Groundwater and Soil Pollution: Bioremediation☆

Anastasios I Zouboulis, Panagiotis A Moussas, and Savvina G Psaltou, Aristotle University of Thessaloniki, Thessaloniki, Greece
© 2018 Elsevier Inc. All rights reserved.

This is an update of AI Zouboulis and PA Moussas, Groundwater and Soil Pollution: Bioremediation, In Encyclopedia of Environmental Health, edited by
JO Nriagu, Elsevier, Burlington, 2011, pp. 1037–1044, ISBN 9780444522726.

Introduction 1
Principles of Bioremediation 2
Microbiology, Nutrition, and Environmental Requirements 3
Bioremediation Strategies and Options 3
In Situ 3
Ex Situ 5
Slurry Bioreactors 7
Vapor Phase Bioreactors (Biofilters) 7
Phytoremediation 7
Electroremediation (Electrokinetic-Enhanced Bioremediation) 10
Microbial Surfactants 11
Further Reading 13

Nomenclature
Abbreviations
EDTA Ethylenediaminetetraacetic acid
TCE Trichloroethylene
UV Ultra violet
VOCs Volatile organic compounds

Introduction

Contamination of soils, waters (groundwater and surface water), sediments, and air with hazardous and toxic compounds is one of
the most serious and difficult problems that the industrialized world faces, and which has immediate impact on the quality of life
on Earth. The problems related to contaminated sites are now attracting increasing attention in many countries, as the estimated
number of such sites continues to grow. The National Priority List of the United States contains over 1200 sites with the potential
sites numbering 32,000. In the United Kingdom, it has been estimated that over 300,000 ha are contaminated due to previous
industrial abuses. It is now recognized that the potential threats of contaminated sites on human health are serious, leading to
national and international efforts for the remediation of these sites, either as a response to the increasing health and environmental
threats or to enable the site to be redeveloped for use.
Conventional remediation strategies may usually include the excavations of contaminated land and transportation to a
(specific) landfill site, or to cap and contain the contaminated areas in site. However, these methods have significant drawbacks.
The former does not actually solve the issue as it simply moves the contamination elsewhere, and it also creates serious problems
during the excavation, handling, transportation of possible hazardous materials, etc. Moreover, the demand for new landfill sites for
the final disposal of contaminated material makes the whole method difficult and cost ineffective. Similarly, the in-place method is
just a temporary solution since the contaminated materials remain in the site, thus requiring intense monitoring and maintenance
of the site, which inevitably increases the operational difficulties as well as the cost. Other remediation techniques focus on the
complete destruction of pollutants or at least on their transformation to less harmful compounds; within these methods the high-
temperature incineration and various types of chemical decomposition, including dechlorination and ultra violet (UV) oxidation,
can mostly be included. Nevertheless, these methods also present certain limitations associated with the elevated cost (especially for
the small-scale applications), the technological complexity, and the low social acceptance; the latter being most significant in the
case of incineration.
The increasing need for remediation of contaminated sites has led to the development of new technologies that emphasize the
biological detoxification and destruction of the (organic) contaminants. Bioremediation is among these technologies that destroy

☆
Change History: May 2018. Anastasios I. Zouboulis, Savvina G. Psaltou involved in preparing the update. Sections In the section “In situ” we add natural
attenuation and biopiling, In section “Phytoremediation” we add the Mycoremediation. Also the sections Electroremediation (Electrokinetic-enhanced bioremediation)
and Microbial surfactants are an update. Tables 2, 3, 5 and Figures 3, 5, 6 were updated.

Encyclopedia of Environmental Health, 2nd Edition https://doi.org/10.1016/B978-0-12-409548-9.11246-1 1

2 Groundwater and Soil Pollution: Bioremediation

or render harmless various contaminants using the biological activity of certain microorganisms. Bioremediation has to frequently
deal with multiphase, heterogeneous environments unlike other conventional waste treatment methods that require homogeneity
and uniform mixing. Owing to these complexities, considerable experience and an interdisciplinary approach, involving different
scientific cooperation such as engineering, microbiology, ecology, geology, and chemistry to design and implement an efficient
bioremediation project, are required. However, bioremediation should not be considered as panacea, as the range of pollutants on
which this method proved effective is rather limited, sometimes the required treatment time is relatively long and the residual level
of contaminants may not always be acceptable. Also, In situ biodegradation can be limited by contaminant bioavailability and
bioaccessibility. These limitations can be overcome by coupling bioremediation with electrokinetics (EK) and by the use of
biosurfactants. Overall, bioremediation is considered as a very promising technology with great potential when dealing with certain
types of contaminated sites. So far, it has been used at an increasing number of sites worldwide, including Europe, with varying
success.

Principles of Bioremediation

Bioremediation is defined as the process whereby (mostly) organic wastes can be biologically degraded under controlled conditions
to an innocuous state, or to levels below the respective concentration limits, as set by the controlling authorities. In other words,
bioremediation employs the living organisms, most notably microorganisms, to degrade the pollutants and convert them into less
toxic or nontoxic form. The suitable organisms can be bacteria, fungi, or plants, which have the physiological abilities to degrade,
detoxify, or render the contaminants harmless. In some occasions, the microorganisms can be already present on the site
(indigenous microorganisms), or can be isolated from elsewhere and added to the treated material, using bioreactors as an
example. Needless to say that as bioremediation actually relies on the microbial growth and activity, its effectiveness is highly
dependent on the applied environmental parameters that influence the microbial growth and the degradation rate. Overall,
bioremediation technique depends on having the right microorganisms in the right place under the suitable environmental
conditions in order for the degradation process to occur successfully. The biological processes involved in bioremediation
technology can be enhanced and focused on the removal of specific hazardous pollutants from soil, water, or atmosphere. It can
mineralize waste material into water, carbon dioxide, biomass, or other nonhazardous products and thus obviate the need for
further treatment. Bioremediation can treat a wide range of compounds. Apart from municipal wastes and process waters, suitable
(for each case) microorganisms may also be applied for the degradation of pesticides, industrial chemicals, components of crude oil,
and even compounds that, until recently, were regarded as nonbiodegradable, such as chlorinated solvents, chlorofluorocarbons,
and other synthetic organic compounds.
Bioremediation technologies can be classified into two general categories: ex situ and in situ. Table 1 summarizes the most
widely used bioremediation techniques. The ex situ techniques require the physical removal of the contaminated material and its
transportation to another area for further treatment by bioreactors, land farming, or composting, whereas in situ technologies
involve treatment of contaminated material in place, such as by bioventing, or biostimulation.
Most bioremediation systems operate under aerobic conditions; however, anaerobic conditions are also applicable, thus
enabling the degradation of recalcitrant molecules by using specific microorganisms. The design, control, and optimized operation
of a bioremediation process is a complex system of several factors including a microbial population suitable for degrading certain
pollutants, the availability of contaminants to the microorganisms, the type of soil (matrix, substrate, etc.), temperature, pH, the
presence of oxygen (or other electron acceptors, such as nitrate or sulfate), and of nutrients (primarily nitrogen and phosphorus).
Bioremediation, as a technique, can offer several advantages over other more conventional treatment methods applicable to
contaminated sites. Firstly, bioremediation, as a (more or less) natural process for the treatment of wastes, is usually acceptable by
the public. Suitable microbial populations can degrade a wide range of contaminants, rendering (transforming) a hazardous
compound to a harmless one. Eventually, the residues of the treatment may include simpler compounds, such as carbon dioxide or
water, but also cell biomass. Therefore, a chance for future hazards in the treatment and disposal of contaminated material is
practically eliminated. Often, bioremediation can be performed on-site, thus reducing the associated transportation costs and
liabilities, and also the potential threats to human health and to the environment due to the transportation of hazardous materials.

