In Situ Treatment Technology - Chapter 7 ppsx

Boettcher, Gary & Nyer, Evan K. "In Situ Bioremediation "
In Situ Treatment Technology
Boca Raton: CRC Press LLC,2001

©2001 CRC Press LLC

CHAPTER

7

In Situ

Bioremediation

Gary Boettcher and Evan K. Nyer

CONTENTS

Introduction
Microbiology and Biochemistry Basics
Microorganisms
Distribution and Occurrence of Microorganisms in the Environment
Soil
Groundwater
Microorganism Biochemical Reactions
Energy Production
Oxidation and Reduction
Aerobic Respiration
Facultative Respiration
Anaerobic Respiration
Microbial Degradation and Genetic Adaptation

Gratuitous Biodegradation
Cometabolism
Microbial Communities
Community Interaction and Adaptation
Genetic Transfer
Growth
Growth Cycle
Important Environmental Factors that Affect Growth
Water
pH
Temperature
Hydrogen Ion Concentration

©2001 CRC Press LLC

Oxygen
Nutrition
Toxic Environments that Affect Growth
Microbial Degradation and Modification
Degradation Rate
Natural Attenuation
Application of Natural Attenuation
Advantages and Disadvantages of Natural Attenuation
Lines of Evidence
Site Characterization
Petroleum Hydrocarbons and Chlorinated Hydrocarbons
Biodegradation of Petroleum Hydrocarbons
Biodegradation of Chlorinated Hydrocarbons
Biodegradation of the Chlorinated Hydrocarbon Used as
an Electron Donor (Carbon and Energy Source)

Biodegradation of the Chlorinated Hydrocarbon Used as
an Electron Acceptor (Reductive Dechlorination)
Cometabolism
Abiotic Degradation
Natural Attenuation Data Collection and Evaluation
Modeling Tools
Case Histories
Petroleum Hydrocarbon Site Application
Chlorinated Hydrocarbon Site Application
Geology and Hydrogeology
Groundwater Quality
Initial Evaluation of Remedial Technologies
Evaluation of Natural Attenuation
Geochemical Study
Reduction of Contaminant Concentrations
Plume Retardation
Reduction of Contaminant Mass
Conclusions
Summary
References

INTRODUCTION

In situ

bioremediation is a true

in situ

technology. In this process, biochemical

reactions destroy or chemically modify compounds such that these compounds are
no longer a threat to human health or the environment. The reactions occur below
ground, making this one of the few in place or

in situ

technologies used today.
Chapters 3, 4, and 5 describe physical and chemical

in situ

technologies that are
designed to actively remediate petroleum hydrocarbons, chlorinated hydrocarbons,
pesticides, or inorganics (including heavy metals). As discussed in those chapters,

©2001 CRC Press LLC

the air used as a carrier during those processes can also stimulate biological reactions.
Bioremediation is an important element of most of these remedial solutions where
groundwater and soil are impacted with organic or inorganic compounds. However,
not all remedial solutions require active soil or groundwater remediation. In recent
years, protocols have been developed that are designed to evaluate the environment’s
ability to naturally attenuate impacts. This is a powerful remediation technology that
will continue to be expanded, refined, and applied where soil and groundwater have
been impacted with organic and inorganic compounds.
This chapter is divided into two main sections in order to understand bioreme-
diation and implement natural attenuation. The first section will focus on biological
and chemical processes that are important to understand when designing or operating
all

in situ

biological remediation systems. The second section will focus on natural
attenuation processes whereby the environmental conditions and these processes are
able to achieve the remediation goals.

MICROBIOLOGY AND BIOCHEMISTRY BASICS

Microorganisms in soil and groundwater complete biochemical reactions. These
reactions often directly or indirectly destroy or modify organic and inorganic chem-
icals. These microorganisms are living creatures, and as such, require favorable
environmental conditions in order to complete their biochemical reactions and reme-
diate organic and inorganic chemicals. Therefore, it is important to have a basic
understanding of microorganisms, metabolism, growth, and microbial degradation
processes in order to design or evaluate

in situ

bioremediation systems. This under-
standing will allow design engineers to exploit biochemical reactions in soil and
groundwater environments and avoid potentially inhibitory conditions.

Microorganisms

Free-living microorganisms that exist on earth include bacteria, fungi, algae,
protozoa, and metazoa. Viruses are also prevalent in the environment; however, these
particles can only exist as parasites in living cells of other organisms and will not
be discussed in this text. Microorganisms have a variety of characteristics that allow
survival and distribution throughout the environment. They can be divided into two
main groups. The eucaryotic cell is the unit of structure that exist in plants, metazoa

animals, fungi, algae, and protozoa. The less complex procaryotic cell includes the
bacteria and cyanobacteria.
Even though the protozoa and metazoa are important organisms that affect soil
and water biology and chemistry, they do not perform important degradative roles.
Therefore, this chapter will concentrate on bacteria and fungi.
Bacteria are by far the most prevalent and diverse organisms on earth. There are
over 200 genera in the bacterial kingdom (Holt 1981). These organisms lack nuclear
membranes and do not contain internal compartmentalization by unit membrane
systems. Bacteria range in size from approximately 0.5 micron to seldom greater
than 5 microns in diameter. The cellular shape can be spherical, rod-shaped, fila-

©2001 CRC Press LLC

mentous, spiral, or helical. Reproduction is by binary fission. However, genetic
material can also be exchanged between bacteria.
The fungi, which include molds, mildew, rusts, smuts, yeasts, mushrooms, and
puffballs, constitute a diverse group of organisms living sometimes in fresh water
and marine water, but predominantly in soil or on dead plant material. Fungi are
responsible for mineralizing organic carbon and decomposing woody material (cel-
lulose and lignin). Reproduction occurs by sexual and asexual spores or by budding
(yeasts).

Distribution and Occurrence of Microorganisms in the Environment

Due to their natural functions, microorganisms are found throughout the envi-
ronment. Habitats that are suitable for higher plants and animals to survive will
permit microorganisms to flourish. Even habitats that are adverse to higher life forms
can support a diverse microorganism population. Soil, groundwater, surface water,
and air can support or transport microorganisms. Since this text focuses on

in situ

treatment, the following briefly describes the distribution and occurrence of micro-
organisms in soil and groundwater only.
Microorganisms found in soil or groundwater represent the part of the entire
population that has flourished under the environmental conditions that are present
during the time of sampling. If the environmental conditions are changed by natural
or man-made influences, then the microbial population will change in response to
the new environment. Chapters 8 and 9 will show how to manipulate the environment
in order to change the microbial population and promote new types of biochemical
reactions. We will limit this chapter to mainly discussing what is naturally found in
the soil and groundwater.

