&EPA
                              United States
                             : Environmental Protection
                         Office of Emergency and
                         Remedial Response
                         Washington, DC 20460
Qffica of
Research and Development
Cincinnati, OH 45368
                                                       EPA/540/S-94/502
                                                  April 1994
Engineering Bulletin
In  Situ   Biodegradation
Purpose

    Section 121(b) of the Comprehensive Environment
Response, Compensation, and Liability Act (CERCLA) man-
dates the Environmental Protection Agency (EPA) to select
remedies that "utilize permanent solutions and alternative
technologies or resource recovery technologies to the maxi-
mum extent practicable" and to prefer remedial actions in
which treatment "permanently and significantly reduces
the volume, toxicity, or mobility of hazardous substances,
pollutants and contaminants as a principal element." The
Engineering Bulletins comprise a series of documents that
summarize the latest information available on  selected
treatment and site remediation technologies and related
issues.  They provide summaries of and references for the
latest information to help remedial project managers, on-
scene coordinators, contractors, and  other site cleanup
managers understand the type of data and site characteris-
tics needed to evaluate a technology for potential applica-
bility to their Superfund or other hazardous waste site.
Those documents that describe individual treatment tech-
nologies focus on remedial investigation scoping needs.
Addenda will be issued periodically to update the original
bulletins.
Abstract

    In situ biodegradation may be used to treat low-to-
intermediate concentrations  of organic contaminants in-
place without disturbing or displacing the contaminated
media. Although this technology has been used to degrade
a limited  number of inorganics, specifically cyanide and
nitrate, in situ biodegradation is not generally employed to
degrade inorganics or to treat media contaminated with
heavy metals.

    During in situ biodegradation, electron acceptors (e.g.,
oxygen and nitrate), nutrients, and other amendments may
be introduced into the soil and groundwater to encourage
the growth of an indigenous population capable of degrad-
ing the contaminants of concern. These supplements are
used to  control  or  modify  site-specific conditions that
                         impede microbial activity and, thus, the rate and extent of
                         contaminant  degradation.   Depending on site-specific
                         cleanup goals, in situ biodegradation can be used as the
                         sole treatment technology or in conjunction with other
                         biological, chemical, and physical technologies in a treat-
                         ment train. In the past, in situ biodegradation has often
                         been used to enhance traditional pump and treat technolo-
                         gies by reducing the time needed to achieve aquifer cleanup
                         standards.

                            One of the advantages of employing an in situ technol-
                         ogy is that media transport and excavation requirements
                         are minimized, resulting in  both reduced potential for
                         volatile releases and minimized material handling  costs.
                         Biological technologies that require the physical displace-
                         ment of media during treatment (e.g., "land treatment"
                         applications involving excavation for treatment in lined
                         beds or tilling of non-excavated soils) assume many of the
                         risks and costs associated with ex situ technologies and
                         cannot strictly be considered in situ applications.

                            As of Fall 1993, in situ  biodegradation was  being
                         considered or  implemented as a component of the remedy
                         at 21 Superfund sites and 38 Resource Conservation and
                         Recovery Act  (RCRA), Underground  Storage Tank (UST),
                         Toxic Substances Control Act (TSCA), and Federal sites with
                         soil, sludge,  sediment,  or  groundwater contamination
                         [1, p. 13]'[2][3].  This bulletin provides information on
                         the technology's applicability, the types of residuals pro-
                         duced, the latest performance data, the site requirements,
                         the  status of the technology, and sources for further
                         information.
                        Technology Applicability

                            In situ biodegradation has been shown to be poten-
                        tially effective at degrading or transforming a large number
                        of organic compounds to environmentally-acceptable or
                        less mobile compounds [4, p. 54][5, p. 103][6][7][8][9].
                        Soluble organic contaminants are particularly amenable to
                        biodegradation; however, relatively insoluble contaminants
                        may be degraded if they are accessible to microbial degrad-
* [reference number, page number]
                                                                                    Printed on Recycled Paper

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v>EPA
                             United States
                             Environmental Protection
                             Agency
                         Office of Emergency and
                         Remedial Response
                         Washington, DC 20460
Office of
Research and Development
Cincinnati, OH 45268
                                                      EPA/540/S«94/50a
                                                 ApriM994
Engineering Bulletin
Ih  Situ   Biodegradation
Treatment
Purpose

    Section  121(b) of the Comprehensive Environment
Response, Compensation, and Liability Act (CERCLA) man-
dates the Environmental Protection Agency (EPA) to select
remedies that "utilize permanent solutions and alternative
technologies or resource recovery technologies to the maxi-
mum extent practicable" and to prefer remedial actions in
which treatment "permanently and significantly reduces
the volume,  toxicity, or mobility of hazardous substances,
pollutants and contaminants as a principal element." The
Engineering  Bulletins comprise a series of documents that
summarize the latest information available on selected
treatment and site remediation technologies and related
issues. They provide summaries of and references for the
latest information to help remedial project managers, on-
scene  coordinators, contractors, and other site cleanup
managers understand the type of data and site characteris-
tics needed to evaluate a technology for potential applica-
bility to  their  Superfund or other hazardous waste site.
Those documents that describe individual treatment tech-
nologies focus on remedial investigation scoping needs.
Addenda will be issued periodically to update the original
bulletins.
Abstract

    In situ biodegradation may be used to treat low-to-
intermediate concentrations of organic  contaminants in-
place without disturbing or displacing the contaminated
media. Although this technology has been used to degrade
a limited number of inorganics,  specifically cyanide and
nitrate, in situ biodegradation is not generally employed to
degrade inorganics or to treat media contaminated with
heavy metals.

    During in situ biodegradation, electron acceptors (e.g.,
oxygen and nitrate), nutrients, and other amendments may
be introduced into the soil and groundwater to encourage
the growth of an indigenous population capable of degrad-
ing the contaminants of concern. These supplements are
used to  control or modify site-specific conditions that
                         impede microbial activity and, thus, the rate and extent of
                         contaminant degradation.  Depending on site-specific
                         cleanup goals, in situ biodegradation can be used as the
                         sole treatment technology or in conjunction with other
                         biological, chemical, and physical technologies in a treat-
                         ment train. In the past, in situ biodegradation has often
                         been used to enhance traditional pump and treat technolo-
                         gies by reducing the time needed to achieve aquifer cleanup
                         standards.

                            One of the advantages of employing an in situ technol-
                         ogy is that media transport and excavation requirements
                         are minimized, resulting in both reduced potential for
                         volatile releases  and minimized material handling  costs.
                         Biological technologies that require the physical displace-
                         ment of media during treatment (e.g., "land treatment"
                         applications involving  excavation for treatment in lined
                         beds or tilling of non-excavated soils) assume many of the
                         risks and costs associated with ex situ  technologies and
                         cannot strictly be considered in situ applications.

                            As  of  Fall 1993, in  situ  biodegradation was  being
                         considered or implemented as a component of the remedy
                         at 21 Superfund sites and 38 Resource  Conservation and
                         Recovery Act (RCRA), Underground Storage Tank (UST),
                         Toxic Substances Control Act (TSCA), and Federal sites with
                         soil, sludge, sediment, or groundwater contamination
                         [1, p. 13]'[2][3].  This bulletin  provides information on
                         the technology's applicability, the types of residuals pro-
                         duced, the latest performance data, the  site requirements,
                         the status of the  technology,  and sources for further
                         information.
                        Technology Applicability

                            In situ biodegradation has been shown to be poten-
                        tially effective at degrading or transforming a large number
                        of organic compounds to environmentally-acceptable or
                        less mobile compounds [4, p. 54][5, p. 103][6][7][8][9].
                        Soluble organic contaminants are particularly amenable to
                        biodegradation; however, relatively insoluble contaminants
                        may be degraded if they are accessible to microbial degrad-
* [reference number, page number]
                                                                                  Printed on Recycled Paper

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 ers. Classes of compounds considered amenable to biodeg-
 radation include petroleum hydrocarbons (e.g., gasoline
 and diesel fuel), nonchlorinated solvents (e.g.,  acetone,
 ketones, and alcohols), wood-treating wastes (e.g., creo-
 sote and pentachlorophenol), some chlorinated  aromatic
 compounds (e.g., chlorobenzenes and biphenyls with fewer
 than five chlorines per molecule), and some chlorinated
 aliphatic  compounds  (e.g.,  trichloroethene  and
 dichloroethene). As advances in anaerobic biodegradation
 continue, many compounds traditionally considered resis-
 tant  to aerobic biodegradation  may eventually be de-
 graded, either wholly or partially, under anaerobic  condi-
 tions. Although not normally used to treat inorganics (e.g.,
 acids, bases, salts, heavy metals, etc.), in situ biodegrada-
 tion has been used to treat water contaminated with ni-
 trate, phosphate, and other inorganic compounds.

