Reviews in Environmental Science and Bio/Technology (2005) 4:115156

DOI 10.1007/s11157-005-2169-4

Springer 2005

Developments in bioremediation of soils and sediments polluted with

metals and radionuclides 1. Microbial processes and mechanisms aecting
bioremediation of metal contamination and inuencing metal
toxicity and transport
Henry H. Tabak1,*, Piet Lens2, Eric D. van Hullebusch2,4,5 & Winnie Dejonghe3
1

US EPA, ORD, National Risk Management Research Laboratory, 26 West Martin Luther King Drive,
Cincinnati, OH, USA; 2Sub-Department of Environmental Technology, Wageningen University, Wageningen,
The Netherlands; 3Flemish Institute for Technological Research, VITO, Belgium; 4Universite de Marne-laVallee, Laboratoire des Geomateriaux, 77454 Marne La Vallee, Cedex, 2 France; 5Universite de Limoges,
Laboratoire des Sciences de lEau et de lEnvironnement, 87060 Limoges Cedex, France (*author for correspondence, e-mail: tabak.henry@epamail.epa.gov)

1. Introduction
1.1. Bioremediation Technologies
Bioremediation technology uses microorganisms
to reduce, eliminate, contain and transform to
benign products, contaminants present in soils,
sediments, water or air. The last 15 years have
seen an increase in the types of contaminants to
which bioremediation is being applied, including
solvents, explosives, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls
(PCBs). Now, microbial processes are beginning
to be used in the cleanup of radioactive and
metallic contaminants.
Bioremediation is an alternative to traditional
remediation technologies such as landlling or
incineration. Bioremediation depends on the presence of the appropriate microorganisms in the
correct amounts and combinations and on the
appropriate environmental conditions. Although
prokaryotes Bacteria and Archaea are usually
the agents responsible for most bioremediation
strategies, eukaryotes such as fungi and algae also
can transform and degrade contaminants. Microorganisms already living in contaminated environments are often well-adapted to survival in the
presence of existing contaminants and to the temperature, pH, and oxidationreduction potential
of the site. These indigenous microbes tend to utilize the nutrients and electron acceptors that are
available in situ, provided liquid water is present.

The bulk of subsurface microbial populations are

associated with both microorganisms and dissolved substances, including contaminants and
their breakdown products.
Bioremediation works by either transforming
or degrading contaminants to non-hazardous or
less hazardous chemicals. These processes are
called, respectively, biotransformation and
biodegradation. Biotransformation is any alteration of the molecular or atomic structure of a
compound by microorganisms. Biodegradation is
the breaking down of organic substance by
microorganisms into smaller organic or inorganic
components.
Unfortunately, metals and radionuclides cannot be biodegraded. However, microorganisms
can interact with these contaminants and transform them from one chemical form to another by
changing their oxidation state through the
addition of (reduction) or removing of (oxidation) electrons. In some bioremediation strategies,
the solubility of the transformed metal or radionuclide increases, thus increasing the mobility of
the contaminant and allowing it to more easily be
ushed from the environment. In other strategies,
the opposite will occur, and the transformed
metal or radionuclide may precipitate out of
solution, leading to immobilization. Both kinds
of transformations present opportunities for
bioremediation of metal and radionuclides in the
environment either to immobilize them in place
or to accelerate their removal.

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1.2. Metal contamination
The challenge posed by metal contaminants lies
in the permanence of their nuclear structures:
stable isotopes are virtually indestructible. They
may be redistributed, however, as well as interconverted among forms of varying chemical and
physical properties; as such metal mobility and
mutability are the sources of both problems
and of many promising solutions.
Most heavy metals exist naturally in the
earths crust at trace concentrations of just a few
parts per million (Bodek et al. 1988), sucient to
provide local biota with trace nutrients, but too
low to cause toxicity. Disposal of wastes from
metal excavation and processing have concentrated these metals to dangerous levels in some
soils and sediments, endangering both wildlife
and people and motivating eorts to detoxify the
soils. EPA standards rarely require that metal
contamination be reduced to background levels,
however, with preliminary remediation goals for
metals varying from 0.2 to 63,000 ppm based entirely on projected risks (Smucker 1996).
Metal contamination of soil is especially
problematic because of the strong adsorption of
many metals to the surfaces of soil particles. Due
to the diculty of desorbing metal contaminants,
some traditional remediation methods simply
immobilize metals in contaminated soils, for
example, by the addition of cement or chemical
xatives, by capping with asphalt, or by in-situ
vitrication. Alternatively, soils are often isolated
by excavation and connement in hazardous
waste facilities (Schleck 1990; Trezek 1990). Although rapid in eect, both of these options are
expensive ($30$300 m)3) and destroy the soils
future productivity (Cunningham et al. 1995).
Soil washing and pump-and-treat technologies
ambitiously attempt to remove contaminating
metals from soils by pumping water or other solvents into soil and extracting the liquid downgradient from the contamination. Metals are
then precipitated from the liquid, which is recycled if possible. The success of these methods is
severely limited by the slow desorption kinetics
of adsorbed metals, with the result that additional agents are often used to promote metal
transfer to the aqueous phase. Typical additives
are acids, chelates, and reductants, which im-

prove cost eectiveness but may introduce further harmful chemicals (Boyle 1993; EPA 1991).
A primary strategy of bioremediation is the
use of similar metal-mobilizing agents in conjunction with soil washing, with the advantage that
they pose no known environmental threat themselves. Biopolymers have been discovered that
bind metals with high anity and travel relatively
unimpeded through porous medium. Certain
microorganisms transform strongly adsorbing
metal species into more soluble forms, and plants
are being recruited that act as self-contained
pump-and-treat systems. Other methods employ
enzymatic ativities to transform metal species into
volatile, less toxic, or insoluble forms. Techniques
for soil bioremediation are usually designed to be
used in-situ, lowering costs; they avoid the use of
toxic chemicals, and in nearly all cases, the soil
structure and potential for productivity are
preserved. Although the understanding of the
processes discussed below is far from complete,
intense research in bioremediation is rapidly
elucidating the mechanisms involved.

2. Microbial processes aecting bioremediation

of metals and radionuclides
Bioremediation of metals and radionuclides relies
on a complex interplay of biological, chemical
and physical processes. A fundamental mechanistic understanding of coupling microbial metabolism, chemical reactions and contaminant
transport is beginning to develop as well as how
these activities could work together to bioremediate metal and radionuclide pollution
Microbes exist in complex biogeochemical
matrices in subsurface sediments and soils. Their
interactions with metals and radionuclides are
inuenced by a number of environmental factors,
including solution chemistry, sorptive reactive
surfaces, and the presence or absence of organic
ligands and reductants (Figure 1).
Microorganisms can interact with metals and
radionuclides via many mechanisms some of
which may be used as the basis of potential
bioremediation strategies. The major types of
interaction are summarized in Figure 2. In addition to the mechanisms outlined, accumulationm
of metals by plants (phytoremediation) warrants

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Figure 1. Abiotic and biotic mechanisms inuence the fate of metals (M) in subsurface environments. From left to right: Organic
material produced by microorganisms can act as ligands (L) and complex with metals, facilitating transport. These ligands can also
be degraded by microbes to relase the metal. Microbes can also directly adsorp/desorb, takeup/excrete, and oxidize/reduce metals.
Oxidation and reduction will change the valence state of the metal either up or down (Mnx). Abiotically, metals can form solution complexes or can complex with the surface of clays (kaolinitepolymer complexes). These mineral complexes can sorb metals
either directly to the mineral or to organic or iron oxide coatings. Metals can also be directly oxidized or reduced to dierence
valence states by the ambient redox conditions or by organic reductants (Lred) or oxidants (Lox). Changing the valence state of
metals will aect their sorption, mobility, precipitation, and toxicity (image courtesy of S. Fendorf, Standford University).

attention as an additional established route for the

bioremediation of metal contamination. The studies of Salt, Raskin and co-workers (Raskin et al.
1997; Salt et al. 1995; Salt et al. 1998) provide a
detailed description of use of plants for (1) phytoextraction; the use of metal-accumulating plants
to remove toxic metals from soil; (2)
rhizoltration; the use of plant roots to remove
toxic metals from polluted waters; and (3) phytostabilization; the use of plants to eliminate the bioavailability of toxic metals in soils. Recently,
comprehensive reviews were published on phytoremediation of metal pollution in soils (Chaney
et al. 1997; Saxena et al. 1999; Vangronsveld 2000;
van der Lelie et al. 2001; Reeves & Barker 2001;
Schwitzguebel et al. 2002; Pulford & Watson 2003).
Iron cycling and associated changes in solidphase chemistry have dramatic implications for
the mobility and bioavailability of heavy metals
and radionuclides. Coupled ow and water
chemistry control the rate and solid phase products of iron hydroxide reduction and provide
critical information in assessing the reactivity of

reduced environments toward metal and radionuclide contaminants.

Bioremediation of soils, sediments, and water
contaminated with metals and radionuclides can
be achieved through biologically mediated changes in the oxidation state (speciation) of those
contaminants biotransformation. Changes in
speciation can alter the solubility of metals and
radionuclides, and therefore their transport properties and toxicity. The latter two characteristics
can determine bioavailability (see van Hullebusch
et al. 2005 for further details). Resistance by subsurface microorganisms to the toxicity of heavy
metals is critical for the bioremediation of contaminated subsurface sites. Remedial action depends on actively metabolizing microbes, and
these microbes might be inhibited by high concentrations of toxic heavy metals.
There are at least three types of microbial
processes that can inuence toxicity and transport of metals and radionuclides: biotransformation, biosorption and bioaccumulation, and
degradation or synthesis of organic ligands that

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Figure 2. Metalmicrobe interactions impacting bioremediation.

aect the solubility of the contaminants. Each

oers the potential for bioremediation of metallic
and radioactive contaminants in the environment.
2.1. Biotransformation
Metal-reducing microorganisms can reduce a
wide variety of multivalent metals that pose environmental problems. The heavy metals and radionuclides subject to enzymatic reduction by
microbes include but are not limited to uranium
(U), technetium (Tc), and chromium (Cr). Direct
enzymatic reduction involves use of the oxidized
forms of these contaminants as electron acceptors. The oxidized forms of U, Tc, and Cr are
highly soluble in aqueous media and are generally very mobile in aerobic ground water, while
the reduced species are highly insoluble and often
precipitate from solution. Direct enzymatic
reduction of soluble U(VI), Tc(VII), and Cr(VI)

to insoluble species has been documented and is

illustrated in Figure 3.
Extracellular precipitation of metals and
radionuclides has been demonstrated in a
number of microbial isolates. For example, the
precipitation of uranium on the cell surface of
the bacterium Shewanella is shown in Figure 4.
Metal-reducing organisms reduce uranyl carbonate, which is exceedingly soluble in carbonatebearing ground water, to highly insoluble U(IV),
which precipitates from solution as the uranium
oxide mineral uraninite.
Signicant advances have been made in understanding the mechanisms of reduction of Fe(III),
U(VI) and Tc(VI) in the subsurface bacterium
Geobacter sulfurreducens, using the tools of biochemistry and molecular biology. A surfacebound c-type cytochrome (mass 40 kDa) that is
involved in the transfer of electrons to extracellular insoluble Fe(III) oxides has been identied

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Figure 3. Direct enzymatic reduction of soluble heavy metals and radionuclides by metal-reducing bacteria. Non-hazardous
organic compounds, such as lactate or acetate, provide electrons used by these microorganisms. Note, however, that if complexed,
the reduced species may become mobile.

Figure 4. Microbes can play an important role in immobilizing radionuclides. This image shows a cross section of the
bacterium Shewanella with uraninite precipitated on the cell
surface (image courtesy of S. Fendorf, Standford University).

and characterized. This protein is not required for

the reduction of U(VI), suggesting that the mechanisms of Fe(III) and U(VI) reduction may be
distinct. Another c-type cytochrome (9.6 kDa),
found in the periplasm, appears to be required
for U(VI) reduction (Figure 5). This protein is
able to reduce U(VI) in vitro, and a mutant
unable to synthesize the protein was unable to
reduce U(VI) eciently. Surprisingly, Tc(VII) is
reduced by yet another mechanism, a periplasmic
Ni/Fe-containing hydrogenase that uses hydrogen
as the electron donor for metal reduction. Moreover, ecient indirect mechanisms may be important in immobilizing Tc in sediments wherein
biologically reduced Fe(III) or U(IV) is able to
transfer electrons directly to Tc(VII).