Soil

Bacteria outnumber the other organisms found in a typical soil. These organisms
rapidly reproduce and constitute the majority of biomass in soil. It is estimated that
surficial soil can contain some 10,000 different microbial species and can have as
many as 109 cells/gm of soil. In addition, cellular biomass can comprise up to
approximately 4 percent of the soil organic carbon (Adriano et al. 1999). Micro-
organisms generally adhere to soil surfaces by electrostatic interactions, London-
van der Waals forces, and hydrophobic interactions (Adriano et al. 1999). Typically,
microorganisms decrease with depth in the soil profile, as does organic matter. The
population density does not continue to decrease to extinction with increasing depth,
nor does it necessarily reach a constant declining density. Fluctuations in density
commonly occur at lower horizons. In alluvial soils, populations fluctuate with
textural changes; organisms are more numerous in silt or silty clay than in inter-
vening sand or course sandy horizons. In soil profiles above a perched water table,
organisms are more numerous in the zone immediately above the water table than
in higher zones (Paul and Clark 1989). Most fungal species prefer the upper soil

profile. The rhizosphere (root zone) contains the most variety and numbers of
microorganisms.

©2001 CRC Press LLC

Groundwater

Microbial life occurs in aquifers. Many of the microorganisms found in soil are
also found in aquifers and are primarily adhered to soil surfaces. Bacteria exist in
shallow to deep subsurface regions but the origins of these organisms are unknown.
They could have been deposited with sediments millions of years ago, or they may
have migrated recently into the formations from surface soil. Bacteria tend not to
travel long distances in fine soils but can travel long distances in course or fractured
formations. These formations are susceptible to contamination by surface water and
may carry pathogenic organisms into aquifer systems from sewage discharge, landfill
leachate, and polluted water (Bouwer 1978).

MICROORGANISM BIOCHEMICAL REACTIONS

Microorganisms responsible for degradation of organic environmental impacts
obtain energy and building blocks necessary for growth and reproduction from
degrading organic compounds. Energy is conserved in the C-C bonds, and during
degradation, the organics are converted to simpler organic compounds while deriving
energy. Ultimately, the organic compounds are degraded (mineralized) to carbon
dioxide or methane, inorganic ions, and water. During the process, microbes use
portions of these compounds as building blocks for new microbial cells.
As discussed above, microorganism populations can be numerous in soil and
groundwater. These populations complete diverse biochemical reactions, and are
able to thrive in wide ranges of environmental conditions. In addition, the presence
of particular microorganisms and the biochemical reactions that they complete are

influenced by the physical and chemical environment. These physical and chemical
environments can be modified by organisms creating favorable conditions for a new
consortium of organisms and biochemical reactions to occur. Often, different envi-
ronmental conditions are created whereby new degradative pathways are induced
resulting in the ability to biochemically degrade different organic pollutants, chem-
ically modify inorganic compounds, or immobilize inorganic compounds such as
heavy metals.
The following sections describe important biochemical reactions that site inves-
tigators and remediation design engineers should understand. Understanding these
reactions will allow remediation teams to determine if biodegradation is likely
occurring or if environmental conditions can be modified to create conditions favor-
able to degrade or modify environmental pollutants. Failure to understand these
concepts can result in remediation systems that limit biological processes, and
therefore minimize effectiveness.

Energy Production

Microorganisms derive energy by degrading a wide variety of organic com-
pounds including man-made (xenobiotic or anthropogenic) compounds. Enzymes
are induced, respiration occurs, organic compounds are cleaved releasing energy,

©2001 CRC Press LLC

intermediate compounds are produced, and growth and reproduction occurs. These
processes allow microorganisms to thrive and contribute to the natural cycling of
carbon throughout the environment (Figure 1). As seen in Figure 1, microorganisms
perform a portion of the overall carbon cycling and it is this portion that bioreme-
diation systems rely on to degrade or modify environmental pollutants.

Oxidation and Reduction

The utilization of chemical energy in microorganisms generally involves what
are called oxidation-reduction reactions. For every chemical reaction, oxidation and
reduction occurs. Oxidation of a compound corresponds to an oxygen increase, loss
of hydrogen, or loss of electrons. Conversely, reduction corresponds to an oxygen
decrease, an increase of hydrogen, or an increase in electrons. This process is coupled
(half-reactions); if a target chemical is oxidized, another compound must be reduced.
In this case, the reactant serves as the electron donor and becomes oxidized, while
the other compound serves as the electron acceptor and becomes reduced. In terms
of energy released, the electron donor is also an energy source (substrate), whereas
the electron acceptor is not an energy source. Once the electron donor has been fully
oxidized (lost all the electrons that it can loose) it is usually no longer an energy
source but may now serve as an electron acceptor.
This is an important concept to understand because biochemical reactions and
the ability to degrade or modify compounds are usually dependant on the oxidation
state of the target compounds and the predominant biochemical processes that are
occurring in soil and groundwater. For example, organic compounds that are in a
reduced state, such as aliphatic hydrocarbons, are more likely to be oxidized in the
environment. Chemicals that are in an oxidized state, such as highly chlorinated

Figure 1

Carbon cycle.

©2001 CRC Press LLC

volatile organic compounds, are more likely to be reduced in the environment. In
addition, because it is often difficult to directly confirm that degradation is occurring
during remediation, it is often necessary to measure indicator parameters in order
to determine the predominant biochemical processes that are occurring.

The types of electron acceptors used by microorganisms affect the quantity of
energy that is available from organics. The energy available from the oxidation-
reduction reaction is expressed as the standard electrode potential (oxidation-reduc-
tion potential [Eh]) (referenced to hydrogen at pH = 7). Common electron acceptors
used to evaluate

in situ

bioremediation processes are shown in Figure 2. The electron
accepting reactions are shown in order of decreasing energy availability. In addition,
common organisms responsible for these reactions are also shown (Adriano et al.
1999 and Brock 1979).

Aerobic Respiration

Aerobic microorganisms have enzyme systems that are capable of oxidizing
organic compounds. The organic compound serves as the electron donor and the
electrons are transferred to molecular oxygen (O

2

). This is the most efficient (less
energy required) biochemical reaction whereby the electron donor (organic substrate)
is degraded producing biomass, carbon dioxide (CO

2

), water, and potentially other
organics as depicted by:
electron donor (organic substrate) + O

2

(electron acceptor)

→

biomass + CO

2

+ H

2

O + metabolites + energy.