Table 1 Common bioremediation technologies

Bioaugmentation Addition of bacterial cultures to a contaminated medium; frequently used in bioreactors and ex situ systems
Biofilters Use of microbial stripping columns to treat (mostly) air emissions or odors (volatile compounds)
Biosparging The injection of air under pressure can enhance biological degradation; usually performed in situ; noninvasive
Biostimulation Stimulation of indigenous microbial populations in soils or groundwater; which can be performed either in situ or ex situ
Bioreactors Biodegradation in a container or reactor; may be used to treat several liquid wastes or slurries; rapid degradation kinetics but relatively high
capital and operational cost.
Bioventing Method of treating contaminated soils by drawing oxygen through the soil to stimulate microbial growth and activity
Composting Aerobic, thermophilic treatment process; can be performed by using static piles, aerated piles, or continuously fed reactors; low cost but
extended treatment time
Land farming Solid-phase treatment system for contaminated soils; may be performed in situ or in a constructed soil treatment cell; cost-efficient
 Groundwater and Soil Pollution: Bioremediation 3

In addition, it is often applied as in situ technology hence the site disruption is minimized, enabling the above ground activities to
be continued. Needless to say that bioremediation as a biological system can often be shown to be less expensive than other relevant
treatment technologies, which can be applied for the same purpose. Finally, bioremediation can be combined with other
technologies into a process chain, thus increasing the efficiency of the whole treatment.
Bioremediation, like any other technology, has certain disadvantages. In particular, it is limited only to those compounds that
are biodegradable. There are also increasing concerns that the bioremediation products may be more persistent or hazardous than
the parent compounds. For example, trichloroethylene (TCE) is converted to vinyl chloride, a known carcinogen, via a series of
biological reactions, resulting in a sequencing removal of chlorine atoms. This process is known as reductive dehalogenation.
Moreover, the effectiveness of bioremediation is highly susceptible to the microbial growth and other environmental parameters of
the site. Finally, bioremediation often requires more time than other treatment options, such as incineration, or excavation and
removal of soil.

Microbiology, Nutrition, and Environmental Requirements

The natural degradation of organic compounds in the environment is processed primarily by two groups of microorganisms:
bacteria and fungi. Owing to a number of their characteristics, which include rapid growth and metabolism, genetic plasticity, and
adjustability to a variety of environments, bacteria are an excellent choice for bioremediation. For efficient degradation, it is
necessary for bacteria and pollutants to be in contact. As bacteria and contaminants are not uniformly spread into the soil,
uniformity of conditions is not easily achieved. Additionally, the bacteria are not always present in adequate numbers for
bioremediation to occur. On such occasions, microbial growth and activity must be stimulated by adding nutrients and oxygen
to the respective contaminated site. Carbon is the basic element of living organisms and must be present in great quantities,
allowing microbes to use and convert it to cell constituents. Simultaneously, energy is released, which eventually promotes the
microbial growth, as the microbes assimilate the carbon and employ the released energy for biosynthesis. Apart from the
requirements for carbon, there are at least 11 essential elements that must be in appropriate ratios and forms within the soil
environment so that the microbial growth can proceed. These include nitrogen, phosphorus, potassium, sulfur, iron, calcium,
manganese, zinc, copper, cobalt, and molybdenum. The latter nine metals are normally present in sufficient (although minor)
concentrations, and only nitrogen and phosphorus may be the limiting factors; thus their supplement is sometimes necessary.
In addition, microbial growth and activity are significantly affected by the existing (or specifically applied) environmental
conditions, namely the pH, temperature, and moisture. Although, it has been noticed that microorganisms can be found in extreme
conditions, the majority of them grow over a narrow pH range of 6–8. Similarly, temperature significantly affects the rate of
biochemical reactions, and it has been found that 10
C rise of temperature can double the kinetic rate. Thus, most of the microbes
can metabolize the organic compounds at temperatures between 0
C and 80
C. Microorganisms are commonly classified into the
following major categories: (1) psychrophiles (at temperatures between 5
C and 15
C), (2) mesophiles (at temperatures between
25
C and 40
C), and (3) thermophiles (at temperatures between 40
C and 60
C). Water availability is another significant factor for
microbial reproduction and growth. Optimal water present in the site to be treated is usually 10%–20% by mass. However, the
presence of excessive water concentration will saturate the soil thus forming anoxic conditions, which are, most of the times,
undesirable.

Bioremediation Strategies and Options

The proper selection of a bioremediation treatment system is highly influenced by several factors, most notably the nature of the
existing contaminant(s), the extent of contamination, the physicochemical properties of contaminated site, the clean-up goals and
the economics of the process. Therefore, a range of different bioremediation approaches have been developed, so as to fulfill the
requirements for each remedial situation. As discussed in the previous section, bioremediation technologies are usually classified
into the in situ and ex situ techniques. The in situ bioremediation technologies refer to the in-place treatment of contaminated site
(Fig. 1).

