In Situ and Ex Situ Biodegradation Technologies 


for Remediation of Contaminated Sites


Index 

1.0 PURPOSE 

2.0 INTRODUCTION 

3.0 TECHNOLOGY DESCRIPTION 

3.1 In Situ Bioremediation 

3.1.1. Intrinsic In Situ Bioremediation 

3.1.2. Enhanced In Situ Bioremediation 

3.2 Ex Situ Bioremediation 

3.2.1. Solids 

3.2.2. Solid-Liquid Mixtures 

3.2.3. Liquids 

4.0 TECHNOLOGY SELECTION FACTORS 

5.0 SUMMARY 

6.0 ACKNOWLEDGEMENTS 

7.0 REFERENCES 

1.0 PURPOSE 

The U.S. Environmental Protection Agency (EPA) Engineering Issues are a new series of technology transfer documents that summarize the latest available information on selected treatment and site remediation technologies and related issues. They are designed to help 

remedial project managers (RPMs), on-scene coordinators (OSCs), 

contractors, and other site managers understand the type of data and 

site characteristics needed to evaluate a technology for potential applicability to their specific sites. Each Engineering Issue document is 

developed in conjunction with a small group of scientists inside the 

EPA and with outside consultants and relies on peer-reviewed literature, EPA reports, Internet sources, current research, and other pertinent information. For this Engineering Issue paper, the reader is assumed to have a basic technical background and some familiarity with 

bioremediation. Those readers interested in a more basic discussion of 

bioremediation should consult the A Citizen’s Guide to Bioremediation 

(EPA, 2001a). 

The purpose of this Engineering Issue paper for biodegradation technologies is to summarize current information on bioremediation and 

to convey that information clearly and concisely to site managers. The 

Table of Contents indicates the types of information covered in this 

Engineering Issue paper, and this information relies, wherever feasible, 

on independently reviewed process performance information. In an 

effort to keep this Engineering Issue paper short, important information is summarized, while references and Internet links are provided 

for readers interested in additional information; these Internet links, 

verified as accurate at the time of publication, are subject to change. 

2.0 INTRODUCTION 

Bioremediation is a grouping of technologies that use microbiota 

(typically, heterotrophic bacteria and fungi) to degrade or transform 

hazardous contaminants to materials such as carbon dioxide, water, 

inorganic salts, microbial biomass, and other byproducts that may 

be less hazardous than the parent materials. Biological treatment has 

been a major component for many years in the treatment of municipal and industrial wastewaters. In recent years, biological mechanisms have been exploited to remediate contaminated ground water 

and soils (EPA, 1998a; EPA, 2000). This Engineering Issue paper focuses on bioremediation technologies for treating contaminated soils, 

sediments, sludges, ground water, and surface water since these are 
the matrices typically found at contaminated sites. Treatments for municipal wastewater, industrial wastewater, 

and storm water will not be discussed. 

During the late 1970s, 1980s, and early 1990s, wastewater treatment1

 and composting technologies were adapted 

to new applications. At that time, bioremediation of any 

hazardous constituents or waste was considered innovative. However, numerous applications of bioremediation 

are now widely accepted as a remedial alternative and are 

in wide use at sites contaminated with petroleum products and/or hazardous wastes. Some bioremediation technologies, such as cometabolic bioventing, are still in development and should be considered innovative. Other 

bioremediation technologies, such as anaerobic bioventing, are current topics of research. Since bioremediation 

technologies that fall into the innovative or research category have limited field implementation and effectiveness 

data, additional site assessment and treatability studies 

may be needed to confi rm that a selected technology will 

be effective at a specifi c site. 

According to the EPA Office of Solid Waste and Emergency Response annual treatment technology status report (10th Edition) covering the period of Fiscal Year 

(FY) 1982 through FY 1999, bioremediation was planned 

or implemented for source control and ground water remediation for 105 Superfund Remedial Action and 51 

Superfund Removal Action projects. In some cases, bioremediation was applied at multiple operable units on a 

site, each of which is included as a project (EPA, 2001b). 

The following contaminants have been bioremediated 

successfully at many sites: 

●	 Halogenated and non-halogenated volatile organic 


compounds (VOCs)


●	 Halogenated and non-halogenated semi-volatile organic compounds (SVOCs). 

Contaminants with a more limited bioremediation performance include: 

●	 Polycyclic aromatic hydrocarbons (PAHs) 

●	 Organic pesticides and herbicides 

●	 Polychlorinated biphenyls (PCBs). 

Although the applications of bioremediation under the 

EPA Superfund Program, as discussed above, involve 

some of the most diffi cult sites and well-documented site 

surveys, these examples represent only a fraction of the 

bioremediation applications nationwide and worldwide. 

Larger numbers of sites are handled under the Resource 

Conservation and Recovery Act (RCRA), state-led remediation programs, leaking underground storage tank 

(LUST) programs, and state voluntary cleanup programs. 

For example, a survey in 2001 showed that biodegradation technologies, such as land farming and biopiles, are 

applied at 33% of the soil LUST sites. The same survey 

showed that in situ bioremediation, biosparging, and technologies that may use bioremediation, such as monitored 

natural attenuation, are used on 79% of the ground water 

plumes at LUST sites (Kostecki and Nascarella, 2003). 

However, applications of various bioremediation technologies at other site types are not summarized, so their use 

is difficult to characterize. 

Bioremediation remains an active field of technology research and development at both the laboratory and fi eld 

scale. For example, applications to chlorinated aliphatic 

hydrocarbons (CAHs), perchlorate, and methyl tert-butyl ether (MTBE) were developed rapidly in recent years. 

Contaminants with a more limited bioremediation performance record include: 

●	 Applications to additional contaminants and ma­


trices, such as (but not limited to) trinitrotoluene 


(TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine 


(RDX), pesticides and herbicides, and dense non­


aqueous phase liquids (DNAPLs)


●	 Delivery of treatment in difficult media (i.e., frac­


tured bedrock or tight clays)


●	 Refinement of strategies for cost-effective system design and operation. 

The field of bioremediation can be divided into several 

broad categories. For example, bioremediation technologies may be applied to in situ or ex situ media. In situ 

processes treat soils and ground water in place, without 

removal. This approach may be advantageous since the 

costs of materials handling and some environmental impacts may be reduced. However, in situ processes may be 

limited by the ability to control or manipulate the physical and chemical environment in place. An example of 

an in situ bioremediation technology is aerobic bioventing, which has been used at many sites to treat subsurface 

soils contaminated by fuels. In aerobic bioventing, air is 

typically injected into the subsurface to facilitate aerobic 

metabolism of hydrocarbons. Ex situ processes involve the 

removal of the contaminated media to a treatment area. 

Historical information on the development of bioremediation can be found in Martin and Gershuny, 1992; Section 2.3 of EPA, 1993a; and Bradley, 2003. 

2 Engineering Issue 

1
Examples of ex situ processes include land treatment and 

composting. In these processes, soils are excavated, mixed 

with amendments, and operated in a manner that facilitates degradation of the contaminants of concern. 

Another way to divide the bioremediation field is based 

on additives to environmental media. Intrinsic bioremediation depends on indigenous microflora to degrade contaminants without any amendments (EPA, 2000). This 

approach is used in situ and takes advantage of pre-existing processes to degrade hazardous wastes. Intrinsic bioremediation requires careful site assessment and monitoring to make sure that the ongoing processes are protective 

of environmental receptors. Temperature, pH, and other 

factors may also be adjusted and monitored to enhance 

bioremediation. Alternatively, enhanced bioremediation 

facilitates biodegradation by manipulating the microbial 

environment, typically by supplying chemical amendments such as air, organic substrates or electron donors, 

nutrients, and other compounds that affect metabolic reactions (EPA, 2000). Enhanced bioremediation may also 

be called biostimulation when only chemical amendments 

are added. Examples of biostimulation include bioventing, land farming or land treatment, biopiles, composting, 

and sometimes anaerobic reductive dechlorination. Biostimulation technologies may be applied to in situ or ex situ 

situations and may be used to treat soil and other solids, 

ground water, or surface water. In some cases, bioaugmentation, which involves the addition of microbial cultures, 

is used to enhance biotreatment. Bioaugmentation may 

be needed for specific contaminants that are not degraded 

by the indigenous organisms. Bioaugmentation is almost 

always performed in conjunction with biostimulation. For 

example, bioaugmentation has been used at some chlorinated solvent sites as a modification of anaerobic reductive 

dechlorination when indigenous microbes were unable to 

completely dechlorinate the contaminants of concern. 

In bioremediation, fundamental biological activities are 

exploited to degrade or transform contaminants of concern. The biological activity to be exploited depends on 

the specific contaminants of concern and the media where 

the contamination is located. For example, in aerobic environments, many microbes are able to degrade organic 

compounds, such as hydrocarbons. These microbes gain 

energy and carbon for building cell materials from these 

biochemical reactions. At many sites with fuel contamination, the amount of oxygen present limits the extent of 

biotreatment. Thus, by adding oxygen in the form of air, 

contaminant degradation proceeds directly. 