    Although in situ  biodegradation  may be  used to
 remediate a specific site, this does not ensure that it will be
 effective at all sites or that the treatment efficiency achieved
 will be acceptable at other sites. The complex contaminant
 mixtures found at many Superfund sites frequently result in
 chemical interactions or inhibitory effects that limit con-
 taminant biodegradability.  Elevated concentrations of pes-
 ticides, highly chlorinated  organics, and  some inorganic
 salts have been known to inhibit microbial activity and thus
 system performance  during in  situ biodegradation.
 Treatability studies should be performed to determine the
 effectiveness of a given in situ biological technology at each
 site. Experts based out of EPA's Risk Reduction Engineering
 Laboratory (RREL) in Cincinnati, Ohio and the Robert S. Kerr
 Environmental Research Laboratory (RSKERL) in Ada, Okla-
 homa may be able to provide  useful guidance during the
 treatability study and design  phases.  Other  sources of
 general observations and average removal efficiencies for
 different treatability groups are contained in the Superfund
 Land Disposal  Restrictions (LDR) Guide #6A, "Obtaining a
 Soil and Debris Treatability Variance for Remedial Actions,"
 (OSWER Directive 9347.3-06FS, September 1990)  [10] and
 Superfund  LDR Guide #6B, "Obtaining a  Soil  and Debris
 Treatability Variance for Removal Actions," (OSWER  Direc-
 tive 9347.3-06BFS, September 1990) [11].
Limitations

    Site- and contaminant-specific factors impacting con-
taminant availability, microbial activity, and chemical reac-
tion rates may limit the application of in situ biodegrada-
tion.  Variations in  media composition and contaminant
concentrations can lead to variations in biological activity
and, ultimately, inconsistent degradation rates. Soil char-
acteristics (e.g., non-uniform particle size, soil type, mois-
ture content, hydraulic conductivity, and permeability) and
the amount, location, and extent of contamination can also
have a profound impact on bioremediation. The following
text expands  upon these factors.

    The biological availability, or bioavailability, of a con-
taminant is a function of the contaminant's solubility in
water and its tendency to sorb on the surface of the soil.
Contaminants with low solubility are less likely to be distrib-
 uted in an aqueous phase and may be more difficult to
 degrade biologically.   Conversely, highly soluble  com-
 pounds may leach from the soil before being degraded. In    ^m^.
 general, however, poor bioavailability can be attributed to    '^^k
 contaminant sorption on the soil rather than a low or high       /
 contaminant solubility.  The tendency of organic molecules
 to sorb  on the soil is  determined by the physical and
 chemical characteristics of the contaminant and soil. In
 general, the leaching potential of a chemical is proportional
 to the magnitude of its adsorption (partitioning) coefficient
 in the soil.  Hydrophobic (i.e., "water fearing") contami-
 nants, in particular, routinely partition from the soil water
 and concentrate in the soil  organic matter, thus limiting
 bioavailability. Additionally, contaminant weathering may
 lead to binding in soil pores, which can limit availability
 even of soluble compounds.  Important contaminant prop-
 erties that affect sorption include:  chemical structure,
 contaminant acidity or basicity (pKa or pKb), water solubil-
 ity, permanent charge, polarity, and molecule size. In some
 situations surfactants (e.g., "surface acting agents") may
 be used  to increase  the bioavailability of "bound" or in-
 soluble contaminants. However, it may be difficult to iden-
 tify a surfactant that is both  nontoxic and not a preferred
 substrate for microbial growth.

    Soil solids, which are comprised of organic and inor-
 ganic components, may contain highly reactive charged
 surfaces that play an important role in immobilizing organic
 constituents, and thus limiting their bioavailability. Certain
 types of  inorganic clays, possess especially  high negative
 charges,  thus exhibiting a high cation exchange capacity.
 Alternatively, clays  may also contain positively charged    •^^
 surfaces, causing these  particles to exhibit a high anion        J
 exchange capacity.  Soil  organic  matter also has many
 highly  reactive charged  surfaces  which  can  limit
 bioavailability [12].

    Bioavailability is also a function of the biodegradability
 of the target chemical, i.e., whether it acts as a substrate,
 co-substrate, or is recalcitrant. When the target chemical
 cannot serve as a metabolic substrate (source of carbon and
 energy) for microorganisms, but is oxidized in the presence
 of a substrate already present or added to the subsurface,
 the process is referred  to  as co-oxidation and  the target
 chemical is defined as the co-substrate [12][1 3, p.4].  Co-
 metabolism occurs when an enzyme produced by an organ-
 ism to degrade a substrate that supports microbial growth
 also degrades another non-growth substrate that is neither
 essential for nor sufficient to support microbial growth. Co-
 oxidation processes are important for the biodegradation
 of high molecular weight polycyclic aromatic hydrocarbons
 (PAHs),  and  some chlorinated  solvents,  including
 trichloroethene (TCE).  However, like  surfactants,
 cometabolites  (e.g.,  acetate and  phenol) may be more
 readily mineralized by the indigenous microorganisms than
the target organics [1 3,  p. 4].

    Microbial activity can be reduced by nutrient, mois-
ture, and oxygen deficiencies, significantly decreasing bio-
degradation rates.  Extreme soil temperatures,  soil alkalin-    ^^^
ity, or soil acidity can limit the diversity of  the microbial    *^N
population and may suppress specific contaminant degrad-
                                                 Engineering Bulletin:  In Situ Biodegradation Treatment

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r
ers.  Spatial variation of soil conditions (e.g., moisture,
oxygen availability, pH, and  nutrient levels) may result in
inconsistent biodegradation due to variations in biological
activity. While these conditions may be controlled to favor
biodegradation, the success  of in situ biodegradation de-
pends in a large part on whether required supplements can
be delivered to areas where they are needed. Low hydraulic
conductivity can hinder the movement of water, nutrients,
aqueous-phase electron acceptors (e.g., hydrogen perox-
ide and nitrate), and, to a lesser extent, free oxygen through
the contamination zone [14, p.  155].  Restrictive layers
(e.g., clay lenses), although more resistant to contamina-
tion, are also  more  difficult to  remediate due to poor
permeability and  low rates of diffusion [1 3, p. 4].  Low
percolation rates may cause amendments to be assimilated
by soils immediately surrounding application points, pre-
venting them from reaching areas that are more remote,
either vertically or horizontally.  During the simultaneous
addition of electron acceptors and donors through injec-
tion  wells, excessive  microbial growth or high  iron  or
manganese concentrations may cause clogging in the well
screen or in the soil pores near the well screen [15]. Variable
hydraulic  conductivities in different  soil  strata within a
contaminated area can also complicate the design of flow
control; minor heterogeneities in lithology can, in some
cases, impede the transfer of supplements to specific sub-
surface locations.

    Microbial activity may also be influenced by contami-
nant concentrations.  Each contaminant  has  a range  of
concentrations at which the potential for biodegradation is
maximized.  Below this range microbial activity may not
occur without the addition  of co-substrate.  Above this
range microbial activity may be inhibited and, once toxic
concentrations are reached,  eventually arrested.  During
inhibition, contaminant degradation generally occurs at a
reduced rate. In contrast, at toxic concentrations, contami-
nant degradation  does not occur. The concentrations  at
which microbial growth is either  supported, inhibited,  or
arrested vary with the contaminant, medium,  and micro-
bial species.  Given long-term exposure,  microbes have
been known to acclimate to very high contaminant concen-
trations and  other conditions inhibiting microbial activity.
However, if prompt treatment is a primary goal, as is the
case during most remedial activities, toxic  conditions may
need to be addressed by pH control, metals control (e.g.,
immobilization), sequential treatment, or  by introducing
microbial strains resistant to toxicants.

    Numerous biological and non-biological mechanisms
(e.g., volatilization, sorption, chemical degradation, migra-
tion, and  photodecomposition) occur during  biological
treatment.  Since  some amendments may react with the
soil, site geochemistry can limit both the form and concen-
tration of any supplements added to the soil.  Thus, care
must be employed when using amendments to "enhance"
biological degradation. For example, ozone and hydrogen
peroxide, which can be added to enhance dissolved oxygen
levels in soil  or groundwater systems, may react violently
with  other compounds  present  in the soil, reduce the
sorptive capacity of the soil  being treated, produce gas
bubbles that block the pores  in the soil matrix, or damage
the bacterial population in the soil [4, p. 43].  Nitrogen and
phosphorus (phosphate) must also be applied cautiously to
avoid excessive nitrate formation [4, p.47] and the precipi-
tation of calcium and iron phosphates, respectively. Exces-
sive  nitrate levels in the groundwater  can  cause health
problems in humans, especially children.  If calcium con-
centrations are high, the added phosphate can be tied up
by the calcium and,  therefore, may not be available to the
microorganisms [16, p.  23].  Lime treatment for soil pH
adjustment is dependent on several soil factors including
soil texture, type of clay,  organic matter  content, and
aluminum concentrations [4, p. 45].  Since changes in soil
pH may also affect the dissolution or precipitation of mate-
rials  within  the soil and may increase the mobility  of
hazardous materials, pH amendments (acid or base) should
be added  cautiously and should be  based  on the soil's
ability to resist changes in pH, otherwise known as the soil's
"buffering capacity" [4, p. 46]. Since the buffering capacity
varies between soils, lime and  acidification  requirements
should be  determined on a site-specific basis.