In Situ
The in situ bioremediation technologies employ the use of microorganisms to degrade pollutants with the least possible disturbance
and therefore are recommended for cases where the excavation of contaminated material is dangerous and cost ineffective. The
effectiveness of the in situ techniques is markedly influenced by the site characteristics, including the site hydrogeology, the soil type
and properties, the presence and types of microorganisms, and the concentration, as well as the physicochemical properties of the
waste material to be treated.
Among the several available in situ technologies, the following can be mentioned:

(a) Natural attenuation is one of the most important processes for contaminant removal. It makes use of natural processes to contain
the spread of contamination from chemical spills and reduce the concentration and amount of pollutants at contaminated sites.
4 Groundwater and Soil Pollution: Bioremediation

Fig. 1 Typical in situ bioremediation technique. From Lees, Z. M. and Senior, E. (1995). Bioremediation: A practical solution to land pollution. In Kirkwood, R. C.
and Longley, A. J. (ed.) Clean Technology and the Environment. London/New York: Blackie Academic & Professional, pp. 120.

Natural attenuation—also referred to as intrinsic remediation, bioattenuation, or intrinsic bioremediation—is an in situ

treatment method. This means that environmental contaminants are left in place while natural attenuation works on them.
The contaminants can be biodegradable under both aerobic and anaerobic conditions. Because the intrinsic bioremediation
rate is limited by in situ environmental factors (e.g., oxygen, nutrients, and electron acceptors), enhanced this method can be
applied to stimulate pollutants biodegradation. Enhanced aerobic bioremediation, such as injecting air at low rate into the
aquifer below the zone of contamination, is an effective mechanism for the removal of petroleum hydrocarbons.
The processes contributing to natural attenuation are typically acting at many sites, but varying rates and degrees of
effectiveness, depending on the types of contaminants present, and the physical, chemical, and biological characteristics of
the soil and ground water. Natural attenuation processes are often categorized as destructive or nondestructive. Destructive
processes destroy the contaminant. Nondestructive processes do not destroy the contaminant but cause a reduction in
contaminant concentrations.
According to the Environmental Protection Agency in the United States, natural attenuation processes may reduce contam-
inant mass (through destructive processes such as biodegradation and chemical transformations, reduce contaminant concen-
trations although simple dilution or dispersion).
During natural attenuation, the pollutants are transformed to less harmful forms or immobilized. Such transformation and
immobilization processes are largely due to biodegradation by microorganisms, and to some extent by the reactions with
naturally-occuring chemicals and sorption on geologic media. The natural attenuation processes are contaminant-specific,
accepted as methods for treating fuel components, but not for many other classes. The time required for natural attenuation
varies considerably with site conditions. According to EPA natural attenuation can degrade PAHs with one, two or three rings in
periods ranging from 16 to 126 days. Many polluted sites may not require an aggressive approach to remediation, and
bioattenuation is efficient and cost-effective. Bioattenuation alone becomes inadequate and protracted in many cases since
many soils are oligotrophic in nature or lack appropriate microorganisms. So, natural attenuation is often used as one part of a
site cleanup that also includes the control or removal of the source of the contamination (Table 2).
(b) Bioventing was one of the first relevant technologies applied in large scale and now is the most common in situ treatment. It
combines the physical soil venting by enhanced biodegradation thereby increasing the degradation rate. The operating
principle of bioventing relies on the supply of air and nutrients through specifically constructed wells to contaminated soil,
so as to stimulate the indigenous microorganisms (Fig. 2). Bioventing technology employs relatively low air flow rates, which
not only provides the oxygen required for biodegradation to occur but also minimizes the volatilization and release of
contaminants to the atmosphere. Bioventing can be classified into active or passive technology. In passive technology the gas
exchange through the vent wells occurs only by the effect of atmospheric pressure, whereas in active technology the air is driven
into the ground with the aid of a blower or a pump. Bioventing is highly efficient especially for hydrocarbon-contaminated
sites.
(c) Bioaugmentation involves the addition of indigenous or exogenous microbial cultures to the contaminated site. It is well
established that bioaugmentation is an efficient technology in both laboratory and field applications for the removal of organic
contaminants, such as benzene, toluene, or chlorinated organic compounds. However, there are two limiting factors that
compromise the efficiency of the technique: (1) The nonindigenous microbial population usually find severe difficulties to

Table 2 A quick look at natural attenuation

• Use naturally occurring environmental processes to clean up sites.

• Is noninvasive and allows the site to be put to productive use while being cleaned up.
• Requires careful study of site conditions and monitoring of contaminant levels.

Source: A Citizen’s Guide to Natural Attenuation, October 1996, EPA 542-F-96-015.

 Groundwater and Soil Pollution: Bioremediation 5

Fig. 2 Application of bioventing technology. From Lees, Z. M. and Senior, E. (1995). Bioremediation: A practical solution to land pollution. In Kirkwood, R. C. and
Longley, A. J. (ed.) Clean Technology and the Environment. London/New York: Blackie Academic & Professional, pp. 120.

compete well enough with an indigenous population. Therefore, the added microbial culture should also have the ability to
withstand different soil conditions and to survive in the presence of other microbial population. (2) Sites that are exposed to
biodegradable wastes for a long time have already developed indigenous microbial population that stiffen the adaptation of the
added microorganisms. However, these indigenous microorganisms may effectively degrade the wastes provided that the land
treatment unit is well managed.
(d) Biosparging is the technology in which air is injected under pressure to groundwater in order to increase the oxygen concentra-
tion, thus enhancing the biological degradation rate by the presence of indigenous microbial populations. The major advantage
of this technology is the relatively easy and low-cost installation of the (especially small diameter) air injector points, which
provides flexibility in the design, construction, and operation of such treatment systems.
(e) Biopiling. Biopiles, also known as biocells, biomounds and compost piles, are used to reduce concentrations of petroleum
constituents in excavated soils through the use of biodegradation. The contaminants are reduced to carbon dioxide and water.