In cometabolism, microbes do not gain energy or carbon 

from degrading a contaminant. Instead, the contaminant 

is degraded via a side reaction. Technologies based on cometabolism are more difficult to use since the microbes do 

not benefit from the desired reactions. Cometabolic bioventing is an example of cometabolism. In this technology, microbes may be fed propane, and they degrade trichloroethylene (TCE) or less chlorinated ethenes as well. 

Depending on the contaminant of concern and the media, a technology may exploit aerobic or anaerobic metabolism. Aerobic metabolism is more commonly exploited 

and can be effective for hydrocarbons and other organic 

compounds. Many organisms are capable of degrading 

hydrocarbons using oxygen as the electron acceptor and 

the hydrocarbons as carbon and energy sources. In some 

cases, contaminants are aerobically degraded to carbon 

dioxide, water, and microbial biomass, but in other cases, 

the microbes do not completely degrade contaminants. 

Aerobic technologies may also change the ionic form of 

metals. If a site contains mixed metal and organic wastes, 

it is necessary to consider whether the oxidized forms of 

the metal species (such as arsenic) will be environmentally acceptable. 

Anaerobic metabolism involves microbial reactions occurring in the absence of oxygen and encompasses many processes including fermentation, methanogenesis, reductive 

dechlorination, sulfate-reducing activities, and denitrifi cation. Depending on the contaminant of concern, a subset 

of these activities may be cultivated. In anaerobic metabolism, nitrate, sulfate, carbon dioxide, oxidized metals, or 

organic compounds may replace oxygen as the electron acceptor. For example, in anaerobic reductive dechlorination, 

chlorinated solvents may serve as the electron acceptor. 

Phytoremediation or phytotechnology, which involves 

the use of plants to remediate contaminated media, is 

not discussed in this Engineering Issue paper because this 

technique can involve a number of physical and chemical 

processes in addition to, or in place of, bioremediation. 

More information on phytoremediation can be found 

at the EPA Web site (http://www.clu-in.org/techfocus/ 

default.focus/sec/Phytoremediation/cat/Overview/) or at 

the Interstate Technology & Regulatory Council (ITRC) 

Web site (http://www.itrcweb.org/gd_Phyto.asp). 

When selecting a bioremediation technology, it is important to consider the contaminants of concern, contaminated matrix, potential biological pathways to degrade 

a contaminant, and current conditions at a site. For exEngineering Issue 3
ample, TCE can be degraded via aerobic and anaerobic 

mechanisms. If ground water is contaminated with TCE, 

current ground water conditions may be helpful in deciding which biological mechanism to exploit. If ground water is already anaerobic, then anaerobic reductive dechlorination may be the best approach. However, if the TCE 

plume is diffuse and the ground water is aerobic, it may 

be possible to use cometabolic technologies. 

A key concept in evaluating all bioremediation technologies is microbial bioavailability. Simply stated, if the contaminant of concern is so tightly bound up in the solid 

matrix (either chemically or physically) that microorganisms cannot access it, then it cannot be bioremediated. 

However, low microbial bioavailability does not imply an 

absence of risk; compounds may be available to environmental receptors depending on the receptor and routes 

of exposure. For example, if a child ingests contaminated 

soil, digestive processes may release contaminants that 

were not available to microbes. 

Many of the guidance documents discussed in this Engineering Issue paper address bioavailability from a practical engineering perspective. A more theoretical summary 

of the implications of bioavailability is presented in Luthy 

et al. (1997) and Alexander (2000). Implementation of 

enhanced bioremediation technologies can, in some cases, 

transfer mass from the solid phase to the mobile (i.e., water) phase through a number of mechanisms, including 

the generation of biosurfactants. 

Thus, when selecting a bioremediation technology for a 

specific site, it is prudent to consider the contaminants of 

concern, potential degradation intermediates and residuals of the contaminants, co-contaminants, environmental 

receptors, routes of exposure, and buffer zones between 

contamination and receptors. Bioremediation technologies have proven to be protective and cost-effective solutions at many sites. However, conditions at a specifi c site 

may not be appropriate. In addition, worker safety issues 

are a consideration in selection, design, and operation of 

bioremediation technologies. These technologies may involve the use of strong oxidants and/or highly reactive, 

potentially explosive chemicals. 

3.0 TECHNOLOGY DESCRIPTION 

This section discusses in situ and ex situ bioremediation. 

Technologies within each of these broad categories are 

presented, including representative process schematics or 

illustrations. Note that there are multiple vendors of some 

technologies, each applying proprietary components or 

processes to their particular technology. The continually 

changing nature of bioremediation and the space limitations of this Engineering Issue paper preclude comprehensive presentation of such information. Additional information on remediation technologies and links to other 

sources are available on the World Wide Web, including: 

●	 Federal Remediation Technologies Roundtable 


(FRTR) at http://www.frtr.gov/


●	 Air Force Center for Environmental Excellence (AFCEE) Technology Transfer Program at 

http://www.afcee.brooks.af.mil/products/techtrans/ 

treatmenttechnologies.asp 

●	 ITRC guidance documents and case studies at 


http://www.itrcweb.org/gd.asp and


http://www.itrcweb.org/successstories.asp.


For information on specific compounds, a Biocatalysis/ 

Biodegradation Database developed by the University of 

Minnesota may be helpful and can be found at 

http://umbbd.ahc.umn.edu/. 

3.1 In Situ Bioremediation 

There are two major types of in situ bioremediation: intrinsic and enhanced. Both rely on natural processes to 

degrade contaminants with (enhanced) or without (intrinsic) amendments. 

In recent years, in situ bioremediation concepts have been 

applied in treating contaminated soil and ground water. 

Removal rates and extent vary based on the contaminant of 

concern and site-specific characteristics. Removal rates also 

are affected by variables such as contaminant distribution 

and concentration; co-contaminant concentrations; indigenous microbial populations and reaction kinetics; and 

parameters such as pH, moisture content, nutrient supply, and temperature. Many of these factors are a function 

of the site and the indigenous microbial community and, 

thus, are difficult to manipulate. Specific technologies may 

have the capacity to manipulate some variables and may be 

affected by other variables as well; these specifi c issues are 

discussed with each technology in the following sections 

(AFCEE, 1996; EPA, 1998a; EPA, 2000; FRTR, 2003). 

When in situ bioremediation is selected as a treatment, sitemonitoring activities should demonstrate that biologically 

mediated removal is the primary route of contaminant 

removal. Sampling strategies should consider appropriate 

analytes and tests, as well as site heterogeneity. In some 

4 Engineering Issue 
cases, extensive sampling may be required to distinguish 

bioremediation from other removal mechanisms or statistical variation. Small-scale treatability studies using samples 

from the contaminated site may also be useful in demonstrating the role that biological activity plays in contaminant removal (EPA, 1995b; EPA, 1998a; EPA, 2000). 

3.1.1 Intrinsic In Situ Bioremediation 

Intrinsic bioremediation relies on natural processes to degrade contaminants without altering current conditions 

or adding amendments. Intrinsic bioremediation may 

play a role in monitored natural attenuation (MNA) sites, 

which is a broader term defined by the National Research 

Council (NRC) and EPA as “biodegradation, dispersion, 

dilution, sorption, volatilization, radioactive decay, and 

chemical or biological stabilization, transformation or destruction of contaminants” (NRC, 2000; EPA, 1999). 

Natural attenuation (NA) relies on natural physical, 

chemical, and biological processes to reduce or attenuate 

contaminant concentrations. Under favorable conditions, 

NA will reduce the concentration, mass, toxicity, mobility, and/or volume of contaminants in soil and ground 

water. Natural processes involved in NA include dilution, 

dispersion, sorption, volatilization, chemical reactions 

such as oxidation and reduction, biological reactions, and 

stabilization. EPA prefers those processes that degrade 

contaminants and expects that NA will be most appropriate for subsurface plumes that are stable. Some processes 

have undesirable results, such as creation of toxic degradation products or transfer of contaminants to other media 

as noted in the Seminar Series on Monitored Natural Attenuation for Ground Water (EPA, 1998a). 

Implementing MNA requires a thorough site assessment 

and development of a conceptual model of the site. After 

determining the presence of a stable or shrinking plume, 

site-specific, risk-based decisions using multiple lines of 

evidence may facilitate implementation of MNA at a 

site. While MNA is somewhat passive in that nothing is 

being added to the contamination zone, MNA requires 

active monitoring, which should be included as part of 

the design plan for a site. In some cases, such long-term 

monitoring may be more expensive than active remediation. MNA is only applicable to carefully controlled and 

monitored sites and must reduce contaminant concentrations to levels that are protective of human health and 

the environment in reasonable timeframes (EPA, 1998a). 

Depending on site-specific conditions, MNA may be a 

reasonable alternative for petroleum hydrocarbons as well 

as chlorinated and non-chlorinated VOCs and SVOCs 

(EPA, 1999a; EPA, 1999b). 

