FEMS Microbiology Reviews 26 (2002) 327^338

www.fems-microbiology.org

Review

Exploiting the genetic and biochemical capacities of bacteria

for the remediation of heavy metal pollution

Marc Valls, V|ctor de Lorenzo

Centro Nacional de Biotecnolog|a CSIC, Campus de Cantoblanco, 28049 Madrid, Spain

Received 14 March 2002; received in revised form 15 May 2002; accepted 25 May 2002
First published online 21 August 2002

The threat of heavy metal pollution to public health and wildlife has led to an increased interest in developing systems that can remove
or neutralise its toxic effects in soil, sediments and wastewater. Unlike organic contaminants, which can be degraded to harmless chemical
species, heavy metals cannot be destroyed. Remediating the pollution they cause can therefore only be envisioned as their immobilisation
in a non-bioavailable form, or their re-speciation into less toxic forms. While these approaches do not solve the problem altogether, they
do help to protect afflicted sites from noxious effects and isolate the contaminants as a contained and sometimes recyclable residue. This
review outlines the most important bacterial phenotypes and properties that are (or could be) instrumental in heavy metal bioremediation,
along with what is known of their genetic and biochemical background. A variety of instances are discussed in which valuable properties
already present in certain strains can be combined or improved through state-of-the-art genetic engineering. In other cases, knowledge of
metal-related reactions catalysed by some bacteria allows optimisation of the desired process by altering the physicochemical conditions of
the contaminated area. The combination of genetic engineering of the bacterial catalysts with judicious eco-engineering of the polluted
sites will be of paramount importance in future bioremediation strategies.
2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Metal accumulation; Bioremediation ; Biocatalyst ; Speciation; Metallothionein

Contents
1.
2.
3.

4.

5.

6.
7.

8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gross responses of bacteria to metals . . . . . . . . . . . . .
Metal biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Bulk removal of metal ions with bacterial biomass
3.2. Genetic engineering of bacterial biosorbents . . . . . .
3.3. Synthesis of novel metal ligands . . . . . . . . . . . . . .
Metal precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Dissimilatory metal reduction . . . . . . . . . . . . . . . .
4.2. Engineering sulde precipitation . . . . . . . . . . . . . .
4.3. Engineering phosphate precipitation . . . . . . . . . . .
Enzymatic transformation of metals and metalloids . . .
5.1. Hg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. As . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Towards in situ bioremediation strategies . . . . . . . . . . .
Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Strain improvement . . . . . . . . . . . . . . . . . . . . . . . .
7.2. Process improvement . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel. : +34 (91) 585 4536; Fax: +34 (91) 585 4506.
E-mail address : vdlorenzo@cnb.uam.es (V. de Lorenzo).
0168-6445 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 4 5 ( 0 2 ) 0 0 1 1 4 - 6

FEMSRE 753 22-10-02

Cyaan Magenta Geel Zwart

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Abstract

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M. Valls, V. de Lorenzo / FEMS Microbiology Reviews 26 (2002) 327^338

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

335

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

335

1. Introduction

FEMSRE 753 22-10-02

2. Gross responses of bacteria to metals

Microbial activity strongly inuences metal speciation
and transport in the environment. Dierent organisms exhibit diverse responses to toxic ions, which confer upon
them a certain range of metal tolerance (see Fig. 1). Eukaryotes are more sensitive to metal toxicity than bacteria
and their typical mechanism for regulating intracellular
metal ion concentrations is the expression of metallothioneins (MTs), a family of metal-chelating proteins. Bacterial MTs that are functionally homologous to the short
(approx. 60 amino acids) cysteine-rich eukaryotic metalbinding proteins have also been reported [3]. Prokaryotic
MTs have been studied in detail only in the cyanobacterium Synechococcus, where they confer resistance to Zn2
and Cd2 [3,4]. In fact, this production of MTs as the
main mechanism of tolerance to metals is exceptional in
the bacterial world. Rather, a number of specic resistance
mechanisms ^ including active eux and sequestration or
transformation to other chemical species ^ become functional at concentrations above the homeostatic or nontoxic levels [1] (Fig. 1). These tolerance mechanisms are
often plasmid-borne, which facilitates dispersion from cell
to cell. A prime example of heavy metal resistance is that
of Ralstonia metallidurans CH34 (previously Alcaligenes
eutrophus CH34) [5,6]. This L-proteobacterium contains
resistance determinants that allow it to grow at millimolar
concentrations of nine dierent toxic metal ions [7].
Sulfate-reducing bacteria (SRB) display a certain degree
of metal tolerance (Fig. 1) as a secondary outcome of their
metabolism. They are anaerobes that produce sulde and
immobilise toxic ions as metal suldes (see below) [8].
Enzymatic metal reduction might also be common in these
bacteria [9]. Finally, the organisms that grow at the highest metal concentrations are the iron- and sulfur-oxidising
bacteria, including the thiobacilli and thermophilic archaea (Fig. 1). The metal resistance of these chemolitho-