Figure 2

Energy tower for different electron acceptors in biodegradation pH = 7 (Adriano
et al. 1999) (Adapted from Brock, 1979).

©2001 CRC Press LLC

Facultative Respiration

In reduced or low molecular oxygen environments, facultative anaerobes are a
class of microorganisms that are able to shift their metabolic pathways and use
nitrate (NO

3
-

) as a terminal electron acceptor. This process is called denitrification
and is generally depicted as follows:
electron donor (organic substrate) + NO

3
-

(electron acceptor)

→

biomass + CO

2

+ H

2

O + N

2

+ metabolites + energy.
The reduction of NO

3

-

to nitrogen gas (N

2

) is completed through a series of
electron transport reactions as follows:
NO

3
-

(nitrate)

→

NO

2
-

(nitrite)

→

NO (nitic oxide)

→

N

2

O (nitrous oxide)

→

N

2

(nitrogen gas)
Most denitrifiers are heterotrophic and commonly occur in soil such as

Pseudomonas, Bacillus,

and

Alcaligenes

genera. A large number of species can
reduce nitritate to nitrite in the absence of oxygen, with a smaller number of species
that can complete the reaction by reducing nitrous oxide to nitrogen gas.

Anaerobic Respiration

Anaerobic respiration is completed by different classes of microorganisms in the
absence of molecular oxygen. The anaerobic organisms important to environmental
remediation include iron and manganese reducing bacteria, and sulfanogenic and

methanogenic bacteria. Anaerobic growth in the environment is not as efficient as
aerobic growth (less energy produced per reaction); however, these organisms com-
plete important geochemical reactions including bacterial corrosion, sulfur cycling,
organic decomposition, and methane production. These reactions are more complex
than aerobic respiration and often rely on a consortium of bacteria to complete the
reactions. In addition, these classes of bacteria are also capable of either degrading
organic pollutants and/or alter environmental conditions whereby chemical reactions
can occur. The following depicts the generalized (and simplified) reactions these
classes of organisms complete:
Iron Reduction:
organic substrate (electron donor) + Fe(OH)

3

(electron acceptor) + H

2
+



→

biomass + CO

2

+ Fe

2+

+ H

2

O + energy
Manganese Reduction:
organic substrate (electron donor) + MnO

2

(electron acceptor) + H

2
+



→

biomass + CO

2

+ Mn

2+

+ H

2

O + energy

©2001 CRC Press LLC

Sulfanogenesis:
organic substrate (electron donor) + SO

4
2-

(electron acceptor) + H

+



→

biomass + CO

2

+ H

2

O + H

2

S + metabolites + energy
Methanogenesis:
organic substrate + CO

2

(electron acceptor) + H

+

(electron donor)

→

biomass + CO

2

+ H

2

O + CH

4

+ metabolites + energy
More detailed information regarding these biochemical reactions can be obtained

by reviewing mircobiological texts such as Brock 1979, Stanier, Adelberg, and
Ingrahm 1979, and Paul and Clark 1989.

Microbial Degradation and Genetic Adaptation

In the preceding sections, the reactions associated with degradation and growth
were discussed. However, the susceptibility of an environmental pollutant to micro-
bial degradation is determined by the ability of the microbial population to catalyze
the reactions necessary to degrade the organics.
Readily degradable compounds have existed on earth for millions of years;
therefore, there are organisms that can mineralize these compounds. Industrial chem-
icals (xenobiotic or anthropogenic) have been present on earth for a short time on
the evolutionary time scale. Many of these compounds are degradable, and many
are persistent in the environment. Some xenobiotic compounds are similar to natural
compounds and bacteria will degrade them easily. Other xenobiotic compounds will
require special biochemical pathways in order to undergo biochemical degradation.
Biodegradation of organic compounds (and maintenance of life sustaining pro-
cesses) is reliant on enzymes. The best way to understand enzyme reactions is to
think of them as a lock and key. Figure 3 shows how only an enzyme with the right
shape (and chemistry) can function as a key for the organic reactions. The lock and
key in the real world are three-dimensional. The fit between the two is precise.
Organic compounds in the environment that are degradable align favorably with
the active site of specific enzymes. The microorganism will not affect compounds
that do not align favorably or compounds that do not bind with the active site of
their enzyme. Degradation of these compounds requires that the microorganism
population adapt in response to the environment by synthesizing enzymes capable
of catalyzing degradation of these compounds.
A few definitions would be helpful here in order to understand different levels
of biological reactions. Biodegradation means the biological transformation of an
organic chemical to another form with no extent implied (Grady 1985). Biodegra-

dation does not have to lead to complete mineralization. Mineralization is the
complete degradation of an organic compound to carbon dioxide or methane and
inorganic ions. Recalcitrance is defined as inherent resistance of a chemical to any
degree of biodegradation and persistence means that a chemical fails to undergo

©2001 CRC Press LLC

biodegradation under a defined set of environmental conditions (Bull 1980). This
means that a chemical can be degradable but due to environmental conditions, the
compounds may persist in the environment. With proper manipulation (or under-
standing) of the environmental conditions, biodegradation of these compounds can
be demonstrated in the laboratory or field.

Gratuitous Biodegradation

Enzymes are typically described as proteins capable of catalyzing highly specific
biochemical reactions. Enzymes are more specific to organic compound functional
groups than to specific compounds. An enzyme will not differentiate between a C-
C bond in a benzene molecule versus a C-C bond in a phenol molecule. The
functional capability of enzymes depends on the specificity exhibited towards the
organic compound. A major enzymatic mechanism used by bacteria to degrade
xenobiotic compounds has been termed gratuitous biodegradation and includes exist-
ing enzymes capable of catalyzing a reaction towards a chemical substrate.
In order for gratuitous biodegradation to occur, the bacterial populations must
be capable of inducing the requisite enzymes specific for the xenobiotic compound.
Often times this occurs in response to similarities (structural or functional groups)
with natural organic chemicals, for example, a bacterium producing the enzymes
for benzene degradation. Chlorobenzene is introduced and is not recognized by
the bacteria (its presence will not induce an enzyme to be produced). However,
the enzymes already produced for benzene will also catalyze the degradation of

chlorobenzene.
The capability of bacterial populations to induce these enzymes depends on
structural similarities and the extent of substitutions on the parent compound. Gen-

Figure 3

Enzymes are represented as a lock and key.

