. of Botany and Microbiology, University

of Oklahoma c Research Microbiologist, Robert S. Kerr Environmental

Research Laboratory

EPA/540/S-92/003

February 1992
2

NAPLs are subdivided into two classes: those that are lighter

than water (LNAPLs density <1.0), and those with a density

greater than water (DNAPLs density >1.0). LNAPLs include

hydrocarbon fuels, such as gasoline, heating oil, kerosene, jet

fuel, and aviation gas. DNAPLs include chlorinated

hydrocarbons, such as 1,1,1-trichloroethene, carbon tetrachloride, chlorophenols, chlorobenzenes, tetrachloroethylene,

PCBs, and creosote.

In this discussion, a technical approach is presented to assess

the potential implementation of bioremediation at a specific

site contaminated with an organic compound. The approach

consists of (1) a site investigation to determine the transport

and fate characteristics of organic waste constituents in a

contaminated aquifer, (2) performance of treatability studies to

determine the potential for bioremediation and to define

required operating and management practices, (3) development of a bioremediation plan based on fundamental

engineering principles, and (4) establishment of a monitoring

program to evaluate performance of the remediation effort.

The pattern of contamination from a release of contaminants

into the subsurface environment, as would occur from an

underground leaking storage tank containing NAPLs, is

complex (Figure 1) (Palmer and Johnson, 1989c; Wilson et al.

1989). As contaminants move through the unsaturated zone,

a portion is left behind, trapped by capillary forces. If the

release contains volatile contaminants, a plume of vapors

forms in the soil atmosphere in the vadose zone. If the

release contains NAPLs less dense than water (LNAPLs),

they may flow by gravity down to the water table and spread

laterally. The oily phase can exist either as a free product,

which can be recovered by pumping, or as a residual phase

after the pore spaces have been drained. Contaminants

associated with NAPLs can also partition into the aquifer's

solid phase or in the vapor phase of the unsaturated zone. If

the release contains DNAPLs, these contaminants can

penetrate to the bottom of an aquifer, forming pools in

depressions. In either case, when ground water comes into

contact with any of these phases, the soluble components are

dissolved into the water phase.

There are a number of techniques available to remediate

ground water contaminated with organic compounds including:

physical containment such as slurry walls, grout curtains, and

sheet pilings (Ehrenfield and Bass, 1984); hydrodynamic

control using pumping wells to manipulate the hydraulic

gradient or interceptor systems (Canter and Knox, 1985);

several methods of free product recovery (Lee and Ward

1986); and (4) extraction of contaminated ground water

followed by treatment at the surface (Keely, 1989; U.S. EPA,

1989b).

Alternatively, contaminated ground water can be treated in

place, without extraction using in-situ chemical treatment or

biological treatment with microorganisms (Thomas et al.,

1987c). An advantage of in-situ treatment strategies is that

treatment can take place in multiple phases.

In-situ chemical treatment techniques are similar to methods

used to treat contaminated materials after withdrawal or

excavation, but are directly applied to the materials in place

(Ehrenfield and Bass, 1984). Chemical treatment may involve

neutralizing, precipitating, oxidizing or reducing contaminants

Figure 1. Regions of contamination in a typical release from an underground storage tank (Wilson et al., 1989).

Fumes

Continuous Phase

Residual

Saturation

Dissolved

Hydrocarbon

Fumes

Water

Table

Leaking Underground

Storage Tank
3

by injecting reactive materials into a contaminated leachate

plume through injection wells, but may be limited by mass

transport and concentration dependence. For treatment of

shallow contaminated aquifers, permeable treatment beds

containing reactive materials such as activated carbon or ion

exchange resins may be constructed downgradient to

intercept and treat the contaminated plume.

Biological in-situ treatment of aquifers is usually accomplished

by stimulation of indigenous microorganisms to degrade

organic waste constituents present at a site (Thomas and

Ward, 1989). The microorganisms are stimulated by injection

of inorganic nutrients and, if required, an appropriate electron

acceptor, into aquifer materials.

Most biological in-situ treatment techniques in use today are

variations of techniques developed by researchers at Suntech

to remediate gasoline-contaminated aquifers. The Suntech

process received a patent titled Reclamation of Hydrocarbon

Contaminated Ground Waters (Raymond, 1974). The process

involves the circulation of oxygen and nutrients through a

contaminated aquifer using injection and production wells (Lee

et al., 1988). Placement of the wells is dependent on the area

of contamination and the porosity of the formation, but are

usually no more than 100 feet apart. The nutrient amendment

consists of nitrogen, phosphorus, and other inorganic salts, as

required, at concentrations ranging from 0.005 to 0.02 percent

by weight. Oxygen for use as an electron acceptor in

microbial metabolism is supplied by sparging air into the

ground water. If the growth rate of microorganisms is 0.02 g/l

per day, the process is estimated to require approximately 6

months to achieve 90 percent degradation of the hydrocarbons present. Cleanup is expected to be most efficient for

ground waters contaminated with less than 40 ppm of

gasoline. After termination of the process, the numbers of

microbial cells are expected to return to background levels.

In addition to stimulating indigenous microbial populations to

degrade organic waste constituents, another technique, which

has not yet been fully demonstrated, is the addition of

microorganisms with specific metabolic capabilities to a

contaminated aquifer (Lee et al., 1988). Populations that are

specialized in degrading specific compounds are selected by

enrichment culturing or genetic manipulation. Enrichment

culturing involves exposure of microorganisms to increasing

concentrations of a contaminant or mixture of contaminants.