A wide range of bacteria reduces the highly

soluble chromate ion to Cr(III), which under
appropriate conditions precipitates as Cr(OH)3.
A number of Cr(VI)-reducing microorganisms
have been isolated from chromate-contaminated
waters, oils, and sediments, including Arthrobacter sp., Pseudomonas aeruginosa S128, some
anaerobic-reducing bacteria, and even several algae. Laboratory experiments with Hanford Site
sediments showed that Cr(VI) concentration in
water decreased signicantly (>66%) in a
month-long incubation in the presence of nitrate
and added dilute molasses as an electron donor.
Thus, the addition of molasses to vadose zone
sediments shows potential to decrease the transport of chromium and nitrate into underlying
aquifers.
Although some microorganisms can enzymatically reduce heavy metals and radionuclides directly, indirect reduction of soluble contaminants
may be possible in sedimentary and subsurface
environments, although this has not been demonstrated under natural conditions, to date. This
indirect immobilization could be accomplished
by metal-reducing or sulfate-reducing bacteria.
One approach would be to couple the oxidation
of organic compounds or hydrogen to the reduction of iron [Fe(III)], manganese [Mn(IV)], or
sulfur [S(IV)] in the form of sulfate, [SO42)]. Iron(III) can be biologically reduced to Fe(II),
Mn(IV) to Mn(III), and S(VI) (sulfate) to S(II)
(hydrogen sulde, H2S). The reduced product
might then, in turn, chemically reduce metals or
radionuclides to yield separate or multicomponent insoluble species (see van Hullebusch et al.
2005 for details).
The most reactive of these reduced species
are ferrous iron [Fe(II)] and H2S. The latter is

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Figure 5. Transmission electron micrograph (TEM) showing extracellular and periplasmic U(IV) precipitates formed by enzymatic
reduction of U(VI) by the subsurface bacterium Geobacter sulfurreducens. (TEM image obtained by S. Glasauer, Guelph University).

generated by the enzymatic activity of ironreducing and some fermentative bacteria, can
reduce multivalent metals such as uranium,
chromium, and technetium (Figure 6a). The reduced forms of these metals are insoluble and
can either precipitate as reduced oxide or
hydroxide minerals, or coprecipitate with Fe(III)
minerals that form during the reoxidation of
Fe(II). In coprecipitation, elements are incorporated in metal oxide minerals as they precipitate
from solution.
Sulfate-reducing bacteria also may be stimulated to produce a chemically reactive redox barrier (Figure 6b). Hydrogen sulde generated by
sulfate-reducing bacteria could chemically reduce

the contaminant to a form that would be stable

for extended periods of time. A study of biolms
in a zinc and lead mine is a good example of indirect immobilization of heavy metals by sulfatereducing bacteria. Sulde produced by sulfate reduces in the lm scavenged zinc and other toxic
metals. X-ray uorescence microbeam analysis revealed that zinc and small amounts of arsenic and
selenium were extracted from ground water and
concentrated in biolms in zinc sulphide precipitates (Figure 7) (Courtesy of K.M. Kenner,
Arrgonne National laboratory and K.M. Baneld, University of California, Berkeley). Thus,
microbial formation of sulde deposits drastically
decreased the migration of contaminant metals.

Figure 6. Indirect mobilization of heavy metals and radionuclides by (a) metal-reducing and (b) sulfate-reducing bacteria.

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Manganese (III/IV) oxides, which are common mineral phases in many soils and sediments,
are also electron acceptors for metal-reducing
bacteria. Manganese oxides are also relatively
strong oxidants and can oxidize insoluble, reduced contaminants such as the mineral uraninite
(UO2), a common product of microbial uranium
reduction. Dierences in the solubility of oxidized Mn (insoluble) and U (soluble) challenge
bacterium predictions of their biogeochemical
behaviour during in situ bioreduction. Results
from laboratory experiments with the subsurface
bacterium Shewanella putrefaciens CN32 quantitatively reduced U(VI) to U(IV), in the form of a
solid UO2. The UO2 was observed in regions
external to the cell as well as in the periplasm,
the region between the inner and outer membrane of the cell, in the presence of the Mn oxides, the reduced U resided exclusively in the
periplasm of the bacterial cells (Figure 8) (Courtesy of J. Fredrickson, of Pacic Northwest
National Laboratory). These results indicate that
the presence on Mn(III/IV) oxides may impede
the in situ biological reduction of U(VI) in
subsoils and sediments. However, the accumulation of U(IV) in the periplasm indicates that the

cell may physically protect reduced U from oxidation by Mn oxides, suggesting that extensive
reduction of soil Mn oxides may not be required
for reductive biomobilization of U(IV).
Natural organic matter (NOM) may play a
role in the reduction of contaminants such as
Cr(VI) and U(VI) in subsurface environments.
NOM consists of a mixture of organic compounds with dierent structures and functional
groups. These groups include aromatic and phenolic moieties, carboxylic and heteroaliphatic hydroxyl functional groups, and free radicals. In the
presence of a metal-reducing bacterium, NOM
eectively mediated the transfer of electrons for
the reduction of Fe(III), Cr(VI), and U(VI), although the reduction rate varied among dierent
NOM samples and among contaminants.
2.2. Bioaccumulation and biosorption
Microorganisms can physically remove heavy
metals and radionuclides from solution through
association of these contaminants with biomass.
Bioaccumulation is the retention and concentration of a substance within an organism. In bioaccumulation, solutes are transported from the

Figure 7. X-ray uorescence trace metal microanalysis of sulfate-reducing bacteria. Results of electron (bottom left) and X-ray (top
right) microprobe analysis of specic biomineralized zinc sulde precipitates. The sensitivity of the X-ray microprobe enables identication of arsenic and selenium constituents in the zinc sulde precipitate on the surface of a sulfate-reducing bacterium.

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Figure 8. TEM images of unstained thin sections from S. putrefaciens CN32 cells incubated with H2, as the electron donor, and
U(VI) in bicarbonate buer in the presence of Mn oxides such as bixbyite or birnessite exhibited an absence of ne-grained extracellular UO2(s) and accumulation of UO2(s) exclusively in the periplasm (images courtesy of J. Fredrickson of Pacic Northwest
National Laboratory).

outside of the microbial cell through the cellular

membrane, into the cell cytoplasm, where the
metal is sequestered. Biosorption describes the
association of soluble substances with the cell
surface. Sorption does not require an active
metabolism. The amount of metal biosorbed to
the exterior of bacterial cells often exceeds the
amount predicted using information about the
charge density of the cell surface. Scientists have
demonstrated that charged functional groups
serve as nucleation sites for deposition of various
metal-bearing precipitates.
Three possible non-reducing mechanisms of
actinide-microbe interactions are shown in
Figure 9. The example shows sorbed U(VI),
which appears to remain in its oxidized state.
These include: (1) sorption on cell surface sites;
(2) additional surface complexation and precipitation of actinides; and (3) precipitation of
actinides with bacterial cell lysates. In Grampositive bacteria, surface complexation occurs
between organic phosphate groups in cell surface teichoic acid and U(VI). Uranium(VI)phosphate solids are the least soluble of all the
U(VI) solid phases. By contrast, Gram-negative
bacteria appear to have a lesser ability to sorb
U, possibly because they lack these cell-surface
organic phosphate groups. One of the most
common surface structures found in both Bacteria and Archaea is a crystalline proteinaceous
surface layer called the S-layer. The S-layer
appears to attenuate the sorption ability of
Gram-positive bacteria.

2.3. Siderophore-mediated uptake by

microorganisms
In aerobic soils, iron exists primarily as Fe(III),
which has low water solubility (10)18) and cannot be acquired as the free ion by soil microbes.
To circumvent this problem, microbes produce
siderophores, low-molecular-weight chelating
agents that bind with iron and transport it into
the cell through an energy-dependent process
(Figure 10) (John et al. 2001). Experiments have
shown that various metals can form complexes
with siderophores and that many of these complexes are recognized by cell uptake proteins.
Study of siderophore actinides and the uptake of
these complexes is an important component in
the understanding of how microbes and actinides
interact in the environment.
Researchers have now demonstrated that a
microorganism can take up plutonium by the
same mechanism it uses to take up iron. The
common soil microorganism Microbacterium
avescens uses siderophores to obtain its nutritionally required iron. Bacteria were incubated
with the siderophore desferrioxamine-(DF)
bound with either plutonium [Pu(VI)], iron
[Fe(III)], or uranium [U(VI), as UO2+2]. Using
transport proteins, the cells took up the Pu-siderophore complex (Figure 10), although at a
much slower rate than they took up the Fe-siderophore complex; however, they did not take
up the U-siderophore complex. Only metabolically active bacteria were capable of taking up

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Figure 9. Three possible non-reductive mechanisms of bacterial cell surface interaction with U(VI) (Courtesy of H. Nitzche and
T. Hazen, Lawrence Berkeley National Laboratory, and S. Clark, Wash. State University).

Figure 10. Siderophore-mediated Pu accumulation by Microbacterium avescens (John et al. 2001).

the Pu siderophore complexes, just as with Fesiderophore uptake. These discoveries could
have wide-ranging implications for future biomediation eorts and for more accurate predictions of how plutonium and other actinides
behave in the environment. Siderophore-mediated uptake and transport could be an important pathway for environmental mobility and Pu
entry into the food chain.

2.4. Microbes and synthetic organic chelators

Organic complexing agents can have a profound
eect on the mobility of metals and radionuclides
in subsurface environments. Synthetic chelators
such as EDTA and NTA can form stable, soluble complexes with heavy metals and radionuclides. These chelators were commonly used as
cleaning agents during industrial processing of

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nuclear fuels and were sometimes co-disposed
with metals and radionuclides. Metalchelate
complexes have entered the environment and
may migrate in ground water. However, the
migration of these complexes can be reduced by
the biodegradation of the organic ligand (Figure 11). The resulting free metal ions are likely to
absorb to mineral surfaces or to form oxide mineral precipitates that would be less mobile in
ground water. The degradation of organic chelators associated with metal or radionuclide contaminants, then, might achieve a desirable
immobilization of contaminants in place.
Some of these chelators can be degraded by
naturally occurring microorganisms. A number of
EDTA- and NTA-degrading organisms have been
isolated and identied. In one study, microbial
degradation of EDTA by the environmental isolate BNC1 was inuenced by the complex metal.
Cobalt(II)EDTA, cobalt (III)EDTA, and nickel(II)EDTA complexes were not degraded,
whereas copper (II)EDTA and zincEDTA
complexes were. The genes and enzymes responsible for EDTA and NTA degradation have been
identied, and the genes have been cloned and sequenced. All the genes necessary to code for degradation of EDTA and NTA occur together in a
gene cluster. Elucidating the genes and enzymes responsible for EDTA and NTA biodegradation will provide an understanding of the
environmental and physiological controls on chelate degradation in bacteria, and provide gene
probes for monitoring this process in the environment. Such fundamental research on the mechanisms of enzymatic degradation of synthetic
chelators is expected to provide useful information for developing bioremediation strategies.
Metal-reducing bacteria also sometimes actually promote the mobilization of insoluble forms
of some heavy metals and radionuclices. It has
been demonstrated that metal-reducing bacteria
can solubilize PuO2, which is insoluble, in the

Figure 11. Immobilization of heavy metals by enzymatic degradation of organic chelators, such as EDTA and NTA.

presence of the synthetic chelator NTA. It is

thought that the bacteria reduced the insoluble
Pu(IV) to Pu(III), which was then complexed by
NTA. This process may provide a means of
mobilizing Pu from contaminated soils and sediments, and could be a step in the removal of this
highly toxic radionuclide from the environment.
However, this approach has not been tested in
the eld.
Organic acids formed by the metabolic activity of microorganisms can lower the pH of the
system to values that interfere with the electrostatic forces that hold heavy metals and radionuclides on the surface of iron or manganese
oxide minerals. Displacement of cations by
hydrogen ions may lead to the solubilization of
the surface-associated metal or radionuclide. In
some cases, the organic metabolites also serve as
complexing agents that can form soluble metal
ligand complexes. These complexing agents
(which include dicarboxylic acids, phenolic compounds, ketogluconic acids, and salicylic acids)
have been shown to promote the dissolution of a
wide range of heavy metals and radionuclides,
including PuO2. Therefore, biogenic production
of complexing agents can accelerate the movement of metals in soils and sediments.

2.5. Microbial metabolism of iron reducing

bacteria
2.5.1. Dissimilatory iron reduction
Iron is extremely abundant in the Earths crust,
primarily in the form of insoluble Fe(III) oxides.
The reduction potential of Fe(III)/Fe(II) is electropositive (Table 1). A number of microorganisms are able to couple oxidation of hydrogen or
organic compounds to the reduction of Fe(III)
and gain energy for growth. The use of iron or
other metals as terminal electron acceptors is
called dissimilatory metal reduction. (Not all dissimilatory metal reduction, however, is linked to
energy conservation.) Geological and microbiological evidence suggests that Fe(III) reduction
was a very early form of respiration on Earth.
A phylogenetically diverse group of Bacteria
and Archaea is known to conserve energy to support growth by oxidizing hydrogen or organic
compounds (including contaminants such as
aromatic hydrocarbons) with the reduction of

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Table 1. Microbially signicant half-reaction reduction potentials
Transformation
O2 depletion
Denitrication
Mn reduction, Mn(IV) to Mn(II)
Fe reduction, Fe(III) to Fe(II)
Sulfate reduction, S(VI) to S(-II)
Methane generation, C(IV) to C(-IV)
H2 generation, H(I) to H(0)

Reaction

Eh, Volts (@ pH 7)
+

0.5O2+2H H2O
NO3)+6H++5e) 0.5N2+3H2O
MnO2+4H++2e) Mn2++2H2O
Fe(OH)3+3H++e) Fe2++3H2O
SO42)+10H++8e) H2S+4H2O
HCO3)+9H++8e) CH4+3H2O
H++e+ 0.5H2

0.82
0.71
0.54
0.01
)0.22
)0.26
)0.41

see Stumm and Morgan (1996).

Fe(III). Such a group includes species from such

genera as Geobacter, Desulfuromonas, Pelobacter,
Shewanella, Ferrimonas, Geovibrio, Geothrix, and
others. These organisms have a broad spectrum
of other metabolic capabilities as well. Many dissimilatory metal reducers such as Geobacter species can reduce soluble U(VI) to insoluble U(IV)
(Figure 12).
Dissimilatory metal-reducing microorganisms
might prevent migration of uranium in ground
water by precipitation and immobilization in the
subsurface. When a simple organic compound
such as acetate is added to the subsurface, aerobic microorganisms quickly consume available
dissolved oxygen and nitrate. Then dissimilatory

metal-reducing microorganisms begin to metabolize acetate, oxidizing it to CO2 while reducing

available metals. While Fe(III) is generally the
most abundant metal electron acceptor in the
subsurface, dissimilatory metal-reducing microorganisms can also simultaneously reduce U(VI) to
U(IV), precipitating it out of ground water. This
has been demonstrated conclusively in laboratory
studies, and is the basis for new strategies of
in situ biomediation.
2.5.2. Mechanics
Mechanisms for ion reduction appear to vary
among dissimilatory metal-reducing microorganisms (Figure 13). Some species use strategies to

Figure 12. Geobacter sulfurreducens growing with insoluble Mn(IV) oxides as the electron acceptor (Image courtesy of D. Lovley,
University Massachusetts).