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Microorganisms have co-existed with metals since early

history. This is reected in the wide range of divalent or
transition metals at the active centres of many enzymes.
The chemical properties of the metal have been recruited
for catalysing key reactions or for maintaining protein
structure. These metals are therefore required in minute
amounts for normal cell metabolism and their intake is
subject to intricate homeostatic mechanisms that ensure
sucient ^ but not excessive ^ acquisition. Many other
metals seem to serve no biologically relevant function. Instead, they cause damage, mostly due to their avidity for
the sulfhydryl groups of proteins, which they block and
inactivate.
Thus, from a physiological point of view, metals fall
into three main categories: (i) those essential and basically
non-toxic (e.g. Ca and Mg), (ii) essential, but harmful at
high concentrations (typically, Fe, Mn, Zn, Cu, Co, Ni
and Mo), and (iii) toxic (e.g. Hg or Cd). Further, interactions with metals depend not only on the particular
element but also on its chemical speciation.
The requirement of living systems to both acquire and
reject metals has led to the selection of a whole repertoire
of mechanisms of interaction which ensure the adaptation
of microorganisms to a changing and frequently hostile
environment. The emergence of metal tolerance determinants would seem to have been a very early event in evolution [1]. More recently, anthropogenic mobilisation from
metal ores has created novel, metal-loaded niches with a
strong selective pressure for metal endurance. Several prokaryotes show specic resistance determinants, tolerating a
wide range in concentration of these elements by a variety
of mechanisms (see Fig. 1). This article surveys the capacities of a variety of bacteria to cope with toxic concentrations of metals generally considered to be environmental
pollutants. Despite the many interesting metal-related
properties of other biological agents (e.g., fungi and
plants), this review focuses only on those bacterial systems
amenable to genetic analysis and, eventually, to genetic
engineering for the enhancement of their metal-associated
abilities. Although, chemically speaking, arsenic (As) is
not a metal, bacteria react to its various molecular species
in a manner not unlike their reaction to metal ions; responses to As will therefore also be briey examined. This
knowledge forms the basis for novel strategies aimed at
remediating (or at least mitigating) intensive and extensive
pollution caused by heavy metal ions. This form of contamination has far more consequences than all the other
types of pollution, of any origin, put together [2].

As discussed below, microorganisms have been successfully exploited to deal with heavy metal pollution in a
variety of schemes. Emerging technologies in this area
rely on enhancing the biosorption of metals into biomass,
or the precipitation of ions by exploiting some metal-related facet of bacterial metabolism (Fig. 2). Due to their
distinct physicochemical properties, mercury and metalloids (As) can sometimes be mobilised away from polluted
sites by microbes that convert them into volatile species,
an alternative to the immobilisation/precipitation approach.

