United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada OK 74820
Research and Development
EPA/SR-93/124 September 1993
&EPA Project Summary
In-Situ Bioremediation of Ground
Water and Geological Material:
A Review of Technologies
Robert D. Morris, Robert E. Hinchee, Richard Brown, Perry L. McCarty,
Lewis Semprini, John T. Wilson, Don H. Kampbell, Martin Reinhard,
Edward J. Bouwer, Robert C. Borden, Timothy M. Vogel, J. Michele Thomas,
and C. H. Ward
Bioremediation off excavated soil, un-
saturated soil, or ground water involves
the use of microorganisms to convert
contaminants to less harmful species
in order to remediate contaminated
sites. Bioremediation sometimes offers
significant advantages over other types
of remediation technologies. This re-
port provides the most recent scien-
tific understanding of the processes
involved with soil and ground-water
bioremediation and discusses the ap-
plications and limitations of the vari-
ous in-situ bioremediation technologies.
In order for the information in this docu-
ment to be of maximum benefit, it is
important that the reader understand
how contaminants are distributed
among the various subsurface compart-
ments. This distribution, or phase par-
titioning of contaminants, is dependent
upon a number of factors including the
characterization of the contaminants
themselves and that of the subsurface
environment. This distribution is exem-
plified in Figure 1 where contaminants
are shown to be associated with the
vapor phase in the unsaturated zone, a
residual phase, or dissolved in ground
water.
This Project Summary was developed
by EPA's Robert S. Kerr Environmental
Research Laboratory, Ada, OK, to an-
nounce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
the back).
Introduction
In-situ bioremediation of subsurface en-
vironments involves the use of microor-
ganisms to convert contaminants to less
harmful products and sometimes offers
significant potential advantages over other
remediation technologies. In order for
these biodegradative processes to
occur, microorganisms require the pres-
ence of certain minerals, referred to as
nutrients, and an electron acceptor. Sev-
eral other conditions, i.e., temperature
and pH, impact the effectiveness of these
processes. The use of biooxidation for
environmental purposes has existed for
many years and has led to considerable
information regarding the biodegradabil-
ity of specific classes of compounds, nu-
trient and electron acceptor requirements,
and degradation mechanisms. Activated
sludge and other suspended growth sys-
tems have been used for decades to
treat industrial and municipal wastes.
Land treatment processes for municipal
wastewater and petroleum refinery and
municipal wastewater sludges have also
been practiced for several decades and
have generated a great deal of informa-
tion on nutrient requirements, degrada-
tion rates, and other critical parameters
affecting biological oxidation.
Printed on Recycled Paper
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Water
Table
Capillary
Frinoe
Dissolved
Contaminants
Flgun 1. Distribution of contaminants in the subsurface.
In the 1970s, tests were conducted to
evaluate biological degradation of petro-
leum hydrocarbons in aquifers. Results
from these tests demonstrated that in-situ
bioremediation could reduce levels of hy-
drocarbons in aquifers and provided con-
siderable information concerning the
processes which take place and the re-
quirements necessary to drive these pro-
cesses.
Although a variety of minerals are re-
quired by the microorganisms, it is usu-
ally necessary to add only nitrogen and
phosphorus. The most common electron
acceptor used in bioremediation is oxy-
gen. Stoichiometrically, approximately
three pounds of oxygen are required to
convert one pound of hydrocarbon to car-
ton dioxide. Nutrient requirements are less
easily predicted. If all hydrocarbons are
converted to cell material, however, it can
be assumed that nutrient requirements of
carbon to nitrogen to phosphorus ratios
are in the order of 100:10:1. In some
cases where the levels of contaminants
are low, sufficient nitrogen and phospho-
rus are naturally present, and only oxy-
gen is required for the biological processes
to proceed.
In-situ bioremediation systems for aqui-
fers typically consist of extraction points
such as wells or trenches, and injection
wells or infiltration galleries. In most cases,
the extracted ground water is treated prior
to the addition of oxygen and nutrients,
followed by subsequent reinjection.