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overcome the need for direct contact with Fe(III)
oxides. For example, Shewanella oneidensis is a
versatile microbe that can use oxygen, nitrate,
uranium, manganese, and iron as electron acceptors. This bacterium appears to release quinones
into the culture medium during growth that serve
as electron shuttles between the bacterium and
the Fe(III) oxide. Shewanella alga and Ceothrix
fermentans (in addition to producing electron
shuttles) solubilize Fe(III) during growth, presumably by releasing one or more Fe(III)-complexing compounds called chelators. By contrast,
Geobacter metallireducens does not release electron shuttles and is highly adapted to contact
with the solid Fe(III) oxide. When growing on
insoluble Fe(III) or Mn(IV) oxides, this microorganism produces agella and uses chemotaxis to
nd the electron acceptor. Pili are also produced
under these conditions, presumably to attach to
insoluble oxides.
Although dissimilatory metal reducers are of
obvious importance to developing strategies for
biomediation of organic contaminants as well as
metals and radionuclides, this process can be

slow. One idea for stimulating their activity in

aquifer sediments is to add humic acids or other
quinone-containing compounds to which Fe(III)reducing microbes can transfer electrons. Electron shuttling via extracellular quinones may
accelerate the rate and extent of biomediation.
2.5.3. Exploring the diversity of iron (III)reducing bacteria in subsurface sediments
Iron(III)-reducing bacteria are thought to catalyze a large number of sedimentary processes
that have important impacts on biomediation.
Many new strains of iron-reducing microorganisms have now been isolated from uranium-contaminated subsurface sediments, expanding our
knowledge of the diversity of this environment.
Gene-sequencing methods have been used to
classify these isolates, which include Gram-positive genera (Clostridium, Camoccus) that were
not closely related to any previously characterized pure cultures of Fe(III)-reducing bacteria.
The Clostridia are well-studied anaerobic bacteria that have been isolated from sediments
since the origin of environmental microbiology.

Figure 13. Mechanisms of Fe(III) reduction by dissimilatory metal-reducing bacteria (c = chelator) (Image courtesy of D. Lovley
and ASM News).

127
However, all of the Clostridia isolated previously
were fermentative organisms incapable of respiration. In contrast, many of the Clostridium strains
described from contaminated subsurface sediments were shown to conserve energy for growth
by coupling the respiration of Fe(III) oxide
minerals to the oxidation of organic acids (acetate or lactate). Several of the bacterial isolates
were also shown to reduce U(VI). Although their
environmental signicance remains to be explored, these newly isolated FeRBs could play an
important role in subsurface biomediation.

3. Microbial mechanisms involved in

bioremediation of metal and radionuclide
contaminated soils and sediments
3.1. Biopolymers and their use in bioremediation
of metal contamination
Microbially produced macromolecules with metal-binding properties include cyclodextrins, exopolysaccharides and amphipathic molecules
termed biosurfactants. Through combinations
of carboxyl, phosphoryl, and hydroxyl groups,
these molecules complex divalent cations,
increasing their aqueous solubility to enhance the
eciency of soil washing. Biopolymers may be
injected with washing solvents or produced within contaminated soil by metal-tolerant microorganisms. The properties of these molecules are
also potentially alterable by genetic engineering
to produce structures with higher metal specicities and complexing strengths.
3.1.1. Biosurfactants
Composed of polar, often phosphate-based
heads and non-polar lipid tails, biosurfactants combine complexation activity with physical sequestration of the complexed ions (Miller,
1995). A surfactant, or surface-active agent,
diminishes the attractive forces among the molecules of a liquid at the liquids surface. Because
the free energy of the surface is thereby lowered,
surfactant molecules tend to concentrate at such
surfaces, including soilwater interfaces (Tinoco
Jr. et al. 1985). This characteristic directly benets the surfactants bioremedial purpose by concentrating the molecules near sorbed metals,

enhancing rates of attainment of complexation

equilibrium. At a suciently high-concentration
of 1200 mg/L, called the critical micelle concentration (CMC), biosurfactant molecules begin to
aggregate into micelles (spherical bilayers),
sequestering the chelated ions within the particles. These particles usually measure less than
50 nm in diameter and are thus expected to
avoid ltration eects in most soils. Adsorption
may still impede their progress, however (Miller
1995).
The most thoroughly studied bio-surfactant is
the anionic rhamnolipid of Pseudomonas aeruginos (Figure 14). The rhamnolipid forms complexes with Cd2+, Zn2+ and Pb2+ in aqueous
solution, with stability constants (Log K) of 6.5,
6.6 and 5.4, respectively (Herman et al. 1995).
These are far lower than the metalEDTA constants (b=105.0 to 106.0) suggesting that the biosurfactant should compete successfully with soil
organic matter for metal complexation.
The rhamnolipids metal-mobilizing capability
has been investigated in a sandy loam soil of minimal (0.11%) organic matter, showing that lead
desorption is the rhamnolipids greatest strength.
In soil samples with adsorbed cadmium, zinc and
lead (total 3.4 mmol kg)1), 12.5 mM rhamnolipd
enhanced lead desorption relative to an electrolyte control solution, while 80 mM rhamnolipid
was needed to improve cadmium and zinc desorption. The variable eectiveness is likely to be due
to properties of the metals: lead is a softer Lewis
acid than cadmium or zinc. Therefore, it tends to
form more stable complexes and is less aected
by ion-exchange processes. These characteristics
are evident in both the notorious diculty of lead
removal from soil and in the measured metal
rhamnolipid stability constants. While a fairly
concentrated (110180 mM) electrolyte solution
released the harder acids by cation exchange,

Figure 14. Rhamnolipid structure of Pseudomonas aeruginosa

ATCC 9027. For C18 monorhamnolipid, m+n=10; for C20,
m+n=12; for C22, m+n=12; and for C24, m+n=12. (Reprinted from Zhang & Miller, 1994, with permission. Copyright 1994 American Society for Microbiology).

128
rhamnolipid complexation was required to mobilize soil-sorbed lead. If rhamnolipids are generally
eective in mobilizing soft Lewis acids, they will
provide a much-needed tool in soil remediation
(Herman et al. 1995).
After complex formation and presumably
after subsurface transport and recovery, the
rhamnolipid can be recovered and recycled:
acidication of a solution containing a cadmiumrhamnolipid complex precipitates the
rhamnolipid, releasing nearly all of the metal
into solution. The rhamnolipid may then be separated by centrifugation, redissolved at neutral
pH and reused, while the metal is recovered
from the supernatant by alkali precipitation or
cations exchange (Tan et al. 1994).
One limitation to rhamnolipid use is the high
concentration needed to release metals from soil,
due to strong adsorption of the rhamnolipid itself. Rhamnolipid-soil sorption is, however,
greatly diminished in solutions of low ionic
strength, suggesting that cation bridging between the anionic head group and cations sorbed
to the soil occurs. Eorts are currently underway
to improve the eciency of rhamnolipid treatment (Herman et al. 1995).
Remarkably, the P. aeruginosa rhamnolipid
can also promote the transport of entire microorganisms through a solid matrix, in this case a
porous sand. Experimentation and modeling
have shown that the rhamnolipid, used at
1501000 mg l)1, acts by decreasing adsorption
of the cells to the particle surfaces, either by
increasing the negative charge density of the surfaces and thus the repulsion between cells and
surfaces, by dissolving extracellular polymers
used in adhesion, or by competing with the
microorganisms for sorption sites. Because in-situ
bioremediation will sometimes require the delivery of microorganisms to sites of contamination
and because most bacteria have low mobility in
soils, this rhamnolipid property may become
quite valuable (Bai et al. 1997).
3.1.2. Cyclodextrins
Cyclodextrins are cyclic oligosaccharides (Figure 15) formed during bacterial degradation of
plant starches (Wang & Brusseau 1993). The central cavity of relatively low polarity allows formation of 1:1 inclusion complexes with non-polar
organic molecules, while the hydrophilic outer

ring enables the complex to remain water-soluble.

The addition of carboxylate groups to the outer
ring has given the cyclodextrins metal-complexing
ability as well: carboxymethyl-b-cyclodextrin, or
CMCD, strongly complexes Cd2+ with a constant
of 103.66 for 1 g/l CMCD (Wang & Brusseau 1995)
and the presence of phenanthrene as a model organic contaminant does not reduce this metalcomplexing capacity (Brusseau et al. 1997).
Like biosurfactants, cyclodextrins do lower
the surface tension of water, but they do not
have a critical micelle concentration (Wang &
Brusseau 1993). Cyclodextrins are small enough
to avoid pore exclusion in soils and experiments
have shown no measurable adsorption of the
cyclodextrin to sandy subsoils and surface soils
(Brusseau et al. 1994), indicating that cyclodextrin mobility in soils should be good. Further,
cyclodextrins are generally non-toxic (Wang &
Brusseau 1995).
In column experiments, mixtures of carboxylated and non-carboxylated cyclodextrins dramatically improved cadmium, nickel, strontium and
phenanthrene elution from soils in comparison to
dilute electrolyte solutions, removing approximately 90% of the metals and 86% of the phenanthrene within 30 pore volumes (Figure 16).
This ability of cyclodextrins to mobilize metal
and organic contaminants simultaneously is a
signicant attribute, suggesting that use of cyclodextrins may greatly enhance pump-and-treat
remediation of sites with mixed metal-organic
contaminants.
3.1.3. Exopolysaccharides
Exopolysaccharides are relatively large molecules,
frequently with molecular weight of 106 daltons
or more, with anionic character due to acidic
functional groups. Exopolysaccharides are typically highly hydrated and exible in solution, and
as metals are bound, they may fold into more
compact structures. Some polysaccharides aggregate into large ocs in the presence of metals,
possibly due to formation of insoluble metal
hydrolysis products that form nuclei for further
precipitation. While useful in the recovery of
metals from waters, this reduced solubility greatly diminishes such a polymers mobility and bioremedial eectiveness in soils (Brierley 1990).
A great diversity of microorganisms produces
free and/or capsular exopolysaccharides, and

129

Figure 15. Structure of hydroxypropyl-b-cyclodextrin (Reprinted from Wang & Brusseau (1993), with permission. Copyright 1993
American Chemical Society).

Figure 16. Elution of a southern Arizona surface soil (0.68% organic carbon, 88.5% sand, 4.3% silt, 10.2% clay, pH 7.5) contaminanted with cadmium (1.6 mg/g soil), nickel, strontium and phenanthrene (7.1 lg/g soil) using CMCD (carboxymethyl-b-cylodextrin) or 0.01 M KNO3. Phenanthrene elution is not shown (Reprinted from Brusseau et al. 1997, with permission. Copyright 1997
American Chemcial Society).

130
most of these are known to possess metal-binding activity. The emulsan of Acinetobacter RAG1, for example, binds up to 240 lg UO2 per mg.
Other exopolysaccharides produced by Pseudomonas, Arthrobacter and Klebsiella have bound
per mg polymer, up to 96 lg uranium, 3.3 lg
cadmium and 22 lg copper, respectively (Miller
1995).
Recently, the rst demonstration has been
made that a expolysaccharide can desorb a metal
bound to a mineral surface and transport the
complexed metal through a porous medium. This
structurally undened polysaccharide, produced
by a Gram negative soil isolate Strain 9702-M4,
actively complexed Cd2+ and sorbed only weakly
to sand particle surfaces. Most importantly, the
polysaccharide released 57% of the (approximately 0.08 ppm) cadmium adsorbed to sand
particles in 1500 pore volumes, compared to nondetectable cadmium release in polymer-free controls. The polymer-facilitated cadmium transport
is an encouraging result, but not necessarily a
general one; in particular, it waits testing in more
nely textured soils (Chen et al. 1995).
3.2. Microbial leaching
Bacteria that leach metals from ores are chemolithotrophic, or rock-eating, meaning that they
obtain energy from the oxidation of inorganic
substances coupled to the respiration (reduction)
of oxygen. Chemolithotrophs of the genera

Thiobacillus and Leptospirillum, the most important in microbial leaching, ourish in metal sulde deposits exposed to air and water. They
oxidize iron and suldes, generating sulfuric acid
and releasing associated metals into aqueous
solution (Brierley 1982). A model of the indirect
leaching mechanism of Thiobacillus ferrooxidans
consists of several steps (Figure 17): the bacteria
bind themselves to a metal sulde substrate with
extracellular polymers, forming biolm of one or
two cell layers. The exopolymers hold 0.55%
iron, needed to overcome the repulsion between
negatively charged sulde minerals and anionic
cell surface macro-molecules (Blake et al. 1994).
The bacteria oxidize the bound iron with molecular oxygen, forming Fe (III) hexahydrates at low
pH, and the Fe(III) hexahydrates in turn oxidize
mineral-surface suldes to soluble thiosulfates.
Thiosulfates may be further oxidized by T. ferroxidans or by commensal bacteria that cannot
oxidize sulde directly, ultimately producing sulfuric acid (Sand et al. 1995).
Thiobacilli are widely exploited: the mining
recovery of metals from low grade ores through
processes known as heap leaching and dump
leaching in which excavated ores are mounded
together, often on an impermeable slope and irrigated with acidied water to promote indigenous, microbial activity. The leachate is collected
at the bottom for metal recovery (Brierly 1982).
The success of microbiological mining has inspired work in recovering metals from other

Figure 17. A model for the indirect leaching attack mechanism as catalyzed by a metal-sulde-attached cell of Thiobacillus ferrooxidans, exaggerating the exopolymer layer to show its importance (Adapted from Sand et al. 1995).