    Finally, high concentrations of metals can have a det-
rimental effect on  the  biological treatment of  organic
contaminants in the same medium. A number of metals can
be oxidized, reduced,  methylated  (i.e., mercury), de-
methylated, or otherwise transformed by various organ-
isms to produce new contaminants. The solubility, volatil-
ity, and  sorption potential of the original soil contaminants
can be greatly changed in the process [1 7, p.  144], leading
to potential significant lexicological effects,  as is the case
during the methylation of mercury. To avoid  these compli-
cations, it is sometimes possible to pretreat or complex the
metals into a less toxic or teachable form.
                                                                   Technology Description

                                                                       During in situ biodegradation, site-specific characteris-
                                                                   tics are modified to encourage the growth of a microbial
                                                                   population capable of biologically degrading the contami-
                                                                   nants of  concern.  Presently, two major types of in situ
                                                                   systems are being employed to biodegrade organic com-
                                                                   pounds present in soils, sludges, sediments, and groundwa-
                                                                   ter: bioventing systems and "traditional" in situ biodegra-
                                                                   dation systems, which usually employ infiltration galleries/
                                                                   wells and recovery wells to deliver required amendments to
                                                                   the subsurface.  In general, bioventing has been  used  to
                                                                   treat contaminants present in the unsaturated zone. Tradi-
                                                                   tional in situ biodegradation, on the other hand, has mostly
                                                                   been used to treat saturated soils and  groundwater. The
                                                                   occasional treatment of unsaturated soil using traditional in
                                                                   situ biodegradation techniques has been generally limited
                                                                   to fairly shallow regions over groundwater that is already
                                                                   contaminated.
                                                                   Traditional In Situ Biodegradation

                                                                       Traditional in situ biodegradation is generally used in
                                                                   conjunction with groundwater-pumping and soil-flushing
                                                                   systems to circulate nutrients and oxygen through a con-
                                                                   taminated aquifer and associated soil. The process usually
        Engineering Bulletin:  In Situ Biodegradation Treatment

-------
involves introducing aerated, nutrient-enriched water into
the contaminated zone through a series of injection wells or
infiltration trenches and recovering the water down-gradi-
ent.  Depending upon local regulations and engineering
concerns, the recovered water can then be treated and, if
necessary, reintroduced to the soil onsite, discharged to the
surface, or discharged to a publicly-owned treatment works
(POTW).  A permit may be required for the re-injection of
treated water. Note that a variety of techniques can be used
to introduce and distribute amendments in the subsurface.
For example, a  lower horizontal well is being used at the
Savannah River Site near Aiken, North Carolina to deliver air
and methane to the subsurface. A vacuum has been applied
to an upper well (in the vadose zone) located at this site to
encourage the distribution of air and methane within the
upper saturated zone and lower vadose  zone [18][19].

    Figure 1 is a general schematic of a traditional in situ
biodegradation system [20, p.  113][16, p. 13].  The first
step in the  treatment process  involves pretreating the
infiltration water, as needed, to remove metals (1). Treated
or contaminated groundwater, drinking  water, or alterna-
tive water sources (e.g., trucked water) may be used as the
water source. If groundwater is used, iron dissolved in the
groundwater may bind phosphates needed for biological
growth. Excess  phosphate may be added to the infiltration
water at  this point in the  treatment process in  order to
complex the iron [20, p. 111 ]. The presence of iron will also
cause a more rapid depletion of hydrogen peroxide, which
is sometimes used  as  an oxygen source.  Surface active
agents  may  also be added at this point in the treatment
process to increase the  bioavailability  of contaminants,
especially hydrophobic or sorbed pollutants, while meth-
ane or other substances may be added to induce the co-
metabolic biodegradation  of certain  contaminants.   In
continuous recycle systems, toxic metals originally located
in the contaminated medium may have to be removed from
the  recycled infiltration  water to prevent inhibition  of
bacterial growth. The exact type of pretreatment will vary
with the water source, contamination problem, and treat-
ment system used.

    Following infiltration water pretreatment, a biological
inoculum can be added to the infiltration water to enhance
the natural microbial population (2). A site-specific inocu-
lum enriched from site samples may be used; commercially
available cultures reported to degrade the contaminants of
concern can also be used (e.g., during the  remediation of
"effectively sterile soils"). Project managers are cautioned
against employing microbial supplements without first as-
sessing the relative  advantages associated  with their use
and potential competition that may occur between the
indigenous and introduced organisms. The ability of mi-
crobes to survive in a foreign  and possibly hostile (i.e.,
toxic) environment, as well as the ability to metabolize a
wide range of substrates should be evaluated.  The health
effects of commercial inocula must also be carefully evalu-
ated, since many products on the market are not carefully
screened or processed for pathogens.  It is essential that
independently-reviewed data be examined before employ-
ing a commercially-marketed microbial supplement [21].

    Nutrient addition can then be employed  to provide
nitrogen and phosphorus, two elements essential  to the
biological activity of both indigenous and  introduced or-
ganisms (3). Optimum nutrient conditions are site-specific.
Trace elements may be added at this stage, but are normally
available in adequate supply in  the soil or groundwater.

    During contaminant oxidation, energy is released  as
electrons are removed. Since oxygen acts as the terminal
electron acceptor during aerobic biodegradation, oxygen
concentrations in the subsurface may become depleted. To
avoid this complication,  air,  oxygen, and  other oxygen
                                                   Figure 1.
                Schematic Diagram of Traditional In Situ Biodegradation of Soil and Groundwater

                                          _^;^_^.     „,,  „        _  n\  I \\  i  i\
                                          ?»,&-$&v ^;^^>,  :s -1\, ,^/Ao or^1J, 1V>• i-V  i 4\
                                          i f ^ ^v"* vv c% %% 'j^ <   •• * K ** ^^^- ^ ''''•"*< ^r
                                                        ,  s  J^t.-/'. \'^u<'A^;«.,^.-XC':*'-;
                                                                Direction of
                                                              Groundwater Flow
                                                                                     Zone of low permeability
                                                                                       (i.e., clay, bedrock)
                                                      ^\
                                                 Engineering Bulletin:  In Situ Biodegradation Treatment

-------
r
 sources (hydrogen peroxide and ozone) can be added to
 the infiltration water (4).  To prevent gas binding in the
 subsurface, and a subsequent reduction in the effective soil
 permeability, oxygen amendment/supplementation meth-
 ods must be carefully selected.  During anaerobic degrada-
 tion, alternative electron acceptors (nitrate, carbonate, or
 sulfate) may be added to the infiltration water in place of
 oxygen. Alternatively, during the co-oxidation of a target
 substrate, a co-substrate (methanol  or  acetate) may be
 added to the infiltration water [22].

    just before the water is added to the soil or groundwa-
 ter, chemical additives may be used to adjust the pH
 (neutral is recommended for most  systems) and other
 parameters that impact biodegradation (5). Care should be
 taken when making adjustments to the pH, since contami-
 nant mobility (especially  of  metals) can be increased by
 changing  the pH [4,  p. 45]. Site managers are also  cau-
 tioned against employing chemical additives that are per-
 sistent in the environment.  The potential toxicity of addi-
 tives and  any synergistic  effects  on contaminant toxicity
 should also be evaluated.

    During in situ bioremediation, amendment concentra-
 tions and application frequencies can  be adjusted to com-
 pensate for physical/chemical depletion and high microbial
 demand.  If these modifications fail to compensate for
 microbial demand, remediation may occur by a sequential
 deepening and widening of the active treatment layer (e.g.,
 as the contaminant is degraded in areas  near the amend-
 ment addition points, and microbial activity decreases due
 to the reduced substrate, the amendments move farther,
 increasing microbial activity in those areas).  Additionally,
 hydraulic fracturing may be employed to improve amend-
 ment circulation within the subsurface.

    The importance  of using  a  well-designed  hydraulic
delivery system and thoroughly evaluating the compatibil-
ity of chemical supplements  was demonstrated at sites in
 Park City, Kansas; Kelly AFB, Texas; and Eglin AFB, Florida.
Air entrainment and iron precipitation  resulted in a contin-
ued loss of injection capacity during treatment at the Park
City site [23][24] and calcium phosphate and iron precipi-
tation resulted in the failure of the two field tests at Kelly
and Eglin AFBs, respectively [25].
        Bioventing

            Bioventing uses relatively low-flow soil aeration tech-
        niques to enhance the biodegradation of soils contami-
        nated with organic contaminants. Although bioventing is
        predominantly used to treat unsaturated soils, applications
        involving the remediation of saturated soils and groundwa-
        ter (e.g., using air sparging techniques) are becoming more
        common [26][27].  Aeration systems similar to those em-
        ployed during  soil vapor extraction are  used to supply
        oxygen to the soil (Figure 2). Typically a vacuum extrac-
        tion, air injection, or combination vacuum extraction and
        air injection system is employed [28]. An air pump, one or
        more air injection or vacuum extraction probes, and emis-
        sions monitors at the ground surface are commonly used.
        Although some systems utilize higher air flow rates, thereby
 combining bioventing with soil vapor extraction, low air
 pressures and  low air flow rates  are generally used to
 maximize vapor retention times in the soil while minimizing
 contaminant volatilization. An interesting modification to
 traditional aeration techniques has been proposed at the
 Picatinny Arsenal  in New jersey.  Here researchers  and
 project managers have proposed collecting TCE vapors at
 the surface,  amending them with degradable hydrocar-
 bons (methane, propane, or natural gas) capable of stimu-
 lating  the cometabolic degradation of vapor-phase TCE,
 and then re-injecting the amended vapors into the unsatur-
 ated  zone  in  an  attempt to encourage  the  in  situ
 bioremediation of the TCE remaining  in the subsurface
 [29][30][31][32][27].

    Off-gas treatment (e.g., through biofiltration or  car-
 bon adsorption) will be needed during most bioventing
 applications to ensure compliance with emission standards
 and to control fugitive emissions.  Off-gas treatment  sys-
 tems similar to those employed during soil vapor extraction
 may be used. These systems must be capable of effectively
 collecting and  treating a vapor stream consisting of the
 original contaminants and/or any volatile degradation prod-
 ucts generated  during treatment.  Although similar vapor
 treatment systems  may be employed during soil vapor
 extraction and bioventing, less concentrated off-gases would
 be  expected  from a bioventing system than from a  soil
 vapor  extraction system employed at the same site. This
 difference in  concentration is attributed to enhanced bio-
 logical degradation within the subsurface.