Critical to the design of an in-situ biore-
mediation system is the ground-water flow
rate and flow path. The ground-water flow
must be sufficient to deliver the required
nutrients and oxygen according to the de-
mand of the organisms, and the amended
ground water should sweep the entire area
requiring treatment. This is a critical point
in that it is often the hydraulic conductivity
of the ground-water system itself or the
variability of the aquifer materials that lim-
its the effectiveness of in-situ technolo-
gies or prevents its utility entirely. The
results of a number of referenced stud-
ies suggest that in-situ bioremediation
of the subsurface is usually limited to
formations with hydraulic conductivities
of 10-4 cm/sec (100 ft/yr) or greater to
overcome the difficulty of pumping fluids
through contaminated formations.
In-situ bioremediation systems are of-
ten integrated with other remediation
technologies either sequentially or simul-
taneously. For example, if free-phase hy-
drocarbons are present, a recovery system
should be used to reduce the mass of
free-phase product prior to the implemen-
tation of bioremediation. In-situ vapor strip-
ping can be used to both physically
remove volatile hydrocarbons and to pro-
vide oxygen for bioremediation. These
systems can also reduce levels of re-
sidual phase hydrocarbons as well as con-
stituents adsorbed to both unsaturated
soils and soils which become unsaturated
during periods when the water table is
lowered.
As a class, petroleum hydrocarbons are
biodegradable. The lighter soluble mem-
bers are generally biodegraded more rap-
idly and to lower residual levels than are
the heavier, less soluble members. Thus
monoaromatic compounds such as ben-
zene, toluene, ethylbenzene, and the xy-
lenes are more rapidly degraded than
the two-ring compounds such as naph-
thalene, which are in turn more easily
degraded than the three-, four-, and five-
ring compounds.
Polyaromatic hydrocarbons are present
in heavier petroleum hydrocarbon blends
and particularly in coal tars, wood treat-
ing chemicals, and refinery waste slud-
ges. These compounds have only limited
solubility in water, adsorb strongly to soils,
and degrade at rates much slower than
monoaromatic hydrocarbons.
Nonchlorinated solvents used in a vari-
ety of industries are generally biodegrad-
able. For example, alcohols, ketones,
ethers, carboxylic acids, and esters are
readily biodegradable but may be toxic to
the indigenous microflora at high concen-
trations due to their high water solubility.
Lightly chlorinated compounds such as
chlorobenzene, dichlorobenzene, chlorin-
ated phenols, and the lightly chlorinated
PCBs are typically biodegradable under
aerobic conditions. The more highly chlo-
rinated analogs are more recalcitrant to
aerobic degradation but more suscep-
tible to degradation under anaerobic con-
ditions.
Chlorinated solvents and their natural
transformation products represent the
most prevalent organic ground-water con-
taminants. These solvents, consisting
primarily of chlorinated aliphatic
hydrocarbons, have been widely used
for degreasing aircraft engines, au-
tomotive parts, electronic compo-
nents, and clothing.
In-situ biodegradation of most of these
solvents depends upon cometabolism and
can be carried out under aerobic or
anaerobic conditions. Cometabolism re-
quires the addition of an appropriate pri-
mary substrate to the aquifer and perhaps
an electron acceptor, such as oxygen or
nitrate, for its oxidation.
In the early 1980s there were few com-
panies that had experience in the biore-
mediation of soil and ground water. Since
that time, many companies have used
bioremediation technologies, although
claims of experience are frequently over-
stated. There now exists a number of
organizations and specialists that are
knowledgeable in the field of in-situ bio-
remediation. Several environmental com-
panies have staffs that are experienced
in the application of this technology. Many
large corporations, especially the oil and
chemical companies, have also devel-
oped in-house expertise. Some of the
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U.S. Environmental Protection Agency
laboratories, as well as Department of
Defense and Department of Energy
groups, have conducted laboratory re-
search and field demonstration studies
concerning bioremediation.