The type of organism (or group of organisms) that is selected

or acclimates to the contaminant depends on the source of the

inoculum, the conditions used for the enrichment, and the

substrate. Examples of changes that may occur during an

acclimation period include an increase in population of

contaminant degraders, a mutation that codes for new

metabolic capabilities, and the induction or derepression of

enzymes responsible for degradation of specific contaminants

(Aelion et al., 1987).

It is important to note that the inoculation of a specialized

microbial population into the environment may not produce the

desired degree of degradation for a number of reasons

(Goldstein et al., 1985; Lee et al., 1988; Suflita 1988b).

Factors that may limit the success of inoculants include

contaminant concentration, pH, temperature, salinity, and

osmotic or hydrostatic pressure. They may act alone or

collectively to inhibit the survival of the microorganisms. The

subsurface environment may also contain substances or other

organisms that are toxic or inhibitory to the growth and activity

of the inoculated organisms. In addition, adequate mixing to

ensure contact of the organism with the specific organic

constituent may be difficult to achieve at many sites.

Successful inoculation of introduced organisms into simpler,

more controllable environments (e.g., bioreactors such as

waste-water treatment plants) to accomplish degradation has

been demonstrated. However, effectiveness of inoculation into

uncontrolled and poorly accessible environments such as the

subsurface is much more difficult to achieve, demonstrate and

assess (Thomas and Ward, 1989).

Genetic manipulation of microorganisms to produce

specialized populations to degrade specific contaminants

involves the acceleration and focusing of the process of

evolution (Kilbane, 1986; Lee et al., 1988). Genetic

manipulation can be accomplished by exposure of organisms

to a mutagen, followed by enrichment culturing to isolate a

population with specialized degradative capabilities, or by the

use of DNA recombinant technology to change the genetic

structure of a microorganism. The use of genetically engineered organisms in the environment is illegal without prior

government approval (Thomas and Ward, 1989). In addition,

the introduction of genetically engineered organisms into the

environment would meet the same kind of barriers to success

as organisms developed by enrichment culturing, or more.

Additional methods that have been suggested to enhance

biodegradation include: cross acclimation, which involves the

addition of a readily degradable substrate to aid in the

biodegradation of more recalcitrant molecules; and analog

enrichment, which involves the addition of a structural analog

of a specific contaminant in order to induce degradative

enzyme activity that will affect both the analog and the specific

contaminant (Suflita, 1989a).

In most contaminated aquifers, the hydrogeologic system is so

complex, in terms of site characteristics and contaminant

behavior, that a successful remediation process must rely on

the use of multiple treatment technologies (Wilson et al.,

1986). The combination of several technologies, in series or

in parallel, into a treatment process train may be necessary to

restore ground-water quality to a required level. Barriers and

hydrodynamic containment controls alone serve as only

temporary plume control measures, but can be integral parts

of withdrawal and treatment or in-situ treatment measures.

A possible treatment train might consist of: (1) source removal

by excavation and disposal; (2) free product recovery to

reduce the mass in order to decrease the amount of

contaminants requiring treatment; and (3) in-situ treatment of

remaining contamination. When applicable, biological in-situ

treatment offers the advantage of partial or complete

destruction of organic contaminants, rather than transfer or

partitioning of contaminants to different phases of the

subsurface.

In-Situ Bioremediation Technical Process

The in-situ bioremediation technical process consists of the

following activities:

1. performance of a thorough site investigation;

2. performance of treatability studies;
4

3. removal of source of contamination and recovery of

free product;

4. design and implementation of the bioremediation

technology; and

5. evaluation of performance of the technology through

a monitoring program (Lee and Ward, 1986; Lee et

al., 1988).

A thorough site investigation in which biological, contaminant,

and aquifer characterization data are integrated, is essential

for the successful implementation of a bioremediation system.

Biological characterization is required to determine if a viable

population of microorganisms is present which can degrade

the contaminants of concern. An assessment of waste

characteristics provides information for determining whether

bioremediation, either alone or as part of a treatment train, is

feasible for the specific contaminants at the site. Aquifer

characteristics provide information on the suitability of the

specific environment for biodegradative processes, as well as

information required for hydraulic design and operation of the

system.

Bioremediation of an aquifer contaminated with organic

compounds can be accomplished by the biodegradation of

those contaminants and result in the complete mineralization

of constituents to carbon dioxide, water, inorganic salts, and

cell mass, in the case of aerobic metabolism; or to methane,

carbon dioxide, and cell mass, in the case of anaerobic

metabolism. However, in the natural environment, a

constituent may not be completely degraded, but transformed

to an intermediate product or products, which may be equally

or more hazardous than the parent compound. In any event,

the goal of in-situ bioremediation is detoxification of a parent

compound to a product or products that are no longer

hazardous to human health and the environment.

In 1973 a review of ground-water microbiology was published

by researchers at the U.S. EPA Robert S. Kerr Environmental

Research Laboratory (RSKERL) (Dunlap and McNabb, 1973)

that stimulated research into microbiology of the subsurface.

Previously, biological activity in the subsurface environment

below the root zone was considered unlikely and that

microbial activity in the subsurface could not be of significant

importance (Lee et al., 1986). However, as methods for

sampling unconsolidated subsurface soils and aquifer

materials without contamination from surface materials

(Dunlap et al., 1977, Wilson et al., 1983, McNabb and Mallard,

1984) as well as methods to enumerate subsurface microbial

organisms (Ghiorse and Wilson, 1988) were developed,

evidence for microbial activity in the subsurface became

convincing.

Bacteria are the predominant form of microorganisms that

have been found in the subsurface, although a few higher life

forms have been detected (Ghiorse and Wilson, 1988; Suflita,

1989a). The majority of microorganisms in pristine and

uncontaminated aquifers are oligotrophic, because organic

materials available for metabolism are likely present in low

concentrations or difficult to degrade. Organic materials that

enter uncontaminated subsurface environments are often

refractory humic substances that resist biodegradation while

moving through the unsaturated soil zone.