Good overviews of natural attenuation are provided by: 

●	 Natural Attenuation for Groundwater Remediation 

(NRC, 2000) 

●	 The MNA page of the AFCEE Technology Transfer 

Program Web site at 

http://www.afcee.brooks.af.mil/products/techtrans/ 

monitorednaturalattenuation/default.asp. 

●	 ITRC in situ bioremediation publications at


http://www.itrcweb.org/gd_ISB.asp.


Detailed protocols for evaluation of natural attenuation at 

sites with petroleum hydrocarbon and CAH contamination can be found at 

http://www.afcee.brooks.af.mil/products/techtrans/ 

monitorednaturalattenuation/Protocols.asp. 

3.1.2 Enhanced In Situ Bioremediation 

Enhanced bioremediation can be applied to ground water, vadose zone soils, or, more rarely, aquatic sediments. 

Additives such as oxygen (or other electron acceptors), 

nutrients, biodegradable carbonaceous substrates, bulking agents, and/or moisture are added to enhance the activity of naturally occurring or indigenous microbial populations (FRTR, 2003). 

3.1.2.1 Vadose Zone Soil Remediation 

While the fundamental biological activities exploited by 

in situ bioremediation may occur naturally, many sites 

will require intervention to facilitate cleanup. For example, the addition of organic substrates, nutrients, or air 

may provide the appropriate environment for specifi c microbial activities or enhanced removal rates. In general, 

hydrocarbons and lightly chlorinated contaminants may 

be removed through aerobic treatment. Highly chlorinated species are degraded primarily through anaerobic treatment. Both anaerobic and aerobic treatment may occur 

through direct or cometabolic pathways (EPA, 2000). 

The primary in situ biological technology applicable to 

the unsaturated zone is bioventing, which is categorized 

as either aerobic, cometabolic, or anaerobic depending on 

the amendments used. 

Engineering Issue 5
3.1.2.1.1 Aerobic Bioventing 

Bioventing has a robust track record in treating aerobically degradable contaminants, such as fuels. In aerobic 

bioventing, contaminated unsaturated soils with low oxygen concentrations are treated by supplying oxygen to facilitate aerobic microbial biodegradation. Oxygen is typically introduced by air injection wells that push air into 

the subsurface (see Figure 3-1); vacuum extraction wells, 

which draw air through the subsurface, may also be used. 

When building foundations or similar structures are close 

to the site, the extraction mode may be used to avoid the 

buildup of contaminated, and possibly explosive, vapors 

in the building basements. Extracted gases may require 

treatment since volatile compounds may be removed from 

the ground. Compared with soil vapor extraction (SVE), 

bioventing employs lower air fl ow rates that provide only 

the amount of oxygen required to enhance removal. Operated properly, the injection of air does not result in the 

release of the contaminants to the atmosphere through 

volatilization because of these low flow rates (AFCEE, 

1996; EPA, 2000; FRTR, 2003). 

Bioventing is designed primarily to treat aerobically biodegradable contaminants, such as non-chlorinated VOCs 

and SVOCs (e.g., petroleum hydrocarbons), that are located in the vadose zone or capillary fringe (EPA, 2000; 

FRTR, 2003). The U.S. Air Force Bioventing Initiative 

and the EPA Bioremediation Field Initiative demonstrated that bioventing was effective under a wide variety of 

site conditions at about 125 sites. The experience from 

bioventing demonstrations at these sites was condensed 

into a manual, Bioventing Principles and Practice (EPA 

1995a), which provides information about the applicability of bioventing and outlines its use and design (AFCEE, 

1996). Data collected during the bioventing demonstrations also provide information about the rates of contaminant removal observed. In addition to the variables 

Appropriate 

gas mix 

Soil gas 

sampling points 

Figure 3-1. Aerobic bioventing in injection mode. (Adapted from 

EPA, 2004c) 

discussed initially, bioventing rates and system design are 

affected by soil gas permeability, soil water content, depth 

to contamination, and oxygen supply and radius of influence (EPA, 2000; FRTR, 2003). The costs for aerobic 

bioventing are about $50/cubic yard (AFCEE, 1996). 

Based on experience gained to date in applying bioventing 

to fuels, site heterogeneity is a principal impediment to 

establishing that biological activity is the principal mode 

of removal at these sites. Measurements of the rate and 

amount of contaminant removed, oxygen supply, and carbon dioxide generation, as well as mass balances relating 

to these three amounts, may be useful in establishing bioremediation as the primary mechanism of removal. For 

sites where other contaminants are to be treated by bioventing, other factors may be considered in establishing 

biological activity as the primary mechanism of removal 

(AFCEE, 1996; EPA, 2000; FRTR, 2003). 

Regulatory acceptance of this technology has been obtained in 30 states and in all 10 EPA regions (FRTR, 

2003). The use of this technology in the private sector is 

increasing following the U.S. Air Force Bioventing Initiative and the EPA Bioremediation Field Initiative. 

In addition to fuels treatment, aerobic bioventing has 

treated a variety of other contaminants including nonhalogenated solvents such as benzene, acetone, toluene, 

and phenol; lightly halogenated solvents such as 1,2-dichloroethane, dichloromethane, and chlorobenzene; and 

SVOCs such as low-molecular-weight PAHs. Since the 

experience with these other types of contaminants is more 

limited, laboratory- and pilot-scale studies may be needed 

to evaluate effectiveness, design the bioventing system, 

and estimate treatment times. 

Bioventing has proven to be a useful technology at many 

sites under a variety of conditions, but like all technologies, bioventing has some limitations. One set of bioventing limitations involves the ability to deliver oxygen to 

the contaminated soil. For example, soils with extremely 

high moisture content may be difficult to biovent because 

of reduced soil gas permeability. Similarly, low-permeability soils also may pose some difficulties for bioventing because of a limited ability to distribute air through 

the subsurface. In both cases, the design of the bioventing 

system may be able to compensate for low permeability. 

Additionally, sites with shallow contamination can pose a 

challenge to bioventing because of the difficulty in developing a system design that can minimize environmental 

release and achieve sufficient aeration. In this situation, 

6 Engineering Issue 
operating the system in the extraction mode may circumvent the difficulty (AFCEE, 1996; FRTR, 2003). 

Another limitation is that bioventing will not stimulate 

contaminant bioremediation if the contaminated zone is 

aerobic. If a soil gas survey measures soil oxygen levels 

consistently above 2–5%, then the soil is suffi ciently aerated for biodegradation to occur and oxygen is not limiting degradation. Bioventing will not enhance bioremediation in this situation. This situation is unusual and, if 

encountered, may indicate that some other contaminants, 

such as metals, are inhibiting degradation (AFCEE, 1996; 

EPA, 1998a). 

While bioventing is relatively inexpensive, bioventing can 

take a few years to clean up a site depending on contaminant concentrations and site-specific removal rates. If a 

quicker cleanup is needed, more intensive ex situ technologies may be more appropriate (AFCEE, 1996; EPA, 

2000; FRTR, 2003). 

3.1.2.1.2 Cometabolic Bioventing 

Cometabolic bioventing has been used at a few sites to 

treat chlorinated solvents such as TCE, trichloroethane 

(TCA), and dichloroethene (DCE). The equipment used 

in cometabolic bioventing is similar to aerobic bioventing, 

but cometabolic bioventing exploits a different biological 

mechanism. Similar to bioventing, cometabolic bioventing 

involves the injection of gases into the subsurface; however, cometabolic bioventing injects both air and a volatile 

organic substrate, such as propane. The concentrations in 

this gas mixture should be well below the lower explosive 

limit (LEL), and should be monitored in soil gas (AFCEE, 

1996; EPA, 1998a; EPA, 2000; FRTR, 2003). 

Cometabolic bioventing exploits competitive reactions 

mediated by monooxygenase enzymes (EPA, 2000). Monooxygenases catalyze the oxidation of hydrocarbons, often through epoxide intermediates, but these enzymes can 

also catalyze the dechlorination of chlorinated hydrocarbons. Thus, by supplying an appropriate organic substrate 

and air, cometabolic bioventing can elicit the production 

of monooxygenases, which consume the organic substrate 

and facilitate contaminant degradation (AFCEE, 1996; 

EPA, 1998a). 

Cometabolic bioventing has been used to treat lightly 

chlorinated compounds in the vadose zone or capillary 

fringe. In addition to the variables discussed in the previous aerobic bioventing section, the degradation rate and 

design of cometabolic bioventing systems are dependent 

on many factors including soil gas permeability, organic substrate concentration, type of organic substrate selected, and oxygen supply and radius of infl uence. Unlike 

many variables that are determined by site conditions, the 

selection and concentration of the organic substrate are 

controllable and can be important to the removal rate. 

Treatability or bench-scale testing can be useful in selecting the organic substrate and concentration for a site. In 

addition, small-scale testing can demonstrate that full dechlorination is observed at a site (AFCEE, 1996; EPA, 

1998a; EPA, 2000; FRTR, 2003). 