131
substrates as well, notably metal contaminated
sewage sludge. Such sludges, generated in abundance by municipal water treatment plants, can
be spread onto agricultural land for decomposition if their metal contents do not exceed regulatory standards. Unfortunately, approximately
50% of the sludges in the USA and Canada do
contain metals, predominantly as metal suldes,
in excess of the standards. Attempts at chemical
dissolution of sludge metals, usually by addition
of acids have proven costly and ineective
(Couillard & Mercier 1991).
To solve the sludge problem, a continuous
ow bioreactor system has been developed
for sludge treatment, using T. ferrooxidans to
catalyze metal dissolution and using industrial
FeSO4 7H2O as an energy source for the bacteria.
With an hydraulic residence time of 18 h and
sludge volume of 30 L, the following metal
solubilizations have been achieved; Cu, 91% of
2500 ppm; Zn, 94% of 1800 ppm; Mn, 93% of
380 ppm; Ni, 67% of 23 ppm and Cd, 67%
of 16 ppm. After treatment, therefore the sludges
easily met the Quebec limits of 1000 ppm Cu,
2500 ppm Zn, 1500 ppm Mn, 180 ppm, Ni and
15 ppm Cd. The biological leaching method reduces the necessary acid input to 15% and the
incubation time to 10% of those required in
batch-type chemical leaching methods (Couillard
& Mercier 1991; Couillard & Mercier 1993). In
further experiments, leaching of Pb was greatly
improved when the FeSO4 7H2O substrate was
replaced with FeCl2 presumably due to the low
solubility of lead sulfate. The bacterial population
adapted well to the FeCl, but not to the substitution of HCl for H2SO4 in initial acidication of
the system, due to either chloride intolerance or to
a sulfate requirement. Further research is needed
to optimize lead solublization by these methods,
but the leaching technique for the other metals
appears ready for implementation (Mercier et al.
1996).
In the past decade, the ability of Thiobacilli
to remove trace metals from soils and sediments
has been studied (Tichy et al. 1998). Operational
conditions studied include soil sediment characteristics (Loser et al. 2004), initial pH (Chen &
Lin 2001a), percentage of inoculum, retention
time, initial solid content (Chen & Lin 2000)
and nutrition (Chen & Lin 2001b). When insoluble elemental sulfur is used as substrate in the

bioleaching process, the microbial oxidation of

the sulfur by Thiobacilli is believed to take place
by the adsorption and growth of bacteria on
the surface of the sulfur particles (Boseker
1997). The adsorption of bacteria to the solid
substrate and plays a vital role in the bioleaching process the sulfur oxidation rate (Chen &
Lin 2001b). Suspension leaching is not economically feasible for treating large amounts of sediment. An alternative is solid bed leaching, as
done with ores. In a solid bed, the density and
particle size distribution of the solid matrix are
important factors regulating mass transport, sulfur oxidation and microbial activity. Improving
the physicochemical properties and the structure
of dredged sediments by pretreatment with
plants enabled leaching of heavy metals in laboratory percolator systems with nearly the same
eciency as suspension leaching (Loser et al.
2001). Nevertheless, good performance in the
laboratory does not guarantee similar eciency
on a large scale since important process parameters can be carefully controlled in the laboratory and uctuations are usually avoided. Some
inuences on process operation under practical
conditions, such as the eects of solid inhomogeneity, channeling, or pH and temperature
gradients can only be studied reliably on a pilot
scale. To be applicable in a commercial plant,
the process must be scaled up without loss of
eciency (Seidel et al. 2004).
3.3. Dissimilatory metal reduction
Respiratory microorganisms obtain energy from
the enzymatically controlled oxidation of electron
donors, such as glucose, acetate, or H2, coupled
to the reduction of electron donors, such as O2,
NO3), Fe(III) and SO42) (Table 2). Recently,
several toxic metals and metalloids have joined
the list of terminal electron acceptors: As(V) as
arsenate, Se(VI) as selenate and U(VI) as uranyl
acetate (Lovley et al. 1991; Ahmann et al. 1994;
Oremland et al. 1994). In addition chromium(VI)
and plutonium(IV) appear to be reduced by respiratory enzymes, although respiratory growth with
these acceptors has not been proven (Lovley and
Phillips 1994; Rusin et al. 1994). The importance
of these reductions for bioremediation lies in the
profound physical and chemical changes that the
metals exhibit following reduction, creating

132
opportunities for detoxication or for separation
of the metallic compounds from soil matrices.
3.3.1. Selenium reduction
Selenium contamination has gained particular
attention in the Central Valley of California and
other areas of the western United States where
naturally selenium-rich soils are irrigated for agriculture. Excess water is drained from the elds
and carried to evaporation ponds, some in ecologically sensitive areas such as the Kesterson
Wildlife Refuge, where the selenium has become
concentrated enough to cause toxicity, deformities, reproductive problems and death in waterfowl (Oremland et al. 1989; Macy 1994). The
selenium problem has inspired investigations into
two bioremedial strategies: microbial reduction of
soluble selenate into insoluble elemental selenium,
discussed below, and microbial methylation of
inorganic species to form volatile products, considered later in this chapter (see 3.4.1).
Selenium is a trace metalloid with a redox
chemistry similar to that of sulfur, possessing primary oxidation states of VI, IV, O and II. Selenate (SeO42)) is the form found most commonly
in soil porewaters (Frankenberger Jr. & Karlson
1994a). Because the reduction potential of selenate (Se(VI)) to elemental selenium is suciently
high, researchers hypothesized that selenate
reduction coupled to hydrogen or acetate oxidation could generate enough energy to support

anaerobic microbial metabolism (Table 2). Three

selenate-respiring bacterial strains have since
been isolated from selenate-rich-sediments: SeS,
SES-3 and Thauera selenatis (Oremland et al.
1994, 1989; Macy et al. 1993). Bacillus and Microbacterium species have also been identied
that reduce, but do not necessarily respire, selenate (Combs et al. 1996).
Selenate and nitrate respirations proceed
simultaneously in T. selenatis. For bioremedial
purposes, the presence of nitrate is desirable,
since nitrate respiration induces the nitrite reductase that catalyzes selenate reduction to elemental
selenium (DeMoll-Decker & Macy 1993). SES-3,
in contrast, prefers nitrate as an electron acceptor and will not respire selenate until nitrate is
depleted (Steinberg et al. 1992). The agricultural
drainage waters contain about 50 mg/L nitrate,
150 times the selenate concentration, making this
an important consideration (Macy et al. 1993).
As the mobile, highly toxic Se(VI) is the dominant species found in the Central Valleys San
Joaquin River and the various evaporation
ponds (Oremland et al. 1989; Thompson-Eagle et
al. 1989), microbial reduction has the potential to
remove selenate from soil porewaters and immobilize it as Se(0) an insoluble, much less bioavailable form. Studies of selenate reduction in
evaporation pond surface sediment slurries have
shown that anaerobic conditions, indigenous
microorganisms are able to remove up to 99.7%

Table 2. Free energies of electron acceptor reduction coupled to H2 oxidation

Reaction

DG* (kcal/mol e))

1/4 O(g) + 1/2 H2 1/2 H2O

1/5NO3) + 1/5H+ +1/2H2 1/10N2(g) + 1/5H2O
1/2MnO2(s) + H+ + 1/2 H2 1/2Mn2+ + H2O
1/2UO22+ + 1/2 H2 H+ + 1/2UO2(s)
1/2SeO42) +1/2H+ + 1/2H2 1/2HSeO3) + 1/2H2O
1/2 CrO42) + 5/3H+ + 1/2 H2 3/2Cr3) + 4/3H2O
Fe(OH)3(s) + 2H+ + 1/2H2 Fe2+ + 3H2O
1/4HSeO3) + 1/4H+ + 1/2 H2 1/4Se + 3/4H2O
1/2H2AsO4) + 1/2H+ + 1/2H2 1/2H2AsO3 + 1/2H2O
1/4SO42)+1/4H++1/2 H2 1/4 HS)+1/2H2O

)23.55a
)20.66a
)22.48b
)18.89c
)15.53b
)10.76b
)10.49b
)8.93b
)5.51d
)0.10a

PH2 = 1066 atm; PN2 = 0.78 atm; PO2 = 0.21 atm; pH = 7.0.
2+
3+
)
2



2



2

]=[UO2
]=[Fe2+]=[HSeO

[NO

2 ]=[Mn
3 ]=[H2 AsO4 ] =[H2AsO3]=[SO4 ]=[HS ]=
2 ]=[SeO4 ]=[HSeO3 ] =[CrO4 ]=[Cr
[10)6] M.
a
Calculated from Zehnder & Stumm 1988.
b
Calculated from Morel & Hering 1993.
c
Calculated from Rusin et al. 1994.
d
Calculated from Vanysek 1995.

133
of the added selenate (approx. 300 lM) from
porewater within 7 days of incubation, with
addition of lactate, acetate and H2 stimulating
reduction rates (Oremland et al. 1989). In pond
sediments, selenate reduction occurs primarily
near the surface, in the same region as denitrication and vertically above sulfate reduction.
Areal rates of selenate removal from pond sediment porewaters have been estimated at up to
300lmol/m2/day (Figure 18).
Bioremediation of selenate contamination by
microbial reduction and precipitation has attracted many advocates; nevertheless, the longterm stability of elemental selenium in oxic soils
is questioned (Frankenberger Jr. and Karlson,
1994b). Because selenite and selenate are soluble
and relatively mobile, aqueous extraction from
soils may be a better alternative. Microbial
reduction might then be used to recover selenium
from the aqueous phase. This technique has been
extremely successful in various wastewaters, consistently removing 9598% of the Se (Lawson
and Macy, 1995; Macy et al. 1993)
3.3.2. Uranium reduction
Uranium contamination of soils and groundwaters has resulted from the release of wastes from
mining, extraction from ores, nuclear fuel reprocessing and ammunitions manufacture. Uranium

exists in most oxygenated wastes as the U(VI)

uranyl ion UO22+, in reducing environments
such as groundwater (Macaskie, 1991). While
U(VI) is soluble and immobile, U(IV) is mobile,
and highly insoluble in most aqueous solutions
(Lovley & Phillips 1992a, b).
Several years ago, the iron-respiring microorganisms Strain GS-15 and Alteromonas purefaciens (later renamed Geobacter metallireducens and
Shewanella purefaciens, respectively) were shown
to respire uranium as well as iron. The group of
uranium-reducing microorganisms now includes
several sulfate-reducing bacteria, and mechanistic
investigation of Desulfovibrio vulgaris has shown
that the respiratory electron transport protein
cytochrome c, catalyzes the U(VI) reduction
(Lovley et al. 1993b). Sulfate-reducing bacteria
do not appear to obtain energy for growth from
the process, however (Lovley et al. 1993a).
Microbial uranium reduction, clearly applicable to precipitation of uranium from wastewaters, has also found a role in uranium removal
from soils. Because bicarbonate ions form very
strong, soluble complexes with U(VI), investigators used a 100 mM solution to extract uranium
from contaminated soils, uranium ores and mill
tailings. The bicarbonate was not able to remove
the uranium completely (2094% recovery) although the extracted uranium might have been

Figure 18. Rates of reduction and porewater concentrations of sulfate, nitrate and selenate in an evaporation pond core from the
San Joaquin Valley. (a) Sulfate reduction; (b) denitrication; (c) selenate reduction. Values indicate the mean of 3 samples (Reprinted from Oremland et al. (1990), with permission. Copyright 1990 American Chemical Society).

134
the most dangerous fraction, as unrelated studies
have shown the bicarbonate removes a much
higher percentage of soil uranium than is bioavailable (Sheppard & Evenden 1992). To this
extract, kept anaerobic under N2 ACO2 (80:20),
H2 was added as an electron donor and Desulfovibrio desulfuricansas as the catalyst. The cells
reduced and precipitated 75100% of the uranium from the leachate within 24 h, yielding a
very pure, highly concentrated uraninite solid.
The reduction was somewhat inhibited by dissolved organic matter in the extract, as evidenced
by a yellow color, but peroxide addition to oxidize the organic matter eliminated the color and
the inhibition (Phillips et al. 1995). A remarkable
feature of this microbial U(VI) precipitation is
that the Desulfovibrio cells can be freeze-dried
and stored under air for up to six months without loss of uranium-reducing activity (Lovley &
Phillips 1992b).
3.3.3. Plutonium reduction
Plutonium, obtained from irradiation of uranium
and assembled into nuclear weapons, is an extensive soil contaminant near the production sites at
Hanford, WA and Rocky Flats, CO (Zorpette
1996). Plutonium is also a product of nuclear energy generation, although much of it can be
reprocessed (Macaskie 1991). Like uranium, plutonium is a redox-active metal, with oxidation
states of III to VI known to exist in aqueous
solution. Oxides and hydroxides of Pu(IV), with
extremely low solubilities estimated at ksp=1056.8
to 1057.8, appear to predominate in contaminated
soils (Rusin et al. 1994).
Pu (III) hydroxide, while still quite insoluble
at Ksp=1022.6, is more soluble than Pu(IV) compounds. Because the reduction of Pu(IV), as hydrous PuO2(s) to Pu3+ is theorectically capable of
supporting bacterial growth, with a reduction potential similar to that of Fe(III), researchers have
investigated the potential for plutonium reduction and dissolution by iron-reducing Bacillus
strains. Experiments showed that cultures of B.
circulans and B. polymyxa did indeed dissolve solid plutonium, amounting to 8591% of the hydrous PuO2(s) added, or 1.25 lmol Pu, in the
presence of 50 mM nitrilotriacetic acid (NTA).
Reduction was the most likely means of dissolution, although this was not conclusively demonstrated: plutonium-dependent growth of the

bacteria, indicating energy gain from the reduction, was also not shown. With further development, including the use of biodegradable
complexing agents in place of NTA, microbial
reduction could provide a new pathway for insitu plutonium remediation (Rusin et al. 1994).