Many subsurface microorganisms are metabolically active and

can use a wide range of compounds as carbon and energy

sources, including xenobiotic compounds (Lee et al., 1988).

Compounds such as acetone, ethanol, isopropanol, tertbutanol, methanol, benzene, chlorinated benzenes,

chlorinated phenols, polycyclic aromatic hydrocarbons, and

alkylbenzenes have been shown to degrade in samples of

subsurface aquifer materials.

The rate and extent of biotransformation of organic

compounds at a specific site are controlled by geochemical

and hydraulic properties of the subsurface (Wilson et al.,

1986). Populations of microorganisms increase until limited by

a metabolic requirement, such as mineral nutrients, substrates

for growth, or suitable electron acceptors. At this point, the

rate of transformation of an organic material is controlled by

transport processes that supply the limiting factor. Since most

subsurface microorganisms are associated with the solid

phase, the limiting factor must be delivered to the microbes by

advection and diffusion through the mobile phases. Below the

water table, all transport must be through liquid phases, and

as a result, aerobic metabolism may be severely limited by the

very low solubility of oxygen in water. As oxygen becomes

limiting, aerobic respiration slows. However, other groups of

organisms become active and continue to degrade contaminating organic materials. Under conditions of anoxia, anaerobic

bacteria can use organic chemicals or several inorganic

anions as alternate electron acceptors (Suflita, 1989a).

Even though microorganisms may be present in a

contaminated subsurface environment and have

demonstrated the potential to degrade contaminants in

laboratory studies, they may not be able to degrade these

contaminants without a long period of acclimation. Acclimation

results in development of the capability to accomplish

degradation.

In summary, the rate of biological activity in the subsurface

environment is generally controlled by:

1. the concentration of required nutrients in the mobile

phases;

2. the advective flow of the mobile phases or the

steepness of concentration gradients within the

phases;

3. opportunity for colonization in the subsurface by

metabolically active organisms or groups of

organisms capable of degradation of the specific

contaminants present;

4. presence, availability, and activity of appropriate

enzymes for degradation of specific contaminants

present; and,

5. toxicity exhibited by the waste or co-occurring

material(s) (Wilson et al., 1986; Suflita, 1989a).

Methods to Collect Biological Samples

Traditionally, unconsolidated soils or sediments are sampled

through a hollow-stem auger with a split-spoon core barrel or

a conventional thin-walled sample tube (Acker 1974, Scalf et

al., 1981; Wilson et al., 1989). The hollow-stem auger acts as

a temporary casing to keep the borehole open until a sample

can be acquired. A borehole is drilled down to the depth to be

sampled and a core barrel is inserted through the annular

opening in the auger and driven or pushed while rotating the

auger into the earth to collect the sample. These tools are

effective in both unsaturated and saturated cohesive
5

materials, but are not as effective in unconsolidated sands as

it is difficult to keep aquifer material out of the hollow stem

auger (a phenomenon referred to as “heaving”) and to keep

the sample in the core barrel while the sample is being

retrieved to the surface. In recent years there have been

many improvements in sampling the subsurface, particularly

with respect to heaving materials (Zapico et al., 1987; Leach

et al., 1988)

Just as it is important to protect the integrity of samples while

coring, it is as important to assure integrity while transferring

sample material to containers which are to be returned to the

laboratory for analysis. To prevent contamination of aquifer

material samples from introduced microorganisms and to

protect samples from the atmosphere to prevent injury of

anaerobic microorganisms, samples are extruded inside a

nitrogen-filled glove box (Figure 2). The glove box is prepared

for sample collection by filling it with the desired number of

sterile sampling jars and sterile paring devices, sealing the

box, and then purging it with nitrogen gas. A slight positive

pressure of nitrogen is maintained in the box by purging during

extrusion and collection of the samples.

Biological Characterization

A wide variety of methods are available to detect, enumerate,

and estimate biomass and metabolic activities of subsurface

microorganisms. These methods include: direct light and

epifluorescence microscopy, viable counts (e.g., plate counts,

most probable number counts, and enrichment culture

procedures), and biochemical indicators of metabolic activity

such as ATP, GTP, phospholipid, and muramic acid (Ghiorse

and Wilson 1988). Levels of microorganisms ranging from 106

to 107 cells/g of dry aquifer material have been reported from

uncontaminated shallow aquifers (Ghiorse and Balkwill, 1985;

Lee et al., 1988). Often the distribution of microorganisms in

aquifers, as it is in soils, is sporadic and nonuniform, indicating

Figure 2. Field sampling glove box (Wilson, et al., 1989).

the presence of micro-environments conducive to growth and

activity.

Waste Characterization

The source of contamination is usually the primary object of

remedial activities (Wilson et al. 1989) as the treatment of

plume areas will not be effective if the source continues to

release contaminants. Information concerning: (1) the areal

location of the source area and contaminant plumes; (2)

amounts of contaminants in the source area; and (3) amounts

of contaminants released into the subsurface are required to

select and apply an appropriate remediation technology and to

determine cost and time requirements for completion of a

remedial action. If in-situ bioremediation is selected as the

remedial technology, information concerning the amount and

distribution of contamination is used in conjunction with

hydrogeological site characteristics to locate injection and

extraction wells and to optimize pumping rates and

concentrations of amendments, such as nutrients and

alternate electron acceptors.