Establishing cometabolic bioventing as the primary mechanism of removal in the field is challenging. Unlike aerobic 

bioventing, the oxygen use and chlorinated solvent removal are not related stoichiometrically because the metabolism of added organic substrates also consumes oxygen. 

As a result, measurements of oxygen use, carbon dioxide 

generation, and contaminant removal cannot be linked 

stoichiometrically. Indirect measures, such as measuring 

chloride ion accumulation in the soil and correlating that 

accumulation to contaminant removal, have been useful 

at some sites. In addition, collecting data to demonstrate 

degradation of the organic substrate (by a shutdown test) 

in the field may be helpful, especially in conjunction with 

laboratory testing using contaminated soil from the site 

(AFCEE, 1996; EPA, 1998a; FRTR, 2003). 

Cometabolic bioventing has been successfully demonstrated at a few sites. The Remediation Technology Development Forum (RTDF) Bioremediation Consortium 

has conducted cometabolic bioventing demonstrations at 

Dover and Hill Air Force Bases. At Dover, a fi eld demonstration of cometabolic bioventing was conducted at 

Building 719—a site contaminated with fuel and solvents 

during engine inspection and maintenance operations. 

The targeted contaminants of the demonstration and 

their concentrations were TCE, as high as 250 mg/kg; 

1,1,1-TCA, 10–1,000 mg/kg; and cis-1,2-DCE, 1–20 

mg/kg. Laboratory tests were used to select propane as 

the cometabolic substrate and to predict that a substrate 

acclimation period would be needed. The test plot was acclimated to propane addition through pulsed propane/air 

injections for three months, then the test plot was operated for 14 months with continuous propane injection. 

Concentrations of TCE, TCA, and DCE were reduced 

to less than 0.25, 0.5, and 0.25 mg/kg, respectively. Soil 

chloride accumulation analysis confi rmed biodegradation 

as the mechanism of removal (EPA, 1998a). 

Engineering Issue 7
As with aerobic bioventing, difficulty in distributing gases 

in the subsurface may make the application of cometabolic bioventing more complicated. In some cases, such as 

high moisture content or low-permeability soils, the design of the cometabolic system may compensate for poor 

permeability. In the case of shallow contamination, designing a cometabolic bioventing system that minimizes 

environmental release and achieves sufficient aeration and 

organic substrate distribution may be diffi cult (AFCEE, 

1996; EPA, 1998a; EPA, 2000; FRTR, 2003). 

Another limitation to cometabolic bioventing is the lack 

of experience with the technology. Since cometabolic bioventing has been demonstrated at a limited number of 

sites, the technology is not as well understood as aerobic 

bioventing. Researchers are still studying which contaminants are amenable to this type of biodegradation and 

what removal rates can be expected. Establishing that biological processes are the primary mechanism for contaminant removal is also more difficult. Finally, regulatory and 

public acceptance is not as strong for cometabolic bioventing as for aerobic bioventing. However, treatability 

testing of samples from the contaminated site and pilotscale testing may alleviate many of these limitations and 

concerns (EPA, 1998a). As more sites are remediated using cometabolic bioventing, these limitations may ease. 

3.1.2.1.3 Anaerobic Bioventing 

While aerobic and cometabolic bioventing are useful for 

degrading many hydrocarbons and lightly chlorinated 

compounds, some chlorinated species are not effectively 

treated aerobically. Microbes may degrade these contaminants directly via anaerobic reductive dechlorination or 

through anaerobic cometabolic pathways. Anaerobic reductive dechlorination is a biological mechanism typically 

marked by sequential removal of chlorine from a molecule. 

Microbes possessing this pathway do not gain energy from 

this process. Anaerobic cometabolism is similar to aerobic cometabolism in that microbes fortuitously degrade 

contaminants while reducing other compounds (cometabolites). Anaerobic bioventing may use both biological 

mechanisms to destroy the contaminants of concern. 

Anaerobic bioventing uses the same type of gas delivery 

system as the other bioventing technologies, but injects 

nitrogen and an electron donor, instead of air, to establish 

reductive anaerobic conditions. The nitrogen displaces 

the soil oxygen, and small amounts of an electron donor gas (such as hydrogen and carbon dioxide) produce 

reducing conditions in the subsurface, thereby facilitating microbial dechlorination. Volatile and semi-volatile 

compounds may be produced during anaerobic bioventing. Some of these compounds may be slow to degrade 

under anaerobic conditions. These compounds may be 

treated in two ways. Volatile compounds may diffuse into 

the soils surrounding the treatment zone, where aerobic 

degradation may occur. SVOCs and VOCs remaining in 

the treatment zone may be treated by following anaerobic 

bioventing with aerobic bioventing. Since aerobic and anaerobic bioventing share similar gas delivery systems, the 

switch can be made by simply changing the injected gas. 

Anaerobic bioventing is an emerging technology that has 

been demonstrated in several laboratory and fi eld studies. This process may be useful in treating highly chlorinated compounds such as tetrachloroethene (PCE), TCE, 

RDX, pentachlorophenol, and pesticides such as lindane 

and dichlorodiphenyltrichloroethane (DDT). Due to the 

limited experience with this technique, laboratory, pilot, 

and field demonstrations are recommended to confi dently apply this technology to remediate a site. 

As with the other bioventing technologies, the ability to 

deliver gases to the subsurface is important. Soils with 

high moisture content or low gas permeability may require careful system design to deliver appropriate levels of 

nitrogen and the electron donor. Sites with shallow contamination or nearby buildings are also a challenge since 

this technology is operated by injecting gases. In addition, 

anaerobic bioventing can take a few years to clean up a 

site depending on the contaminant concentrations and 

site-specifi c removal rates. If a quicker cleanup is needed, 

other technologies may be more appropriate. 

3.1.2.2 Surficial Soil Remediation 

If contamination is shallow, soil may be treated in place 

using techniques similar to land treatment or composting. Variations of these technologies involve tilling shallow soils and adding amendments to improve aeration 

and bioremediation. This process is similar to the land 

farming and composting discussed later in the Ex Situ 

Bioremediation section of this Engineering Issue paper, 

except that the soils are not excavated. 

Since these treatments do not include an impermeable 

sublayer, contaminant migration may be a concern depending on the contaminants of concern and treatment 

amendments. A more prudent approach would be to excavate soils and treat them in lined beds. 

8 Engineering Issue 
This technology will generally require special permission 

from the applicable regulatory agency. Frequently, some 

type of monitoring for contaminant migration is required. 

3.1.2.3 Ground Water and Saturated Soil Remediation 

In situ bioremediation techniques applicable to ground 

water and saturated soil include dechlorination using anaerobic reducing conditions, enhanced aerobic treatment, 

biological reactive barriers that create active remediation 

zones, and bioslurping/biosparging techniques that promote aerobic degradation. 

3.1.2.3.1 Anaerobic Reductive Dechlorination 

Anaerobic reductive dechlorination has been used at 

many sites where the ground water has been contaminated with chlorinated solvents, such as TCE or PCE. In 

this treatment, organic substrates are delivered to the subsurface where they are fermented. The fermentation creates an anaerobic environment in the area to be remediated and generates hydrogen as a fermentation byproduct. 

The hydrogen is used by a second microbial population 

to sequentially remove chlorine atoms from chlorinated 

solvents (AFCEE, 2004). If PCE were degraded via reductive dechlorination, the following sequential dechlorination would be observed: PCE would be converted to 

TCE, then to DCE, vinyl chloride (VC), and/or dichloroethane (EPA, 1998a). 

Anaerobic dechlorination may also occur via cometabolism where the dechlorination is incidental to the metabolic activities of the organisms. In this case, contaminants 

are degraded by microbial enzymes that are metabolizing 

other organic substrates. Cometabolic dechlorination 

does not appear to produce energy for the organism. At 

pilot- or full-scale treatment, cometabolic and direct dechlorination may be indistinguishable, and both processes may contribute to contaminant removal. The microbial 

processes may be distinguished in the more controlled environment of a bench-scale system (EPA, 1998a). 

Anaerobic reductive dechlorination is primarily used to 

treat halogenated organic contaminants, such as chlorinated solvents. As well as the variables discussed initially, the 

treatment rate and system design are dependent on several factors including site hydrology and geology, type and 

concentration of organic substrates, and site history. As 

with cometabolic bioventing, the selection of organic substrate and the concentration used are controllable and can 

be important to the removal rate. Treatability or benchscale testing can be useful in selecting the best organic substrate and concentration for a site. In addition, small-scale 

testing can demonstrate that full dechlorination is possible 

at a site. In some cases, dechlorination may stall at DCE 

despite the presence of sufficient electron donors. If a site 

does not demonstrate full dechlorination (either as part 

of site assessment or in microcosm testing), a combined 

treatment strategy, such as anaerobic treatment followed 

by aerobic treatment, may be successful. Alternatively, 

bioaugmentation may improve the dechlorination rate 

(AFCEE, 1996; EPA, 1998a; EPA, 2000; FRTR, 2003). 