3.3.4. Arsenic reduction

Microbial arsenic reduction, like plutonium
reduction, transforms a relatively immobile species into a more mobile one and thus has the potential to operate in conjunction with soil
washing to mobilize soil-bound arsenic. Inorganic arsenic in neutral aqueous solution occurs
as As(V), arsenate (H2AsO4), and As(III), arsenite (H3AsO3). As(-III), occurring as arsine
(AsH3), is a volatile reactive species formed by
certain fungi and bacteria (Cheng & Focht 1979).
Arsenate adsorbs strongly to iron and manganese
oxyhydroxides and is thus very dicult to remove from soils and sediments. Arsenite, although more toxic, is more mobile, suggesting
that reduction may promote arsenic mobilization
(Bodek et al. 1988; Kuhn & Sigg 1993).
Recently, several species of arsenate-respiring
bacteria have been isolated from arsenic-rich substrates, including Strain MIT-13 (renamed Geospirillum arsenophius) (Ahmann et al. 1994). Its
close relatives that also reduce selenate, are
Strain SES-3 (Laverman et al. 1995) Desulfotomaculum auripigmentum (Newman et al. 1997)
and Chrysiogenes arsenatis (Macy et al. 1996).
Arsenic reduction is rapid in these microorganisms, measured at 25 mmol As-ml culture1 day1
in both G. arsenophilus (Ahmann et al. 1994) and
in Strain SES-3 (Laverman et al. 1995). The
respiratory action of G. arsenophilus is able to
dissolve arsenic from contaminated sediments
(approximately 1000 ppm As) and other arsenical
solids (Ahmann et al. 1997), and continuous-ow
pilot-scale studies are in progress to maximize
the arsenic-mobilizing potentials of the microorganism and others indigenous to contaminated
soils.

3.3.5. Chromium reduction

Cr(III) found at trace concentrations of
490 ppm in pristine areas, is the most stable
and abundant form of chromium in most soils.

135
Kinetically it is quite inert, forming long-lived
aqueous complexes with ammonia, sulfates, halides and organic acids. At slightly acidic to alkaline pH, however, ionic Cr(III) species tend to
precipitate as amorphous Cr(OH3) if Fe3+ is
present, with the result that much of the Cr(III)
in soils is insoluble and immobile (Krishnamurthy & Wilkens 1994). Although Cr(III) is an
essential trace nutrient, it does not readily enter
living cells. Industrially-produced and -discharged Cr(VI), in contrast occurs as soluble,
highly toxic and carcinogenic chromate and dichromate. Hexavalent chromium is freely taken
up by cells and reduced intracellularly to Cr(III),
which appears to mutagenize. Because Cr(III) is
signicantlly less toxic and less mobile than
Cr(VI)-contaminated soil, numerous studies have
focused on reducing Cr(VI) in-situ, by chemical
(e.g., ferrous iron) or biological means. The
intent is thus to x the chromium in the soil
in a harmless form. This approach requires
careful management, since reoxidation of Cr(III)
to Cr(VI) is catalyzed by manganese oxides
and by molecular oxygen. The oxidation appears
to be quite slow, primarily limited by solubility
and mobility of the Cr(III)-laden soil is still
quite possible under oxidizing conditions. The
two essential requirements for safe chromium
remediation by reduction, therefore, are (1) a
permanent reducing environment and (2) immobilization, by precipitation or by complexation
with degradation-resistant organic polymers, of
the reduced chromium (Bartlett 1991; Krishnamurthy & Wilkens 1994; Losi et al. 1994a).
Bioremediation eorts to date have focused
on the study of Cr(VI)-reducing microorganisms.
Numerous such bacteria have been isolated,
including species of Bacillus, Desulfovibrio,
Achromobacter, Aeromonas, Escherichia, Enterobacter and Pseudomonas (Lovley & Phillips 1994;
Turick et al. 1996). Mechanisms of chromate
reduction in anaerobic bacteria appear to involve
respiratory electron-transport enzymes (Bopp &
Ehrlich 1988; Shen & Wang 1993), with convincing evidence for catalysis by cytochrome c3 in
Desulfovibrio vulgaris (Lovley & Phillips 1994).
While it is thermodynamically possible for chromate reduction to generate enough energy to
support respiration (Table 2), true chromate respiration remains to be discovered (Lovley & Phillips 1994; Shen & Wang 1993; Turick et al.

1996). In aerobic chromate-reducers, reduction

may be fortuitously catalyzed by enzymes that
have other natural substrates (Cervantes 1991).
The presence of reactive electron donors appears to be the single greatest factor promoting
chromate reduction both chemical and biological,
in soils. In one illustrative set of experiments,
sterile soils alone reduced almost half of the
800 lg/kg Cr(VI) present within 8 days. Addition
of organic matter (cattle manure) improved the
reduction to approximately 63%, and native
microbial activity in organic-amended samples
resulted in over 96% reduction, emphasizing the
eectiveness of biological catalysis (Figure 19).
Studies simulating eld conditions have supported these results, conrming (1) the ubiquity
of indigenous Cr-reducing microorganisms in a
variety of soil, both contaminated and clean, (2)
the importance of irrigation to maintain reducing
conditions and (3) the proportionality between
organic matter loading and Cr(VI) reduction
(Cifuentes et al. 1996; Losi et al. 1994a, b), suggesting that Cr(VI) bioremediation by reduction
in soil holds great promise in cases where reoxidation can be permanently prevented.
3.4. Volatilization
3.4.1. Selenium volatilization
Microbial transformation of selenium into volatile methylated species, principally dimethylselenide (CH3)2Se with smaller amounts of
dimethyldiselenide (CH3)2(Se)2 and dimethylselenone (CH3)2 (SeO2), removes selenium from soil
and disperses it into the atmosphere. Airborne
dimethylselenide (DMSe) reacts with hydroxyl
radicals and ozone to yield oxidized products
which are then thought to become associated
with particles or aerosols that have residence
times of several days, during which they may be
dispersed over great distances (Frankenberger Jr.
& Karlson 1994a). Microbial volatilization was
rst recognized as an important part of the selenium biogeochemical cycle in 1987, and it is now
estimated that soils and plants emit 1700
3300 metric tons of selenium into the atmosphere
in the Northern hemisphere each year (Cooke &
Bruland 1987; Frankenberger Jr. & Karlson
1995).
Selenium methylation produces substances of
greatly reduced toxicity and is thus thought to

136

Figure 19. Reduction of Cr(VI) over time in moist soils under sterile and non-sterile conditions, amended with cattle manure (OM)
at 0 and 50 tons ha)1. Final values followed by dierent letters are statistically dierent (Reprinted from Losi et al. 1994a,b, with
permission. Copyright 1994 Society for Environmental Toxicology and Chemistry).

protect the methylators: dissolved dimethylselenide is 500700 times less toxic than selenate
and selenite, while inhaled dimethylselenide is
non-toxic in concentrations at up to 8034 ppm.
Selenium volatilization activity appears to be
widespread among aerobic microorganisms,
including the native microbiota of the Kesterson
evaporation ponds and additional fungi of the
genera Alternaria, Scopulariopsis, Fusarium, Acremonium, Pencillium and Ulocladium (Frankenberger Jr. & Karlson 1995). The biomethylation
pathway has not been fully elucidated, but is
thought to require reduction of selenium species to
Se2), followed by methylation; as a result, selenite
(Se(IV)) is volatilized more rapidly than is selenate
(Se(VI)) (Frankenberger Jr. & Karson 1994a).
Selenium volatilization is controlled both by
enzymatic activities and vapor pressures of the
methylated gases, with the result that the process
is highly temperature-dependent (Q10=1.7 from
453 C). The process thus shows pronounced
seasonal uctuations, with average emission rates
of 75100 lg Se/h/m2 in the summer and
1050 lg Se/h/m2 in the winter in managed
dewatered evaporation ponds containing only
indigenous microorganisms. Other factors were
also important in these pilot study plots; soil
moisture contents of at least 70% optimized Se

emissions, although saturation, which could have

created anoxia or leached selenium into deeper
soil layers, was avoided. Carbon addition in the
form of cattle manure also increased volatilization rates and frequent tillage improved contact
between air and the soil solution, presumably
releasing methylated compounds and stimulating
aerobic metabolism. Nearly 60% of the selenium
in the soils (mean original content: 11.4 ppm)
was removed in 22 months, and 18-fold enhancement over removal from untreated plots. The
time needed to reach the cleanup goal of 4 ppm
was estimated to be 2.6 years (Frankenberger Jr.
& Karlson 1994a, 1995).
Microbial volatilization of selenium is a
promising method of bioremediation and one
which may be further improved when the relevant molecular mechanisms are understood. An
important limitation, however, is the microbial
ability to metabolize only water-soluble selenium,
apparently because only these forms may be taken up by microorganisms (Frankenberger Jr. &
Karlson 1994a). While this pool may represent
the most hazardous selenium present, signicant
amounts of soil-bound but labile selenium may
resist volatilization, necessitating long treatment
periods or the assistance of solubilizing agents to
reach cleanup standards.

137
3.4.2. Mercury volatilization
Mercury resistance systems have been found in
all bacterial groups tested and consistently include an operon of six genes: merR, a regulatory
gene: merD, a second regulator, merP and merT,
encoding proteins that transport mercuric ions
into the cell; merA, encoding a mercuric reductase that transforms Hg2+ to volatile Hg0 and
merB, encoding an organomercurial lyase that
cleaves Hg from organomercury compounds
(Figure 20). The bioremedial potential of this
system lies in the ability of the bacteria to remove highly toxic mercuric ions from a substrate, releasing them as elemental mercury of
greatly reduced toxicity that can then be trapped
or dispersed into the atmosphere. The system is
quite well understood and although it is ubiquitous among bacteria, it is showing greatest promise within transgenic plants for mercury removal
from soils (Rugh et al. 1996; Silver 1994).

4. Metal-microbe interactions and their impact

on bioremediation of metal contaminated soils
and sediments
Estimates of the global market for the cleanup
and prevention of metal contamination vary, but
conservative calculations suggest that the current
market for metal bioremediation may rise to
$200 billion in the US alone by 2005. The emerging market for the clean up of radioactive contamination in the US may already be worth as
much as $300 billion (McCullough et al. 1999).

Unfortunately, existing chemical techniques are

not always economical or cost eective for the
remediation of water or land contaminated with
metals and radionuclides. Current strategies for
land contaminated with metals include the use of
dig and dump approaches that only move the
problem to another site, are expensive and
impractical for large volumes of soil or sediment.
Likewise soil washing, which removes the smallest particles that bind most of the metals, is useful but can be prohibitively expensive for some
sites. Pump and treat technologies rely on the
removal of metals from the site in an aqueous
phase which is treated ex situ (e.g. above land).
These approaches can cut down on excavation
costs but are still expensive, and metal removal
can be inecient. A potentially economical alternative is to develop biotechnological approaches
that could be used in the sediment or soil (in
situ) to either extract the metals or stabilise them
in forms that are immobile or non-toxic.
In addition to developing biotechnologies to
treat contaminated land, there is also considerable
interest in more eective techniques that can be
used to treat water contaminated with metals
from a range of industrial processes. Problems
inherent in currently used chemical approaches include a lack of specicity associated with some ion
exchange resins, or the generation of large quantities of poorly settling sludge through treatment
with alkali or occulating agents. This section
gives an overview of metalmicrobe interactions,
and describes how they could be harnessed to
clean up metal contaminated, soil and sediments.

Figure 20. Genes and polypeptides of mercury resistance systems from Gram-negative Serratia sp. Aa, length of gene products in
amino acids; P/O, promoter/operator region (Adapted from Ji & Silver 1995, with permission of the Society for Industrial Microbiology).