The use of conventional monitoring wells can generally

accurately define the geometry of the ground-water plume

(Palmer and Johnson, 1989a; Wilson et al., 1989). However,

there are important factors that control the quality of

information collected from a network of monitoring wells,

which include the amount of well purging done prior to

sampling (Barcelona and Helfrich, 1986), method of sampling

(Stolzenburg and Nichols, 1985), and method of well

construction and installation (Keely and Boateng, 1987).

Methods for ground-water sampling are presented by Scalf et

al. (1981), Ford et al. (1984), and Barcelona et al. (1985).

Other methods used for detecting contaminant plumes in the

subsurface include geophysical techniques such as surface

resistivity and electromagnetic surveys, chemical time-series

sampling tests (Palmer and Johnson, 1989a), and vapor

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA

AAAAAAAAAA

AAAAAAAAAAAAAA

AAAAAAAAAAAAAA

AAAA

AAAAAAAAAAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAA AAA AAA AAA

AAA AAA AAA AAA AAA AAA AAA AAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA

AAAAAAAAAA

AAAAAAAAAAAAAA

AAAAAAAAAAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

A

A

A

A

A

A

A

A

A

A

A

A

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

A

A

A

A

A

A

A

A

A

A

A

A

A

A

AAA

AAA

AAA

AAA

AAA

AAA

AAA AAA

AAA

AAA

AAA

AAA

AAA AAA

AAA

AAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA

AAA AAA AAA AAA AAA

AAA AAA AAA AAA AAA AAA AAA

AAA AAA AAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA

AAAAAAAAAA

AAAAAAAAAAAAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAAAAAA

AAAAAAAAAAAAAAAAAA

AAAAAAAAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAA AAA AAA AAA AAA

AAA AAA AAA AAA AAA AAA AAA

AAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAAAAAA

AAAAAAAAAAAAAAAAAA

AA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA

AAAAAAAA

AAAAAAAAAAAAAAAAAA

AAAAAAAAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

AAAA AAAA AAAA AAAA AAAA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA

AAAA

Flushing Vent

Flow Regulator

and Indicator

Sample Tube

from Extruder

Iris Port

Sample Head

Space Analysis

Vent
6

monitoring wells (Devitt et al., 1988; Palmer and Johnson,

1989b).

The distribution of the source area and the extent of

contamination should also be characterized by collecting

cores of the solid aquifer materials. Precise information is

required to define the vertical extent of contamination so that

nutrients, oxygen and other amendments injected into the

aquifer contact the contaminants. Injection into a clean part of

the aquifer is a wasted effort and may give the false

impression that the region of aquifer between the injection and

recovery wells is clean (Figure 3).

Additional characteristics of waste contaminants present at a

specific site that should be considered are related to their

environmental fate and behavior in specific aquifer materials

(Armstrong, 1987; Johnson et al., 1989). These characteristics include physical and chemical properties that determine

recalcitrance, reactivity, and mobility of contaminants at the

site. Information concerning partitioning of contaminants

between aquifer solids and water is especially important. This

information is used to evaluate the extent and rate of release

of contaminants into the ground water, their mobility, and the

quantity of electron acceptors and inorganic nutrients that

must be supplied to support in-situ bioremediation.

Aquifer Characterization

Important geological characteristics of an aquifer that should

be considered during a site investigation include the

composition and heterogeneity of aquifer material, specific

yield, hydraulic connections to other aquifers, magnitude of

water table fluctuations, ground-water flow rate and direction,

hydraulic conductivity distribution, permeability, bulk density,

and porosity (Lee et al., 1988; Palmer and Johnson, 1989a).

Hydraulic conductivity (K) is an especially important

characteristic since the aquifer must be permeable enough to

allow the transport of electron acceptors and inorganic

nutrients to the microorganisms in the zone of contamination.

Permeable aquifer systems, i.e., aquifers with K values of 10-4

cm/sec or greater, are usually considered good candidates for

in-situ bioremediation (Thomas and Ward, 1989).

Hydraulic conductivity of an aquifer can be determined by a

variety of methods (Thomas et al., 1987b, Palmer and

Johnson, 1989a). Knowledge of K values at multiple locations

is necessary because of the heterogeneity of aquifer

materials. Laboratory methods are also available for

determining hydraulic conductivity, but field-measured values

represent average properties over a larger volume and utilize

less disturbed materials (Palmer and Johnson, 1989a).

Aquifer characteristics play an extremely important role in

determining the effectiveness of in-situ bioremediation. Even

in the presence of organisms acclimated to the specific waste

constituents present in an aquifer, biodegradation of

contaminants may be limited by unfavorable aquifer

characteristics that affect microbial activity including:

1. insufficient concentrations of dissolved oxygen for

aerobic metabolism of compounds susceptible to

aerobic degradation;

2. excessive oxygen that inhibits anaerobic

biodegradation of many halogenated compounds in

the subsurface;

Figure 3. The value of accurately locating the contaminated interval (Wilson et al., 1989).

Contaminated

Interval Direction of Flow

Wasted

Land Surface

Injection

Well

Water

Table
7

3. lack of a suitable alternative electron acceptor, if

oxygen is unavailable or not usable;

4. insufficient inorganic nutrients, such as nitrogen,

phosphorus, and trace minerals;

5. presence of toxic metals or other toxicants; and

6. other aquifer characteristics, such as pH, buffering

capacity, salinity, osmotic or hydrostatic pressures,

radiation, sorptive capacity, and temperature

(Armstrong, 1987; Lee et al., 1988).