M. Valls, V. de Lorenzo / FEMS Microbiology Reviews 26 (2002) 327^338

trophs is an adaptation to very acidic environments, where

metal solubility is high. Interestingly, when the growth of
Thiobacillus ferrooxidans is dependent on Fe(II), this bacterium is highly resistant to Al, Cu, Co, Ni, Mn and Zn
(0.1^0.3 M), although it remains quite sensitive to other
metal ions [10].
Bacteria thus show a panoply of responses to metal ions
and diverse bacterial groups have developed abilities to
cope with these toxic elements in a variety of environments
(e.g. anaerobic, aerobic, thermophilic). With respect to
pollution control, these activities show promise with regard to (i) hastening the mobilisation of metals (with a
possible application in biomining) [11,12], (ii) designing
metal-tolerant strains that are better adapted to performing biodegradation of organic pollutants, and (iii) metal
bioremediation or mitigation through the breeding of natural or engineered strains. This paper focuses on the latter.

may then be regenerated by alkali treatment for reuse in

further sorption^desorption cycles [20,21]. The main drawback in the use of biomaterials is that existing ionexchange synthetic resins provide a similar performance
and their use is already well established. Nevertheless, biosorption methods seem to be more eective than their
physicochemical counterparts in removing dissolved metals at low concentrations (below 2^10 mg l31 ) [18]. In
addition, the higher specicity of biosorbents may avoid
an important problem encountered with ion-exchange resins: the overloading of binding sites by alkaline-earth metals present in polluted euents. Finally, biological systems
oer the potential of genetic modication to further increase the specicity towards certain metal ions or the
bioaccumulation yield. To this end, metal-binding proteins
found in nature as well as novel metal-chelating peptides
obtained in the laboratory have been introduced into bacteria.
3.2. Genetic engineering of bacterial biosorbents
The rst studies in the genetic engineering of biosorbents involved the cloning of eukaryotic MTs for their
intracellular expression in bacteria. As mentioned above,
MTs are a family of low molecular mass proteins able to
coordinate a variety of heavy metal ions. They are thought
to constitute the main mechanism by which eukaryotes
protect themselves from these contaminants [22]. Cytoplasmic production of human MT fused to araB in Escherichia coli brought about a 3^5-fold increase in Cd and Cu
bioaccumulation [23]. These gures were more remarkable
when the dead biomass of modied bacteria ^ in this case

3. Metal biosorption
3.1. Bulk removal of metal ions with bacterial biomass
One way to perform metal recovery is through the use
of plant, algal or microbial biomass ^ sometimes treated
with a strong base to enhance metal-binding ability ^ to
remove metal species from aqueous solutions [13,14]. The
term biosorption is frequently used to describe the uptake
or binding of heavy metals or radionuclides to cellular
components. However, the use of living bacteria [12,15]
and biopolymers [16] has been recently incorporated into
the concept of biosorption. Biosorbents may be viewed as
natural ion-exchange materials that primarily contain
weakly acidic and basic groups, the chelation process
being unspecic [17]. The metals can be stripped from
the matrix after loading by sulfuric or hydrochloric acid,
sodium hydroxide or complexing agents, whether dead
biomass or live bacteria are used [18,19]. The system

FEMSRE 753 22-10-02

Fig. 2. How bacteria cope with toxic concentrations of heavy ions. The
scheme summarises the various means by which bacteria react to the
presence of metals (M2 ) in the medium, with reference to the cellular
compartment that harbours the response. These mechanisms include the
intra- or extracellular binding (and thus immobilisation) of the metal
with a cognate protein (frequently a metallothionein) or a matching
anion, the biotransformation of the toxic ion into a less noxious or
more volatile form, and the dissimilatory reduction of the metal.

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Fig. 1. Bacterial capacities and mechanisms of tolerance to heavy metals. Microorganisms cited in the text and the molecular strategies they
exhibit to cope with metal ions are indicated. Organisms are situated in
the scheme according to their tolerance towards typical toxic ions such
as Cd(II) or Ni(II). Concentrations shown at the top are only indicative
since metal resistance varies widely within taxonomic groups and according to the element considered (adapted from [121]).