©2001 CRC Press LLC

erally, as the number of substitutions increases, biodegradability decreases unless a
natural inducer is present to permit synthesis of required enzymes. To overcome
potential enzymatic limitations, bacteria populations often induce a series of
enzymes that coordinately modify xenobiotic compounds. Each enzyme will modify
the existing compound such that a different enzyme may be specific for the new
compound and capable of degrading it further. Eventually, the original xenobiotic
compound will not be present and the compound will resemble a natural organic
compound and enter into normal metabolic pathways. This concept of functional
pathways is more likely to be completed through the combined efforts of mixed
communities than by any one single species.

Cometabolism

Cometabolism has been defined as "the transformation of a nongrowth substrate
in the obligate presence of a growth substrate” (Grady 1985). A nongrowth substrate
cannot serve as a sole carbon source that provides energy to support metabolic
processes. A second compound is required to support biological processes allowing
transformation of the nongrowth substrate. This requirement is added to make a
distinction between cometabolism and gratuitous biodegradation.
During cometabolism, the organism receives no known benefit from the degra-

dation of the organic compound. In fact, the process may be harmful to the micro-
organism responsible for the production of the enzyme (McCarty and Semprini
1994). Cometabolism of chlorinated ethenes (with the exception of perchloroethene
[PCE]) has been reported to occur in aerobic environments and it is believed that
the rate of cometabolism increases as the degree of dechlorination decreases (Murray
and Richardson 1993, Vogel 1994, and McCarty and Semprini 1994).

Microbial Communities

Complete mineralization of a xenobiotic compound may require more than one
microorganism. No single bacterium within the mixed culture contains the complete
genome (genetic makeup) of a mixed community. The microorganisms work together
to complete the pathway from organic compound to carbon dioxide or methane.
These associations have been called consortia, syntrophic association, and synergis-
tic associations and communities (Grady 1985). We need to understand the impor-
tance of the community when we deal with remediation. Conversely, we need to
understand the limitations of laboratory work with single organisms. This work does
not represent the real world of degradation. Reviewing the strengths of the commu-
nities will also reveal the limitations of adding specialized bacteria that have been
grown in the laboratory.

Community Interaction and Adaptation

Microbial communities are in a continuous state of flux and constantly adapting
to their environment. Population dynamics, environmental conditions, and growth

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substrates continually change and impact complex interactions between microbial
populations. Even though microorganisms can modify environmental disturbances,

microbial ecosystems lack long-term stability and are continually adapting (Grady
1985). It is important to understand the complexities and interactions within an
ecosystem to prevent failure when designing a biological remediation system.
Mixed communities have greater capacity to biodegrade xenobiotic compounds
due to greater genetic diversity of the population. Complete mineralization of xeno-
biotic compounds may rely on enzyme systems produced by multiple species.
Community resistance to toxic stresses may also be greater due to the likelihood
that an organism can detoxify the ecosystem.
Community adaptation is dependent upon evolution of novel metabolic path-
ways. A bacterial cell considered in isolation has a relatively limited adaptive poten-
tial and adaptation of a pure culture must come from mutations (Grady 1985).
Mutations are rare events. These mutations are generally responsible for enzymes
that catalyze only slight modifications to the xenobiotic compound. An entire path-
way can be formed through the cooperative effort of various populations. This is
due to the greater probability that an enzyme system exists capable of gratuitous
biodegradation within a larger gene pool. This genetic capability can then be trans-
ferred to organisms lacking the metabolic function that enhances the genetic diversity
of the population. Through gene transfer, individual bacteria have access to a larger
genetic pool allowing evolution of novel degradative pathways.

Genetic Transfer

Genes are transferred throughout bacterial communities by three mechanisms
called conjugation, transformation, and transduction (Brock 1979, Stanier, Adelberg,
and Ingrahm 1976, Moat 1979, Grady 1985, and Rittman, Smets, and Stahl 1990).
Conjugation appears to be the most important mechanism of gene transfer in the
natural environment. Conjugation involves the transfer of DNA from one bacterium
to another while the bacteria are temporarily joined. The DNA strands that are
transferred are separate from the bacterial chromosomal DNA and are called plas-
mids (Brock 1979, Stanier, Adelberg, and Ingrahm 1976, Moat 1979, and Rittman,

Smets, and Stahl 1990). Plasmids exist in cells as circular, double-stranded DNA
and are replicated during transfer from donor to recipient. Unlike chromosomal DNA
that encodes for life sustaining processes, plasmid genes encode for processes that
enhance growth or survival in a particular environment. Examples of functions that
are encoded on plasmids include antibiotic resistance, heavy metal resistance, and
certain xenobiotic degradation enzymes (such as toluene) (Rittman, Smets, and Stahl
1990).
There are many natural processes that the microorganisms employ to expand the
type of compounds that they can use as an energy source. We can create environments
and provide growth factors that facilitate these processes, or data can be collected
that documents that the biochemical reactions are occurring without modifying the
environment. The rest of this section will discuss these various processes.

©2001 CRC Press LLC

GROWTH

Growth is defined as an increase in the quantity of cellular constituents, struc-
tures, or organisms (biomass). Growth is controlled by a complex interaction between
food sources (usually organics), inorganic nutrients and cofactors, terminal electron
acceptors, predators, physical conditions, and chemical conditions. It is the design
engineer’s objective to optimize these conditions in order to maximize the biological
treatment system’s effectiveness.

Growth Cycle

A microorganism growth cycle can be divided into several phases called the lag
phase, exponential phase, stationary phase, and death phase (Figure 4). It is the
remediation engineer’s objective to design biological systems that maintain a high
growth rate until the environmental pollutant has been degraded or modified. At this

point, organic carbon (food) usually becomes limiting and the microorganism pop-
ulation proceeds into the death phase.

Important Environmental Factors that Affect Growth

As discussed earlier in this chapter, organic compound degradation and growth
is completed in aerobic and/or anaerobic environments. These reactions occur only
when the physical, chemical, and biological environment are conducive to supporting
these reactions.
The following sections describe the most important factors that must be consid-
ered for every biological remediation design. Failure to include these factors in all

Figure 4

Typical growth curve for a bacterial population.