Treatability Study

A treatability study is designed to determine if bioremediation

is possible at a specific site, and whether there are any

biological barriers to attaining clean-up goals. Even though

the scientific literature may indicate that a specific chemical is

likely to biodegrade in the environment, a treatability study

using site specific variables should be used to confirm that

contention (Suflita, 1989a). Microcosms are generally used to

conduct treatability studies. Pritchard (1981) defined a

microcosm as “a calibrated laboratory simulation of a portion

of a natural environment in which environmental components,

in as undisturbed a condition as possible, are enclosed within

definable physical and chemical boundaries and studied under

a set of laboratory conditions.” Microcosms may range from

simple batch incubation systems to large and complex flowthrough devices (Suflita, 1989a).

Results of a treatability study can also provide an estimate of

the rate and extent of remediation that can be attained if

microorganisms are not limited by the rate of supply of an

essential growth factor or by the presence of an unfavorable

environmental factor.

Treatability studies to determine inorganic nutrient and

electron acceptor requirements of subsurface microorganisms

present at a specific site should be conducted using samples

of subsurface solids as well as the ground water. Nutrient and

electron acceptor requirements that will enable indigenous

microorganisms to efficiently degrade organic contaminants

present at a specific site can be determined by incubating

contaminated subsurface materials with combinations of levels

of inorganic nutrients and electron acceptors. Studies should

be performed under conditions that simulate field

environmental conditions. Results of the studies are used to

design the bioremediation program as well as to optimize the

treatment strategy.

Design and Implementation of an In-Situ

Bioremediation System

Before implementation of an in-situ bioremediation system, the

source of contamination in the soil and in the ground water

should be removed as much as possible. In the case of a

liquid fuel spill, source removal may consist of recovery of

LNAPL free product from the ground water. Depending on the

characteristics of the aquifer and contaminants, free product

can account for as much as 91 percent of the spilled

hydrocarbon, with the remaining hydrocarbon (accounting for

9-40 percent of the spill) sorbed to the soil or dissolved in the

ground water (Lee et al., 1986).

Physical recovery techniques, based on the fact that LNAPL

hydrocarbons are relatively insoluble in and less dense than

water, are used to remove free product from a contaminated

site. Physical recovery often accounts for only 30 to 60

percent of spilled hydrocarbon before yields decline.

Continued pumping of recovery wells may be used to contain

a spill while in-situ bioremediation is being implemented. If a

spill is comprised of DNAPLs, which may sink to the bottom of

the aquifer, physical recovery may be considerably more

difficult to achieve.

Information from the performance of site characterization and

treatability studies may be integrated with the use of

comprehensive mathematical modeling to estimate the

expected rates and extent of treatment at the field scale

(Javandel, 1984; Keely, 1987). The specific model chosen

should incorporate biological reaction rates, stoichiometry of

waste transformation, mass-transport considerations, and

spatial variability in treatment efficiency (U.S. EPA, 1989a).

After assessment of site characterization and treatability

studies, if results indicate that in-situ bioremediation is

applicable to the site and will be an effective clean-up

technology, the information collected is used to design and

implement the system.

When in-situ bioremediation of a contaminant ground-water

plume involves using methods to enhance the process, such

as the addition of nutrients, additional oxygen sources, or

other electron acceptors, the use of hydraulic controls to

minimize migration of the plume during the in-situ treatment

process may be required (Thomas et al., 1987c; U.S. EPA,

1989a). In general, hydraulic control systems are generally

less costly and time consuming to install than physical

containment structures such as slurry walls. Well systems are

also more flexible, for pumping rates and well locations can be

altered as the system is operated over a period of time.

Pumping-injection systems can be used to: (1) create

stagnation zones at precise locations in a flow field; (2) create

gradient barriers to pollution migration; (3) control the

trajectory of a contaminant plume; and (4) intercept the

trajectory of a contaminant plume (Shafer, 1984). The choice

of a hydraulic control method depends on geological

characteristics, variability of aquifer hydraulic conductivities,

background velocities, and sustainable pumping rates (Lee et

al. 1988). Typical patterns of wells that are used to provide

hydraulic controls include: (1) a pair of injection-production

wells; (2) a line of downgradient pumping wells; (3) a pattern

of injection-production wells around the boundary of a plume;

and (4) the “double-cell” hydraulic containment system. The

“double-cell” system utilizes an inner cell and an outer

recirculation cell, with four cells along a line bisecting the

plume in the direction of flow (Wilson, 1984).

Well systems also serve as injection points for addition of the

materials used for enhancement of microbial activity and for

control of circulation through the contaminated zone. The

system usually includes injection and production wells and

equipment for the addition and mixing of the nutrients (Lee et

al., 1988). A typical system in which microbial nutrients are

mixed with ground water and circulated through the

contaminated portion of the aquifer through a series of

injection and recovery wells is illustrated in Figure 4 (Raymond

et al., 1978; Thomas and Ward, 1989).

Materials can also be introduced to the aquifer through the

use of infiltration galleries (Figure 5) (Brenoel and Brown,
8

Figure 4. Typical schematic for aerobic subsurface bioremediation (Thomas and Ward, 1989).

AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAA AAAAAAAA AAAAAAA

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAA A

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAA

A

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAA

A

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAA AAAAAAA AAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAA

AAAA AAAAAAA AAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAA AAAAAAAA AAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A AAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAA AAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAA

A

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A AAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAA AAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAA AAAAAAAAAAAAAAAAAAAA A AAAAAAAAAAAA AAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAA

AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAA AAA AAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAA AAA AAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA AAAAAAAA AAAAAAAA AAAAAAA AAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAA AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAA AAA AAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAA AAAAAAAAAAAAA

AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAAAAAAAA AAAAAAAAAAAAA AAAAAAAAAAAAA AAAAAAAAAAA AAAAAAAAAA AAAAAAAAA

AAAA AAAAAAAA AAAAAAAA AAAA AAAAAAAA AAAAAAAA AAAAAAAAAA AAAAAAAA AAAAAAA AAAAAAA

Nutrient

Addition

Tank To Sewer or

Recirculate

Air Compressor

Water Supply

Injection We

Sparger

Coarse Sand

Water Table

Production Well

Spilled Materials

Clay

Figure 5. Use of infiltration gallery for recirculation of water and nutrients in in-situ bioremediation (Thomas and Ward, 1989).