The basic biopile system (Fig. 3) includes a treatment bed, an aeration system, an irrigation/nutrient system and a leach ate
collection system. Moisture, heat, nutrients, oxygen and pH are controlled to enhanced biodegradation. The irrigation/nutrient
system is buried under the soil to pass air and nutrients either by vacuum or positive pressure. Soil pipes can be up to 20 ft high
and may be covered with plastic to control runoff, evaporation and volatilization, and to promote solar heating. If volatile
organic compounds (VOCs) in the soil volatilize into the air stream, the air leaving the soil may be treated to remove or destroy
the VOCs before they are discharged into the atmosphere. Treatment time is typically 3–6 months.
Biopiles are similar to landfarms in that they are either above-ground, engineered systems that use oxygen, generally from
air, to stimulate the growth and reproduction or aerobic bacteria which, in turn, degrade the petroleum constituents absorbed to
soil. While landfarms are aerated by tilling or plowing, biopiles are aerated most often by forcing air to move by injection or
extraction through slotted or perforated piping placed throughout the pile. Biopiles, like landfarms, have been proven effective
in reducing concentrations of nearly all the constitutes of petroleum products typically found at underground storage tank sites.
Lighter (more volatile) petroleum products (e.g., gasoline) tend to be removed by evaporation during aeration processes (i.e.,
air injection, air extraction or pile turning) and, to a lesser extent, degraded by microbial respiration. Table 3 summarizes the
advantages and disadvantages of biopiles.

Ex Situ
The ex situ bioremediation technologies involve the removal of contaminated material from the site to another location for further
treatment. The most important and common techniques falling in this category include:

(a) Land farming is a simple technology, in which contaminated soil is removed and spread over a prepared surface area and
periodically tilled, until natural degradation occurs. Unlike conventional land farming techniques, soil bioremediation
treatment systems do not employ large surface areas for spreading the contaminated material. Instead, the treated material is
placed in windrows or lined cells or treatment beds, and the required oxygen is supplied by tilling or forced aeration. When
necessary, inorganic nutrients are simultaneously supplied to the system. The goal of this technology is to stimulate the
indigenous microbial populations for the aerobic degradation of the pollutants. In general, the active area of microorganisms is
limited to the superficial 15–30 cm of soil. Soil nutrients, pH, buffer capacity (lime requirements), and moisture are constantly
monitored and adjusted favorably to the biodegradation. It is worth mentioning that inadequate moisture disturbs the osmotic
balance between the microbial organism and the medium, whereas excess moisture compromises the air transport due to water
clogging. The advantages of this technology include mainly: (1) a significant reduction of surface area required for treatment,
(2) reduced remediation time due to improved design, and (3) ease of applied treatment.
(b) Composting is a biological process that is described as the combination of contaminated material with nonhazardous organic
amendments, such as manure or agricultural wastes. This mixture facilitates the development of a rich microbial population,
6 Groundwater and Soil Pollution: Bioremediation

Fig. 3 Typical biopile system. From EPA 510-B-16-005. (2016). How To Evaluate Alternative Cleanup Technologies For Underground Storage Tank Sites, 1–3.

Table 3 Advantages and disadvantages of biopiles

Advantages Disadvantages

Relatively simple to design and implement.
Short treatment times: usually 6 months to 2 years under optimal conditions.
Cost effective: $ 30–90/ton of contaminated soil.
Effective on organic constituents with slow biodegradation rates.
Requires less land area than landfarms.
Can be designed to be a closed system; vapor emissions can be controlled.
Can be engineered to be potentially effective for any combination of site
conditions and petroleum products.
Concentration reductions >95% and constituent concentrations <0.1 ppm
are very difficult to achieve.
May not be effective for high constituent concentrations (>50,000 ppm total
petroleum hydrocarbons).
Presence of significant heavy metal concentrations (>2,500 ppm) may inhibit
microbial growth.
Volatile constituents tend to evaporate rather than biodegrade during
treatment.
Requires a large land area for treatment, although less than landfarming.
Vapor generation during aeration may require treatment prior to discharge.
May require bottom liner if leaching from the biopile is a concern.

EPA 510-B-16-005 (2016). How to Evaluate Alternative Cleanup Technologies for Underground Storage Tank Sites, 1–3.

consisting mainly of mesophilic and thermophilic microorganisms. Therefore, the optimal composting environment is
characterized by elevated temperatures (50
C), excessive nutrients, high moisture level, nonlimiting oxygen, and neutral pH.
The major advantage of composting, when compared to land farming, is that it enables the improved control and optimization
of the process, so that the rate and extent of microbial activity become significantly better than those in land farming.
Composting is also considered a feasible option for detoxifying, degrading, or inactivating hazardous wastes. The composting
methodology in that case does not differ greatly from the methodology applied for nonhazardous materials.
(c) Bioreactors are used for the ex situ bioremediation treatment of contaminated soil, or of water coming from a contaminated
plume. This technology offers through the control of critical parameters, such as microbial population, nutrients, pH, and
moisture, the near-perfect environment for biodegradation. Unlike the in situ technologies, the environment of bioreactors is
totally controllable, as the matrix—that is, soil, sediment, sludge, and water—does not determine the progress and govern the
success of the process. In simple words, a bioreactor is a reaction vessel equipped with a mixing system, a system supplying
oxygen and nutrients and influent and effluent pumps. It can be run in batch or continuous mode. There are several types of
bioreactors, namely submerged fixed-film, plug flow, fluidized bed, sequencing batch, slurry reactors, and vapor phase
bioreactors (similar to biofilters). Overall, the rate and extent of microbial activity and consequently of biodegradation are
much higher in this case than the in situ technologies, due to the controllable and manageable environment. However, there are
also some inevitable disadvantages: (1) excavation of soil and pumping of groundwater are necessary, (2) during the
application of treatment a certain amount of sludge (treated material plus biomass) and a volume of gases (e.g., carbon
dioxide, methane, hydrogen sulfide) are produced, considered eventually as secondary pollutants, which require further
treatment, thus considerably increasing the cost. The major types of bioreactors are discussed briefly below.
 Groundwater and Soil Pollution: Bioremediation 7

Slurry Bioreactors

Slurry phase bioremediation involves the mixing of excavated soil with water to form slurry that is mechanically aerated in a vessel.
The environment of the slurry reactor is optimized for enhancing microbial activity, by providing excess nutrients and adjusting
appropriately the pH, moisture, and temperature. In addition, other amendments, such as surfactants and materials supporting the
microbial growth area, are also added for improving the biodegradability of the substrate. After the completion of the treatment the
slurry is dewatered, further treated, or disposed of. The produced process water may be treated prior to disposal or recycled into the
reactor for the next treatment cycle. Vapor emission treatment may also be required, depending on the specific characteristics of
contaminated material.