©2001 CRC Press LLC

biological designs can significantly limit remedial effectiveness, as these factors can
control the type of bacteria that are prominent and the biodegradation rate. In addition
to these factors, the physical environment associated with soil is important; however,
these considerations are not included in this text. The authors highly suggest other
sources such as Paul and Clark 1989 and Adriano et al. 1999 be reviewed to better
understand biological associations in soil.

Water

Water is an important factor for biochemical reactions. In saturated and unsat-
urated conditions, the bacteria may have to expend energy in order to acquire the
water that they require. In the aquifer, the availability of water to microorganisms

can be expressed in terms of water activity, which is related to vapor pressure of
water in the air over a solution (relative humidity). Water activity in freshwater and
marine environments is relatively high and lowers with increasing concentrations of
dissolved solute (Brock 1979). Bacteria can grow well in the saltwater of an ocean
(or 3.5 percent dissolved solids). Therefore, groundwater, even from brine aquifers,
will not pose any problems for bacterial growth.
In soil, water potential is used instead of water activity and is defined as the
difference in free energy between the system under study and a pool of pure water
at the same temperature and includes matrix and osmotic effects. The unit of mea-
surement used is the

MPa

. As with water activity, this determines the amount of
work that the cell must expend to obtain water. Generally, activity in soil is optimal
at -0.01 MPa (or 30 to 90 percent of saturation) and decreases as the soil becomes
either waterlogged near zero or desiccated at large, negative water potentials (Paul
and Clark 1989).

pH

Microorganisms have ideal pH ranges that allow growth. Within these ranges,
there is usually a defined pH optimum. Generally, the optimal pH for bacteria is
between 6.5 and 7.5 standard units, which is close to the intracellular pH. A bacteria
cell contains approximately 1000 enzymes and many are pH dependent (Paul and
Clark 1989). Most natural environments have pH values between 5 and 9. Only a
few species can grow at pH values of less than 2 or greater than 10 (Brock 1979).
In environments with pH values above or below optimal, bacteria are capable of
maintaining an internal neutral pH by preventing H

+

ions from leaving the cell or
by actively expelling H

+

as they enter. The most important factor with pH is to not
allow major shifts in pH during remediation.

Temperature

As the temperature rises, chemical and enzymatic reaction rates in the cell
increase. For every organism there is a minimum temperature below which growth
no longer occurs, an optimum temperature at which growth is most rapid, and a
maximum temperature above which growth is not possible. The optimum tempera-

©2001 CRC Press LLC

ture is always nearer the maximum temperature than the minimum. Temperature
ranges for microorganisms are wide. Some microorganisms have optimum temper-
atures as low as 5

o

to 10

o

C and others as high as 75

o

to 80

o

C. The temperature range
in which growth occurs ranges from below freezing to boiling.
No single microorganism will grow over this entire range. Bacteria are frequently
divided into three broad groups: thermophiles, which grow at temperatures above
55

o

C; mesophiles, which grow in the midrange temperature of 20

o

to 45

o

C; and
psychrophiles, which grow well at 0

o

C. In general, the growth range is approximately
30 to 40 degrees for each group. Microorganisms that grow in terrestrial and aquatic

environments grow in a range from 20

o

to 45

o

C. Figure 5 demonstrates the relative
rates of reactions at various temperatures. As can be seen in Figure 5, microorganisms
can grow in a wide range of temperatures.
In general, biological reactions will occur year round in the aquifer due to the
relatively constant temperature. Rates will be faster in warmer climates due to higher
temperatures. Biological reactions in surface soils will be affected by temperature.
Biological surface remediation will slow or not occur during the winter months in
colder climates.

Hydrogen Ion Concentration

Hydrogen is a key component of anaerobic respiration and the terminal electron-
accepting process. During the early stages of organic degradation, hydrogen is
produced by a wide variety of microorganisms as part of normal metabolism. As
the hydrogen is produced, anaerobic bacteria oxidize the hydrogen and reduce

Figure 5

Relationships of temperature to growth rate of a psychrophile, a mesophile and a
thermophile.

©2001 CRC Press LLC

terminal electron acceptors. The rapid turnover of the hydrogen pool has been termed
interspecies hydrogen transfer (Lovley and Goodwin 1988).
Nitrate- Fe(III)-, Mn(IV)-, sulfate-, and CO

2

-reducing (methanogenic) microor-
ganisms exhibit different efficiencies in using the H

2

that is continually produced.
Nitrate reducers are highly efficient H

2

utilizers and maintain low steady-state H

2

concentrations. Fe(III) reducers are slightly less efficient and thus maintain some-
what higher H

2

concentrations. Sulfate reducers and methanogenic bacteria are
progressively less efficient and maintain even higher H

2

concentrations. These ter-
minal electron accepting processes generally result in characteristic H

2

concentra-
tions in groundwater systems (USEPA 1998).

Oxygen

Oxygen is the most thermodynamically favored electron acceptor used by micro-
organisms to degrade organic compounds. Generally, an oxygen atmosphere in soil
of less than 1 percent will change the predominant respiration reaction from aerobic
to anaerobic (Paul and Clark 1989). In aqueous environments, oxygen concentration
less than approximately 0.5 to 1.0 mg/l can switch metabolism from aerobic to
anaerobic (Tabak 1981).

Nutrition

Up to this point we have discussed biochemical reactions responsible for deriving
energy, namely respiration (organic degradation). These processes are called dissim-
ilatory reactions where the chemical energy stored in C-C bonds is broken by
enzymes producing energy and metabolites used to build biomass.
Microorganism growth also requires assimilatory reactions where the organism
gathers carbon (C), nitrogen (N), phosphorous (P), sulfur (S), and micronutrients.
The remainder of this section describes the role of nutrients in the degradation
process.
Molecular composition of bacterial cells is fairly constant and indicates the
requirements for growth. Water constitutes 80 to 90 percent of cellular weight and

is always a major nutrient. The solid portion of the cell is made of carbon, oxygen,
nitrogen, hydrogen, phosphorus, sulfur, and trace elements. The approximate ele-
mentary composition is shown in Table 1.
As can be seen from Table 1, the largest component of bacteria is carbon. The
organic pollutants that we wish to destroy can provide this element. After carbon,
oxygen is the highest percentage of the cell. When oxygen requirements of new
cells are added to the required oxygen as an electron acceptor, large amounts of
oxygen may be utilized in biological degradation.
The other major nutrients required by the microorganisms are nitrogen and
phosphorous. The three main forms of nitrogen found in microorganisms are pro-
teins, microbial cell wall components, and nucleic acids. The most common sources
of inorganic nitrogen are ammonia and nitrate. Ammonia can be directly assimilated
into amino acids. When nitrate is used, it is first reduced to ammonia and is then
synthesized into organic nitrogen forms.