Air Compressor or

Hydrogen Peroxide

Tank

Nutrient Addition

Infiltration Gallery

Trapped Hydrocarbons

Water Table

Recovery Well

Monitoring Well

Recirculated Water & Nutrients
9

1985; Thomas and Ward, 1989). Infiltration galleries allow

movement of the injection solution through the unsaturated

zone as well as the saturated zone, resulting in potential

treatment of source materials that may be trapped in the pore

spaces of the unsaturated zone.

Amendments to the aquifer are added to the contaminated

aquifer in alternating pulses. Inorganic nutrients are usually

added first through the injection system, followed by the

oxygen source. Simultaneous addition of the two may result in

excessive microbial growth close to the point of injection and

consequent plugging of the aquifer. High concentrations of

hydrogen peroxide (greater than 10%) can be used to remove

biofouling and restore the efficiency of the system.

Operations Monitoring

Both the operation and effectiveness of the system should be

monitored (Lee et al., 1988). Operational factors of importance include the delivery of inorganic nutrients and electron

acceptor, the point of the delivery within the aquifer in relation

to the contaminated portion of the plume, and the effectiveness of containment and control of the contaminated plume.

Measurements of dissolved oxygen and nutrient levels in

ground-water samples are recommended to assess whether

or not bioremediation is being accomplished. Increases in

microbial activities in samples of aquifer materials may be

quantified relative to plume areas prior to treatment, areas

within the plume that did not receive treatment, and control

areas outside the plume. Carbon dioxide levels in groundwater samples may also be a useful indicator of microbial

activity (Suflita, 1989b).

Measurement of contaminant levels should indicate that

concentrations of contaminants are decreasing in areas

receiving treatment and remaining relatively unchanged in

areas that are not. If degradation pathways of specific

contaminants are known, measurement of presence and

concentrations of metabolic products may be used to

determine whether or not bioremediation is occurring. Both

soil and ground-water samples should be collected and

analyzed to develop a thorough evaluation of treatment

effectiveness. The use of appropriate control samples, e.g.,

assays of untreated areas or areas outside the plume, is

highly recommended to confirm the effectiveness of the

bioremediation technology (Suflita, 1989b).

The frequency of sampling should be related to the time

expected for significant changes to occur along the most

contaminated flow path (U.S. EPA, 1989a). Important

considerations include time required for water to move from

injection wells to monitoring wells, seasonal variations in water

table elevation or hydraulic gradient, changes in the

concentration of dissolved oxygen or alternative electron

acceptor, and costs of monitoring.

Advantages and Limitations in the Use of In-Situ

Bioremediation

There are a number of advantages and disadvantages in the

use of in-situ bioremediation (Lee et al., 1988). Unlike other

aquifer remediation technologies, it can often be used to treat

contaminants that are sorbed to aquifer materials or trapped in

pore spaces. In addition to treatment of the saturated zone,

organic contaminants held in the unsaturated and capillary

zones can be treated when an infiltration gallery is used.

The time required to treat subsurface pollution using in-situ

bioremediation can often be faster than withdrawal and

treatment processes. A gasoline spill was remediated in 18

months using in-situ bioremediation, while pump-and-treat

techniques were estimated to require 100 years to reduce the

concentrations of gasoline to potable water levels (Raymond

et al., 1986). In-situ bioremediation often costs less than other

remedial options. The areal zone of treatment using

bioremediation can be larger than with other remedial

technologies because the treatment moves with the plume

and can reach areas that would otherwise be inaccessible.

There are also disadvantages to in-situ bioremediation

programs (Lee et al., 1988). Many organic compounds in the

subsurface are resistant to degradation. In-situ

bioremediation usually requires an acclimated population of

microorganisms which may not develop for recent spills or for

recalcitrant compounds. Heavy metals and toxic

concentrations of organic compounds may inhibit activity of

indigenous microorganisms. Injection wells may become

clogged from profuse microbial growth resulting from the

addition of nutrients and oxygen.

In-situ bioremediation is difficult to implement in lowpermeability aquifers that do not permit the transport of

adequate supplies of nutrients or oxygen to active microbial

populations. In addition, bioremediation projects require

continuous monitoring and maintenance for successful

treatment.

References

Aelion, C. M., C. M. Swindoll, and F. K. Pfaender. 1987.

Adaptation to and Biodegradation of Xenobiotic Compounds

by Microbial Communities from a Pristine Aquifer. Appl.

Environ. Microbiol. 53:2212-2217.

Acker, W. L., Ill. 1974. Basic Procedures for Soil Sampling and

Core Drilling. Acker Drill Co. Scranton, PA.

Armstrong, J. 1987. Some Problems in the Engineering of

Groundwater Cleanup. p. 110-120. In: N. Dee, W. F.

McTernan, and E. Kaplan (eds.) Detection, Control, and

Renovation of Contaminated Ground Water. Am. Soc. Civil

Eng., New York, NY.

Barcelona, M.J. and J.A. Helfrich. 1986. Well Construction and

Purging Effects on Ground-Water Samples. Environ. Sci.

Technol. 20:1179-1184.

Barcelona, M.J., J.P. Gibb, J.A. Helfrich, and E.E. Garske.

1985. Practical Guide for Ground-Water Sampling. EPA/600/

2-85/104, Robert S. Kerr Environmental Research Laboratory,

U.S. Environmental Protection Agency, Ada, OK.