    Nutrient addition may be employed during bioventing
 to enhance biodegradation.   Nutrient addition can be
 accomplished by surface application, incorporation by till-
 ing into surface soil, and transport to deeper layers through
 applied irrigation water.  However, in some field applica-
 tions to date,  nutrient additions have been found to provide
 no additional benefits [33], Increasing the soil temperature
 may also  enhance  bioremediation, although in general
 high temperatures should be avoided since they can  de-
 crease  microbial population and activity. Heated air, heated
 water,  and low-level radio-frequency heating are some of
 the techniques which can be used to modify soil tempera-
 ture.  Soil core  analyses can be performed  periodically to
 assess system performance as determined by contaminant
 removal. A control plot located near the bioventing system,
 but not biovented, may also be used to obtain additional
 information to assess system performance.


 Process Residuals

    During in situ biodegradation, limited but potentially
 significant process residuals may be generated.  Although
 the majority of wastes requiring disposal are generated as
 part of pre- and  post-treatment activities, process residuals
directly arising from in situ biological activities may also be
generated. These process residuals may include: 1) par-
tially degraded  metabolic by-products, 2) residual con-
tamination, 3) wastes produced during groundwater pre-
and post-treatment activities, and 4) volatile contaminants
that are either  directly released into the atmosphere or
        Engineering Bulletin:  In Situ Biodegradation Treatment

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 collected within add-on emission controlXtreatment sys-
 tems.  The following text expands upon the specific types
 of process residuals, their control, and their  impact on
 disposal requirements.

    Ultimately biological technologies seek to mineralize
 hazardous contaminants into relatively innocuous by-prod-
 ucts, specifically carbon dioxide, water, and inorganic salts.
 However, a number of site- and contaminant-specific fac-
 tors may cause the partial degradation or "biotransforma-
 tion" of a contaminant and the generation of an intermedi-
 ate by-product.   These metabolic by-products may be
 located in either the saturated or  unsaturated zones.  The
 identity, toxicity, and mobility of these partially degraded
 compounds should be determined since intermediate deg-
 radation products can be as toxic or more toxic than the
 parent compound. Since metabolic by-products can accu-
 mulate in the soil and groundwater, future remedial actions
 may be necessary.

    In addition to intermediate degradation by-products,
 residual contamination  may persist in the soil following
 treatment.  Microbes are capable of degrading only that
 fraction of  the contamination  that is readily available for
 microbial incorporation. As a result, biologically resistant
 contaminants and contaminants that remain sorbed to the
 soil and sediment during the  remedial action cannot be
 degraded.  Depending on the nature of the contaminants
 and media, the "bound" fraction may slowly desorb over
 long  periods of times (months to years), potentially re-
 contaminating "treated" media near the residual contami-
 nation  [34][35].  Additionally, fluctuations in  the water
table may  result  in the recontamination  of previously
remediated soils if groundwater contamination, specifically
                                                      contamination associated with the presence of a light non-
                                                      aqueous  phase  layer (LNAPL),  has not been effectively
                                                      addressed.

                                                          Above-ground activities taken to ensure that the reme-
                                                      dial action  complies with regulatory requirements and
                                                      adequately  guards  against cross-contamination and un-
                                                      controlled releases may result in the generation of a signifi-
                                                      cant volume of  waste requiring disposal.  For example,
                                                      when groundwater  is used to deliver amendments to the
                                                      subsurface,  it may  be necessary to  pre-treat the water
                                                      before it can be re-introduced to the subsurface. Addition-
                                                      ally, in order to protect water quality outside of the treat-
                                                      ment zone from  contaminant or amendment  migration, a
                                                      down-gradient groundwater recovery and  treatment sys-
                                                      tem designed to collect and treat amendment- and con-
                                                      taminant-laden groundwater may be needed. The residu-
                                                      als produced  by these add-on  treatment  processes will
                                                      eventually require disposal.

                                                          Significant volatile emissions may also be produced
                                                      during in situ biodegradation (e.g.,  bioventing). Depend-
                                                      ing on their concentration, toxicity, and total volume, these
                                                      emissions, which may consist of the original contaminant or
                                                      any volatile  degradation products produced during treat-
                                                      ment, may  need to be controlled, collected, or treated.
                                                      Ultimately, the by-products of  an  emissions treatment/
                                                      control system will require disposal.


                                                      Site Requirements

                                                         In situ biodegradation normally requires the installa-
                                                      tion of wells or infiltration trenches; therefore, adequate
                                            Figure 2.  Bioventing
                                       Low rate
                                     air injection
                                                             Surface monitoring
                                                                 to ensure
                                                                no emissions
                                Air extraction
                                                               Air extraction
                                                              Biodegradation
                                                             of contaminated
                                                                   soils
  Monitoring of soil
gas to assess vapor
   biodegradation
               Biodegradation of vapors
                                               Engineering Bulletin: In Situ Biodegradation Treatment

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           access roads are required for heavy equipment such as well-
           drilling rigs and backhoes.  Soil-bearing capacity, traction,
           and soil stickiness can limit vehicular traffic [17, p. 61].

              In general, the area required to set up mixing equip-
           ment is not significant.  However,  space requirements
           increase as the  complexity of the various pre- and post-
           treatment systems increases.  During the installation of
           infiltration galleries and wells, several hundred up to several
           thousand square feet of clear surface area will be required.
           Climate can also influence site requirements.  If periods of
           heavy rainfall or extremely cold conditions are expected, a
          cover may be required.

              Electrical requirements will  depend on the  type  of
          technology employed. Standard 220V, three-phase electri-
          cal service may  be used to supply power  to pumps and
          mixing equipment.  Since water is used for a variety  of
          purposes during biological treatment, a readily available
          water supply will be needed at most sites. Municipal water
          or clean groundwater may be used. Contaminated ground-
          water may be used if permitted by the appropriate regula-
          tory agency. The quantity of water  needed is site- and
          process-specific. Waste storage is not normally required for
          in situ biodegradation.

              Onsite analytical equipment for conducting pH and
          nutrient analyses will help improve operation efficiency and
          provide better information for process control.  During
          bioventing applications, air emissions monitors at the ground
          surface are commonly used.


          Regulatory Considerations  and
          Response Actions

             Federal mandates can have a significant impact on the
          application of in situ  biodegradation.  RCRA  LDRs that
          require treatment of wastes to best demonstrated available
          technology (BDAT) levels prior to land disposal may some-
          times be determined  to be applicable or relevant and
          appropriate requirements (ARARs) for CERCLA response
          actions. The  in situ biodegradation technology can pro-
         duce a treated waste that meets treatment levels set by
          BDAT, but may not reach these treatment levels in all cases.
         The ability to meet required treatment levels is dependent
         upon the specific waste constituents and the waste matrix.
         In cases where in  situ biodegradation does not meet these
         levels, it still may, in certain situations, be selected for use
         at the site if a treatability variance establishing alternative
         treatment levels  is  obtained.  Treatability  variances are
         justified for handling complex soil and debris matrices. The
         following guides describe when and how to seek a treatability
         variance for soil and debris: Superfund LDR Guide #6A,
         "Obtaining a Soil and Debris Treatability Variance for Reme-
         dial Actions" (OSWER Directive 9347.06FS, September 1990)
         [10], and Superfund LDR Guide #6B, "Obtaining  a Soil and
         Debris Treatability Variance for Removal Actions" (OSWER
         Directive 9347.06BFS,  September 1990) [11].   Another
^^    approach could be to use other treatment techniques with
//2P^    in situ biodegradation to obtain desired treatment levels,
                                                            for example, carbon treatment of recovered groundwater
                                                            prior to re-infiltration into the subsurface.

                                                               When determining performance relative to ARARs and
                                                            BDATs, emphasis should be placed  on  assessing the risk
                                                            presented by a bioremediation technology. As part of this
                                                            effort, risk assessment schemes, major metabolic pathways
                                                            of selected hazardous pollutants, human health protocols
                                                            for metabolite and pathogenicity tests, and fate protocols
                                                            and issues  for microorganisms and  metabolites must be
                                                            assessed [36]. A detailed summary of the findings of the
                                                            June 17-18, 1993 EPA/Environment Canada Workshop in
                                                            Duluth, Minnesota addressing Bioremediation Risk Assess-
                                                            ment should be available in  early 1994.