Vapor Phase Bioreactors (Biofilters)

Biofiltration is a proven technology and has become a suitable option for dealing with volatile organic compounds (VOCs), that is,
air emissions produced during the in situ bioremediation, composting, and bioreactor’s off-gas. It is a very cost-effective treatment
method, especially for high-volume emissions with low concentration of contaminants, and it is considered as a low-energy
consumptive technology. The bioreactors are usually packed with an appropriate solid material (mostly compost or wood chips and
sand), and the microbial population is attached on its surface. The treated gases are driven through the compost pile where the
microorganisms degrade the organic compounds of the vapor. There are two main types of biofilters: the soil filter and the treatment
bed/disk. The major advantage of this technology is the no-sludge production.
Overall, the application of bioremediation of contaminated sites can produce several benefits to the society, economic
prosperity, environmental sustainability, let alone the human health, because contaminated soils or waters are considered as
potential threats to human health. Bioremediation may act as a response to these threats by rendering harmless a series of toxic and
persistent compounds, enabling the site to be redeveloped for reuse, improving the environmental quality and thereby ensuring the
human health.
However, when protecting human health, the possibility of potential adverse effects should always be under careful consider-
ation. For example, under certain circumstances, nonpathogenic microorganisms may behave as pathogens, whereas there are some
concerns that occasionally the intermediate or the final products of biodegradation may be more toxic than the parent compounds.
Although not all potential health outcomes can be predicted, the potential adverse actions of microorganisms could be identified
via the following steps: (1) use of previous knowledge and information, (2) use of screening tests to identify the potential risks, and
(3) use of human (epidemiological) studies to determine the magnitude of any potential problems.

Phytoremediation

Phytoremediation is an emerging technology that uses certain higher plants to remove pollutants from contaminated environments
(mostly from soil or from shallow groundwaters). In other words, phytoremediation is associated with the use of green plants and
their microbiota in combination with specific soil amendments and agronomic techniques to remove or limit and render harmless
several hazardous pollutants. Phytoremediation has gained wider acceptance, especially during the past 10 years, as a cost-effective,
low-invasive, and attractive alternative treatment technique, usually complementary to many of the currently practiced ex situ and
in situ engineering-based technologies. The major advantages of this method include: low capital and maintenance cost, low
environmental impact, noninvasiveness, easy start-up, easy large-scale applicability, high public acceptance, and the pleasant
landscape that eventually results as a by-product. Phytoremediation technologies have been tested as alternative options to remove
several categories of inorganic and organic pollutants, including heavy metals, chlorinated solvents, aromatic and petroleum
hydrocarbons, pesticides, etc. In view of this, phytoremediation techniques have been gaining increasing attention for the
rehabilitation of contaminated sites, including Municipal Solid Waste (MSW) landfills and mine-degraded soils. Fig. 4 demon-
strates the common processes involved in phytoremediation. The various phytoremediation techniques can be classified into
several types according to the contaminant fate and the surface medium. Table 4 summarizes the key features of phytoremediation.
A short description of the major relevant treatment techniques is presented in the following text:
Phytostabilization: This phytoremediation technique is mainly applied to metal-contaminated soils. Plant species that can grow
on metal-contaminated and nutrient-deficient soils with deep or fibrous roots are commonly employed in this case. This technique
reduces the mobility of metals through the accumulation by roots, adsorption onto roots, and precipitation within the rhizosphere,
thus preventing the reentrance of the contaminants into the soil environment. In addition, the respective metal bioavailability for
reentry into the food chain is also substantially reduced. There is evidence that the efficiency of phytostabilization can be increased
by combining the plant species with appropriate soil amendments, such as zeolites, beringite, or hydroxyapatite, thus reducing the
mobility of metals in the polluted soil.
Phytoextraction (or phytoaccumulation): This method involves the uptake of metals onto the roots and their translocation to the
above ground shoots or leaves. These plants must be, in turn, harvested and treated, preventing the recycle of removed pollutants,
when the plant decomposes. Further treatment options may include incineration, disposal, or composting for metal recycling. For
8 Groundwater and Soil Pollution: Bioremediation

Fig. 4 Processes involved in phytoremediation. From Nagendran, R., Selvam, A., Joseph, K., and Chiemchaisri, C. (2006). Phytoremediation and rehabilitation of
municipal solid waste landfills and dumpsites: A brief review. Waste Management 26, 1357–1369.

Table 4 Overview of phytoremediation applications

Treatment method Plant mechanism Surface medium

Phytoextraction Uptake and concentration of metals via direct uptake into the plant tissue with the subsequent Soils
removal of the plants
Phytotransformation Plant uptake and degradation of organic compounds Surface water, groundwater
Phytostabilization Root exudates cause metal to precipitate and become less available Soils, groundwater, mine
tailings
Phytodegradation/ Enhances microbial degradation in plant/rhizosphere Soils, groundwater within
Rhizodegradation rhizosphere
Rhizofiltration Uptake of metals into plant roots Surface water and water
pumped
Phytovolatilization Plants evapotranspirate selenium, mercury, and volatile hydrocarbons Soils and groundwater
Vegetative cap Rainwater is evapotranspirated by plants to prevent leaching contaminants from disposal sites Soils

Cunningham, S. D. and Ow, D. W. (1996). Promises and prospects of phytoremediation: Update on biotechnology. Plant Physiology 110, 715–719; Vidali, M. (2001). Bioremediation:
An overview. Pure Applied Chemistry 73(7), 1163–117, with permission from International Union of Pure and Applied Chemistry.

attaining high remediation efficiency, plant species must fulfill the following prerequisites: high metal accumulation ability,
tolerance against soil pollution, and production of high quantity of biomass. Lately, some plants, which were found to accomplish
a highly abnormal level of accumulating metals to the extent that can be described as “hyperaccumulators,” have been gaining
increasing attention. However, the efficiency of phytoextraction can be influenced by a number of important factors, including the
sorption of metals onto the surface of soil particles and their low solubility. These drawbacks can be compensated for by the
addition of appropriate chemical agents, such as synthetic chelators (e.g., ethylenediaminetetraacetic acid, EDTA), which can
increase the metal solubility and hence promote the uptake from roots. This process is also known as assisted or induced
phytoextraction. Other possible options for enhancing phytoextraction are the selective breeding and application of genetically
engineered plants, which contain specific hyperaccumulator genes. This treatment technique is considered to be particularly cost
effective, as it allows the accumulation of low levels of contaminants from a widespread area.
Phytofiltration or Rhizofiltration: Rhizofiltration refers to the removal of contaminants from flowing water. The latter can be
achieved by the sorption of pollutants onto or into the roots, as well as by the microorganisms that are present in the rhizosphere.
The range of contaminants that can be removed by this technique includes mainly metals, radionuclides, and hydrophobic organic
compounds. Certain varieties of sunflowers and floating plants, such as water hyacinth and duckweed, can be used in this method.
Duckweed has been used with success in a large-scale treatment plant of municipal wastewater in Asia. The efficiency of this
technology can be significantly increased by the addition of organic amendment, which in turn can facilitate the establishment and
growth of such plants.
 Groundwater and Soil Pollution: Bioremediation 9