Brenoel, M., and R.A. Brown. 1985. Remediation of a Leaking

Underground Storage Tank with Enhanced Bioreclamation.

Proc., Fifth National Symp. on Aquifer Restoration and Ground

Water Monitoring. National Water Well Assoc., Worthington,

OH.
10

Canter, L.W., and R.C. Knox. 1985. Ground Water Pollution

Control. Lewis Publishers, Chelsea, MI.

Devitt, D.A., R.B. Evans, W.A. Jury, T.H. Starks, B. Eklund, A.

Ghalsan. 1988. Soil Gas Sensing for Detection and Mapping

of Volatile Organics. EPA/600/8-87/036, Environmental

Monitoring and Support Laboratory, U.S. Environmental

Protection Agency, Las Vegas, NV.

Dunlap, W.J. and J.F. McNabb. 1973. Subsurface Biological

Activity in Relation to Ground Water Pollution. EPA-660/2-73-

014, Robert S. Kerr Environmental Research Laboratory, U.S.

Environmental Protection Agency, Ada, OK.

Dunlap, W.J., J.F. McNabb, M.R. Scalf, and R.L. Cosby. 1977.

Sampling for Organic Chemicals and Microorganisms in the

Subsurface. EPA-600/2-77-176, Robert S. Kerr Environmental

Research Laboratory, U.S. Environmental Protection Agency,

Ada, OK.

Ehrenfield, J. and J. Bass. 1984. Evaluation of Remedial

Action Unit Operations of Hazardous Waste Disposal Sites.

Pollution Technology Review No. 110, Noyes Publications,

Park Ridge, NJ.

Ford, P.J., P.J. Turina, and D.E. Seely. 1984. Characterization

of Hazardous Waste Sites - A Methods Manual, Volume II:

Available Sampling Methods. EPA/600/4-84/076. Robert S.

Kerr Environmental Research Laboratory, U.S. Environmental

Protection Agency, Ada, OK.

Ghiorse, W.C., and D.L. Balkwill. 1985. Microbiology of

Ground Water Environments. In: G. E. Januer (ed.) Progress

in Chemical Disinfection II: Problems at the Frontier. State

Univ. of New York (SUNY) at Binghamton, Binghamton, NY.

Ghiorse, W.C., and J.T. Wilson. 1988. Microbial Ecology of the

Terrestrial Subsurface. Adv. Appl. Microbiol. 33:107-172.

Goldstein, R.M., L.M. Mallory, and M. Alexander. 1985.

Reasons for Possible Failure of Inoculation to Enhance

Biodegradation. Appl. Environ. Microbiol. 50:977-983.

Javandel, I., C. Doughty, and C.F. Tsang. 1984. Groundwater

Transport: Handbook of Mathematical Models. Water

Resources Monograph No. 10, Am. Geophysical Union,

Washington, DC.

Johnson, R.L., C.D. Palmer, and W. Fish. 1989. Subsurface

Chemical Processes. In: Transport and Fate of Contaminants

in the Subsurface. EPA/625/4-89/019, Robert S. Kerr

Environmental Research Laboratory, U.S. Environmental

Protection Agency, Ada, OK.

Keely, J.F. 1987. The Use of Models in Managing GroundWater Protection Programs. EPA/600/8-87/003. Robert S.

Kerr Environmental Research Laboratory, U.S. Environmental

Protection Agency, Ada, OK.

Keely, J.F. and K. Boateng. 1987. Monitoring Well Installation,

Purging, and Sampling Techniques. Part II: Case Histories.

Ground Water 25:427-439.

Kilbane, J.J. 1986. Genetic Aspects of Toxic Chemical

Degradation. Microbiol. Ecol. 12:135-145.

Leach, L.E., F.P. Beck, J.T. Wilson, and D.H. Kampbell. 1988.

Aseptic Subsurface Sampling Techniques for Hollow-Stem

Auger Drilling. Proc., Second National Outdoor Action

Conference on Aquifer Restoration, Ground Water Monitoring

and Geophysical Methods 1:31-51.

Lee, M.D. and C.H. Ward. 1986. Ground Water Restoration.

Report submitted to JACA Corporation, Fort Washington, PA.

Lee, M.D., J.M. Thomas, R.C. Borden, P.B. Bedient, J.T.

Wilson, and C. H. Ward. 1988. Biorestoration of Aquifers

Contaminated with Organic Compounds. CRC Critical Rev.

Environ. Controls 18:29-89.

McNabb, J.F., and G.E. Mallard. 1984. Microbiological

Sampling in the Assessment of Groundwater Pollution. In: G.

Bitton and C.P. Gerba (eds.), Groundwater Pollution

Microbiology. John Wiley & Sons, New York, NY.

Palmer, C.D., and R.L. Johnson. 1989a. Determination of

Physical Transport Parameters. In: Transport and Fate of

Contaminants in the Subsurface. EPA/625/4-89/019, Robert

S. Kerr Environmental Research Laboratory, U.S.

Environmental Protection Agency, Ada, OK.

Palmer, C.D., and R.L. Johnson. 1989b. Physical Processes

Controlling the Transport of Contaminants in the Aqueous

Phase. In: Transport and Fate of Contaminants in the

Subsurface. EPA/625/4-89/019, Robert S. Kerr Environmental

Research Laboratory, U.S. Environmental Protection Agency,

Ada, OK.

Palmer, C.D., and R.L. Johnson. 1989c. Physical Processes

Controlling the Transport of Non-Aqueous Phase Liquids in

the Subsurface. In: Transport and Fate of Contaminants in the

Subsurface. EPA/625/4-89/019, Robert S. Kerr Environmental

Research Laboratory, U.S. Environmental Protection Agency,

Ada, OK.