©2001 CRC Press LLC

Phosphorus in the form of inorganic phosphates is used by microorganisms to
synthesize phospholipids and nucleic acids. Phosphorous is also essential for the
transfer of energy during organic compound degradation.
Numerous studies have been completed to determine the ideal C/N/P ratio of
macronutrients to maintain or accelerate biodegradation. These studies evaluated
microorganism composition, laboratory treatability studies, and field studies. In
general, nutrients should be present in soil and groundwater and their approximate
ratio should be 100/10/1. This ratio corresponds to the approximate ratio of these
macronutrients in microorganisms. This ratio represents the macronutrient require-
ments for new microorganisms. When we do not need to grow new bacteria, then
the requirements for macronutrients are much lower. Most natural attenuations do
not require added nutrients. However, when large quantities of organics are present,
then the addition of macronutrients will increase the rate of bacterial growth and

the subsequent rate of organic destruction.
Micronutrients are also required for microbial growth. There are several micro-
nutrients that are universally required such as sulfur, potassium, magnesium, cal-
cium, and sodium. Sulfur is used to synthesize two amino acids, cysteine and
methionine. Inorganic sulfate is also used to synthesize sulfur containing vitamins
(thiamin, biotin, and lipoic acid) (Brock 1979). Several enzymes including those
involved in protein synthesis are activated by potassium. Magnesium is required for
activity of many enzymes, especially phosphate transfer and functions to stabilize
ribosomes, cell membranes, and nucleic acids. Calcium acts to stabilize bacterial
spores against heat and may also be involved in cell wall stability.
Additional micronutrients commonly required by microorganisms include iron,
zinc, copper, cobalt, manganese, and molybdenum. These metals function in
enzymes and coenzymes. These metals (with the exception of iron) are also consid-
ered heavy metals and can be toxic to microorganisms.
All of these factors are necessary to maintain a microorganism's metabolic
processes. Often macronutrients (N and P) are limited in soil and groundwater, and

Table 1 Molecular Composition

of a Bacterial Cell
Element
Dry Weight
(%)

Carbon 50
Oxygen 20
Nitrogen 14
Hydrogen 8
Phosphorus 3
Sulfur 1

Potassium 1
Sodium 1
Calcium 0.5
Magnesium 0.5
Chlorine 0.5
Iron 0.2
Other ~0.3

©2001 CRC Press LLC

it may be necessary to add these nutrients to enhance or accelerate biodegradation.
Micronutrients, however, are usually present in soil and groundwater and amendment
is usually not necessary. The design engineer should evaluate if nutrient amendments
are required to complete soil or groundwater remediation. If amendments are
required, the organic carbon should be the limiting factor in the biochemical reaction
such that organic degradation occurs more completely.

Toxic Environments that Affect Growth

Many factors can render an environment toxic to microorganism. Physical agents
such as high and low temperatures, high and low pH, sound and radiation, and
chemical agents such as heavy metals, halogens, organic pollutants, and oxidants
can inhibit microbial growth. In addition, oxygen, water, and nutrients can be toxic
if added in too high of concentrations.
Chemical agents such as heavy metals and halogens can disrupt cellular activity
by interfering with protein function. Mercury ions combine with SH groups in
proteins, silver ions will precipitate protein molecules, and iodine will iodinate
proteins containing tyrosine residues preventing normal cellular function. The effects
of various metals in soil has been described (Dragun 1988)

and is affected by the
concentration and pH of the soil. Oxidizing agents such as chlorine, ozone, and
hydrogen peroxide oxidize cellular components destroying cellular integrity.
It is also possible that the environmental pollutant will induce toxicity to micro-
organisms. These compounds can destroy cellular components such as cell walls,
cause mutational changes and inhibit reproduction, or inhibit assimilatory or dis-
similatory biochemical reactions. Often these toxic effects can be mitigated by
reducing the concentrations of the toxicant such that the biochemical reactions will
occur. It is important that the design engineer evaluate potentially toxic conditions,
and if necessary, incorporate steps into the remediation process designed to reduce
toxicity before relying on biochemical reactions to complete soil and groundwater
remediation.

MICROBIAL DEGRADATION AND MODIFICATION

Much research has been completed to determine degradation pathways. Infor-
mation sources such as Adriano et al. 1999 should be reviewed to obtain more
information regarding specific studies and pathways. Many petroleum hydrocarbons,
halogenated hydrocarbons, pesticides, and other anthropogenic organic compounds
can be degraded biologically. In addition, microorganisms can also chemically
modify inorganic compounds. As discussed in earlier sections, the ability of micro-
organisms to degrade or modify compounds depends on the ability to produce
requisite enzymes and ideal environmental conditions for the reactions to occur. In
addition, sufficient biomass and communication between the pollutant and the
enzymes (intracellular or extracellular) is necessary.
As described in Adriano et al. 1999 (and others), degradation of organic com-
pounds can be divided into three groups as follows (Figure 6).

©2001 CRC Press LLC
1. Biodegradation starts immediately and the compounds are readily used as sources
of energy and growth (immediate degradation).
2. Biodegradation starts slowly and requires a period of acclimation before more rapid
degradation occurs.
3. The compounds are persistent and biodegradation is slow or does not occur.

There are general rules-of-thumb regarding degradation that remediation engi-
neers should understand. Table 2 presents important physical, chemical, and struc-
tural elements that usually determine if an organic compound can be degraded.
Historically,

in situ

bioremediation has focused on treating organic pollutants.
However, biological processes are also being used to modify inorganic compounds,
particularly heavy metals, such that the compounds can be physically removed, made
less toxic, or rendered immobile, and therefore, exposure is reduced.
The form of the metal (e.g., elemental, oxide, sulfide, ionic, inorganic complex,
organic complex, coprecipitate), the availability of electron donors (C), nutrients (N
and P), the presence of electron acceptors (O

2

, NO

3
-

, Fe

3+

, Mn

4+

, SO

4
2-

, organic
compounds), and environmental factors (pH, Eh, temperature, moisture) affect the
type, rate, and extent of microbial activity, and hence, transformation of metals
(Adriano 1999).
Oxidizing and reducing environments influence the mobilization and immobili-
zation of metals. For example, in an anaerobic environment, certain metals are
reduced enzymatically from a higher oxidation state to a lower one and this affects
their solubility and bioavailability. The reduction of Fe