Phytotransformation or Phytodegradation: This method involves the sorption or uptake of contaminants from soil, sediments, or
water, and their subsequent transformation into more stable, less toxic, and less mobile forms. Organic pollutants, including
nitroaromatics and aliphatic organic compounds, are, most notably, removed via this technique. For instance, the hexavalent
chromium anions can be reduced to the trivalent (cationic) form, which is less mobile and less carcinogenic. Various trees and
grasses are suitable for an efficient phytotransformation. Like other phytoremediation techniques, phytotransformation can be
improved by the addition of soil amendments, or by the application of appropriate plant species.
Phytovolatilization: This technique refers to the uptake of certain metals, such as mercury or selenium, from soil or groundwater
and their subsequent conversion to volatile chemical species. In addition, VOCs can also be removed through plant evapotrans-
piration. Various types of trees and wetland plants can be applied for the removal of VOCs, existing in groundwater, as well as of Hg
or Se from soils, respectively.
Overall, phytoremediation techniques can be described as a cost-effective and practicable solution in respect of treating large
field sites, where other remediation methods might fail, especially at sites with low concentration of contaminants. Inevitably,
phytoremediation has also some limitations that need to be carefully considered before this process could be selected for site
remediation. These drawbacks mainly include: (1) long period of time (probably several years) required for effective remediation,
(2) potential harmful interference with the vegetation and food chain, (3) difficulties in establishing and maintaining desirable
levels of suitable vegetation at sites that are phytotoxic.
The application of bioremediation and phytoremediation technologies can offer significant benefits to environmental pollution
abatement and, more importantly, to human health. In particular, the remediation of contaminated soils or waters can successfully
eliminate the potential threats to human health by rendering harmless most toxic compounds or pathogenic microorganisms. The
improvement of the environmental quality, by enabling sites to be redeveloped and reused, indirectly secures the human health.
Additionally, the avoidance of waste transporting eliminates further the potential threats to human health that may arise during
transportation. However, the risk of adverse health effects should always be under consideration. The great variability of contam-
inants and toxic compounds may increase the potential threats to human health. For example, under certain conditions, a
microorganism, which is generally considered as nonpathogenic, may behave as pathogen. Additionally, the major concern
about the use of bioremediation agents is the potential for biotransformation that may lead to the formation of intermediate
metabolites or to final products, which are occasionally more hazardous than the parent compounds. Finally, the application of
these technologies may produce a potential harmful interference with the vegetation and subsequently with the food chain.
Mycoremediation: Most research within the field of bioremediation has focused on bacteria, with fungal bioremediation
(mycoremediation) attracting interest just within the past two decades. Fungi are a diverse group of organisms and are ubiquitous
in the environment. They can exist and survive in almost every habitat. Fungi play vital roles in all ecosystems and are capable of
regulating the flow of nutrients and energy through their mycelia networks. Fungi are microscopic eukaryotic organisms exhibiting
growth on various substrates and are capable of continuing their function almost indefinitely. These organisms, including the
molds, yeasts and filamentous fungi, are unique microorganisms that can be employed in the remediation of wastes and
wastewaters.
White rot fungi is a physiological grouping of fungi that can degrade lignin (and lignin-like substances). The main mechanism of
biodegradation employed by this group of fungi is the lignin degradation system of enzymes. These extracellular lignin-modifying
enzymes (LMEs) have very low substrate specificity so they are able to mineralize a wide range of highly recalcitrant organopollu-
tants that are structurally similar to lignin. The fact that these fungal enzymes work extracellularly allows them to access many of the
nonpolar, nonsoluble toxic compounds that intracellular processes (such as cytochrome P450) cannot.
Fungi are known to degrade, or cause to deteriorate, a wide variety of materials and compounds, processes known as
mycodegradation and mycodeterioration. The degradative activities of fungi have been recognized in various situations where
they destroy different types of wood, stored paper, textiles, plastics, leather and electro insulating and various wrapping materials.
Chemicals white rot fungi are able to degrade include many pesticides, polyaromatic hydrocarbons (PAHs), polychlorinated
biphenyls (PCBs), and other halogenated aromatics (including dioxins), some dyes, 2.4.6-trinitrotoluene (TNT) and other nitro
explosives, and other toxic chemicals, such as cyanides, azide, carbon tetrachloride, and pentachlorophenol (PCP). Fungi are
especially well-suited to PAH degradation relative to other bacterial decomposers for a few reasons. They can degrade high
molecular-weight PAHs, whereas bacteria are best at degrading smaller molecules. They also function well in nonaqueous
environments where hydrophobic PAHs accumulate; a majority of other microbial degradation occurs in aqueous phase. Also,
they can function in very low-oxygen conditions that occur in heavily PAH-contaminated zones. Sometimes the same fungi that
degrade PAHs have been found to remediate toxic metals as well, which are commonly found in the same polluted sites and can
reduce the effectiveness of some degradative microorganisms.
Although the use of fungi in several cases led to better bioremediation efficiencies than substrate addition to contaminated soil,
the results also point to some of the major limitations. Contrary to technologies using bacteria or bioaugmentation, fungal
bioremediation requires on site approach instead of in situ treatment. The soil thus has to be collected, homogenized and
supplemented with nutrients. Also, the fungus does not grow well in a suspended cell system and enzyme induction is negatively
affected by mixing actions and the ability of the fungus to effectively attach itself to a fixed medium is poor.
10 Groundwater and Soil Pollution: Bioremediation

Electroremediation (Electrokinetic-Enhanced Bioremediation)