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3.3. Synthesis of novel metal ligands

Protein design and selection of novel metal-binding peptides are useful strategies to increase ligand stability and to
improve the anity or selectivity towards certain heavy
metal ions. Protein design based on MTs has produced
variable results. In one approach, synthetic polypeptides
containing tandem repeats of the N. crassa MT fused to
the maltose-binding protein were constructed [37]. The
modied proteins were targeted to the periplasm of
E. coli, endowing the modied strains with enhanced
109
Cd2 -binding capacity at low metal concentrations.
Up to a 6.5-fold enhancement in metal uptake was
achieved in comparison with cells expressing an MT
monomer [37]. However, the introduction of Cys to His
mutations in mouse and lobster MTs that were meant to
improve their stability in oxygenated conditions rendered
dierent proteins species with lower metal coordination
capacities [38,39]. Finally, the generation of dimeric and
tetrameric human MTs increased binding activities stoichiometrically, but the resulting proteins were insoluble
when expressed in E. coli [40].
Other designed metal ligands include peptides with high
contents of Cys or His, the amino acids able to establish
coordination bonds with metal ions. A peptide designed to
show high anity for cadmium ions by incorporation of
Cys-Gly and Cys-Cys-Gly repeats was expressed in E. coli
as a fusion to the maltose-binding protein. The modied
cells showed enhanced binding of Hg2 and Cd2 [41].
Surface display on E. coli of other cysteine- and histidine-rich metal-binding peptides engineered into the
LamB protein increased the Cd2 -binding activity 4-fold
[42]. The E. coli outer membrane proteins LamB and
OmpC have been exploited to surface-expose poly-His
peptides, conferring, respectively, 15-fold and 3-fold
more metal adsorption than control cells [43,44]. Increased
Cd2 and Ni2 binding was also obtained by expressing
poly-His peptides on Gram-positive bacteria such as
Staphylococcus species [45]. Finally, synthetic genes encoding several (Glu-Cys)nGly peptides linked to an lpp-ompA
fusion gene, were cloned into E. coli [46,47]. These peptides emulate the structure of phytochelatins, metal-chelating molecules that play a major role in metal detoxication in plants and fungi. The phytochelatin analogues were
presented on the bacterial surface, enhancing Cd2 and
Hg2 bioaccumulation by 12-fold and almost 20-fold, respectively [34,46,47].
A singular approach for obtaining completely novel
metal-binding proteins by design is the introduction of
metal-binding sites into the framework of proteins of determined structure. The NMR structure of the B1 domain
of streptococcal protein G was used to identify, by computer search, positions at which the Zn(II) ion could be
bound with tetrahedral geometry and that allowed mutagenesis without aecting protein folding. These positions
where then mutated to amino acids that could coordinate

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E. coli containing the Neurospora crassa MT ^ was incubated with solutions of low metal content [24]. In addition,
the chelating eciency of MT was proven to be higher
when targeted to the periplasmic space [24]. Because of
this observation, and in order to circumvent the problems
associated with cytoplasmic expression (i.e. metal uptake
limitations, toxicity associated with intracellular metal accumulation, interference with the redox state of the cytosol), later studies directed MTs to the periplasmic space or
to outer membrane compartments of E. coli [25^31]. Expression on the cell surface might facilitate the use of nonviable cells for metal accumulation and the ecient desorption of the bound metal to regenerate the system.
An E. coli strain expressing MT fused to the outer membrane maltose protein (LamB) showed a 15^20-fold rise in
Cd2 binding, as compared to its wild-type counterpart
[27]. A similar Cd2 -binding performance was obtained
through the expression of MT on the surface of E. coli,
R. metallidurans and Pseudomonas putida as a fusion to
the L domain of the IgA protease autotransporter
[30,31]. Other fusions of MT to bacterial membrane proteins have resulted in only a minor enhancement of bioaccumulation potential, probably due to protein instability
or the inappropriate location of the fusion protein [26,28].
An eective alternative to the surface display of metalcoordinating moieties is cytoplasmic expression combined
with the introduction of specic heavy metal membrane
transporters [32]. This approach overcomes metal uptake
limitations across the cell membrane, but is restricted to
those metals for which there are active import systems
(mercury, copper, lead, nickel, etc.). The cloning of pea
or yeast MTs fused to glutathione S-transferase in
E. coli, together with a nickel transporter from Helicobacter pylori, produced a 3-fold increase in Ni accumulation, with respect to cells expressing MT but not the transporter [33]. Similarly, genetically engineered bacteria
coexpressing the MerT^MerP mercury transporter with
MTs or metal-binding peptides in the cytoplasm showed
an Hg bioaccumulation comparable to that of cells directly expressing the binding peptides on the cell surface
[32,34]. One of these strains was assayed for its use as a
biosorbent in a medium-scale hollow bre bioreactor. At
low concentrations, the system was capable of removing
and recovering Hg2 eectively, reducing a 2 mg l31 solution to about 5 Wg l31 [35]. Cytoplasmic expression of
metal-binding polypeptides (e.g., phytochelatins, see below) is also an eective system for cellular detoxication
of some metals, another advantage of this strategy [3,4].
A few reports describe the importance of engineering
lipopolysaccharides, surface structures in Gram-negative
bacteria with a major role in metal biosorption. It has
been proposed that Pseudomonas aeruginosa PAO1
A-band and B-band lipopolysaccharide mutants dier in
their binding specicities towards iron and lanthanum,
suggesting that the design of biosorbents with higher metal
specicity may be feasible [36].