                                                            Performance Data

                                                               Performance data for Superfund sites are limited. The
                                                           first record of decision (ROD) selecting in situ biodegrada-
                                                           tion as a component of the remedy was in FY87. Since then,
                                                           in situ biodegradation of soil  or groundwater contaminants
                                                           has either been considered or selected at 22 Superfund sites
                                                           and 30 RCRA, UST, TSCA, and Federal sites [1][2][3]. The
                                                           following two subsections address traditional in situ and
                                                           bioventing  applications, respectively; a  third subsection
                                                           has been included to briefly address information sources
                                                           and data concerns related to remedial efforts performed in
                                                           the private sector.
                                                           Traditional In Situ Bioremediation

                                                               Methane and phenol were employed during a series of
                                                           stimulus-response studies investigating the co-metabolic
                                                           degradation  of TCE,  cis-dichloroethene (c-DCE), trans-
                                                           dichloroethene (t-DCE), and vinyl chloride (VC) at the
                                                           Moffet Field site in  California. Both sets of experiments
                                                           used indigenous bacteria and were performed under the
                                                           induced gradient conditions of injection and extraction.
                                                           During the first set of experiments, methane, oxygen, and
                                                           TCE (from 50 to  100  ^ig/L), c-DCE, t-DCE, and VC  were
                                                           added to the soil to stimulate methanotrophic degradation
                                                           of the injected chlorinated aliphatic compounds. Approxi-
                                                           mately 20 percent of  the TCE added to the system was
                                                           degraded  within the 2-meter hydraulically-controlled
                                                           biostimulated zone.  Approximately 50 percent of the c-
                                                           DCE, 90 percent of the t-DCE, and 95  percent of the VC
                                                          were also degraded.  During the second set of tests, meth-
                                                          ane was replaced with phenol in order to stimulate growth
                                                          of an indigenous  phenol-utilizing population.  During 4
                                                          weeks of testing, the concentration of TCE injected into the
                                                          subsurface was raised from an initial concentration of 62
                                                          jig/L to a final concentration  of 1000  ng/L  A bromide
                                                          tracer was used to determine transformation extent. Up to
                                                          90 percent of the TCE in the 2-meter biostimulated zone
                                                          was degraded, demonstrating  that even at relatively  high
                                                          TCE concentrations significant removal efficiencies can be
                                                          achieved in situ through phenol and dissolved oxygen (DO)
                                                          addition. During the course of the project, transformation
                                                          yields (i.e., grams of TCE per grams of phenol) ranging from
                                                          0.0044 to 0.062 were obtained for varying concentrations
Engineering Bulletin:  In Situ Biodegradation Treatment

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 of phenol and TCE. Future studies at the site will determine
 whether a  compound more environmentally acceptable
 than methane or phenol can be used to induce an indig-
 enous population that effectively degrades TCE [37][7][8].

     A 40- by 120-foot test zone in an aquifer that receives
 leachate from an industrial landfill at the Du Pont Plant near
 Victoria, Texas was used to demonstrate the in situ biotrans-
 formation  of  tetrachloroethene  (PCE), TCE, DCE,
 chloroethane, and VC to ethane and ethylene using micro-
 bial reductive dehalogenation under sulfate-reducing con-
 ditions.  Croundwater from  this zone  was alternately
 amended with either benzoate or sulfate and circulated
 through the aquifer. Initially PCE and TCE concentrations
 were approximately 10 and 1 micro-mole (uM),  respec-
 tively.  After a year of treatment the halogenated com-
 pounds were reduced to concentrations near or below 0.1
 ^iM.  PCE and TCE degraded to DCE rapidly following the
 introduction of benzoate. A decrease in sulfate concentra-
 tions led to increases in the vinyl chloride concentrations.
 Therefore, sulfate concentrations were kept above 10 mg/
 L until  the DCE was further biodegraded.  After approxi-
 mately  6 months  of treatment, most  of the DCE,
 chloroethane, and VC biodegraded to produce ethane and
 ethylene [38].

    A field-scale in situ bioremediation system, consisting
 of down-gradient groundwater extraction wells and an up-
 gradient infiltration  system, was  installed at a gasoline-
 contaminated site owned by the San Diego Gas and Electric
 Company.  [Note: extracted groundwater was amended
 with nutrients (nitrate and phosphate) prior to re-infiltra-
 tion into the subsurface].  Due to the relatively low rate of
 groundwater extraction (approximately 800 to 900 gallons
 per day) and the low hydraulic gradient at the site (0.004),
 it took nearly 2 years (until June/July 1991) for the added
 nitrate to reach the down-gradient well and overtake the
 xylene (BTX) plume.  BTX concentrations, which ranged
 from  25 to 50  mg/L for the  preceding 2-year  period,
 dropped markedly as nitrate levels in the groundwater
 increased.   By late August 1991, benzene and toluene
 concentrations had  dropped below  the  detection limit
 (0.01  mg/L), and total xylene concentrations had dropped
 to 0.02 mg/L. The coincident occurrence of nitrate appear-
 ance and BTX  loss in the aquifer, as well as an eight-fold
 increase in the percentage  of denitrifiers present  in the
 groundwater (from 1 to 8 percent), points to a potential
 stimulatory effect nitrate may have on BTX loss in situ [5].

    An  in situ bioremediation  system consisting of four
 injection and three recovery wells was employed to treat
 gasoline contamination present in  the saturated zone at a
 former service station in Southern California. During treat-
 ment, recovered groundwater was amended with hydro-
 gen peroxide (from 500 to 1,000 mg/L) and nutrients and
 re-injected into the aquifer.  Prior to treatment, total fuel
 hydrocarbons in the saturated clay soils ranged from below
 detection limits to 32 mg/kg as BTX. Maximum groundwa-
 ter concentrations were 2,700 ^ig/L for benzene; 6,600 ^ig/
 L for toluene; 4,100 ng/L for xylene; and 45,000 ng/L for
TPH [4].  After 10 months,  BTX  and  TPH  levels in the
groundwater and saturated soils had dropped below the
           detection limits.  Roughly 1,350 kilograms of hydrogen
           peroxide were introduced to the aquifer over 10 months,
           roughly two times the estimated requirements based on the
           estimated mass of hydrocarbon in the saturated zone (i.e.,
           110 kg of fuel hydrocarbon and 2 to  3 kg of dissolved
           hydrocarbons). After 34 months of treatment, soil hydro-
           carbon concentrations ranged from below the detection
           limit to 321  ppm as TPH; benzene was not detected in any
           samples [39].

              Following successful laboratory treatability  testing,
           General Electric performed a  10V2-week field study  to
           investigate the biodegradation of polychlorinated biphe-
           nyls (PCBs) in  the Hudson River sediment.   Initial  PCB
           concentrations in the sediment ranged between 20 and 40
           ppm. The study attempted to enhance the aerobic bacteria
           native to  the upper Hudson  River.  Six caissons were
           installed at the Hudson River  Research  Station  (HRRS)  to
           isolate sections of the  river bottom for this field study.
           Because of extensive, naturally occurring dechlorination,
           approximately 80 percent of the total PCBs encountered in
           the sediments were  mono-, di-, and trichlorobiphenyls.
           Biodegradation was stimulated using oxygen and nutrient
           addition. Mixing was employed to enhance the dispersal of
           oxygen and nutrients within the sediment. Between 38 and
           55  percent  of the PCBs  present in the sediment were
           removed  by aerobic degradation during  the study.
          This corresponds to the percentage  biologically available
           PCBs [9].
          Bioventing

              In May 1992, the U.S. Air Force began a Bioventing
          Initiative to examine bioventing as a remedial technique at
          contaminated sites across the country.  The Air Force's
          decision to examine bioventing on such a large  scale was
          prompted by a successful demonstration of the technology
          at Tyndall AFB,  Florida,  where bioventing coupled with
          moisture addition removed one-third of the TPH and nearly
          all of the BTEX in JP-4  contaminated soils during  7 months
          of treatment.  The Bioventing Initiative targets  138 sites
          with diesel fuel, jet fuel, or fuel oil in soil. In selecting sites
          for the initiative, the  Air Force looked for characteristics
          appropriate for bioventing, such as deep vadose soil, heavy
          hydrocarbon contamination, and  high  air  permeability.
          The chosen sites  represent  a wide range of depths to
          groundwater,  hydrocarbon concentrations,  and soil tex-
          tures.   Preliminary testing has been completed and 33
          systems have  been installed  at Battle  Creek Air National
          Guard Base and  the following AFBs: Beale, Eglin, Eielson,
          F.E. Warren, Galena, Hanscom, Hill, K.I. Sawyer, McGuire,
          Newark, Offutt, Plattsburgh, Robins, Vandenberg, and
          Westover. According to the Air Force, initial results are very
          promising  with  degradation  rates  measured  as high as
          5,000 mg/kg per year [40][41].

             The EPA RREL, in collaboration with the U.S. Air Force,
          initiated two  3-year pilot-scale bioventing field studies in
          mid-1991  at JP-4 contaminated fuel sites located at Eielson
          AFB near Fairbanks, Alaska and at Hill  AFB near  Salt Lake
          City,  Utah.  Four  soil  plots  are  being used  to  evaluate
          passive, active, and buried heat tape soil-warming  methods
   8
Engineering Bulletin: In Situ Biodegradation Treatment

-------
during the Eielson study. The fourth plot was vented with
injected air but not artificially heated. Roughly 1 acre of soil
is contaminated from a depth of 2 feet to the water table at
6 to 7 feet. At the Hill site, a series of soil gas cluster wells
capable of obtaining samples up to 90 feet deep is being
used with a single air injection  well and two groundwater
wells to  remediate  JP-4 contamination found  at  depths
ranging from 35 feet to perched water at approximately 95
feet. Inert gas tracer studies, regular soil gas measurements
at several locations and depths, and periodic in situ respirom-
etry tests to measure in  situ oxygen uptake rates are being
performed.  Final soil hydrocarbon analyses will be con-
ducted at both sites in  mid-1994 and compared with the
initial soil data.  In situ respirometry data from the Hill site
(Table 1) indicate that petroleum hydrocarbons are being
removed at a significant rate.  Intermediate respirometry
data from the test  and control  plots at  the Eielson site
indicate that higher biodegradation  rates are  being ob-
tained at higher soil temperatures.[42][43].
                        Table 2.
     Groundwater Quality After Seven Months of
  Biosparging at the U.S. Coast Guard Air Station in
               Traverse City, Michigan
Well
Depth (ft)
Control
16
17.5
20.5
22
Sparge Plot
15
18
19.5
21
Benzene
(US/L)

9.9
228
70
57

1.9
<1
<1
<]
Xylenes
(ng/L)

19
992
38
7.7

5.3
5.0
<1
<"•
Total Fuel
Carbon (u,g/L)

2,880
4,490
956
783

559
<6
<6
<6
                       Table 1.
  Rates of Biodegradation, Averaged Over Depth, at
               Three Wells at Hill AFB

Well
CW-1
CW-2
CW-3
Depths
(ft)
20-90
60-90
10-90
Rate (mg/kg/day)
September 1991
0.97
0.59
0.56
September 19921
0.30
0.36
0.32
    1   Since bioventing is being performed on a sandy soil, with
little to no naturally occurring organic matter, a biodegradation
rate approaching zero would indicate that biodegradation had
finished.