Electroremediation is a green and powerful remediation technology that has been extensively studied for use in organic and metal
contamination. The electrokinetics is the use of electric fields of about 0.2–2.0 V cm1 to soil and the basic phenomena which make
up electrokinetic remediation are diffusion, electrolysis, electroosmosis, electrophoresis and electromigration. However, electroki-
netic remediation changes the soil properties, including the soil pH, moisture and microbial biomass, these effects limit the current
that can be applied, which limits the efficiency of electrokinetic remediation in long-term treatment processes. This technology can
be applied to contaminant concentration ranging from small amount of ppm to concentrations greater than 10,000 ppm. However,
it may not be effective for treating multiple contaminants that have significantly different concentrations.
An electric current between electrodes inserted into the soil generates hydrogen ions at the anode and hydroxyl ions at the
cathode, which move into the soil by electromigration, forming pH gradient. When hydroxyl ions are titrated with acid at the
cathode and hydrogen ions acidify the soil, cationic metal species can be eluted from their interactions with soil particles. These
metals move by electromigration towards the cathode, where they are removed and collected. Anionic metal complexes, inorganic
anions and negatively charged organics will migrate toward the anode. Dipolar interactions between water molecules and soil
surfaces lead to an electroosmotic flow of water toward the cathode, which is able to transport uncharged organics. The incorpo-
ration of surfactants has proved to be effective in solubilizing more hydrophobic organics. Fig. 5 demonstrates the effect of
electrokinetic phenomena on porous soil.
There are limitations with electrobioremediation technology that need to be overcome, and these include: (a) solubility of the
pollutant and its desorption from the soil matrix, (b) the availability of the right type of microorganisms at the site of contami-
nation, (c) the ratio between target and nontarget ion concentrations, (d) requirement of conducting pore fluid to mobilize
pollutants, (e) heterogeneity or anomalies found at sites, such as large quantities of iron or iron oxides, large rocks or gravel, and, (f )
toxic electrode effects on microbial metabolism or dielectric cell membrane breakdown or changes in the physicochemical surface
properties of microbial cell. Table 5 summarizes the influence of electrokinetic processes on in situ bioremediation.
(a) Bioattenuation is a low impact and cost effective remediation technique, but is limited to sites where the contaminant is
biodegradable and there is adequate mixing of contaminants, electron acceptors and microorganisms for biodegradation to
occur at suitable rate. Natural mixing can be enhanced with electrokinetic and optimized by increasing the number or

Fig. 5 The effect of electrokinetic phenomena on porous soil. The application of an electric current generates hydroxide ions (OH) and hydrogen gas (H2) at
cathode and hydrogen ions (Hþ) and oxygen gas (O2) at the anode. Subsequent diffusion of OH and Hþ introduces a pH gradient throughout the affected subsurface
that in turn facilitates electrokinetic migration of soil constituents. Microbes and PAHs (example with phenanthrene) migrate the cathode by electroosmosis (EO).
Electronegative microbes also migrate to the anode electrophoretically (EP). Whereas electromigration (EM) dictates the migration of ions (such as sodium and
chlorine) and heavy metals (HM). From Acuña, A. J., Pucci, O. H. and Pucci, G. N. (2012). Electrobioremediation of hydrocarbon contaminated soil from patagonia
argentina. In: Jorge Salgado Gomes (ed.) New technologies in the oil and gas industry. Croatia: InTech doi: 10.5772/50872. Available from: http://www.intechopen.
com/books/new-technologies-in-the-oil-and-gasindustry/electrobioremediation-of-hydrocarbon-contaminated-soil-from-patagoniaargentina
 Groundwater and Soil Pollution: Bioremediation 11

Table 5 Potential influence of electrokinetic processes on in situ bioremediation

Bioremediation process and limitations Electrokinetic influence

Bioattenuation-transformation of pollutants by natural means, mixing limited by
hydrodynamic dispersion in situ
Biostimulation-increased biodegradation by the addition of nutrients and/or
oxygen or other electron acceptors to optimize the P:K:N ratio, improve redox
conditions and increase bioavailability. Limited by hydraulic delivery and
access to low permeability zones
Bioaugmentation-introduction of allochthonous or cultured microbial species
adapted to the biodegradation of a particular contaminant. Limited by hydraulic
delivery and access to low permeability
Phytoremediation-use of plants and/or microbes in root zones to effect
remediation. Limited by access to contaminants
EK-Bioattenuation- EK transport processes increase bioavailability of
contaminants and naturally occurring nutrients and electron acceptors
EK-Biostimulation- EK transport processes allow addition and delivery of
nutrients, electron acceptors and surfactants into contaminated zones
regardless or permeability to increase bioavailability of limiting substances.
EK Bioaugmentation- EK transport of bacterial population to specific zones
regardless of permeability where indigenous community is not adapted
EK Phytoremediation- EK transport processes increase the bioavailability of
contaminants

Gill, R. T., Harbottle, M. J., Smith, J. W. N. and Thornton, S. F. (2014). Chemosphere Electrokinetic-enhanced bioremediation of organic contaminants : A review of processes and
environmental applications. Chemosphere 107, 31–42.