Bioventing
Bioventing is the process of supplying
air or oxygen to soil to stimulate the aero-
bic biodegradation of contaminants. This
technology is applicable to contaminants
in the vadose zone and contaminated re-
gions of an aquifer just below the water
table. This in-situ process may be applied
to the vadose zone as well as an ex-
tended unsaturated zone caused by de-
watering. Bioventing is a modification of
the technology referred to as soil vacuum
extraction, vacuum extraction, soil gas ex-
traction, and in-situ volatilization.
Laboratory research and field demon-
strations involving bioventing began in the
early 1980s, with particular emphasis on
the remediation of soil contaminated with
hydrocarbons. Early on, researchers con-
cluded that venting would not only re-
move gasoline by physical means but
would also enhance microbial activity and
promote the biodegradation of gasoline.
Much of the success of this technology is
because the use of air as a carrier of
oxygen is 1,000 times more efficient than
water. It is estimated that various forms of
bioventing have been applied to more than
1,000 sites worldwide; however, little ef-
fort has been given to the optimization of
these systems.
Bioventing is potentially applicable to
any contaminant that is more readily bio-
degradable aerobically than anaerobically.
Although most applications have been to
petroleum hydrocarbons, applications to
PAH, acetone, toluene, and naphthalene
mixtures have been reported. In most ap-
plications, the key is biodegradability ver-
sus volatility. If the rate of volatilization
significantly exceeds the rate of biodegra-
dation, removal essentially becomes a
volatilization process.
In general, low-vapor pressure com-
pounds (less than 1 mm Hg) cannot be
successfully removed by volatilization and
can only be biodegraded in a bioventing
application. Higher vapor pressure com-
pounds (above 760 mm Hg) are gases at
ambient temperatures and therefore vola-
tilize too rapidly to be biodegraded in a
bioventing system. Within this intermedi-
ate range (1 - 760 mm Hg) lie many of the
petroleum hydrocarbon compounds of
regulatory interest, such as benzene, tolu-
ene, and the xylenes, that can be treated
by bioventing.
In addition to the normal site character-
ization required for the implementation of
this or any other remediation technology,
additional investigations are necessary.
Soil gas surveys are required to deter-
mine the amount of contaminants, oxy-
gen, and carbon dioxide in the vapor
phase; the latter are needed to evaluate
in-situ respiration under site conditions.
An estimate of the soil gas permeability
along with the radius of influence of vent-
ing wells is also necessary to design full-
scale systems, including well spacing
requirements, and to size blower equip-
ment.
Although bioventing has been performed
and monitored at several field sites, many
of the effects of environmental variables
on bioventing treatment rates are still not
well understood. In-situ respirometry at
additional sites with drastically different
geologic conditions has further defined en-
vironmental limitations and site-specific
factors that are pertinent to successful
bioventing. However, the relationship be-
tween respirometric data and actual
bioventing treatment rates has not been
clearly determined. Concomitant field
respirometry and closely monitored field
bioventing studies are needed to deter-
mine the type of contaminants that can
successfully be treated by in-situ
bioventing and to better define the envi-
ronmental limitations to this technology.
Air Sparging
Air sparging is the injection of air under
pressure below the water table to create
a transient air-filled porosity by displacing
water in the soil matrix. Air sparging is a
remediation technology applicable to con-
taminated aquifer solids and vadose zone
materials. This is a relatively new treat-
ment technology which enhances biodeg-
radation by increasing oxygen transfer to
the ground water while promoting the
physical removal of organics by direct vola-
tilization. Air sparging has been used ex-
tensively in Germany since 1985 but was
not introduced to the United States until
recently.
When air sparging is applied, the result
is a complex partitioning of contaminants
between the adsorbed, dissolved, and va-
por states. Also, a complex series of re-
moval mechanisms are introduced,
including the removal of volatiles from the
unsaturated zone, biodegradation, and the
partitioning and removal of volatiles from
the fluid phase. The mechanisms respon-
sible for removal are dependent upon the
volatility of the contaminants. With a highly
volatile contaminant, for example, the pri-
mary partitioning is into the vapor phase,
and the primary removal mechanism is
through volatilization. By contrast, contami-
nants of low volatility partition into the
adsorbed or dissolved phase, and the pri-
mary removal mechanism is through bio-
degradation.