    In November 1991, a pilot-scale bioventing  system
originally used to treat gasoline-contaminated vadose soils
at the U.S. Coast Guard Air Station in Traverse City, Michi-
gan was converted into a groundwater biosparging pro-
cess.  Eight 2-inch diameter sparge wells were installed to
a depth of 10 feet below the water table.  A control plot
located in the vicinity of the contaminated plume, but not
biosparged, was established  to help assess the system's
performance. After 12 months of biosparging, one-third of
the oily phase residue below the water table, as  well as
almost all the BTEX initially present within the groundwater
plume, was removed. (See Table 2 for groundwater quality
data after 7 months of biosparging.) The globular nature of
the oily residue limited the surface area in contact with the
introduced air, thus restricting the  biodegradation and
vaporization of the oily-phase contaminants [44][45].


Non-Superfund Sites

    In situ biodegradation has been applied at many sites
in the private sector. Those interested in accessing informa-
tion generated in the private sector may want to refer to the
following EPA Publications:

        U.S. Environmental Protection Agency.
        Bioremediation Case Studies: Abstracts.
        EPA/600/R-92/044, March 1992.

        U.S. Environmental Protection Agency.
       Bioremediation Case Studies: An Analysis of
       Vendor Supplied Data. EPA/600/9-92/043,
       March 1992.

    Most of the data contained in  these resources were
directly supplied by the vendor and have not been techni-
cally reviewed by EPA. Since independently-reviewed data
are not always available from privately sponsored remedial
efforts, in part due to proprietary issues [46, p. 1 -1 ], readers
should use these data cautiously. Often the quality of the
data used to determine system effectiveness has not been
substantiated by the  scientific community.  Thus, many
vendor claims of effectiveness, specifically regarding intro-
duced organisms and surface-active agents,  are not sup-
ported within the scientific literature. Furthermore, many
bioremediation firms have only limited experience working
with the complex wastes normally associated with Super-
fund sites. Typically these firms deal only with gasoline and
petroleum product leaks and spills.  Additionally, many of
the systems currently on the market involve the use of in situ
biodegradation in combination  with other above-ground
treatment technologies such as  carbon adsorption,  air
stripping, and biological reactors. In situ biodegradation is
believed  to  enhance  the total removal efficiency of the
system. However, in many cases, it is unclear how much of
the degradation occurred as a result of biological  or non-
biological mechanisms (volatilization,  chemical  destruc-
tion, etc.).  How much biodegradation actually takes place
in the soil or groundwater, in contrast to ex situ biodegra-
dation, is not always clear.
Engineering Bulletin:  In Situ Biodegradation Treatment

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Technology Status

    In situ biodegradation either has been considered or
selected as the remedial technology at 21  Superfund sites,
as well as 38 RCRA, UST, TSCA, and Federal sites[l][2][3].
Table 3 lists the location, primary contaminants, treatment
employed, and status of these sites. Information has also
been included on three in situ biotechnology demonstra-
tions presently being performed under the U.S. EPA Super-
fund Innovative Technology Evaluation (SITE) Program and
seven sites selected for performance evaluations under the
U.S. EPA Bioremediation Field Initiative. The data obtained
during the SITE demonstrations and Bioremediation Field
Initiative performance evaluations will be  used to develop
reliable cost and performance information  on biotreatment
technologies and applications.

    The majority of the information found in Table 3 was
obtained from the August 1993 version of  "Bioremediation
in the Field" [1],  These sites have been sorted numerically
by  Region and then alphabetically by site name.  Sites
employing "in situ  land  treatment" were not included in
this list since these  applications typically involve a signifi-
cant amount of material handling. Additionally, some of
the information was modified based on phone calls made to
the various site project  managers. This resulted in  the
removal of the American Creosote Works site in Florida and
four pesticides sites (i.e., the Joliet Weed Control District
site in the Joliet, Montana; the Lake County Weed Control
site in Ronan,  Montana; the Miles Airport site in Miles City,
Montana; and the Richey Airport site in Richey, Montana)
[47], which are no longer considering in situ treatment.
Quarterly updates of this information can be obtained from
subsequent versions of "Bioremediation in the Field".

    Most of the hardware components of  in situ biodegra-
dation systems are  available off-the-shelf and present no
significant availability problems.  Selected cultures, nutri-
ents, and  chemical/biological additives  are  also readily
obtainable.

    Bioremediation, particularly in situ applications, which
avoid excavation and emissions control costs, are generally
considered cost effective. This can be attributed in part to
low operation and maintenance requirements. During set
up and operation, material handling requirements are mini-
mal, resulting  in lowered worker exposures and reduced
health impacts. Although in situ technologies are generally
slow and somewhat difficult to control, a  large volume of
soil may be treated at one time.

    It is difficult to  generalize about treatment costs since
site-specific characteristics  can significantly impact costs.
Typically, the greater  the number of variables  requiring
control during biological treatment, the more problematic
the implementation and the higher the cost. For example,
it is less problematic to implement a technology in which
only one parameter (e.g.,  oxygen availability)  requires
modification than to implement  a remedy that requires
modification of multiple factors (e.g., pH, oxygen levels,
nutrients, microbes, buffering agents, etc.). Initial concen-
trations  and volumes, pre- and  post-treatment require-
ments, and air emissions and control systems will impact
         final treatment costs. The types of amendments employed
         (e.g., hydrogen peroxide) can also impact capital cost and
         costs associated with equipment and manpower required
         during their application.

             In  general, however, in situ bioremediation is consid-
         ered to be a relatively low-cost technology, with costs as
         low as 10 percent of excavation or pump and treat costs [7,
         p. 6-16].  The cost of soil venting using a field-scale system
         has been reported to be approximately $50  per ton as
         compared to incineration, which was estimated to be more
         than ten times this amount. A cost estimate of about $15
         per cubic yard for  bioventing sandy soil at a JP-4 jet fuel
         contaminated site has been reported by Vogel [48]. Exclu-
         sive of site characterization, the biological remediation of
         JP-4 contaminated  soils at the Kelly Air Force Base site was
         estimated to  be $160 to $230 per gallon  of residual fuel
         removed from the aquifer [9]. At the French Limited site in
         Texas, the cost of bioremediation is projected to be almost
         three times less expensive than incineration. Because of the
         large amount of material requiring treatment at this site, it
         has been projected that cleanup goals will be achieved in
         less time by using bioremediation rather than incineration.


         EPA  Contact

             Technology-specific questions regarding  in situ  bio-
         degradation may be directed to:

                Steve Safferman,  EPA-RREL
                Cincinnati, Ohio
                (513)569-7350

                John  Matthews, EPA-RSKERL
                Ada,  Oklahoma
                (405) 436-8600


         Acknowledgments

             This bulletin was prepared for the U.S. Environmental
         Protection Agency, Office of Research  and Development
         (ORD), Risk Reduction Engineering Laboratory (RREL), Cin-
         cinnati, Ohio, by Science Applications International Corpo-
         ration (SAIC) under Contract No. 68-C8-0062 and Contract
         No. 68-CO-0048.   Mr. Eugene Harris served as the  EPA
         Technical Project Monitor. Mr. Jim  Rawe served as SAIC's
         Work Assignment Manager. This bulletin was authored by
         Mr. Rawe and Ms. Evelyn Meagher-Hartzell of SAIC.

             The following other Agency and contractor personnel
         have contributed their time and comments by participating
         in the expert review meetings or independently reviewing
         the document:
                 Mr. Hugh Russell
                 Ms. Tish Zimmerman
                 Mr. Al Venosa
                 Dr. Robert Irvine
                 Dr. Ralph Portier
                 Mr. Clyde Dial
EPA-RSKERL
EPA-OERR
EPA-RREL
University of Notre Dame
Louisiana State University
SAIC
  w
Engineering Bulletin: In Situ Biodegradation Treatment

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                                      Table 3. Superfund, RCRA, UST, TSCA, and Federal Sites
         Site Location
          (Regions)
        Primary Contaminants
           Status/Cost
                                                                                  Treatment
    Chariestown Navy Yarc
    Boston, MA (t)

    General Electric -
    Woods Pond
    Pittsfield, MA(1)

    FAA Technical Center -
    AreaD
    Atlanta County, Nj (2)


    General Electric -
    Hudson River, NY (2)


    Knispel Construction
    Site
    Horsehead, NJ (2)

    Picatinny Arsenal
    NJ(2)


    Plattsburgh AFB
    Plattsburgh, NY (2)

    ARC
     ainesville, VA (3)

    Dover AFB
    Dover, DE (3)
    .A. Clarke & Son
    redericksburg, VA (3)


    Charleston AFB
    Iharieston, SC (4)
    glin AFB
    L(4)

    avannah River Site
    Uken, NC (4)

    tallworth Timber
    eatrice, AL (4)
    Hied Chemical
    onton, OH (5)


    moco Production Co.
    alaska, Ml (5)

    &F Trucking
    ompany
    ochester, MN (5)

    endixCorp./Allied 1
    utomotive Site
    . Joseph, Ml (5)
 Sediments: wood preserving (PAHs).
 Sediments: PCBs.
 Volume: 250 gallons.