Research methods used to establish that anaerobic dechlorination is occurring at a site are similar to those discussed 

in previous sections of this Engineering Issue paper. 

Regulatory considerations for this technology involve the 

Safe Drinking Water Act and RCRA hazardous waste regulations, as well as state and local regulations. At the time 

that this Engineering Issue paper was written, judgments 

about the applicability of this technology were made on 

a case-by-case basis. The regulations can impact the design and operation of the treatment system as well as the 

overall applicability. Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents: Fundamentals and 

Field Applications (EPA, 2000) provides more detailed 

information about regulatory concerns and applicability 

(EPA, 1998a). 

Additional information on anaerobic reductive dechlorination is available from the following source: Principles 

and Practices of Enhanced Anaerobic Bioremediation of 

Chlorinated Solvents (AFCEE, 2004). 

3.1.2.3.2 Aerobic Treatment 

Similar to bioventing, enhanced in situ aerobic ground 

water bioremediation processes are used in situations 

where aerobically degradable contaminants, such as fuels, 

are present in anaerobic portions of an aquifer. In these 

situations, air or other oxygen sources are injected into 

the aquifer near the contamination (see Figure 3-2). As 

the oxygenated water migrates through the zone of contamination, the indigenous bacteria are able to degrade 

the contaminants (EPA, 1998a; EPA, 2000). 

Aerobic treatment may also be used to directly or cometabolically degrade lightly chlorinated species, such as DCE 

or VC. In the direct aerobic pathway, air is injected into 

the aquifer. The microbes appear to generate energy by 

oxidizing the hydrocarbon backbone of these contaminants, resulting in the release of chloride (EPA, 2000). 

Engineering Issue 9
This process has been used to complete contaminant removal following anaerobic treatment at several sites (EPA, 

1998a; EPA, 2000). 

Nutrient 

adjustment 

pH 

adjustment 

To further 

treatment discharge 

or recharge 

Vadose zone 

Injection Saturated zone 

well 

Contaminated 

ground water 

Ground water 

extraction wells 

Air blower 

Submersible 

pump 

Figure 3-2. Aerobic treatment. (Adapted from FRTR, 2003) 

Cometabolic aerobic treatment is founded on the same 

biological principles as cometabolic bioventing and involves the addition of oxygen and organic substrates, 

such as methane, to the aquifer. As with other cometabolic processes, these organic substrates are metabolized 

by enzymes that incidentally degrade the contaminant. In 

this treatment, sufficient oxygen must be present to fuel 

the oxidation of both the substrate and contaminant (AFCEE, 1996; EPA, 1998a). 

3.1.2.3.3 Amendment Delivery 

In situ ground water treatment, either aerobic or anaerobic, may be configured as direct injection of air or aqueous streams or as ground water recirculation. In direct injection, amendments, such as organic substrates, oxygen 

sources, or nutrients, are directly injected into the aquifer. 

For example, oxygen may be sparged into the aquifer as 

a gas. Lactate or hydrogen peroxide may be injected as 

a liquid stream; when using hydrogen peroxide, caution 

should be used as it may act as a disinfectant. In some cases, both liquids and gases are added. The ground water recirculation configuration involves extracting ground water, amending it as needed, and then re-injecting it back 

into the aquifer. Recirculation may also be conducted below the ground surface by extracting ground water at one 

elevation, amending it in the ground, and re-injecting it 

into another elevation (EPA, 1998a; EPA, 2000). 

In addition to the variables discussed initially, the 

treatment rates and system design are the result of 

several factors including site hydrology and geology, amendment to be added, solubility of air or 

oxygen sources, and site history. The low solubility of air in water often limits reaction rates and 

may make this process impractical if cleanup time 

is short (AFCEE, 1996; EPA, 1998a; EPA, 2000; 

FRTR, 2003). 

Careful attention also should be given to co-contaminants, especially metals. When an aquifer environment is converted from an aerobic to an anaerobic environment, a variety of chemical species 

may become soluble. Therefore, it is important to 

check for changes in co-contaminants such as arsenic, which may be solubilized during the treatment process (AFCEE, 1996; EPA, 1998a; EPA, 

2000; FRTR, 2003). 

3.1.2.3.4 Biological Reactive Barriers 

Biological reactive barriers consist of an active bioremediation zone created in the contamination zone. These 

barriers may be constructed to exploit aerobic or anaerobic processes depending on the contaminant of concern 

and site needs. A trench is excavated and filled with sand 

pre-mixed with nutrient-, oxidant-, or reductant-rich 

materials to form a bioremediation zone (see Figure 3­

3). Alternatively, a bioremediation curtain can be formed 

by injection of amendments or recirculation of amended 

ground water at the toe of the plumes (EPA, 2000). Contaminants biodegrade as they pass through the permeable 

reactive barrier (PRB). 

Trenches are dug with a backhoe or similar device and 

are filled with permeable materials, such as sand or bark 

mulch, that are mixed together prior to placement. Nutrients, degradable carbonaceous substrates (e.g., manure, compost, and wheat straw), and other additives 

are introduced into the permeable layer. As ground water flows through the treatment zone, indigenous microbes are stimulated to improve natural biodegradation 

(NFESC, 2000). Biological PRBs have been studied by 

the PRB RTDF, and further information, including case 

studies, may be found at http://www.rtdf.org/public/ 

10 Engineering Issue 

permbarr/pbar_qa.htm and http://costperformance.org/ 

search.cfm. 
GW flow 

Permeable 

reactive barrier 

Clay lens 

Figure 3-3. Permeable reactive barrier. (Adapted from EPA, 2000) 

3.1.2.3.5 Biosparging and Bioslurping 

Biosparging (similar to air sparging) involves the injection 

of a gas (usually air or oxygen) and occasionally gas-phase 

nutrients, under pressure, into the saturated zone to promote aerobic biodegradation (GWRTAC, 1996). In air 

sparging, volatile contaminants also can be removed from 

the saturated zone by desorption and volatilization into 

the air stream. Emphasis on the biological degradation rate 

over physical removal, as well as lower rates of air injection, 

are what distinguishes this technology from air sparging. 

Typically, biosparging is achieved by injecting air into a 

contaminated subsurface formation through a specially 

designed series of injection wells. The air creates an inverted cone of partially aerated soils surrounding the injection 

point. The air displaces pore water, volatilizes contaminants, and exits the saturated zone into the unsaturated 

zone. While in contact with ground water, oxygen dissolution from the air into the ground water is facilitated and 

supports aerobic biodegradation. 

A number of contaminants have been successfully addressed with biosparging technology, including gasoline 

components such as benzene, toluene, ethylbenzene, and 

xylenes (BTEX) and SVOCs. Biosparging is most often 

recommended at sites impacted with mid-weight petroleum hydrocarbon contaminants, such as diesel and jet 

fuels. Lighter contaminants, such as gasoline, tend to be 

easily mobilized into the unsaturated zone and physically 

removed. Heavier contaminants, such as oils, require longer 

remedial intervals because of reduced microbial bioavailability with increasing carbon chain length (EPA, 2004b). 

Care must be taken to determine whether contaminant 

concentrations in soil gas and released vapors resulting 

from biosparging require treatment. For this reason, biosparging may be implemented along with SVE or bioventing as a remedy for increased contaminant concentrations 

in the unsaturated zone. The SVE wells are designed to 

capture the introduced air and contaminant vapors (EPA, 

2004b). Figure 3-4 depicts a typical biosparging system 

with optional SVE system. Alternatively, a lower-flow 

bioventing system may be added to facilitate bioremediation of volatilized contaminants in the vadose zone. 

Atmospheric 

discharge 

Blower 

Vapor 

treatment 

Compressor 

UST 

Air 

flow 

Air 

flow 

Air 

flow 

Vapor phase 

Adsorbed phase 

Dissolved phase 

Water table 

Figure 3-4. Biosparging system (used with soil vapor extraction). 

(Adapted from NMED, 2004) 

One specialized form of biosparging involves the injection of organic gases into the saturated zone to induce cometabolic biodegradation of chlorinated aliphatic hydrocarbons, and this is analogous to cometabolic bioventing 

discussed in this Engineering Issue paper. The injection 

of gases below the water table distinguishes biosparging 

from bioventing. 

In contrast to cometabolic bioventing, the solubility of 

organic gases in water limits delivery of the primary substrate during cometabolic biosparging applications. This 

solubility limitation affects the economics of cometabolic 

biosparging applications since the interaction between 

bacterial cometabolite consumption and cometabolite water solubility directly determines the number of methane 

biosparging injection wells required at a given site. Safety precautions similar to those required for cometabolic 

bioventing apply to cometabolic biosparging (Sutherson, 

2002; AFCEE, 1998). 