3+



→

Fe

2+

increases its solu-
bility, while reduction of U

6+



→

U

4+

or Cr

6+



→ Cr
3+
decreases their solubility
(Adriano et al. 1999). These concepts along with mechanisms of microbial dissolu-
tion, stabilization, and recovery are explored in Adriano et al. 1999 with an emphasis
on exploiting these processes for remediating impacted soil.
Figure 6 Degradation of organic compounds (Adriano et al. 1999).
©2001 CRC Press LLC
DEGRADATION RATE
The ultimate goal of an effective in situ remediation design and operation is

maximizing the rate at which organic compounds are degraded or inorganic com-
pounds are modified. Maximizing the degradation rate is often balanced against
economics and time requirements in order to implement the most cost effective
solution.
The kinetics associated with degradation rates have been described in the liter-
ature (Suthersan 1997, and Suarez and Rifai 1999). In nature, degradation processes
are complicated, variable, and depend on the physical, chemical, and biological
properties of the environment. It is unlikely that degradation can be represented by
one precise and consistent mathematical expression. Therefore, based on laboratory
and field experience, it is commonly accepted that degradation rates are estimated
using Monod kinetics, and zero- and first-order mathematical expressions. These
expressions are used to simplify degradation rates and provide input for modeling
the effectiveness of remediation systems.
Table 2 Physical, Chemical, and Structural Properties That Influence Degradability
of an Organic Compound
Property
Degradability
More Easily Less Easily
Solubility in Water Soluble in Water Insoluble in Water
Size Relatively Small Relatively Large
Functional Group Substitutions Fewer Functional Groups Many Functional Groups
Compound More Oxidized In Reduced Environment In Oxidized Environment
Compound More Reduced In Oxidized Environment In Reduced Environment
Created… Biologically Chemically by Man
Aliphatics Aliphatic up to 10 C-
chains
High Molecular Weight
Alkanes
Straight Chains Branched Chains
Aromatic Compounds

with One or Two Nuclei
Polyaromatic
Hydrocarbons
Substitution on Aromatic Rings -OH, -COOH, -CHO, -CO -F, -Cl, -NO
2
-OCH
3
, -CH
3
-CF
3
, -SO
3
H, -NH
2
Substitutions on Organic
Molecules
Alcohols, Aldehydes,
Acids, Esters, Amides,
Amino Acids
Alkanes, Olefins, Ethers,
Ketones, Dicarboxylic
Acids, Nitriles, Amines,
Chloroalkanes
Substitution Position p-position m- or o-position
o- or p-disubstituted
phenols
m-disubstituted phenols
Number of Hydroxy Group Increasing Decreasing
Biphenyl and Dioxins One or Less Halogens Two or More Halogens

Halogenated Alkanes Few Halogenated
Substitutions and Away
from the C
Many Halogen
Substitutions or Directly
at C
Substitution of Halogen
Derivatives
Asymmetrical Substitution Symmetrical Substitution
©2001 CRC Press LLC
Degradation rates are reported in various forms in the literature. In addition, site-
specific degradation rates can be derived from laboratory, investigation, or remediation
data. Half-life, rate constants, and percent disappearance are all used. These values
are often used to determine if compounds are amenable to biodegradation under a
defined set of conditions, and to predict the amount of time that may be required to
complete the reactions. Therefore, it is important to understand and document that
the environmental conditions from which these values were derived are representative
of site conditions so degradation rates are not over or underestimated.
NATURAL ATTENUATION
Natural attenuation is, or should be, a component of all remedial solutions where
groundwater is impacted. Few, if any, remediation technologies can achieve final
site-specific remediation objectives like natural attenuation. Therefore, it is important
to understand the basics of biochemical reactions, physical attenuation mechanisms,
the regulatory basis for the technology, how natural attenuation should be applied,
advantages and disadvantages, and the evaluation process.
The United States Environmental Protection Agency (USEPA) (USEPA 1998),
United States Air Force, United States Navy, American Society for Testing Materials,
and many state and local regulatory agencies have developed protocols for evaluating
natural attenuation as a groundwater remedial solution. In addition, the Interstate
Technology and Regulatory Cooperation Work Group, In Situ Bioremediation Work

Team is a state led, national coalition of personnel from the regulatory and technol-
ogy programs devoted to deploying and improving innovative environmental tech-
nologies. They have also produced technical requirements for evaluating natural
attenuation (ITRC 1999). For the most part, all of these protocols are similar. In
fact, many of the same experts in the field have contributed directly (or indirectly)
to protocol development.
Natural attenuation includes several processes including: biodegradation, disper-
sion, sorption, and volatilization. When these processes are shown to be capable of
attaining site-specific remediation objectives in a time period that is reasonable
compared to other alternatives, they may be selected alone or in combination with
other more active remediations as the preferred remedial alternative (USEPA 1998).
Monitored natural attenuation (MNA) is a term that refers specifically to the use
of natural attenuation processes as part of overall site remediation. The USEPA
(USEPA 1998) defines MNA as follows:
The term “monitored natural attenuation,” …, refers to the reliance on natural atten-
uation processes (within the context of a carefully controlled and monitored cleanup
approach) to achieve site-specific remedial objectives within a time frame that is
reasonable compared to other methods. The “natural attenuation processes” that are
at work in such a remediation approach include a variety of physical, chemical, or
biological processes that, under favorable conditions, act without human intervention
to reduce the mass, toxicity, mobility, volume, or concentration of contaminants in
soil and ground water. These in situ processes include, biodegradation, dispersion,
©2001 CRC Press LLC
dilution, sorption, volatilization, and chemical or biological stabilization, transforma-
tion, or destruction of contaminants.
Monitored natural attenuation is appropriate as a remedial approach only when it can
be demonstrated capable of achieving a site’s remedial objectives within a time frame
that is reasonable compared to that offered by other methods and where it meets the
applicable remedy selection program…. EPA, therefore, expects that monitored nat-
ural attenuation typically will be used in conjunction with active remediation mea-