138
4.1. Metal and radionuclide contamination
The US EPA (http://www.epa.gov) lists four
main groups of priority compounds which include polychlorinated biphenyls (PCBs), base
neutrals and acids (e.g. phenol and naphthalene)
and volatile organic compounds (VOCs), including carbon tetrachloride and BTEX contaminants. Trace metals that are recoverable and
dissolved are also listed; these include antimony,
chromium, mercury, silver, arsenic, copper, nickel, thallium, beryllium, lead, selenium, zinc and
cadmium. In addition to these toxic metals, radionuclides are priority contaminants at nuclear
installations worldwide, and will require treatment. For example, in the US Department of
Energys 120 sites contains 1.7 trillion gallons of
contaminated ground water and 40 million cubic
metres of contaminated soil and debris. More
than 50% of the sites are contaminated with radionuclides, with the priority radionuclides being
cesium-137, plutonium-239, strontium-90, technetium-99 and uranium-238 and uranium-235, in
addition to toxic heavy metals including chromium, lead and mercury (McCullough et al.
1999). The US DOE Natural and Accelerated
Bioremediation (NABIR) program is notable in
its support of emerging biotechnological approaches for dealing with this legacy waste. In
Europe, the EU Water Framework Directive
(2000/60/EC) is the principal driver for environmental legislation, and also recognizes metals
amongst the 32 substances on the priority list.
These include lead, nickel, mercury cadmium and
tin and their compounds.
4.2. Biosorption
The term biosorption is used to describe the
metabolism-independent sorption of heavy metals
and radionuclides to biomass. It encompasses
both adsorption; the accumulation of substances
at a surface or interface and absorption; the almost uniform penetration of atoms or molecules
of one phase forming a solution with a second
phase (Gadd & White 1989). Both living and
dead biomass are capable of biosorption and ligands involved in metal binding include carboxyl, amine, hydroxyl, phosphate and
sulfhydryl groups. Biosorption of metals has
been reviewed extensively (McHale & McHale

1994; Tobin et al. 1994; Volesky & Holan 1995;

Beveridge et al. 1997a,b; Lloyd & Macaskie
2000). Biosorption is generally rapid and unaffected over modest temperature ranges and in
many cases can be described by isotherm models
such as the Langmuir and Freundlich isotherms
(Volesky & Holan 1995). Gadd and White
(1989), however, noted that more complex interactions are dicult to model because the adsorption of solutes by solids is aected by factors
including diusion, heterogeneity of the surface
and pH. An additional isotherm, the Brunauer
EmmettTeller (BET) isotherm, which assumes
multilayer binding at constant energy has also
been used to describe metal biosorption (de
Rome & Gold 1991; Andres et al. 1993). This
model assumes that one layer need not necessarily be completely lled before another is commenced. Further insight is oered by Andres
et al. (1993), who summarize that each adsorption layer of the BET model can be reduced to
Langmuir behavior with homogeneous surface
energy, in contrast to the adsorption energy
requirements of the Freundlich isotherm. Other
studies have used complex multistage kinetic approaches to model biosorption (Weidemann et al.
1981; Treen-Sears et al. 1984).
Ultimately, however, the amount of residual
metal remaining in solution at equilibrium is
governed by the stability constant of the metal
ligand complex (Macaskie 1991), and the only
way to change the equilibrium position is to
modify the binding ligand to one which has a
greater binding anity for the given metal, or to
transform the metal from a poorly sorbing species to one which has a higher ligand-binding
anity, e.g. by a change of metal valence. Alternatively some new studies have applied the tools
of molecular biology to enhance metal sorption
and do warrant attention. For example, a mouse
metallothionein was targeted to the outer membrane of a metal-resistant Ralstonia eutropha isolate (Valls et al. 2000). The engineered strain
accumulated more Cd2+ than its wild type counterpart, and oered tobacco plants some protection from Cd2+ when inoculated into
contaminated soil (Valls et al. 2000). A surface
display technique has also been used successfully
to generate ZnO binding peptides fused to mbrae on the surface of cells of Escherichia coli
(Kjaergaard et al. 2000). Finally, Gram-positive

139
bacteria (Staphylococci) have also been engineered to produce surface exposed peptides, able
to bind Hg2+ and Cd2+ (Samuelson et al. 2000).
Enhanced uptake of cadmium and mercury by
Escherichia coli expressing a metal-binding motif
has also been reported (Pazirandeh et al. 1998).
Future studies could usefully compare the
performance of such engineered systems with
other more traditional biosorbants. However, it
has been noted that despite the relatively long
time during which biosorption has been studied,
and considerable early interest, there has been reduced interest in commercialising this type of
technology, and the authors are aware of no current commercial applications of this type of
technology.
4.3. Metabolism-dependent bioaccumulation
Energy-dependent metal uptake has been demonstrated for most physiologically important metal
ions, and some toxic metals and radionuclides
enter the cell as chemical surrogates using
these transport systems. Monovalent cation
transport, for example K+ uptake, is linked to
the plasma membrane-bound H+-ATPase via the
membrane potential, and is, therefore, aected
by factors that inhibit cell energy metabolism.
These include the absence of substrate, anaerobiosis, incubation at low temperatures and the
presence of respiratory inhibitors such as cyanide
(White & Gadd 1987). The requirement for metabolically active cells may, therefore, limit the
practical application of this mode of metal uptake to the treatment of metals and radionuclides
with low toxicity and radioactivity. For example,
in the study of White and Gadd (1997), increasing metal concentrations inhibited H+ pumping,
potentially deenergizing the cell membrane and
reducing cation uptake. Although the presence of
multiple transport mechanisms of diering anities may cause added complication, metal inux
frequently conforms to MichaelisMenten kinetics (Borst-Pauwels 1981).
Once in the cell, metals may be sequestered by
cysteine-rich metallothioneins (Higham et al.
1984; Turner & Robinson 1995) or, in the case of
fungi, compartmentalized into the vacuole (Gadd
& White 1989; Okorov et al. 1977). In this context, it should be emphasized that the uptake of
higher mass radionuclides, e.g. the actinides into

microbial cells has been reported sporadically

and remains poorly characterised (Lloyd & Macaskie 2002; Lloyd & Macaskie 2000). Metabolism-dependent bioaccumulation of metals by
microorganisms has not been used commercially
for bioremediation purposes.
4.4. Enzymatically catalysed biotransformation
Microorganisms can catalyze the transformation
of toxic metals to less soluble or more volatile
forms. For example, the microbial reduction of
Cr(VI) to Cr(III), Se(VI) to Se(0), V(V) to V(III),
Au(III) to Au(0), Pd(II) to Pd(0), U(VI) to
U(IV) and Np(V) to Np(IV) results in metal precipitation under physiological conditions and has
been reviewed in detail recently (Lloyd 2003). In
many cases the high valence metal can be used as
an electron acceptor under anoxic conditions. A
good example here is the reduction of soluble
U(VI) to insoluble U(VI) by Fe(III)-reducing
bacteria (Lovley et al. 1991), which has been harnessed recently to remediate sediments contaminated with uranium in situ. Metal detoxication
is also possible through biotransformations of
toxic metals. The bioreduction of Hg(II) to relatively non-toxic Hg(0) is perhaps the best studied
example. Finally, in addition to redox biotransformations, biomethylation may also increase the
volatility of metals, with the methylation of mercury, cadmium, lead, tin, selenium and tellurium
recorded (Diels et al. 1995). These mechanisms
could also oer potential use for the in situ remediation of contaminated soil (Losi & Frankenberger 1997).
As mentioned above, microbes have evolved
sophisticated approaches to deal with toxic metal (Bruins et al. 2000), often involving redox
transformations of the toxic metal. Perhaps the
best studied metal resistance system is encoded
by genes of the mer or mercury resistance operon (Hobman et al. 2000). Recent studies have
conrmed the biotechnological potential of this
widespread resistance determinant. Hg(II) is
bound in the periplasm of Gram-negative bacteria by the MerP protein, transported into the
cell via the MerT transporter, and detoxied by
reduction to relatively non-toxic volatile elemental mercury by an intracellular mercuric reductase (MerA). Mer proteins are expressed under
the regulation of the activator protein, MerR,

140
which binds Hg(II) and activates gene expression. Mercury-resistant bacteria and the proteins
that they encode have been used recently for the
bioremediation of Hg-contaminated water and
in the development of biosensors for bioavailable concentrations of Hg(II) (Bontidean et al.
2002). Such sensors may prove very useful in
identifying the need for metal remediation,
which will be dictated in many cases by the
concentration of bioavailable toxic metals in a
given soil or water matrix, and also in dening
the end point for bioremediation eorts. Focusing on Hg biosensors, several dierent components of the mer system have been used in
prototype sensors, including the NADPH-dependent mercuric reductase (MerA) in an enzymelinked biosensor (Eccles et al. 1997), the mer
regulatory region in a whole cell biosensor (Geiselhart et al. 1991), and the MerR protein in a
capacitance biosensor (Bontidean et al. 2000).
4.5. Biomineralization via microbially generated
ligands
Microorganisms are able to precipitate metals
and radionuclides as carbonates and hydroxides
via plasmid-borne resistance mechanisms, whereby proton inux countercurrent (antiport) to metal eux results in localized alkalinization at the
cell surface (Diels et al. 1995; Van Roy et al.
1997). Alternatively, metals can precipitate with
enzymatically generated ligands, e.g. sulde
(Barnes et al. 1991), or phosphate (Macaskie
et al. 1992). The concentration of residual free
metal at equilibrium is governed by the solubility
product of the metal complex (e.g. 10)20 to 10)30
for the suldes and phosphates, higher for the
carbonates). Most of the metal should be removed from solution if an excess of ligand is
supplied. This is dicult to achieve using chemical precipitation methods in dilute solutions; the
advantage of microbial ligand generation is that
high concentrations of ligand are achieved in juxtaposition to the cell surface, that can also provide nucleation foci for the rapid onset of metal
precipitation; eectively the metals are concentrated uphill against a concentration gradient.
This was demonstrated using the gamma isotope
241
Am supplied at an input concentration of
approximately 2.5 parts per billion; approximately 95% of the metal was removed as bio-

mass-bound phosphate (Macaskie et al. 1994),

with the use of gamma-counting permitting
detection at levels below those of most analytical
methods. In many cases, the production of the ligand can also be ne-tuned by the application of
MichaelisMenten kinetics.
Sulde precipitation, catalysed by a mixed culture of sulfate-reducing bacteria, has been utilized rst to treat water co-contaminated by
sulfate and zinc (Barnes et al. 1991) and also,
soil leachate contaminated with sulfate alongside
metal and radionuclides (Kearney et al. 1996).
Ethanol was used as the electron donor for the
reduction of sulfate to sulde in both examples.
A later study has conrmed the potential of integrating the action of sulfur cycling bacteria
(White et al. 1998). In the rst step of a twostage process, sulfur-oxidising bacteria were used
to leach metals from contaminated soil via the
generation of sulfuric acid, and in the second
step the metals were stripped from solution in an
anaerobic bioreactor containing sulfate-reducing
bacteria (Kearney et al. 1996). The ubiquitous
distribution of sulfate-reducing bacteria in acid,
neutral and alkali environments (Postgate 1979),
suggests that they have the potential to treat a
variety of euents, while the ability of the
organisms to metabolize a wide range of electron
donors, may also allow co-treatment of other
organic contaminants. Recent work by Paques
BV of the Netherlands have conrmed the potential of sulfate-reducing bacteria to treat metal
waste in ex situ bioreactors for the treatment of
a wide range of metal-contaminated waters.
Bioprecipitation of metal phosphates via
hydrolysis of stored polyphosphate by Acinetobacter spp. is dependent upon alternating aerobic
(polyphosphate synthesis) and anaerobic (polyphosphate hydrolysis and phosphate release)
periods (Boswell et al. 2001). This obligately aerobic organism is fairly restricted in the range of
carbon sources utilized, but the preferred substrates (acetate and ethanol) are widely and
cheaply available. The best-documented organism
for metal phosphate biomineralization, a Citrobacter sp., (now reclassied as a Serratia sp. on
the basis of molecular methods, the presence of
the phoN phosphatase gene and the production
of pink pigment under some conditions
(Pattanapipitpaisal et al. 2002) grows well on
cheaply available substrates and viable cells are

141
not required for metal uptake since this relies on
hydrolytic cleavage of a supplied organic phosphate donor (Macaskie et al. 1992). The expense
of adding organic phosphate was calculated to be
the single factor which limited the economic viability of this approach (Roig et al. 1995); moreover organophosphorus compounds are often
highly toxic. A possible alternative phosphate
donor, tributyl phosphate (TBP), is used widely
as a cheap solvent and plasticizer but its degradation is more dicult because this compound is
a phosphate triester, with three cleavage events
per molecule required to liberate one molecule of
phosphate. TBP is biodegradable; this activity
was unstable (Thomas & Macaskie 1998) but
biogenic phosphate from TBP hydrolysis was
harnessed to the removal of uranium from solution in a ow-through system (Thomas & Macaskie 1996). The enzymatic activity responsible for
TBP hydrolysis remains obscure but a recent report of TBP hydrolysis by enzymatic degradation
was studied using cell free extracts of Serratia
odorifera (Berne 2004).
As an alternative to TBP and glycerol 2-phosphate the possibility exists for the harnessing of
phytic acid degrading organisms to metal accumulation. Phytic acid, widely and cheaply available as a natural plant product, accounts for up
to 50% of the total organic P in soils (Rodriguez
& Fraga 1999). The additional advantage of phytic acid is that it contains 6 phosphates/molecule
(c.f. 1 phosphate/molecule in TBP and G2P).
Several phytases have been cloned and characterized, including one from the related organism E.
coli (Greiner et al. 1993) with the phytase reaction mechanism described. Preliminary studies
have suggested that E. coli phytase can be applied to metal removal (PatersenBeedle et al.
unpublished).

target metal precipitation reactions (Macaskie et

al. 1996). The priming deposit is made initially
by the sulde or phosphate biomineralization
routes described above.
With addition of Fe as the precipitant metal
to sulfate-reducing bacteria producing H2S, cellbound FeS then acts as a sorbent for target
metals (Ellwood et al. 1992; Watson & Ellwood
1994, 1988). The use of FeS is notable because
this provides the mechanism for rapid biomass
separation from the liquor via the magnetic properties of Fe in a high-gradient magnetic separator
(Ellwood et al. 1992; Watson & Ellwood 1994;
Watson & Ellwood 1988).
Metal phosphate can also be used as the
priming deposit, in two ways. LaPO4 pre-deposited onto metal-accumulating Citrobacter sp. via
biogenic phosphate release can be used as the
priming deposit for subsequent deposition of
actinide phosphates (Macaskie et al. 1994). Also,
by use of a priming deposit with an appropriate,
well-dened cystalline lattice, the target metal
can be intercalated within the host lattice, eectively by a mechanism of bioinorganic ion-exchange, well-established as a chemical process
(Cleareld 1988; Pham-Thi & Columban 1985;
Pozas-Tormo et al. 1987) and best-characterized
biologically in the case of Ni2+ removal into biomass-bound HUO2PO4nH2O (Bonthrone et al.
1996). Within the context of nuclear waste remediation, the approach of MECHM, using biogenic hydrogen uranyl phosphate as the host
matrix, and using either intercalative ion exchange or co-crystallisation, has been applied to
the removal of Cs, Co and Sr from surrogate
solutions and the isotopes 137Cs, 60Co and 90Sr
from real nuclear wastes in South Korea (PatersonBeedle et al. unpublished).