Engineering Issue 11
Bioslurping (also known as multi-phase extraction) is effective in removing free product that is floating on the water table (Battelle, 1997). Bioslurping combines the two 

remedial approaches of bioventing and vacuum-enhanced 

free-product recovery. Bioventing stimulates aerobic bioremediation of contaminated soils in situ, while vacuumenhanced free-product recovery extracts light, nonaqueous-phase liquids (LNAPLs) from the capillary fringe and 

the water table (AFCEE, 2005). Bioslurping is limited to 

25 feet below ground surface as contaminants cannot be 

lifted more than 25 feet by this method. 

A bioslurping tube with adjustable height is lowered into 

a ground water well and installed within a screened portion at the water table (see Figure 3-5). A vacuum is applied to the bioslurping tube and free product is “slurped” 

up the tube into a trap or oil water separator for further 

treatment. Removal of the LNAPL results in a decline in 

the LNAPL elevation, which in turn promotes LNAPL 

flow from outlying areas toward the bioslurping well. As 

the fluid level in the bioslurping well declines in response 

to vacuum extraction of LNAPL, the bioslurping tube 

also begins to extract vapors from the unsaturated zone. 

This vapor extraction promotes soil gas movement, which 

in turn increases aeration and enhances aerobic biodegradation (Miller, 1996). 

Recent improvements in bioslurping technology and application assessments for this technology are contained in 

Air discharge 

Air treatment 

Hydrocarbon/ 

water separator 

Water discharge 

Hydrocarbon discharge 

Surface seal 

Backfill/grout 

Bentonite seal 

Vacuum pump 

Movement of 

hydrocarbon to well 

Aerobic 

biodegradation 

Slurping tube 

Casing 

Ground surface 

Water 

treatment 

Air 

Liquid hydrocarbon layer 

Water table 

Wall screen Filter pack 

reports by the U.S. Navy (NFESC, 2003) and the U.S. 

Army Corps of Engineers (USACE, 1999). It is widely 

accepted in the industry that source removal, such as that 

offered by proper application of the bioslurping technology, should be part of most remedial strategies at sites involving separate product phases. 

3.2 Ex Situ Bioremediation 

Ex situ bioremediation technologies can most easily be 

classified by the physical state of the medium to which 

they are typically applied. The following discussion is 

organized accordingly, with descriptions of bioremediation processes for various solids, solid–liquid mixtures, 

and liquids. 

Also common to the ex situ remediation technologies are 

the processes for removing contaminated materials for 

treatment. Contaminated media are excavated or extracted (e.g., ground water removal by pumping) and moved 

to the process location, which may be within or adjacent 

to the contamination zone. Special handling required for 

excavation and preparation of sediments is described in 

detail in Selecting Remediation Techniques for Contaminated Sediment (EPA, 1993b), as well as Physical Separation (Soil Washing) for Volume Reduction of Contaminated 

Soils and Sediments: Processes and Equipment (Olin et al., 

1999). 

3.2.1 Solids 

The most common types 

of solids bioremediation 

are (1) land farming or 

land treatment, (2) composting, and (3) biopiles, 

cells, or mounds. In practice, these types are not 

rigidly divided, but the 

subdivision is useful for 

this discussion. Table 3-1 

presents a comparison of 

the characteristics of various solid-phase bioremediation technologies. 

Figure 3-5. Bioslurping technology. (Adapted from USAEC, 2004) 

12 Engineering Issue 
Table 3-1. Comparison of the Characteristics of Ex Situ Bioremediation Technologies for Solids 

Characteristic Land Treatment Composting Biopiles 

Static Pile In-Vessel Windrow 

Liner Utilized Yes Yes No Yes Yes 

Containerized Bermed Pad Yes Pad Pad 

Bulking Agent Sometimes Yes Yes Yes Sometimes 

Mechanical Mobile Equipment No In-Vessel Mobile Equipment Mobile Equipment 

Aeration2 Mechanical Mixing Pressure Vacuum Mechanical Pressure/Vacuum 

Nutrient3

 Added Sometimes Yes Yes Yes Yes 

Temperature Ambient 54°–65°C 54° 65°C Ambient/Mesophilic 

1 	 Mechanical mixing may be necessary to provide thorough distribution of nutrients and other additives, promote aeration, and enhance biodegradation. Mechanical 

mixing may use mobile equipment (e.g., bulldozers) or may occur within a reactor vessel. 2	 Aeration involves the introduction of oxygen into ex situ material to promote aerobic degradation. Aeration mechanisms may include mechanical mixing, pressure, and 

vacuum, as well as natural draft. “Typical” aeration mechanisms are shown. 3	 Nutrients may not be required or may be supplied by bulking agents alone, or additional nutrients may be supplied. 

3.2.1.1 Land Treatment 

Land treatment, also called land farming, is useful in treating aerobically degradable contaminants. This process is 

suitable for non-volatile contaminants at sites where large 

areas for treatment cells are available. Land treatment of 

site-contaminated soil usually entails the tilling of an 8-

to 12-inch layer of the soil to promote aerobic biodegradation of organic contaminants. The soils are periodically tilled to aerate the soil, and moisture is added when 

needed. In some cases, amendments may be added to 

improve the tilth of the soil, supply nutrients, moderate 

pH, or facilitate bioremediation. Typically, full-scale land 

treatment would be conducted in a prepared-bed land 

treatment unit (see Figure 3-6)—an open, shallow reactor with an impermeable lining on the bottom and sides 

to contain leachate, control runoff, and minimize erosion 

and with a leachate collection system under the soil layer 

(EPA, 1993). In some cases, hazardous wastes (such as 

highly contaminated soils) or process wastes (such as distillate residues) may be treated in land treatment units. In 

these cases, the waste may be applied to a base soil layer. 

The performance of land treatment varies with the contaminants to be treated. For easily biodegradable contaminants, such as fuels, land treatment is inexpensive and 

effective. Contaminants that are difficult to degrade, such 

as PAHs, pesticides, or chlorinated organic compounds, 

are topics of research and would require site-specifi c treatability testing to verify that land treatment can meet desired endpoints. 

Existing 

ground 

surface 

Leachate 

collection pipe 

Compacted 

subgrade 

surface 

Compacted 

sand 

Polyethylene 

geomembrane 

Microbes’ 

nutrients 

Contaminated 

soils 

Figure 3-6. Land treatment. (Adapted from FRTR, 2003) 

3.2.1.2 Composting 

Composting is a controlled biological process that treats 

organic contaminants using microorganisms under thermophilic conditions (40°–50°C). For some practitioners, 

the creation of thermophilic conditions is the primary 

distinction between composting and biopiles (which operate at less than 40°C), although others use composting as a term that encompasses both temperature ranges 

(NFESC, 2005). 

In composting, soils are excavated and mixed with bulking agents and organic amendments, such as wood chips 

and vegetative wastes, to enhance the porosity of the mixture to be decomposed. Degradation of the bulking agent 

heats up the compost, creating thermophilic conditions. 

Oxygen content, moisture levels, and temperatures are 

monitored and manipulated to optimize degradation. 

Oxygen content usually is maintained by frequent mixing, such as daily or weekly turning of windrows. SurEngineering Issue 13
face irrigation often is used to maintain moisture content. 

Temperatures are controlled, to a degree, by mixing, irrigation, and air flow, but are also dependent on the degradability of the bulk material and ambient conditions 

(FRTR, 2003). 

There are three designs commonly applied for composting: 

1.	 Aerated static piles—Compost is formed into piles 

and aerated with blowers or vacuum pumps. 

2. 	 Mechanically agitated in-vessel composting—Compost 

is placed in a reactor vessel, in which it is mixed and 

aerated. 

3. 	 Windrow composting—Compost is placed in long, 

low, narrow piles (i.e., windrows) and periodically 

mixed with mobile equipment. 

Windrow composting is the least expensive method, 

but has the potential to emit larger quantities of VOCs 

(FRTR, 2003). In-vessel composting is generally the most 

expensive type, but provides for the best control of VOCs. 

Aerated static piles, especially when a vacuum is applied, 

offer some control of VOCs and are typically in an intermediate cost range, but will require offgas treatment 

(FRTR, 2003). 

Berms may also be needed to control runoff during composting operations. Runoff may be managed by retention 

ponds, provision of a roof, or evaporation. 

Composting has been successfully applied to soils and 

biosolids contaminated with petroleum hydrocarbons 

(e.g., fuels, oil, grease), solvents, chlorophenols, pesticides, herbicides, PAHs, and nitro-aromatic explosives 

(EPA, 1998b; EPA, 1997; EPA, 2004b). For TNT, complete mineralization has been diffi cult to demonstrate via 

composting. TNT may bind to soil, resulting in low microbial bioavailability and apparent disappearance (Rodgers and Bunce, 2001). Composting is not likely to be successful for highly chlorinated substances, such as PCBs, 

or for substances that are difficult to degrade biologically 

(EPA, 1998b). 