sures (e.g., source control), or as a follow-up to active remediation measures that
have already been implemented.
The intent of this section is to provide an overview of natural attenuation as it
pertains to in situ bioremediation. Dispersion, sorption, and volatilization are impor-
tant elements of natural attenuation; however, biodegradation (in most cases) is likely
the most effective process and is the focus of this text.
This text is not intended to replace or reiterate all elements contained within the
various protocols. Interested readers should at least obtain and review the USEPA
protocol as a supplement to this text. A word of caution must be stated about the
protocols, however. It is the authors’ and others’ (Norris 1999) opinions that the
protocols should be used as a guideline, and that practitioners must have a complete
understanding of the physical, chemical, and biological processes that control natural
attenuation. If the processes are not understood, aspects of the protocols can be
easily misapplied. For example, some protocols recommend that site conditions be
scored. Inaccurate scores can lead to an erroneous decision regarding natural atten-
uation even when primary lines of evidence, such as the presence of degradation
products, mass reduction, and plume stabilization data, indicate that natural attenu-
ation is occurring. In addition, monitoring well placement, construction, and data
collection procedures can introduce errors that if not considered, can lead to another
erroneous score. Therefore, knowledge and common sense must be used to provide
sound and defensible opinions regarding the technology.
Application of Natural Attenuation
Currently, natural attenuation is most routinely applied at sites where ground-
water is impacted by petroleum hydrocarbon fuels and chlorinated hydrocarbons.
These compounds are most frequently detected at impacted sites and their atten-
uation processes are best understood. Natural attenuation of other organic com-
pounds and heavy metals can also occur, and the same protocols can be used to
evaluate these processes. In addition, applying natural attenuation to soil is emerg-
ing and it is believed that MNA will become an important remedial solution for
impacted soil.

Advantages and Disadvantages of Natural Attenuation
Natural attenuation has several advantages and disadvantages as compared to
other remediation technologies. The advantages include the following:
©2001 CRC Press LLC
• Less remediation waste is generated and cross-media transfer of contamination and
human exposure is less as compared to typical ex situ technologies
• Less intrusion and few surface structures are required
• Can be applied to all or part of a site depending on-site conditions and cleanup
objectives
• Can be used in conjunction with, or as a follow up to other remedial technologies
• Can lower overall remediation costs as compared to costs associated with active
remediation
There are also potential disadvantages of natural attenuation as follows:
• Remediation time may be longer than time frames achieved by a more active
remedial solution
• Characterization costs may be more complex and costly
• Degradation of parent compounds may result in the production of more toxic
degradation products
• Long-term monitoring is often required
• Institutional controls may be required to ensure risk protection
• Contamination migration and/or cross-media transfer of contaminants can poten-
tially occur if the site’s hydrology and geochemistry changes
• There may be a negative public perception regarding the technology and public
outreach and education may be required before the technology is accepted
While all of these advantages and disadvantages have to be considered, in the
end, the designer may not have a choice but to use natural attenuation. As we
discussed in Chapter 1, there are geological limitations to all active remediations.
However, natural attenuation occurs throughout the aquifer. The bacteria are located
everywhere (variable concentrations depending on the environment), and do not
suffer from geological limitation. Once the active processes have accomplished all

that they can, natural attenuation may be the only method to eliminate the last of
the contaminants. During the diffusion controlled (Chapter 2) portion of the project,
natural attenuation, and the enhancements discussed in Chapters 8 and 9, are the
only remediation methods that can be successfully applied.
Lines of Evidence
Lines of evidence are used to evaluate natural attenuation. Lines of evidence are
used because multiple processes can be effectively treating constituents, and it is
difficult to prove that any one process is responsible for all treatment. Therefore,
three primary lines of evidence are used to evaluate if natural attenuation is effec-
tively treating groundwater impacts as follows (USEPA 1997 and 1998):
1. Historical groundwater and/or soil chemistry data that demonstrate a clear and
meaningful trend of decreasing contaminant mass and/or concentration over time at
appropriate monitoring or sampling points. In the case of a groundwater plume,
decreasing concentrations should not be solely the result of plume migration. In the
case of inorganic contaminants, the primary attenuating mechanism should also be
understood.
©2001 CRC Press LLC
This line of evidence is important and should be the first evaluation step. Ground-
water concentrations must be stable or decreasing, and/or the dissolved contaminant
plume no longer advancing. The processes controlling the plume may include vol-
atilization, dilution, dispersion, advection, or biodegradation. Sufficient monitoring
data over a period of time necessary to document anthropogenic or seasonal events
must be collected. The intent of evaluating the first line of evidence is to demonstrate
that the plume is stable, not to document the physical, chemical, or biological
processes affecting plume stability.
In addition to understanding the fate and transport processes associated with the
impacted plume, it is important to also use these data to evaluate potential human
or ecological exposure pathways that may exist for current or future receptors. This
is important because a natural attenuation remedial solution may not be the most
expedient option, and if current or future human or ecological receptors are being

exposed, a more active remedial solution may be required before natural attenuation
is used to complete the process.
2. Hydrogeologic and geochemical data that can be used to demonstrate indirectly
the type(s) of natural attenuation processes active at the site, and the rate at which
such processes will reduce contaminant concentrations to required levels. For exam-
ple, characterization data may be used to quantify the rates of contaminant sorption,
dilution, or volatilization, or to demonstrate and quantify the rates of biological
degradation processes occurring at the site.
The second line of evidence builds upon the first. Once it has been determined
that the dissolved contaminant plume is stable, no longer migrating, concentrations
are decreasing, or the contaminant mass is decreasing, then the mechanism by which
the attenuation is occurring must be determined. Therefore, it is necessary to evaluate
the likely mechanisms by which the contaminants are being destroyed. Biological
degradation is likely the most predominant and important attenuation process; how-
ever, abiotic mechanisms must also be considered.
In order to evaluate if the impacts are being degraded, the second line of evidence
is usually divided into two parts. The first includes completing mass balance calcu-
lations which include determining the likely environmental conditions and respira-
tory pathways occurring, and correlating concentrations of electron donors and
acceptors to determine if it is likely that the processes will occur to completion.
Computer modeling can be used as a tool, and many of these relatively new models
are briefly described in the Modeling Tools section of this chapter. The second portion
includes estimating the biodegradation rate constants that are important to predict
when remediation will be complete. There are several methods to determine the
biodegradation rate constant including comparing site conditions to published liter-
ature conditions and values, tracer studies, or using actual site data collected across
a defined flow path.
3. Data from field or microcosm studies (conducted in or with actual contaminated
site media) which directly demonstrate the occurrence of a particular natural atten-
uation process at the site and its ability to degrade the contaminants of concern

(typically used to demonstrate biological degradation processes only).