One of the problems in applying air
sparging is controlling the process. In ei-
ther bioventing or ground-water extrac
tion, the systems are under control
because contaminants are drawn to the
point of collection. By contrast, air sparging
systems cause water and contaminants to
move away from the point of injection
which can accelerate and aggravate the
spread of contamination. Changes in li
thology can profoundly affect both the di
rection and velocity of air flow. A second
problem in air sparging is accelerated va
por travel. Since air sparging increases
the vapor pressure in the vadose zone
any exhausted vapors could be drawn into
receptors such as basements. As a result
in areas with potential vapor receptors, air
sparging should be done with vent sys
terns which allow an effective means o-
capturing sparged gases.
As with any technology, there are limi
tations to the utility and applicability of air
sparging. The first is associated with the
type of contaminants to be removed. Fo<-
air sparging to work effectively, the con-
taminant must be relatively volatile and
relatively insoluble. If the contaminant is
soluble and nonvolatile, it must be biode-
gradable. The second limitation to the USB
of air sparging is the geological character
of the site. The most important geological
characteristic is the homogeneity of the
site. If significant stratification is present,
there is a danger that sparged air could
be held below an impervious layer and
spread laterally, thereby resulting in the
spread of contamination.
Another constraint of concern is depth
related. There is both a minimum and
maximum depth for a sparge system. A
minimum depth of 4 feet, for example,
may be required for a sufficient thickness
to confine the air and force it to "cone out"
from the injection point. A maximum depth
of 30 feet might be required from the
standpoint of control. Depths greater thai
30 feet make it difficult to predict where
the sparged air will travel.
Alternate Electron Acceptors
Bioremediation using electron acceptors
other than oxygen is potentially advanta-
geous for overcoming the difficulty in sup-
plying oxygen for aerobic processes.
Nitrate, sulfate, and carbon dioxide are
attractive alternatives to oxygen because
they are more soluble in water, inexpen-
sive, and nontoxic to microorganisms. The
demonstration of this technology in the
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field is limited; therefore, its use as an
alternate electron acceptor for bioremedi-
ation must be viewed as a developing
treatment technology.
Some compounds are only transformed
under aerobic conditions, while others re-
quire strongly reducing conditions, and
still others are transformed in both aero-
bic and anaerobic environments. In the
absence of molecular oxygen, microbial
reduction reactions involving organic con-
taminants increase in significance as en-
vironmental conditions become more
reducing. In this environment, some con-
taminants are reduced by a biological pro-
cess known as reductive dehalogenation.
In reductive dehalogenation reactions, the
halogenated compound becomes the elec-
tron acceptor. In this process, a halogen
is removed and is replaced with a hydro-
gen atom.
Bioremediation with alternate electron
acceptors involves the stimulation of mi-
crobial growth by the perfusion of elec-
tron donors, electron acceptors, and
nutrients through the formation. Addition
of alternate electron acceptors other than
nitrate for bioremediation has not been
documented at field scale but has been
widely studied at laboratory scale. Nitrate
as an electron acceptor has been used
for bioremediation of benzene, toluene,
ethylbenzene, and xylenes in ground wa-
ter and on aquifer solids. As for other in-
srtu remediation technologies, formations
with hydraulic conductivities of 10/4 cm/
sec (100 ft/sec) or greater are most ame-
nable to bioremediation.
The combination of an anaerobic pro-
cess followed by an aerobic process has
promise for the bioremediation of highly
chlorinated organic contaminants. Gener-
ally, anaerobic microorganisms reduce the
number of chlorines on a chlorinated com-
pound via reductive dechlorination, and
susceptibility to reduction increases with
the number of chlorine substitutes. Con-
versely, aerobic microorganisms are more
capable of transforming compounds with
fewer chlorinated substitutes. With the re-
moval of chlorines, oxidation becomes
more favorable than does reductive
dechlorination. Therefore, the combina-
tion of anaerobic and aerobic processes
has a potential utility as a control technol-
ogy for chlorinated solvent contamination.