 Soil (saturated sand)/groundwaten
 petroleum (Jet fuel, NAPLs).
 Volume: 33K cubic yards.


 Sediments: PCBs, cadmium,
 chromium, lead.
 Volume: 150 cubic feet.
 Soil/groundwater: petroleum.
Soil (vadose)/soil vapors: solvents
CTCE).


Groundwater: petroleum.
 oil: solvent (chlorobenzene).
Volume: 2,000 cubic yards.


Soil (vadose sand and silt)/ground-
water: petroleum, PAHs, TCE, solvents,
metals (lead, iron, manganese).
 'olume: 365K cubic yards.

Sediments/soil: wood preserving.
/olume: 119K cubic yards.


 oil (vadose sand): petroleum (jet
uel), solvents (1,1 -DCE; 1,1,1 -TCA;
TCE; VC; trans-1,2-DCE; PCE; and
 ichloromethane), lead.
/olume: 25 cubic yards.

 oil (vadose): petroleum (jet fuel).


 oil(yadose)/groundwater/sediments:
 hlorinated solvents (TCE and PCE).

 oil (sand, silt)/groundwater: wood
 reserving (PCP).
ediments (coal and coke fines): PAHs,
rsenic,
olume: 500K cubic yards.

oil (saturated)/groundwater: BTEX.


oil (vadose and saturatedrg)/ground-
ater: petroleum (lube oil).
olume: 700 cubic yards.


roundwater: solvents (TCE, DCE,
CA, VC).
 Design: pilot scale TS underway.


 Design: lab scale TS underway.
 Design: pilot scale TS completed
 8/927
 Expected cost: capital, S286K;
 O&M, S200K

 Predesign: lab scale TS
 completed.
 Incurred cost: S2.6M.

 Completed: full scale 10/89
 Start date: 01/89.
 ncurred cost: O&M, $25K.

 Design:  lab scale studies
 completed.


 Design: pilot scale.
 Start date (est.): 3/94.

 Completed: full scale 6/91.
 "tart date: 10/89.


 Four separate processes are
 planned.  Field and lab TS results
 are expected 2/94 and 11/94.


 Design: pilot scale TS started
 7/92
 Expected cost: $23M.

 >ilot scale TS started 11/92.
 Expected completion 12/93.
Completed field scale study.


Operational: pilot scale research
tudy.

 redesign.
 esign: pilot scale TS study
 ompleted.
 xpected cost: $26M

 lot scale TS completed.


 perational: full scale.
 tart Date: 4/91.
 curredcost: J341K.


 edesign: lab scale TS underway.
 In situ treatment.  Ex situ treatment
 Aerobic and anaerobic.

 Anaerobic treatment, confined treatmen
 facility, nutrient addition.

 Nutrient addition (soil, water).
 Groundwater re-injection.
 Aerobic treatment.  Less than 1% of site
 underwent bioremediation.


 Aerobic treatment, hydrogen peroxide,
 nutrient addition (water).  100% of site
 underwent bioremediation [25].

 Aerobic treatment, bioventing.
 Co-metabolic degradation (methane,
 propane, or natural gas) [27].

 Aerobic treatment, bioventing.
Aerobic treatment, bioventing.
Exogenous organisms. 5% of the site
underwent bioremediation.

Aerobic treatment, bioventing, air
 parging. Ex situ land treatment.
 T situ treatment, creosote recovery.
 5% of site will undergo bioremediation.


 ierobic treatment, bioventing.  Less than
 0% of the site under bioremediation.
 Aerobic treatment, bioventing.  Nutrient
 nd hydrogen peroxide addition [27].

 erobic treatment, horizontal wells,
methane addition [18][19],

n situ aerobic treatment, nutrient
 ddition.  Ex situ treatment, activated
 udge, continuous flow.  Exogenous and
 idigenous organisms.  100% of site will
 ndergo bioremediation.

 erobic treatment 50% of site will
 ndergo bioremediation.


 erobic treatment, air sparging [49].


  situ treatment.  Ex situ treatment
equencing batch reactor, continuous
ow. AeroDic conditions. 75% of site
nder bioremediation.

erobic and anaerobic treatment.
Engineering Bulletin: In Situ Biodegradation Treatment
                                                                                                    11

-------
                           Table 3. Superfund, RCRA, UST, TSCA, and Federal Sites (continued)
       Site Location
         (Regions)
     Primary Contaminants
                                           Status/Cost
                                                                                                         Treatment
 n-named site2
 uchanan, Ml (5)
 alesburg/Kopper
 alesburg, IL (5)

 entchells
 raverse City, Ml (5)
 enworth Truck Company
 hillicothe, OH (5)
 J. Sawyer AFB
vlarquette, Ml (5)

vlayville Fire Department
vlayville, Ml (5)

vlichigan Air National Guard
-attle Creek, Ml (5)
Newark AFB
Newark, OH (5)
Onalaska Municipal Landfill
Lacrosse County, Wl (5)
Parke-Davis
Holland, Ml (5)

Reilly Tar & Chemical 1«2
St. Louis Park, MN (5)
Sheboygan River and Harbor
Sheboygan, IL (5)
West K&L Avenue e Landfill 1
Kalamazoo, Ml (5)
Wright-Patterson AFB
Dayton, Ohio (5)

Dow Chemical Company
Plaquemine, LA (6)
 French Limited
 Crosby, TX (6)
 Kelly AFB
 San Antonio, TX (6)
 roundwater: BTEX, PCE, TCE, DCE.


 oil: phenols, chlorophenol, PNAs,
 CP, PAHs.

 oil/groundwater: petroleum.

 oil (vadose)/groundwater:
 olvents (BTEX, acetone, TPH).
 oil (vadose sand): petroleum.


 iroundwater: petroleum.
>oil (vadose: sand, silt): petroleum,
heavy metals.
Soil (vadose: silt, clay): petroleum
 gasoline).
volume: 60 cubic yards.
Soil (vadose and saturated sand):
solvents (TCE), petroleum (total
 lydro-carbons), wood preserving
 naphthalene).
Volume: 5,000 cubic yards.

Soil/groundwater: petroleum,
solvents, arsenic, chloride, zinc.

Soil (vadose loam): wood preserving
(PAHs).
 Sediments (sand, silt, clay): PCBs.
 Volume: 2,500 cubic yards.
 Croundwater: solvents (acetone;
 TCE; trans-1,2-DCE; 1,2-DCA;
 1,1-DCA; BTEX; VC; methyl isobutyl
 ketone; MEK.

 Soil (vadose: sand, silt, clay):
 petroleum (jet fuel).
 Volume: 7.5K cubic yards.
 Croundwater: solvents (1,1-DCA;
 1,2-DCA;1,1,1-TCA;1,1-DCE,
 chloroethane).
 Volume: 90K cubic yards.

 Sediments (sand, silt)/sludge/soil
 (sand, silt, clay)/groundwater:
 PCBs, arsenic, roleum (BAP, VOCs),
 arsenic.

 Soil (vadose clay): petroleum (jet
 fuel), solvents (Pc£ TCE, VC, DCE).
 ilot field study started 3/93.
 xpected completion 3/94.

 redesign.
 tart date (est): 12/92.

 )perational: full scale.
 tart date: 9/85.
 esign: lab scale TS completed.
 ull scale system being installed.
 ield TS report expected 10/93.


Operational: full scale since 5/90.
Completion date (est): 1 /94.

Design: pilot scale TS started
->/92
 tart date (est): 9/93
Expected cost: capital, $3,000;
O&M, $1,268.
Design: pilot scale TS started
8/92. Expected completion
8/94.
Expected cost: capital, $35K;
O&M, $2K.
Design: lab scale TS completed
3/92.
Expected cost: capital, $400K;
O&M, $20K.


 'redesign.


Design: pilot scale TS started
 11 /92. Expected completion
11/95.
 ncurred cost: $25K.
Expected cost: $70K.
Lab and pilot scale TS are being
conducted.
 Design: pilot and lab scale TS
 ongoing.
 Predesign: pilot scale studies
 planned.
 Expected completion 3/94.

 Design: pilot scale started 3/93.
 Expected cost: capital, $1M;
 O&M, $50K.
 Incurred cost: capital, $250K;
 O&M, $10K.

 Operational: full scale since 1 /92
 Expected cost: $90M.
 Operational: full scale since 2/93
 Completion data (est): 9/94.
 erobic treatment.


Nutrient addition. 100% of site
 nder bioremediation.

 erobic treatment, biosparging.

  situ aerobic treatment, hydrogen
 eroxide, nutrient addition
 nitrogen, phosphorus). Ex situ
 reatment, CAt bioreactor. 100% of
 te will undergo bioremediation.

Aerobic treatment, bioventing.


Aerobic treatment, air sparging.
 00% of site will undergo
 lioremediation.
 icrobic treatment, bioventing.
 00% of site will undergo
 jioremediation.
Aerobic treatment, bioventing. 40%
of site under bioremediation.
Aerobic treatment, bioventing. 20%
of site will undergo bioremediation.
 n situ treatment. Ex situ treatment,
 ixed film.