Pritchard, P.H. 1981. Model Ecosystems. In: R.A. Conway

(ed.), Environmental Risk Analysis for Chemicals. Van

Nostrand Reinhold, New York, NY.

Raymond, R.L. 1974. Reclamation of Hydrocarbon

Contaminated Ground Waters. U.S. Patent Office,

Washington, DC. Patent No. 3,846,290. Patented

November 5, 1974.

Raymond, R.L., R.A. Brown, R.D. Norris, and E.T. O’Neill.

1986. Stimulation of Biooxidation Processes in Subterranean

Formations. U.S. Patent Office, Washington, DC. Patent No.

4,588,506. Patented May 13, 1986.

Raymond, R.L., V.W. Jamison, J.O. Hudson, R.E. Mitchell,

and V.E. Farmer. 1978. Field Application of Subsurface

Biodegradation of Gasoline in a Sand Formation. API Publication 4430, American Petroleum Institute, Washington, DC.

Scalf, M.R., J.F. McNabb, W.J. Dunlap, R.L. Cosby, and J.

Fryberger. 1981. Manual of Ground Water Sampling

Procedures. National Water Well Assoc., Worthington, OH.

Schafer, J.M. 1984. Determining Optimum Pumping Rates for

Creation of Hydraulic Barriers to Ground Water Pollutant

Migration. In: D.M. Nielsen (ed.), Proc., Fourth National Symp.
11

Environmental Research Laboratory, U.S. Environmental

Protection Agency, Ada, OK.

Wilson, J.T., J.F. McNabb, D.L. Balkwill, and W.C. Ghiorse.

1983. Enumeration and Characterization of Bacteria

Indigenous to a Shallow Water-Table Aquifer. Ground Water

21:134-142.

Wilson, J.T., L.E. Leach, M.J. Henson, and J.N. Jones. 1986.

In Situ Biorestoration as a Ground Water Remediation

Technique. Ground Water Monitoring Rev. 6:56-64.

Zapico, M.M., S. Vales, and J.A. Cherry. 1987. A Wireline

Piston Core Barrel for Sampling Cohesionless Sand and

Gravel Below the Water Table. Ground Water Monitoring Rev.

7(3):74-82.

on Aquifer Restoration and Ground Water Monitoring. National

Water Well Assoc., Worthington, OH.

Stolzenburg, T.R. and D.G. Nichols. 1985. Preliminary Results

on Chemical Changes in Groundwater Samples Due to

Sampling Devices. EPRI Report No. EA-4118, Electric Power

Research Institute, Palo Alto, CA.

Suflita, J.M. 1989a. Microbial Ecology and Pollutant

Biodegradation in Subsurface Ecosystems. In: Transport and

Fate of Contaminants in the Subsurface. EPA/625/4-89/019,

Robert S. Kerr Environmental Research Laboratory, U.S.

Environmental Protection Agency, Ada, OK.

Suflita, J.M. 1988b. Microbiological Principles Influencing the

Biorestoration of Aquifers. In: Transport and Fate of

Contaminants in the Subsurface. EPA/625/4-89/019, Robert

S. Kerr Environmental Research Laboratory, U.S.

Environmental Protection Agency, Ada, OK.

Thomas, J.M., and C.H. Ward. 1989. In Situ Biorestoration of

Organic Contaminants in the Subsurface. Environ. Sci.

Technol. 23:760-766.

Thomas, J.M., M.D. Lee, and C.H. Ward. 1987a. Use of

Ground Water in Assessment of Biodegradation Potential in

the Subsurface. Environ. Toxicol. Chem. 6:607-61 4.

Thomas, J.M., H.J. Marlow, R.L. Raymond, and C.H. Ward.

1987b. Hydrologic Considerations for In Situ Biorestoration.

US/USSR Symposium on Fate of Pesticides and Chemicals in

the Environment, Oct. 12-16, Iowa City, IA (under review by

John Wiley & Sons, Inc., New York, NY).

Thomas, J.M., M.D. Lee, P.B. Bedient, R.C. Borden, L.W.

Canter, and C.H. Ward. 1987c. Leaking Underground Storage

Tanks: Remediation with Emphasis on In Situ Biorestoration.

EPA/600/2-87/008, Robert S. Kerr Environmental Research

Laboratory, U.S. Environmental Protection Agency, Ada, OK.

U.S. Environmental Protection Agency. 1989a. Bioremediation

of Hazardous Waste Sites Workshop: Speaker Slide Copies

and Supporting Information. CERI-89-11, Center for

Environmental Research Information, U.S. Environmental

Protection Agency, Cincinnati, OH.

Wilson, J.L. 1984. Double-cell Hydraulic Containment of

Pollutant Plumes. In: D. M. Nielsen (ed.), Proc., Fourth

National Symp. on Aquifer Restoration and Ground Water

Monitoring. National Water Well Assoc., Worthington, OH.

Wilson, J.T., and B.H. Wilson. 1985. Biotransformation of

Trichloroethylene in Soil. Appl. Environ. Microbiol. 49:242-243.

Wilson, J.T. and C.H. Ward. 1987. Opportunities for

Bioreclamation of Aquifers Contaminated with Petroleum

Hydrocarbons. Dev. Industrial Microbiol. (J. Industrial

Microbiol. Suppl. 1 ) 27:109-116.

Wilson J., L. Leach, J. Michalowski, S. Vandegrift, and R.

Callaway. 1989. In Situ Bioremediation of Spills from

Underground Storage Tanks: New Approaches for Site

Characterization, Project Design, and Evaluation of

Performance. EPA/600/2-89/042, Robert S. Kerr