4.6. Microbially enhanced chemisorption of heavy

metals

4.7. Metal remediation through biodegradation of

associated organic compounds

Microbially enhanced chemisorption of heavy

metals (MECHM) is a generic term to describe a
class of reactions whereby microbial cells rst
precipitate a biomineral of one metal (priming
deposit). The priming deposit then acts as a
nucleation focus, or host crystal for the subsequent deposition of the metal of interest (target metal), acting to promote and accelerate

Citrate has found use as a chelating agent in

decontamination operations forming highly soluble metalcitrate complexes, which can be degraded
by
microorganisms
resulting
in
subsequent re-precipitation of metals (Francis
1994). Early work suggested that the type of
complex formed between the metal and citric
acid plays an important role in determining its

142
biodegradability, with the binuclear uraniumcitrate complex being recalcitrant to microbial degradation (Gadd & White 1989), but subsequent
photodegradation was possible for these problematic complexes. This opened up the way for a
multi-stage process for the biodegradation of
mixed metalcitrate complexes via microbiological and photochemical steps (Francis 1994). Cultures of Pseudomonas aeruginosa and P. putida
were also able to grow using a range of metal
citrate complexes as carbon, with metal precipitation promoted by the addition of inorganic phosphate (Thomas et al. 2000). A unique aspect of
this study was the use of P. putida to treat Ni
citrate waste generated by cleaning a bioinorganic ion exchange column previously used to remove Ni. Another industrial chelating agent
ethylenediaminetetraacetate (EDTA) also forms
very strong complexes with di- and trivalent metals that are degraded by a mixed microbial population (Thomas et al. 1998) and by bacterial
strain DSM 9103 (Satroutdinov et al. 2000). The
biodegradation of toxic organotin compounds
used as biocides and antifouling agents has also
received recent attention (e.g. the degradation of
tributyl tin to di- and monobutyl tin reviewed in
Gadd (2000)).
4.8. In-situ versus ex-situ remediation
Although most of the processes described thus
far have been developed primarily for ex situ
treatment of contaminated water in situ remediation oers the potential for low cost treatment
of groundwater contamination with metals. Ex
situ remediation strategies such as pump and
treat systems remain a method of choice for
metal-contaminated sites and can be eective at
removing signicant quantities of contaminant
metals from heavily contaminated (and highly
concentrated) areas. For low contaminant concentration scenarios in situ immobilization of
contaminants has been proposed as an attractive
potential treatment option (Lovley et al. 1991;
Abdelouas et al. 1998a, b, 1999; Finneran et al.
2002).
In situ immobilization of contaminant metals
relies on the stimulation of anaerobic microbial
communities within the subsurface. Anaerobic
microorganisms enzymatically reduce contaminant metals such as uranium, chromium, techne-

tium, vanadium and metalloids such as selenium

thereby converting these metals to less soluble
forms (Lloyd et al. 2000; Lovley & Coates 2000;
Anderson & Lovley 2002; Carpentier et al. 2003;
Ortiz-Bernard et al. 2004). Stimulation of metal
reduction within the subsurface oers a relatively
simple method to remove metal contaminants
from groundwater, thereby limiting or greatly
reducing further transport of contaminants downgradient (Lovley et al. 1991; Lovley 1993; Anderson & Lovley 2002). Eorts to understand and
control in situ metal reduction are currently active
areas of research within the U.S. Department of
Energy (U.S. Department of Energy, O.o.B.a.E.R
2004) particularly in light of the recent focus on
the potential ecological impacts of ex situ remediation practice (Whicker et al. 2004).
A key issue in the development of in situ bioremediation systems for metal contaminants is
the identication of target groups of microorganisms within the subsurface whose known physiology has the potential to aect contaminant metal
mobility. Both Fe(III)- and sulfate-reducing
organisms are known to enzymatically reduce
contaminant metals such as U(VI), Cr(VI),
Co(III) and Tc(VII) in laboratory cultures (Lovley et al. 1991; Lovley & Phillips 1992a, b; Gorby
& Lovley 1992; Lovley 1993; Caccavo Jr. et al.
1994; Lloyd & Macaskie 1996; Gorby & Bolton
1998; Tebo & Obraztsova 1998; Lloyd et al. 2000)
but for remediation purposes it is necessary to
demonstrate that the appropriate microorganisms
are present within the contaminated subsurface.
Members of the Geobacteraceae (d-proteobacteria) are of particular note as these organisms have
been identied as a dominant group within sediments upon the stimulation of Fe(III)-reducing
conditions (Snoeyenbos-West et al. 2000; Finneran et al. 2002; Holmes et al. 2002; Anderson
et al. 2003) and in contaminated sediment where
Fe(III) reduction is a dominant process (Anderson et al. 1998; Roling et al. 2001; Cummings
et al. 2003). Geobacteraceae were also recently detected within the groundwater of an uraniumcontaminated aquifer during a test of stimulated
in situ U(VI) reduction (Anderson et al. 2003).
Geobacteraceae, while best known as dissimilatory Fe(III)-reducing bacteria, also couple the
oxidation of simple organics to the reduction of
contaminant metals such as uranium, chromium,
technetium and vanadium (Chen & Hao 1998;

143
Lloyd et al. 2000; Anderson & Lovley 2002; Oritz-Bernad et al. 2004). Additionally, the product
of dissimilatory Fe(III) reduction, Fe(II), is a potential abiotic reductant for contaminant metals
such as Tc(VII), V(V) and Cr(VI) (Fendorf et al.
2000; Lloyd et al. 2000; Oritz-Bernad et al. 2004).
The demonstrated enrichment of Geobacteraceae
within an aquifer during an in situ trial of stimulated uranium reduction and the potential abiotic
benets of in situ Fe(II) production suggest Geobacteraceae to be a target group of microorganisms within the subsurface for removing metals
from groundwater. Further investigation of stimulated subsurface metal reduction will likely reveal other groups of bacteria, such as sulfate
reducers, that could also play an important role
in contaminant metal reduction and stabilization
within subsurface environments.

5. Role of biolms in bioremediation of metal

polluted soils and sediments
5.1. Biolms in natural environment
Bacteria are widespread in soil and aquatic environments and are predominantly found in biolm communities. A biolm may be dened as a
gelatinous extracellular polymeric substance
(EPS) of biological origin containing a consortium of bacteria, fungi, and algae. Biolms are
present at the solidaqueous interface throughout
the aquatic environment (Lappin-Scott et al.
1993) and typically at the sedimentwater interface (Davison et al. 1997; Forstner, 2004). Biolms form when bacterial consortia attach to
mineral surfaces and produce lms of hydrated
extracellular polymers (Jackson et al. 1999). Once
biolms form, the bacterial-mineral micro-aggregates create complex interfaces with the surrounding aqueous solution. The biolms may act
as an insulating layer between the solution and
the mineral surface or form micro-environments in which the local solution conditions are
dierent than those in the bulk solution (Glazer
et al. 2002). Reactive functional groups, such as
carboxyl, hydroxyl, amino and phosphoryl moieties, present on the bacterial surfaces and exopolysaccharide matrix, can provide a large array
of binding sites for metals (Flemming & Leis
2002). For instance, autotrophic biolms have

been observed to sequester metals at 45 orders

of magnitude above the concentration in the surrounding waters (Liehr et al. 1994). Biolms may
simultaneously block surface sites on the underlying substrate (Templeton et al. 2003a,b). In
addition, bacterial activity may catalyze the
trans-formation of toxic metals into less (or
more) toxic species or enhance the dissolution of
the underlying mineral substrate and bulk solution surrounding of the biolm (Beyenal et al.
2004; Labrenz & Baneld 2004). All of these
combined interactions may strongly aect the
mechanisms and kinetics of metal sorption reactions in soils and aquatic environments (Newman
& Baneld 2002; Gadd 2004).
However, biolms that form on solid phases
in natural contaminated environment sites may
dier signicantly in form and function from
those found in other environments described to
date. So far biolms have been mainly used to
remove inorganic contaminants from aqueous
systems (industrial wastewaters) (Diels et al.
2003, Remoudaki et al. 2003; Wagner-Dobler
2003; van Hullebusch et al. 2003; Bender et al.
2004, Bender & Phillips 2004). However, they
been also shown to inuence the mobility of
inorganic contaminants in subsurface environment such as soils and sediments. Ecological success of biolms and their broad array of
microbial activities suggest that these microbial
ecosystems might be useful to bioremediation of
environmental pollutants as described in the
following subsection.
5.2. Biolm and metals/radionuclides
immobilization
Only the main mechanisms involved in the
immobilization of metal and radionuclides, as
shown Figure 21, will be described in the following subsection.
5.2.1. Passive processes: biosorption of metals by
biolms
Microorganisms adsorb and concentrate many
metallic cations through electrostatic interactions
with anionic carboxyl and phosphoryl groups in
the cell wall. Biosorption consists of several
mechanisms, mainly ion exchange, chelation,
adsorption, and diusion through cell walls and
membranes. Biosorption encompasses those

144
physico-chemical mechanisms by which metal
species and radionuclides are removed from an
aqueous solution. This phenomenon is often
attributed to the binding of metals onto the bacterial cell surface (White et al. 1995; Cox et al.
1999). In addition, however, the EPS that surround cells are also very reactive and can readily
bind metals such as iron (Konhauser et al. 1993,
1994). A growing body of literature shows evidence that EPS bind and concentrate a range of
inorganic contaminants. EPS is a highly hydrated
material (99% water) possessing an extremely
porous brillar structure that renders it highly
adsorptive. The EPS consist of polysaccharides,
proteins, lectins, lipids, nucleic acids, etc... The
structure of the EPS-matrix is considerably
stronger in the presence of dierent cations,
which interact with exposed carboxyl groups on
the EPS. EPS can bind a wide variety of metals,
including Pb, Sr, Zn, Cd, Co, Cu, Mn, Mg, Fe,
Ag, and Ni (Ferris et al. 1989). The sorption
capacity of biolms can be attributed to the
complex or chelate formation of the EPS-matrix
with dierent cations and the uptake of cations

by algae and bacteria living in the biolms. The

binding capacity of a given EPS-matrix also depends on its physical state (gel versus slime versus dissolved state) and the pH of the water. In
order to reach stronger binding of the metal ions,
the gel structure and higher pHs (basic conditions) are favourable. Ferris et al. (1989) reported that near neutral pH, microbial biolms
concentrated metals up to 12 orders of magnitude higher than observed under acidic conditions. Neutral to alkaline microenvironments are
commonly produced within microbial mats and
biolms through such processes as photosynthesis and sulfate reduction (Krumbein 1979).
5.2.2. Microbial mineralization
Minerals formed in association within biolms
are commonly referred to as biominerals, e.g.
sulfate, phosphate, carbonate, sulde or silicate
ions (Schultze-Lam et al. 1996). Given the ability
of EPS to concentrate various metals, it is not
surprising that bacteria have been implicated in a
wide variety of biomineralization processes. As a
result of cellular metabolism, microorganisms

Figure 21. Biolmmetal interactions which have an impact on bioremediation of metal contaminated soils and sediments (Modied from van Hullebusch et al. 2003).

145
alter the chemical microenvironment around the
cell, modulating the pH, as well as the concentration of a variety of organic and inorganic solutes. This can induce the large-scale precipitation
of authigenic minerals in natural environments
(Geesey et al. 1989; Beveridge et al. 1997a,b;
Douglas and Beveridge, 1998; Langley & Beveridge 1999). For instance, stromatolites, made of
calcium carbonate precipitates, are intimately
associated with biolms that comprise an abundant mix of cyanobacteria, bacteria and diatoms
(Douglas & Beveridge 1998; Arp et al. 2001).
Sometimes nanomineral phases can even form directly in the bacterial cytoplasm as has been described for acanthite (Ag2S) (Klaus et al. 1999)
or amorphous iron hydroxide (Fortin 2004). The
tolerance of biolms to high metal concentrations may also be due to their ability to precipitate insoluble metal salts outside the cells as
suldes, phosphates, carbonates, oxides or
hydroxides (Bender et al. 1995; Douglas & Beveridge 1998).
5.2.3. Oxidation and reduction
Inorganic contaminant entrapment and adsorption
by iron and manganese oxides.
A microbial biolm consortium catalyses oxidation of Fe(II) in acidic and near neutral solutions
to produce iron (Fe(III)) hydroxides (Brown
et al. 1998b, 1999). Iron oxide precipitates associated with biolm EPS were identied as ferrihydrite in both pH conditions but the precipitate
morphology and minor elemental composition
was pH dependent (Ferris et al. 1989). Investigation of biolm consortia from groundwater by
Transmission Electronic Microscopy (TEM)
showed that the accumulation of iron on the cell
surfaces was stepwise and that iron precipitates
were found at the cell surfaces and throughout
the biolm matrix (Brown et al. 1998a). Bacterial
cell walls and EPS are generally negatively
charged, which allows positively charged metal
ions to be adsorbed. These metal ions may than
act as nucleation sites for the deposition of more
iron from solution as shown by Brown et al.
(1998a). Many investigations report that the
sorptive capacities of Fe/Mn oxides for metals
are increased in the presence of microorganisms
(Haack & Warren 2003; Nelson et al. 2002). The
iron oxide present in the biolm can adsorb zinc,
lead and cadmium in the biolm (Baldi et al.