Natural Bioremediation
The basic concept behind natural bio-
remediation is to allow naturally occurring
microorganisms to degrade contaminants
that have been released into the subsur-
face. It is not a "no action" alternative, as
in most cases it is used to supplement
other remediation techniques. In some
cases, only the removal of the primary
source may be necessary. In others, con-
ventional ground-water remediation tech-
niques such as pump-and-treat may be
used to reduce contaminant concentra-
tions within the aquifer.
Natural bioremediation is capable of
treating contaminants aerobically in the
vadose zone and at the margins of plumes,
where oxygen is not limiting. Some sites
have shown that anaerobic bioremedia-
tion processes also occur naturally and
can significantly reduce contaminant con-
centration on aquifer solids and in ground
water. Benzene, toluene, ethylbenzene,
and xylene can be removed anaerobi-
cally in methanogenic or sulfate-reduc-
ing environments; highly chlorinated
solvents can undergo reductive dechlori-
nation in anaerobic environments.
While there are no "typical" sites, it may
be helpful to consider a hypothetical site
where a small release of gasoline has
occurred from an underground storage
tank (Figure 2). Rainfall infiltrating through
the hydrocarbon-contaminated soil will
leach some of the more soluble compo-
nents including benzene, toluene, and xy-
lenes. As the contaminated water migrates
downward through the unsaturated zone,
a portion of the dissolved hydrocarbons
may biodegrade. The extent of the bio-
degradation will be controlled by the size
of the spill, the rate of downward move-
ment, and the appropriateness of requi-
site environmental conditions. Dissolved
hydrocarbons that are not completely de-
graded in the unsaturated zone will enter
the saturated zone and be transported
downgradient within the water table where
they will be degraded by native microor-
ganisms to an extent limited by available
oxygen and other subsurface conditions.
The contaminants that are not degraded
will move downgradient under anaerobic
conditions. As the plume migrates, dis-
persion will mix the anaerobic water with
oxygenated water at the plume fringes.
This is the region where most natural
aerobic degradation occurs.
One of the major factors controlling the
use of natural bioremediation is the ac-
ceptance of this approach by regulators,
environmental groups, and the public. The
implementation of these systems differs
from conventional techniques in that a
portion of the aquifer is allowed to remain
contaminated. This results in the neces-
sity of obtaining variances from regula-
tions, and some type of risk evaluation is
usually required. Even when public health
is not at risk, adjoining land owners may
have strong concerns about a contami-
nant plume migrating under and poten-
tially impacting their property. Therefore,
control of plume migration at these sites,
usually using some type of hydraulic sys-
tem, is often necessary. Although natural
bioremediation imposes few costs other
than monitoring and the time for natural
processes to proceed, the public may per-
ceive that this is a "no action" alternative.
These various factors may generate op-
position to selecting natural bioremedia-
tion rather than conventional technologies.
Aerobic— Uncontaminated Ground Water
Flgun 2. Profile of a hypothetical hydrocarbon plume undergoing passive bioremediation.
4
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There is almost no operating history to
judge the effectiveness of natural biore-
mediation. In addition, there are currently
no reliable methods for predicting its ef-
fectiveness without first conducting exten-
sive field testing. This is often the primary
reason why natural bioremediation is not
seriously considered when evaluating re-
medial alternatives. At many low priority
sites, regulators may have assumed that
natural bioremediation would control the
migration of dissolved contaminants. Of-
ten, these sites have not been adequately
characterized nor have they been moni-
tored to determine the effectiveness of
this remediation technology. At present,
there are no well-documented, full-scale
investigations of natural bioremediation.
Such investigations will be necessary to
define site and contaminant characteris-
tics conducive to this remediation alterna-
tive before it can be accepted with
confidence by practitioners, regulators, and
the public.