electrodes, reversing the electrode polarity, placing electrodes in a radial configuration and rotating the polarity. This is in order
to increase the diversity and connectivity of flow-paths for potential contact and mixing between microorganisms and
contaminants, maintain more uniform pH and moisture conditions as well as microorganism distribution and increase the
area over which the electric field and enhanced biodegradation is effective.
An important control on biodegradation under electrokinetic is the voltage gradient that limits the migration rate of
substances in situ.
(b) Biostimulation. Electrokinetics can enhance the delivery of nutrients (e.g., phosphate), electron donors (e.g., lactate), and
electron acceptors (e.g., nitrate and sulfate) through different materials at rates greater than diffusion.
Demonstrated and potential applications of EK-stimulation include:
• In a contaminated groundwater plume the concentrations of the contaminant and substance limiting biodegradation are
inversely proportional to each other. EK could regardless of the subsurface permeability.
• Contaminants can diffuse into low permeability matrices rendering them inaccessible and difficult treat. Once sequestered
the contaminant can diffuse back into the host matrix and pose a pollution risk over long timescales, extending the
remediation period. EK-biostimulation could add an amendment at a rate greater than diffusion to stimulate bioremedi-
ation in these low permeability matrices.
(c) Bioaugmentation. EK can enhance the migration of microorganisms through low permeability soils, despite potential occlusion
of cells due to small pore throat size in fine grained materials. This enhancement has been attributed to movement of
microorganisms preferentially along a flow path through macropores within the soil by electroosmotic flow.
EK-bioaugmentation has been used within sequential treatment of contaminated soil conditioned with an active degrader
species. EK-bioaugmentation can also be effective at redistributing bacteria as a pretreatment step for soils contaminated with
heavy metals.
(d) Phytoremediation for recalcitrant organic contaminants in the shallow subsurface (soil and root zone) requires a symbiotic
relationship between the plant and the soil microbial community. Most EK-phytoremediation studies focus on treatment of
heavy metal-polluted soils where the electric field accumulates contaminants around the plant roots increasing bioavailability.
Electrokinetic remediation has been in use for a while to clean up sites contaminated with heavy metals as well for ground
improvement in laboratory and field scale. For instance, in previous studies the effectiveness of electrokinetic remediation field
application in the removal of copper from a contaminated site with an average removal of 85%. Many researchers have conducted
laboratory test using electrokinetic bioremediation. On the other hand, field applications are very limited. For example, Lasagna
technique was used to remove trichloroethylene (TCE) from a contaminated site, in Paducah Kentuky. At this site, EK was successful
in cleaning up TCE from clay soil with removal between 95% and 99%. However, very few field studies have been conducted in
electrokinetic bioremediation. The first field application of electrokinetic bioremediation was conducted in Denmark in 2012 to
degrade perchloroethylene (PCE) from clay soil. The results show that electrokinetics can be used successfully to deliver microor-
ganisms capable of degrading perchloroethylene (PCE).

Microbial Surfactants

Many surfactants can be of natural origin, being part of several cellular structures and biological membranes. Most of these
surfactants are synthesized by living organisms, such as: saponins, produced by plants; glycolipids, by microorganisms; and bile
salts, from animals. These compounds with surfactant properties produced by microorganisms are called biosurfactants.
Most of the biosurfactants are anionic or nonionic; the structure is characteristic of the microorganism producing the surfactant
under the specific growth conditions. The biosurfactants can be toxic or even utilized preferentially by the pollutant-degradating
12 Groundwater and Soil Pollution: Bioremediation

microorganisms. But, the application of biosurfactant-producing and pollutant-degradating microorganisms offers dual advantages
of a continuous supply of biodegradable surfactant and the ability to degrade pollutant(s).
Biosurfactants have countless advantages in comparison with chemical surfactants, especially regarding biodegradability,
compatibility with the environment, low toxicity, high selectivity and their activity even in extreme temperature, pH and salinity
conditions.
These compounds can be roughly divided into two main classes: low-molecular-weight compounds called biosurfactants, such
as lipopeptides, glycolipids, proteins, and high-molecular-weight polymers of polysaccharides, lipopolysaccharides proteins or
lipoproteins that are collectively called bioemulsans or bioemulsifiers. The former group includes molecules which can efficiently
reduce surface and interfacial tension, while the latter are amphiphilic and polyphilic polymers which are usually more effective in
stabilizing emulsions of oil-in-water but do not lower the surface tensions as much.
The best studied microbial surfactants are glycolipids. Among these, the best known compounds are rhamnolipids (Fig. 6),
trehalolipids, sopholipids, and mannosylerythritol lipids (MELs), which contain mono- or disaccharides, combined with long
chain aliphatic acids or hydroxyaliphatic acids.
To overcome the expensive cost constrains associated with biosurfactant production, two basic strategies are generally adopted
worldwide to make it cost effective: (1) the use of inexpensive and waste substrates for the formulation of fermentation media which
lower the initial raw material costs involved in the process; (2) development of efficient and successfully optimized bioprocesses for
maximum biosurfactant production and recovery.
The application of biosurfactants in the remediation of organic compounds, such as hydrocarbons, aims at increasing their
bioavailability (biosurfactant-enhanced bioremediation) or mobilizing and removing the contaminants by pseudosolubilization
and emulsification in a washing treatment. The application of biosurfactants in the remediation of inorganic compounds such as
heavy metals, on the other hand, is targeted at chelating and removal of such ions during a washing step facilitated by the chemical
interactions between the amphiphiles and the metal ions.
The main industrial application of biosurfactants is in the field of oil recovery and processing. Since traditional oil recovery
technologies can only recover approximately 40%–45% of the oil present in the reservoir, some technologies, collectively defined as
enhanced oil recovery, have been developed. Among these, microbial EOR (MEOR), which takes advantage of microbial production
of surface active compounds, is considered to have the most effective potential. Three strategies have been identified for MEOR:
biosurfactant production in offsite reactors and subsequent addition to the oil reservoir; biosurfactant production by injected
allochthonous microorganisms; injection of nutrients into the reservoir to stimulate biosurfactant production in situ by indigenous
bacteria.
Microbial surfactants can promote the growth of bacteria on hydrocarbons by increasing the surface area between oil and water
and through emulsification and increasing pseudosolubility of hydrocarbons through partitioning into micelles. High molecular
weight biosurfactants (bioemulsifiers) have great potential for stabilizing emulsions between liquid hydrocarbons and water, thus
increasing the surface area available for bacterial biodegradation. For low molecular weight biosurfactants, above the Critical
Micelle Concentration (CMC), a significant fraction of the hydrophobic contaminant partitions in the surfactant micelle cores. In
some cases, this results in a general increase in the bioavailability of contaminants for degrading microorganisms. It is well known
that the presence of a surfactant can detrimentally affect biodegradation. Micelle cores can trap organic contaminants creating a
barrier between microorganisms and organic molecules, resulting in the potential substrate becoming less rather than more
available. Another proposed role of biosurfactants in hydrocarbon uptake is the regulation of cell attachment to hydrophobic
and hydrophilic surfaces by exposing different parts of cell-bound biosurfactants, thus changing cell-surface hydrophobicity.

Fig. 6 Microbial surfactants: Chemical structure of two rhamnolipids. Dobler, L., Vilela, L.F., Almeida, R.V., Neves, B.C. (2016). Rhamnolipids in perspective: Gene
regulatory pathways, metabolic engineering, production and technological forecasting. New Biotechnology 33(1), 123–135. doi: 10.1016/j.nbt.2015.09.005.
 Groundwater and Soil Pollution: Bioremediation 13

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