3.2.1.3 Biopiles 

Biopiles involve the mixing of excavated soils with soil 

amendments, with the mixture placed in a treatment area 

that typically includes an impermeable liner, a leachate 

collection system, and an aeration system. Biopiles are 

typically 2–3 meters high, and contaminated soil is often 

placed on top of treated soil (see Figure 3-7). Moisture, 

nutrients, heat, pH, and oxygen are controlled to enhance 

biodegradation. This technology is most often applied to 

readily degradable species, such as petroleum contaminants. Surface drainage and moisture from the leachate 

collection system are accumulated, and they may be treated and then recycled to the contaminated soil. Nutrients 

(e.g., nitrogen and phosphorus) are often added to the 

recycled water. Alkaline or acidic substances may also be 

added to the recycled water to modify or stabilize pH to 

optimize the growth of select microbes capable of degrading the contaminants of concern (FRTR, 2003). 

Berm 

Air inlet/ 

exhaust Leachate collection 

and treatment (optional) 

Nutrient and 

moisture addition 

Soil vapor 

monitoring probes 

Contaminated soil 

Air injection 

(or extraction) 

Figure 3-7. Typical biopile system. (Adapted from EPA, 2004b) 

An air distribution system is buried in the soil as the biopile is constructed. Oxygen exchange can be achieved utilizing vacuum, forced air, or even natural draft air fl ow. 

Low air fl ow rates are desirable to minimize contaminant 

volatilization. If volatile constituents are present in significant concentrations, the biopile may require a cover 

and treatment of the offgas. 

Biopile treatment lasts from a few weeks to a few months, 

depending on the contaminants present and the design 

and operational parameters selected for the biopile (FRTR, 

2003). Biopiles are typically mesophilic (10°–45°C). 

Additional information on ex situ biological soil treatment 

is available from the following sources: 

●	 U.S. Navy Biocell Application Guidance (NFESC, 


1998)


●	 U.S. Navy Biopile Design and Construction Manual


(Battelle, 1996a)


●	 U.S. Navy Biopile Operations and Maintenance Manual (Battelle, 1996b) 

●	 U.S. Army Environmental Center Multiple Biotechnology Demonstration of Explosives-Contaminated Soils 

(USAEC, 2005) 

14 Engineering Issue 
●	 Chapters 13 and 14 in Biodegradation of Nitroaromatic Compounds and Explosives (Spain et al., 2000) 

●	 On-site Bioremediation of Oil and Grease Contami­


nated Soils (Vance, 1991).


3.2.2 Solid–Liquid Mixtures 

Solid-liquid mixtures consist of materials such as slurries 

and sludges. One technology for treating such mixtures is 

discussed below. 

3.2.2.1 Slurry Bioreactors 

Slurry bioreactors are utilized for soil, sediments, sludge, 

and other solid or semi-solid wastes. Slurry bioreactors are 

costly and, thus, are likely to be used for more diffi cult 

treatment efforts. 

Typically, wastes are screened to remove debris and other 

large objects, then mixed with water in a tank or other 

vessel until solids are suspended in the liquid phase. If 

necessary, further particle size reduction can be accomplished before the addition of water (by pulverizing and/ 

or screening the wastes) or after the addition of water 

(through use of a sheering mixer). Suspension and mixing of the solids may increase mass transfer rates and may 

increase contact between contaminants and microbes capable of degrading those contaminants (EPA, 1990). 

Mixing occurs in tanks or lined lagoons. Mechanical mixing is generally conducted in tanks. Typical slurries are 

10–30% solids by weight (FRTR, 2003). Aeration, with 

submerged aerators or spargers, is frequently used in lagoons and may be combined with mechanical mixing to 

achieve the desired results. Nutrients and other additives, 

such as neutralizing agents, surfactants, dispersants, and 

co-metabolites (e.g., phenol, pyrene) may be supplied to 

improve handling characteristics and microbial degradation rates. Indigenous microbes may be used or microorganisms may be added initially to seed the bioreactor or 

may be added continuously to maintain proper biomass 

levels. Residence time in the bioreactor varies with the 

matrix as well as the type and concentration of contaminant (EPA, 1990). 

Once contaminant concentrations reach desired levels 

on a dry-weight basis, the slurry is dewatered. Typically, a clarifier is utilized to dewater the slurry by gravity. 

Other dewatering equipment may be used depending 

on slurry characteristics and cost considerations (Olin 

et al., 1999). Water, air emissions from all process steps, 

and oversize materials may require additional treatment. 

More information on this technology is available from the 

Naval Facilities Engineering Service Center (NFESC) at 

http://enviro.nfesc.navy.mil/erb/erb_a/restoration/ 

technologies/tech_transfer/ttweb.asp?id=3. 

3.2.3 Liquids 

Liquids, such as surface water, ground water, mine drainage, and effluent from other treatment operations, can 

undergo ex situ bioremediation in constructed wetlands. 

Note that surface water and ground water have important 

differences, such as concentrations of contaminants and 

degradable organic material, than may be found in waste 

streams from other treatment operations. 

3.2.3.1 Constructed Wetlands 

Constructed wetlands provide for biological assimilation, 

breakdown, and transformation of contaminants; chemical breakdown and transformation of contaminants; and 

physical sedimentation and filtration (USDA and EPA 

1994a), as shown in Figure 3-8. Biological processes associated with wetlands include bioremediation (microbially-based remediation) and phytoremediation (plant-based 

remediation). Microbes attached to the surfaces of plants, 

plant litter, and the wetland substrate degrade and/or sorb 

the organic substances present in the water undergoing 

treatment (USDA and EPA, 1994a). Phytoremediation 

uses plants to remove, transfer, stabilize, or destroy contaminants through biological, chemical, and physical processes that are influenced by plants and their roots (i.e., 

rhizosphere) that include degradation, extraction through 

accumulation in plant roots/shoots/leaves, metabolism 

of contaminants, and immobilization of contaminants at 

the interface of roots and soil (EPA, 2004a). 

Sediment Landfill 

Water 

inflow 

Water 

outflow 

Gas 

discharge 

Sediment 

accumulation 

recovery 

Plant 

uptake 

Microbial 

oxidation and 

reduction 

Algae uptake 

and disposition 

Man-made wetlands


(organic soil, microbial fauna,


algae, plants, microorganisms)


Figure 3-8. Constructed wetland. (Adapted from FRTR, 2003) 

Engineering Issue 15
Wetlands inherently have a higher rate of biological productivity/activity than many other natural ecosystems and 

are thus capable of efficiently and economically transforming many common contaminants to harmless byproducts 

(Kadlec and Knight, 1996). Constructed wetlands have 

been applied successfully to remove contaminants such as 

metals, petroleum hydrocarbons, and glycols; to decrease 

metal concentrations via chemical or microbial precipitation; and to neutralize acidity (ESTCP, 2004a; USDA 

and EPA, 1994b). Recent research also has demonstrated applicability to explosive-contaminated water (Bader, 

1999). However, wetlands are sensitive to high ammonia 

levels, herbicides, and contaminants that are toxic to the 

plants or microbes (USDA and EPA, 1994a). 

Constructed wetlands are well suited for the treatment of 

contaminated ground water emerging from surface and 

mine seeps, pump-and-treat waste streams with low concentrations of easily biodegradable contaminants, and contaminated surface waters (EPA, 2001c). Constructed wetlands may also be used to pretreat contaminated water prior 

to conventional treatment or to further treat a waste stream 

prior to disposition or discharge (USDA and EPA, 1994b). 

However, applicability to highly acidic waste streams may 

not be cost-effective (USDA and EPA, 1994b). 

Discharges must meet applicable effluent limitations and 

related regulatory requirements. Discharges that do not 

meet these requirements may be required to undergo further treatment or may be found suitable for recycling into 

the wetland as a supplemental water source (USDA and 

EPA, 1994a). 

There are various types of constructed wetlands, depending on the type of flow (surface or subsurface), contaminant of concern, or type of substrate, which can include 

limestone, organic material such as compost, or gravel 

(USDA and EPA, 1994a; USDA and EPA, 1994b; Bader, 

1999). The chemical and microbial processes may proceed either in an anaerobic or aerobic environment. 

Since constructed wetlands function both as macroscopic and microscopic ecosystems to promote contaminant 

treatment, the biological characteristics of the system 

must be taken into account during the design phase. The 

chemistry of the waste stream and how the passive chemical, physical, and biological processes affect this or are, in 

turn, affected by the waste stream are important design 

factors (USDA and EPA, 1994b). The chemical characteristics of the waste stream can affect sizing of the system for adequate retention time and whether the waste 

stream may require pretreatment to (1) address concentration, ammonia, nutrient, and organic loads that may 

damage vegetation, or (2) remove solids or materials, such 

as grease, that may clog the wetland (USDA and EPA, 

1994a). In addition, pH adjustment may be necessary, 

either prior to waste stream treatment or through use of 

limestone substrate (USDA and EPA, 1994b). 

Climatic and seasonal circumstances as well as waste 

stream characteristics are important considerations when 

selecting the types of plants to use in a constructed wetland. Salinity, either in the waste stream or as a result of 

treatment, can harm or destroy the wetland vegetation if 

the plants are not salt tolerant. In addition, cold weather 

can reduce microbial activity, and hail or other weather 

events can damage the plants (USDA and EPA, 1994a). 