 Aerobic treatment, bioventing,
 nutrient addition [50].
 n situ treatment, capping of
 sediments. Ex situ treatment,
 confined treatment facility (tank).
 Aerobic and anaerobic conditions.

 Anaerobic treatment under sulfate
 reducing conditions.
 Aerobic treatment, bioventing.
 100% of site will undergo
 bioremediation.
 Anaerobic treatment, nutrient
 addition. Less than 1% of site under
 bioremediation. Experiencing;
 nutrient dispersion problems [46].


 Aerobic treatment, pure oxygen
 dissolution system, nutrient addition
 (soil, water, sediments). 100% of
 site under bioremediation.

 Aerobic treatment, bioventing.
   12
                             Engineering Bulletin:  In Situ Biodegradation Treatment

-------
                              Table 3. Superfund, RCRA, UST, TSCA, and Federal Sites (continued)
      Site Location
        (Regions)
        Primary Contaminants
                            Status/Cost
               Treatment
  Fairfield Coal & Gas
  Faiffield,IA(7)
  Offutt AFB
  LaPlatte, NE (7)


  ParkCrty1
  Park City, KS (7)


  Burlington Northern Tie
  Plant
  Somers, MT (8)

  Geraldine Airport
  Geraldine, MT (8)
  Idaho Pole Company
  Bozeman, MT (8)
 Hill AFB1
 Salt Lake City, UT (8)

 Libby Groundwater Site1
 Libby, MT (8)
 Public Service Company1
 Denver, CO (8)


 Beale AFB
 Marysville, CA (9)
 Converse/Montabello
 Corporation Yard
 Montabello, CA (9)

 :ormer Service Station
 Los Angeles, CA (9)
 Orvi
   ppers Company, inc.
 Marine Corps Air/
 Ground Combat Center
 Twenty-Nine Palms,
 CA(9)

 ^aval Air Station Fallen
 allon, NV (9)
 slaval Weapons Station
 >eal Beach, CA (9)

 Oakland Chinatown
 Oakland, CA (9)
 Soil (saturated: sand, silt
 clay)/groundwater: coal tar (BTEX,
 PAHs):

 Soil (vadose: sand, silt): petroleum
 (TRPH), arsenic, barium, lead, zinc.
 Volume: 700 cubic yards.

 Groundwater: petroleum (lube oil),
 benzene.
 Volume: 700K cubic feet.

 Soil/groundwater: wood preserving
 (PAHs).
 Volume: 82K cubic yards.
 Soil (vadose: sand, silt, loam, clay):
 pesticides (aldrin; dieldrin; endrin;
 chlordane; toxaphene; b-BHC;
 4,4'-DDE; 4,4'-DDT; 4,4'-DDD);
 herbicides (2,4-D).
 Sediments/soils/groundwater: PCP,
 PAHs, dioxins/furans.
 Soil: petroleum (JP-4 jet fuel).


 Soil (vadose and saturated)/
 groundwater: wood preserving (PAHs,
 Dyrene, PCP, dioxin, naphthalene,
 anenanthrene, benzene, arsenic).
 /olume: 45K cubic yards.


  roundwater: petroleum.
 Volume: 12M gallon.


 Soil (vadose silty clay): petroleum
 [gasoline,  diesel), solvents (TCE), lead.
 volume: 163K cubic yards.
Soil (vadose silt): petroleum (gas,
diesel).


 oil/groundwater: petroleum.
Volume: 3,000 cubic yards.
Soil (vadose: sand, clay, gravel,
cobbles): wood preserving (PCP,
PAHs, dioxins/furans), arsenic,
chromium.
Volume: 110K cubic yards.
Soil: petroleum (jet fuel, gasoline,
diesel, aviation fluid,
 luid).
transmission
Soil (vadose and saturated
 ilt)/groundwater: petroleum (jet fuel,
 >-xylene. naphthalene, 1-methyl
naphthalene, n-butylbenzene),
 irsenic.
 Jroundwater: petroleum.


 oil (saturated sand): groundwater:
petroleum.
                  Design: pilot scale TS started
                  12/91. Expected completion
                  12/93.
                  Expected cost: 51.6M.

                  Design: pilot scale TS started
                  8/92


                  Design: pilot scale TS completed.
                  Incurred cost: J275K.
                  Expected cost: J650K.

                  Installed: full scale.
                  Start date (est): 7/92.
                  Expected cost: J11M.

                  Predesign.
                  Predesign.
                  Operational: full scale since 9/91.
                  Completion date (est): 9/94.

                  Operational: three pilot scale
                  efforts ongoing.
                  ncurred cost: $4M.
                  TS results available (est): 8/93
                  and 4/94.


                  Completed: full scale 3/92.
                  Start date: 06/89.
                  incurred cost: S500K.

                  Seven processes are planned.
                  4 are in design (pilot  scale),
                  2 are in predesign (full scale), and
                  1 is presently operating
                  completion date 7/95).
                  Lxpected cost: capital J500K;
                  O&M, 136K.

                  Design: pilot scale TS started
                  5/93
                  Expected completion  12/93.

                  Completed: full scale  3/91
                  Start date: 11/88.
                  ncurred cost: 51.6M.


                  'redesign; pilot scale  TS planned.
                  Ixpected completion  11 /94,
                  Expected cost: capital, S4.5M;
                  O&M, S7.7M,


                  Design: full scale.
                 Design: pilot scale TS started
                  0/92.
                  S conducted or in progress:
                 aboratory scale.

                  'olume: 10K cubic yards.
                 Completed: full scale 8/90.
                 >tart date: 3/89.
 Aerobic treatment, injection and
 extraction wells, hydrogen peroxide,
 nitrate addition.
 Aerobic treatment, bioventing.  10% of
 site under bioremediation.
 Groundwater: in situ treatment. Possible
 bioventing for soils.  Anaerobic and
 aerobic conditions [23][24].

 In situ treatment.  Ex situ land treatment.
 Aerobic conditions. 80% of site will
 undergo bioremediation.

 In situ treatment. Ex situ treatment.
 Aerobic and anaerobic conditions.
 In situ treatment, oxygen enhancement,
 nutrient addition.  Ex situ treatment,
 fixed film, slurry reactor. Aerobic
 conditions.

 Aerobic treatment, bioventing.  100% of
 site under bioremediation [4(5].

 In situ treatment (groundwater), ex situ
 and treatment (soil), nutrient addition
 [soil, water).  Also, treatment of
 groundwater in bioreactor.  Aerobic
 :onditions. 75% of site under
 bioremediation.

 Aerobic treatment, hydrogen peroxide,
 nutrient addition, combined bioprocess.


 n situ aerobic treatment, bioventing.
 :x situ aerobic treatment, pile. Results of
 4 bioventing TS expected 2/94.
Aerobic treatment, bioventing, nutrient
addition.  10% of site under
bioremediation.

Aerobic treatment, hydrogen peroxide,
nutrient addition (water), closed loop
system. 65% of site underwent
 )ioremediation.

Aerobic treatment, nutrient addition.
30% of site will undergo bioremediation.
'20 year remedial effort)
Aerobic treatment, bioventing.
Aerobic treatment, bioventing, nutrient
addition (soil), oil/water separation.
Aerobic and anaerobic treatment.
Aerobic treatment, hydrogen peroxide
 nd nutrient addition.
Engineering Bulletin: In Situ Biodegradation Treatment
                                                                                                       13

-------
                          Table 3. Superfund, RCRA, UST, TSCA, and Federal Sites (continued)
    Site Location
      (Regions)
      Primary Contaminants
         Status/Cost
                                                                          Treatment
San Diego Cas and
Electric
San Diego, CA (9)
Williams AFB2
Phoenix, AZ (9)

East 15th Street Service
Station
Anchorage, AK (10)
Eielson AFB'
Fairbanks, Alaska (10)

FairchildAFB
Spokane, WA (10)
Soil (sand): petroleum (gasoline).
Volume: 1,200 cubic yards.

Soil (vadose): petroleum (JP-4 jet fuel).
Soil: petroleum (TPH diesel).
Volume: 1,500 cubic yards.

Soil (sand/silt): petroleum QP-4 jet
fuel).

Soil (vadose and saturated
silt)/groundwater: petroleum, solvents
(TCE).
Completed: full scale 4/93.
Start date: 10/89.
Pilot field testing started 5/92.
Test completed 6/93.


Operational: full scale since 6/92.
Incurred cost: J75K.
Expected cost: $200K.
Operational: pilot full scale.
Start date: 9/91.
Completion date (est): 9/94.
3 separate processes are planned.
The first process is in pre-design;
a pilot scale TS should start 1/95.
The remaining two started pilot
scale TSs in 4/93.
Aerobic treatment. 100% of site
underwent bioremediation [5].

In situ treatment, bacterial
supplementation (non-indigenous micro
aerofilic bacteria).

Aerobic treatment, bioventing. 20% of
site under bioremediation.

Aerobic treatment, bioventing, soil
warming [42]

Aerobic treatment, bioventing, nutrient
addition.
TS - Treatability Study
1  Bioremediation Field Initiative
2  Superfund Innovative Technology Evaluation (SITE) Demonstration
                                                    REFERENCES
1.   U.S. Environmental Protection Agency. Bioremediation
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2.   U.S. Environmental Protection Agency. Superfund Inno-
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3.   U.S. Environmental Protection Agency.  Bioremediation
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                                         8.
                                         9.
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                                Engineering Bulletin:  In Situ Biodegradation Treatment

-------
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