Introduced Organisms
Historically, the movement of microor-
ganisms in the subsurface was first dis-
cussed in the mid-1920s in relation to the
enhanced recovery of oil by the produc-
tion of biological surfactants and gases.
Later, the transport of bacteria through
soil was studied to measure the effective-
ness of soil-based sewage treatment fa-
cilities such as pit latrines and septic tanks
in terms of the removal of pathogens. In
recent years, research has been directed
toward the introduction of microorganisms
to soil and ground water to introduce spe-
cialized metabolic capabilities, to degrade
contaminants which resist the degradative
processes of indigenous microflora, or
when the subsurface has been sterilized
by contaminants. In these attempts to in-
troduce microorganisms to the subsur-
face, it is often difficult to differentiate
their activities from indigenous popula-
tions. The use of introduced microorgan-
isms has proven most successful in
surface bioreactors when treating ex-
tracted ground water in closed-loop recir-
culation systems.
For added organisms to be effective in
contaminant degradation, they must be
transported to the zone of contamination,
attach to the subsurface matrix, survive,
grow, and retain their degradative capa-
bilities. There are a number of phenom-
ena which affect the transport of microbes
in the subsurface including grain size,
cracks and fissures, removal by sorption
in sediments high in clay and organic
matter, and the hydraulic conductivity.
Many other factors affect the movement
of microorganisms in the subsurface in-
cluding their size and shape, concentra-
tion, flow rate, and survivability.
The use of microorganisms with spe-
cialized capabilities to enhance biore-
mediation in the subsurface is an
undemonstrated technique. However,
research has been conducted to de-
termine the potential for microbial
transport through subsurface materi-
als, public health effects, and micro-
bial enhanced oil recovery.
Summary
This report has been prepared by lead-
ing soil and ground-water remediation sci-
entists in order to present the latest
technical, institutional, and cost consider-
ations applicable to subsurface remedia-
tion systems. It is aimed at scientists,
consultants, regulatory officials, and oth-
ers who are, in various ways, working to
achieve efficient and cost-effective reme-
diation of contaminants in the subsurface
environment.
The document contains detailed infor-
mation about the processes, applications,
and limitations of using remediation tech-
nologies to restore contaminated soil and
ground water. Field tested as well as new
and innovative technologies are discussed.
In addition, site characterizations require-
ments for each remediation technology are
discussed along with the costs associated
with their implementation. A number of
case histories are presented, and knowl-
edge gaps are pointed out in order to
suggest areas for which additional research
investigations are needed.
&U.S. GOVERNMENT PRINTING OFFICE: I*M • 5SO-M7/M227
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Robert D. Norris is with Eckenl'elder, Inc., Nashville, Tennessee 37228. Robert £
Hinchee is with Battelle Memorial Institute, Columbus, Ohio 43201-2693. Richard
Brown is with Groundwater Technology, Inc., Trenton, New Jersey 08691. Perry L
McCarty and Lewis Sempriniare with Stanford University, Stanford, California 94305-
4020. The EPA authors, John T. Wilson and Don H. Kampbell, are with the U.S.
Environmental Protection Agency, Robert S. Kerr Environmental Research Labora-
tory, Ada, Oklahoma 74820. Martin Reinhard is with Stanford University, Stanford,
California 94305-4020. Edward J. Bouwer is with the Johns Hopkins University,
Baltimore, Maryland21218. Robert C. Borden is with North Carolina State University,
Raleigh, North Carolina 27695-7908. Timothy M. Vogel is with the University of
Michigan, Ann Arbor, Ml 48109-2125. J. Michele Thomas and C. H. Ward are with
Rice University, Houston, Texas 77251.
John E. Matthews is the EPA Project Officer (see below).
The complete report, entitled "In-Situ Bioremediation of Ground Water and Geological
Material: A Review of Technologies, "(OrderNo. PB93-215564; Cost $36.50) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
Ada, OK 74820
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
Official Business
Penalty for Private Use
$300
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EPA
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