The low cost, passivity (i.e., lack of dependence on power or mechanical components), and efficacy for treating 

many common contaminants are key advantages of constructed wetland treatment systems. Constructed wetlands 

are often visually attractive, but can require more space 

than other remedial systems. The wetlands should be sized 

with an understanding that both plant-based and bacterial-based remediation will decline during colder seasons. A 

key design element is sizing to achieve adequate retention 

time to enable the biological, chemical, and physical processes to be effective (USDA and EPA, 1994a). Seasonal, 

climatological, and waste stream factors that control the 

water balance in the wetland also must be considered during design to achieve project goals (FRTR, 2003). For example, photosynthesis of TNT colors the treated water 

red, which negatively impacts plant growth (Bader, 1999), 

and pH affects the kinetics of the abiotic and biotic processes, including solubility of metal oxides or hydroxides, 

oxidation, and hydrolysis (USDA and EPA 1994b). In 

addition, animals such as tadpoles or deer may defoliate 

plant material, thereby affecting treatment (Bader, 1999). 

Constructed wetlands require a continuous supply of water. While tolerant of fl uctuating flows, constructed wetlands cannot withstand complete drying. A slow water 

flow must be maintained to prevent the development of 

stagnant water that can lead to performance and vector 

difficulties. Recycling wetland water can supplement inflow, but this can increase salinity over time, which can 

affect design and cost (USDA and EPA, 1994a). 

More information on constructed treatment wetlands is 

available in the Interstate Technical and Regulatory Guidance Document for Constructed Wetlands (ITRC 2003). 

16 Engineering Issue 
4.0 TECHNOLOGY SELECTION FACTORS 

Table 4-1 summarizes the general applicability of each 

technology type for the contaminant classifi cations discussed in this Engineering Issue. The table presents information for contaminant treatment in soil, sediment, 

sludge, ground water, surface water, and leachate. The 

analysis of technology applicability is based on published 

literature and expert judgment. Note that the technologies may be applicable to some contaminants within a 

contaminant group but not to others. Site- and contaminant-specific treatability studies may be required to determine the actual efficacy of any one technology on the 

site-specific soils, contaminants, and conditions. 

Applicability of biologically based remedies is highly influenced by the type of microbiological community that 

is best suited for the biodegradation of the particular contaminant or mix of contaminants. Of primary importance 

in gaining an initial understanding of the applicability of 

a biological remedy to contaminants of concern is the 

baseline oxidation/reduction potential of the site to be remediated. The presence or absence of oxygen is a signifi ­

cant determining factor that defines the microbiological 

community characteristics. Some contaminants are best 

treated under anaerobic conditions, and others can only 

be treated aerobically. Some contaminants can be treated 

both anaerobically and aerobically, but there are technical implications to be considered and contaminant halflives may vary between the two processes. Therefore, the 

baseline oxidation/reduction potential is of primary importance to the practitioner who is evaluating a contaminated site for a biological remedy (Rottero et al., 2004). 

Table 4-1. Demonstrated Effectiveness of Biological Treatment Technologies for Soil, Sediment, Bedrock, and Sludge 

Contaminant 

Type 

In Situ Treatment Technologies Ex Situ Treatment Technologies1 

Intrinsic Enhanced 

Solids 

Solid– 

Liquid 

Mixtures 

Liquids Soil / 

Ground 

Water 

Vadose 

Zone 

Surfi cial 

Soil 

Ground Water / 

Saturated Soil 

Monitored NaturalAttenuationAerobic BioventingCometabolic BioventingAnaerobic BioventingLand TreatmentCompostingAnaerobic ReductiveDechlorinationAerobic TreatmentBiological ReactiveBarriersBiosparging/BioslurpingLand TreatmentCompostingBiopilesSlurry BioreactorsConstructed Wetlands 

Non-halogenated VOCs ♦ ♦ ■ ■ ♦2 ♦2 ■ ♦ ♦ ♦ ♦2 ♦2 ♦2 ♦2 ♦2 

Halogenated VOCs ♦ ■ ♦ ▲ ■ 2 ■ ♦ ■ ♦ ♦ ■ 2 ■ 2 ■ 2 ■ 2 ■ 2 

Non-halogenated SVOCs ▲ ♦ ■ ■ ♦ ♦ ■ ♦ ▲ ▲ ♦ ♦ ♦ ♦ ♦ 

Halogenated SVOCs ▲ ■ ▲ ▲ ■ ■ ▲ ■ ♦ ■ ■ ■ ■ ■ ■ 

Fuels ♦ ♦ ■ ■ ♦2 ♦2 ■ ♦ ♦ ♦ ♦2 ♦2 ♦2 ♦2 ♦2 

Inorganics ■ ● ● ■ ■ ■ ■ ■ ♦ ■ ■ ■ ■ ■ ♦ 

Radionuclides ■ ■ ■ ■ ■ ■ ■ ■ ♦ ■ ■ ■ ■ ■ ■ 

Explosives ■ ▲ ■ ▲ ♦ ♦ ■ ▲ ♦ ■ ♦ ♦ ▲ ♦ ▲ 

1 Not generally applicable to rocks and bedrock. 2 Volatilization must be controlled. 

♦ Demonstrated Effectiveness: Successfully treated at pilot or full scale and 

verified by an independent agency. 

▲	 Potential Effectiveness: Successfully treated at laboratory or bench scale, or 

similar contaminant types have been successfully demonstrated at pilot or full 

scale. 

■	 No Expected Effectiveness: No successful treatments documented at any 

scale, and expert opinion notes that the contaminant in question is not likely to 

be effectively treated by the technology. 

●	 Potential Adverse Effects: Adverse effects are documented at any scale, 

or expert opinion notes that the treatment technology may result in adverse 

effects to the environment. 

Adapted from information in EPA (1998a, 2000, 2004b, 2004c), FRTR (2003, 2004), 

ESTCP (2001, 2004a, 2004b), ITRC (2004), and AFCEE (1996). 

Site characterization and long-term monitoring are necessary to support 

system design and sizing as well as to verify continued performance. There 

are also regulatory requirements to be addressed regarding system design, 

implementation, operation, and performance, including the disposition of liquid 

effluents and other wastes resulting from the treatment process. 

Engineering Issue 17
5.0 SUMMARY 

In situ and ex situ biodegradation technologies are increasingly selected to remediate contaminated sites, either alone or in combination with other source control 

measures. Bioremediation technologies have proven effective in remediating fuels and VOCs and are often able to 

address diverse organic contaminants including SVOCs, 

PAHs, CAHs, pesticides and herbicides, and nitro-aromatic compounds (such as explosives), potentially at lower cost than other remediation options. Some bioremediation techniques are also able to address heavy metal 

contamination. Bioremediation continues to be an active 

area of research, development, and demonstration for its 

applications to diverse contaminated environments. 

A unique feature of bioremediation is the diversity of its 

application to solids, liquids, and liquid–solid mixtures, 

involving both in situ and ex situ environments. Amendments may be necessary to support or enhance the biodegradation processes to improve the timeframe involved 

to achieve cleanup goals. 

Site characterization and long-term monitoring are necessary to support system design and sizing as well as to 

verify continued performance. There are also regulatory 

requirements to be addressed regarding system design, 

implementation, operation, and performance, including 

the disposition of liquid effluents and other wastes resulting from the treatment process. 

6.0 ACKNOWLEDGMENTS 

This Engineering Issue paper was prepared for the U.S. 

Environmental Protection Agency, Office of Research 

and Development, National Risk Management Research 

Laboratory (NRMRL) by Science Applications International Corporation (SAIC) under Contract No. 68-C-02­

067. Doug Grosse served as the EPA Work Assignment 

Manager. Paul McCauley (NRMRL) acted as the Technical Project Manager. Lisa Kulujian was SAIC’s Work 

Assignment Manager, and Jim Rawe and Virginia Hodge 

served as SAIC’s technical leads and primary authors 

of this Engineering Issue paper, with additional technical input provided by Carolyn Acheson (NRMRL) and 

Chris Lutes and David Liles from ARCADIS. Review 

and comments were provided by the following members 

of the EPA Engineering Forum: Jon Josephs (EPA Region 

2), Ed Mead (USACE), Bernie Schorle (EPA Region 5), 

Charles Coyle (USACE), Michael Gill (EPA Region 9), 

and Jon Bornholm (EPA Region 4). David Reisman and 

Ann Keely also reviewed drafts of this document. 

For additional information, contact the ORD Engineering Technical Support Center (ETSC): 

David Reisman, Director 

U.S. EPA Engineering 

Technical Support Center 

26 W. Martin Luther King Drive MLK-489 

Cincinnati, OH 45268 

(513) 487-2588 

Reference herein to any specifi c commercial products, 

process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the 

United States Government. The views and opinions of 

authors expressed herein do not necessarily state or refl ect 

those of the United States Government, and shall not be 

used for advertising or product endorsement purposes. 

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Engineering Issue 19
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20 Engineering Issue 
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Engineering Issue 21
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