2001). Lack et al. (2002a, b) showed that the

anaerobic bio-oxidation of 10 mM Fe(II) by Dechlorosoma species and precipitation of Fe(III)
oxides by these microorganisms resulted in rapid
adsorption and removal of 55 lM uranium and
81 lM cobalt from solution.
Metal reductive precipitation. Bioreduction is
generally recognized as an important metabolic
process controlling the fate and transport of heavy metals and radionuclides, as well as the over
all geochemistry of subsurface and sedimentary
environments. Bioreduction of multivalent metals
can convert dissolved, oxidized forms of multivalent heavy metals and radionuclides (Cr, Mo,
Se, U, Pd, Tc, Au), such as U(VI) and Cr(VI), to
reduced forms that readily precipitate from solution. The mobility of most of these elements in
the environment is strongly dependent on their
chemical oxidation state. Under oxidizing conditions, they occur as highly soluble and mobile
ions, however, under reducing conditions they
usually form insoluble phases (Abdelouas et al.
2000). A recent review gives a detailed understanding of some of these transformations at a
molecular level (Lloyd 2003). For instance,
molybdenum (VI) reduction by D. desulfuricans
in the presence of sulde resulted in the extracellular precipitation of the mineral molybdenite
MoS2(S) (Tucker et al. 1997). Lloyd et al. (2001)
show that SRB reduce Tc(VII) and this metal
precipitates as TcO2 at the periphery of the cell.
Microbial reduction of toxic Se(VI) and Se(IV)
to the colloidal elemental selenium [Se(0)] can be
used to remove Se from wastewater (Lloyd et al.
2001; Hockin & Gadd 2003). Elemental selenium
has not been localized on the bacterial cells
(Lloyd et al. 2001) but was released in the bulk
solution. SRB biolms have been shown to reduce Cr(VI) to Cr(III), which precipitates as
insoluble hydroxides (Cr(III)) (Smith & Gadd
2000).

5.2.4. Microbial precipitation

Sulde precipitation. Sulfate-reducing bacteria
(SRB) are the important physiological group for
sulde production. SRB are commonly used for
the bioremediation of metal-contaminated soil
or water. White et al. (1998), for instance,
described an integrated microbial process for the

146
bioremediation of soil contaminated with toxic
metals using microbially catalyzed reactions. In
this process, bioleaching of Cd, Co, Cr, Cu,
Mn, Ni, and Zn via sulfuric acid produced by
sulfur-oxidizing bacteria was followed by precipitation of the leached metals as insoluble suldes
by the action of SRB. Microorganisms have
been used in the remediation of metal pollution
in very specic cases. The most successful has
been the reduction and the eventual precipitation of soluble metal sulphates as insoluble suldes in liquid wastes by the use of the
microorganisms that are part of the sulfur cycle,
especially sulfate-reducing bacteria that use sulfate as their ultimate electron acceptor to produce hydrogen sulde, which binds with the
metals to give metal sulde. This procedure is
used nowadays not only for the treatment of
surface waters but also for massive cleaning up
of underground water and is available in many
formats, even as commercial large-scale treatment plants. Bioprecipitation of metals by
microbiologically produced sulde (SRB) is then
an eective way to immobilise a wide range of
metals, including Zn, Cu, Pb, Ni and Cd. The
metals precipitate as highly insoluble suldes
(entrapment, nucleation and crystallisation of
insoluble suldes), e.g. ZnS, CdS, CuS, CoS,
NiS and FeS (White & Gadd 1998; White &
Gadd 2000; Labrenz et al. 2000; Wang et al.
2001; Drzyzga et al. 2002; Valls & de Lorenzo,
2002; Utgikar et al. 2002; White et al. 2003;
Krumholz et al. 2003). Sulde precipitation is an
ecient means of removing toxic metals from
solution, while immobilisation in a biolm is
also an aective method to reduce the mobility
of the metals, which is of value in bioremediation of anoxic sediment and wetlands (White
et al. 1998; Kaksonen et al. 2003; Labrenz &
Baneld 2004). Sharma et al. (2000) studied a
highly cadmium resistant Klebsiella planticola
strain that is able to induce the precipitation of
cadmium sulde. This strain is a good candidate
for the accelerated bioremediation of systems
contaminated by high levels of cadmium. Edenborn (2004) showed that the addition of both
polylactic acid (PLA) and gypsum in constructed wetlands and reactive barrier walls is
required for stimulating indigenous bacteria to
lower redox potential, raise pH, and enhance the
bacterial sulfate reduction activity.

Phosphate precipitation. Microorganisms can produce phosphate that precipitates metals as phosphates and it is eective for a range of metals. A
mixture of accumulative and chemisorptive mechanisms contribute to the overall process. Biologically produced metal precipitates as phosphates is
eective for a range of metals and radionuclides
(Renninger et al. 2001; Basnakova et al. 1998, See
Macaskie et al. 2004 for a detailed review). A Citrobacter sp. is able to accumulate the uranyl ion
(UO22+) via precipitation with phosphate liberated from organic phosphate substrates by phosphatase activity. Accumulation of uranium as
HUO2PO4 by Citrobacter sp. was observed in the
presence of excess inorganic phosphorus (Renninger et al. 2001). Cell-bound HUO2PO4 facilitated
also Ni2+ removal by intercalative ion exchange
into the polycrystalline lattice and promoted also
zirconium removal (Basnakova et al. 1998).
Carbonate precipitation. Bacterial calcication is
another example for the precipitation of inorganic material by biolms. The formation of calcium carbonates particles in microbial mats and
other aggregates was suggested to be, at least in
part, because of heterotrophic bacteria (Hammes
& Verstraete 2002). Recently, interest in bacterially associated calcium carbonate precipitation
has grown due to strategic bioremediation approaches involving solid phase capture of radionuclide and trace element contaminants by
carbonates in groundwater systems (Curti 1999;
Warren et al. 2001; Fujita et al. 2004). Fe(III)
oxides are commonly thought of as the most
important sorbents responsible for sequestration
of many metals and radionuclides in soils and
groundwater systems (Warren & Haack 2001).
However, in subsurface anoxic aquifers, Fe(III)
(hydr)oxides can be dissolved through microbial
reactions involving reduction of Fe (III) to Fe
(II). Such dissolution is accompanied by release
of adsorbed or coprecipitated metals or radionuclides. Hence, if bacteria can promote precipitation of other geochemically reactive, non
redox-sensitive solid phases such as carbonates,
then the bacteria may provide an important bioremediation tool for solid phase sequestration of
radionuclide or trace element contaminants in
anoxic environments. Microbial degradation of
urea was investigated as a potential geochemical
catalyst for Ca carbonate precipitation and asso-

147
ciated solid phase capture of common groundwater contaminants (Sr, UO2, Cu) in laboratory
batch experiments (Warren et al. 2001). Bacterial
degradation of urea increased pH and promoted
Ca carbonate precipitation in both bacterial control and contaminant treatments. Associated solid phase capture of Sr was highly eective,
capturing 95% of the 1 mM Sr added within
24 h (Warren et al. 2001). Because bacterial ureolysis can generate high rates of calcite precipitation, the application of this approach is
promising for remediation of Sr contamination in
environments where calcite is stable over the long
term (Fujita et al. 2004).
5.3. In-situ utilization of biolms for
bioremediation
As a process for immobilizing metals, precipitation of metals as suldes has already achieved
large-scale application. Although much research is
currently taking place in metal bioadsorption and
the microbial reductive precipitation of metal and
radionuclides contaminants, the fact is that these
techniques are not yet ready for immediate use in
extensive cleaning up operations. Table 3 gives
some examples of bacterial strains isolated from
the environment that can be used for the bioremediation of contaminated subsurface environment
through microbial reductive precipitation process.
However, understanding the bacterially mediated
precipitation processes at a mechanistic level is
still a requisite for properly applying these technologies to complex systems.
Most bacteria (>90%) in subsurface or sedimentary environments are attached to particulate
phases. Knowledge of the arrangement of surface-associated microorganisms is necessary in
order to more fully understand the factors (e.g.,
electron donor and acceptor availability, pH, enzyme kinetics, diusional limitations) that control the biogeochemistry of redox-sensitive
minerals. This knowledge will allow to determine
the role of bacteria in inuencing or controlling
the fate and transport of heavy metals and radionuclides in contaminated environments. The current understanding of sorption and accumulation
of heavy metals and radionuclides by bacteria attached to mineral surfaces compromises our ability to predict microbiological immobilization of

contaminants in subsurface and sedimentary

environments.

6. Summary
The bioremediation of metal-contaminated soils
is a multi-faceted enterprise that applies a wide
variety of biological activities, from single molecules to entire macroorganisms, to the substantial
problem of detoxifying hazardous land. Several
techniques attempt to remove the tightly bound,
slowly desorbing metals; complexation and facilitated transport by bio-surfactants, cyclodextrins
and exopolysaccharides; acid leaching by the
chemolithotrophic activity of Thiobacillus; reductions of arsenic and plutonium to more soluble
species and reduction of mercury to volatile Hg0,
methylation of selenium to volatile dimethylselenide; and extraction of cadmium, zinc, lead, selenium and other metals into living plant tissue.
The reductions of selenium and chromium, in
contrast, have as their goal the detoxication and
immobilization of the metals in-situ, with the
understanding that continuous careful management will be required to maintain reducing conditions in such environments.
An important question in each of these methods is the source of the bioremedial organisms.
Some of the more unusual activities, such as plutonium reduction, are known to be catalyzed by
only a few microorganisms and it may be necessary to introduce specic microbes for in-situ
treatment. Distributing bacterial cells through
soils is itself a signicant undertaking, although
progress is being made in this area (Bai et al.
1997) and the survival and activity of non-native
microbes in a new community is never assured.
In several cases, including chromate reduction,
selenate reduction, selenium volatilization and
mercury volatilization, the desired activities are
conveniently ubiquitous, indigenous to contaminated areas and easily stimulated by hydrogen,
water, warmth, and various forms of organic
matter. In-situ treatments requiring whole-cell
activities thus appear most straightforward when
native microorganisms can be recruited. In
phytoremediation, in contrast, native plants do
not often appear to be the most eective; because
of the comparative ease of plant cultivation,

148
however, maintaining the desired organisms
poses little diculty.
All attempts at restoration of contaminated
land face the similar, internally conicting goals
of speed, cost eciency, and generation of a
clean, productive patch of earth. The earliest
brute-force methods of solidication/stabiliza-

tion, excavation and landlling, and capping

were markedly constrained by the rst two goals,
at the expense of the third. Developing chemical
technology increased the eciency of soil washing and pump-and-treat methods, greatly
improving the fate of the treated land but still
requiring the addition of further hazardous

Table 3. Studies on metalmicrobe interaction in soils and sediments that have impact on bioremediation of metal contamination
Contaminant

Comments

References

Uranium Mine
sediment
Soil and
sediment

Uranium

Suzuki et al. 2003

Sediment

Selenium
reduction

Sediment

Chromate
reduction

Subsurface
environment

Uranium
immobilization

Soils and
sediments

Cadmium

Subsurface
sediment

Uranium

Selenite polluted
site
Subsurface
sediment
Soil

Selenium

Desulfosporosinus sp. Reduced U(VI) to U(IV). This organism may

be stimulated with addition of sulfate and nutrients uranium.
Anaerobic Bio-oxidation of 10 mM Fe(II) and precipitation of
Fe(III) oxides by Dechlorosoma sp. Resulted in rapid adsorption
and removal of 55 lM uranium and 81 lM cobalt. The results of
this study demonstrate the potential of this novel approach for
stabilization and immobilization of heavy metals and radionuclides
in the environment
A selenite respiring bacterium, Bacillus selenitrieducens, produced
signicant levels of Se()II) and is also able to reduce selenite
through Se(0) to Se ()II). The microorganism showed a great
potential to immobilize selenium in anaerobic environments
Consortium of marine sulfate-reducing bacteria was shown to
3+
. These indigenous SRB might
reduce chromate (CrO2

4 ) into Cr
have potential application in bioremediation of metal contaminated sediments.
Immobilisation of hexavalent uranium [U(VI)] by biolm of the
sulfate-reducing bacterium Desulfovibrio desulfuricans G20. The
hexavalent uranium was immobilized following enzymatic or
chemical reduction reaction. The chemical reduction of uranium
was due to the presence of dissolved sulde species.
Highly cadmium resistant Klebsiella planticola strain that is able to
induce the precipitation of cadmium sulde. This strain is a good
candidate for the accelerated bioremediation of systems contaminated by high levels of cadmium
Stimulation of dissimilatory metal reduction activity of an enrichment of microorganisms closely related to Pseudomonas and Desulfosporisinus sp. which promote the reductive precipitation of
uranium
Ralstonia metallidurans CH34 can reduce selenite to elemental red
selenium, that is highly insoluble
Immobilization of cobalt by sulde precipitates

Subsurface
environments

Uranium

Bio-oxidation
of Fe(II)

Cobalt
Mercury

Microbial reduction of soluble ionic mercury, Hg(II), to volatile

Hg(0). The reduced Hg(0) can then ux out of the environment and
be diluted in the atmosphere
Biolm of the sulfate reducing bacterium Desulfovibrio desulfuricans attached to hematite (a-Fe2O3) surface can accumulates both
U(VI) and U(IV) and thus can limit the transport of uranium in
subsurface environments

Lack et al. 2002a, b

Herbel et al. 2003

Cheung and Gu 2003

Beyenal et al. 2004

Sharma et al. 2000

Nevin et al. 2003

Roux et al. 2001

Krumholz et al. 2003
Hobman et al. 2000

Neal et al. 2004

149
substances. Research in bioremediation is now
opening the virtually innite possibilities inherent
in enzymatic systems. Because biological techniques are being developed to cost as little or less
than the best available conventional methods, to
operate in a timely manner, employ biodegradable and (with the exception of metal-laden phytoremedial shoots) non-toxic substances, and to
yield functional soils and sediments, bioremediation may soon provide the tools to restore metalcontaminated soils rapidly, inexpensively and
permanently.
Driven by the realization that large areas of
land contaminated with metals and radionuclides
cannot be economically remediated using conventional chemical approaches, signicant resources
have become available for the area of bioremediation of metal contamination. Supported by
genomics-enabled studies ongoing in many laboratories worldwide (Lovley 2003) we can expect this
research area to develop further, delivering more
robust techniques for the bioremediation of metal
contaminated waters, soils and sediments. Exciting developments in the use of microorganisms for
the recycling of metal wastes, with the formation
of novel biominerals with unique properties are
also predicted for the near future.

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