M. Valls, V. de Lorenzo / FEMS Microbiology Reviews 26 (2002) 327^338

4. Metal precipitation
In addition to their use as biosorbents, bacteria can be
used to eciently immobilise certain heavy metals through
their capacity to reduce these elements to a lower redox
state, producing less bioactive metal species. However,
precipitation and biosorption are sometimes overlapping
phenomena and it can be dicult to assign the contribution of each to metal immobilisation [53]. Microbiological
metal precipitation is a widespread activity that is either
the result of a dissimilatory reduction or the secondary
consequence of metabolic processes unrelated to the transformed metals. For instance, Cr(VI) reduction to insoluble
Cr(III) by SRB may be due to bacterial respiration or to
indirect reduction by Fe2 and sulde [54]. Indirect reduction by formation of metal suldes and phosphates is the
strategy that has received most interest in the biotechnology of metal precipitation and has already been subject to
genetic manipulation. However, dissimilatory metal reduction can also be eectively utilised for decontamination.
4.1. Dissimilatory metal reduction
Dissimilatory processes are those in which the transformation of the target metal is unrelated to its intake by the
microbial catalyst and, thus, the chemical species that result from the cognate biological activity generally end up
in the extracellular medium. Dissimilatory reduction of
uranium, selenium, chromium, technetium, gold and possibly other metals is performed by a number of microorganisms under various environmental conditions and
could be used not only in waste treatment but also in
metal concentration from low-grade ores [55^57]. For instance, Cr(VI) reduction occurs both in aerobiosis and

FEMSRE 753 22-10-02

anaerobiosis, with NADH and electron transport systems

serving as the respective electron donors [58^60]. Microbially driven uranium reduction has also been well documented. While U(VI) compounds are readily soluble, the
reduced U(IV) species, like the hydroxide or carbonate,
have a low solubility and form precipitates at neutral
pH. Several genera are able to foster U(VI) reduction
and this activity has been used in soil remediation in combination with chemical extraction [61,62].
SRB have received much attention because they enzymatically mediate the reductive precipitation of toxic metals including U(VI), Cr(VI), Tc(VI) and As(V) [9,63^65].
For instance, Desulfovibrio desulfuricans has been reported
to couple the oxidation of a variety of electron donors to
the reduction of Tc(VII) by a periplasmic hydrogenase
[66]. This is a clear example of a bacterium immobilising
heavy metals by promoting their precipitation at the cell
periphery as an insoluble low-valence oxide and suggests
the possible use of this organism in the treatment of metalcontaminated wastewaters. In one case, hexavalent chromium reduction to insoluble Cr(III) by a sulfate-reducing
bacterial biolm removed around 90% of the metal from
solution in 48 h [65].
Dissimilatory metal-reducing activities are catalysed by
membrane-bound enzymes that are either c-type cytochromes or associated with such cytochromes [9,64,66].
When the gene encoding one of these multihaem proteins
(cytochrome c7 from Desulfuromonas acetoxidans) was
transferred to D. desulfuricans, its metal-reductive activity
was clearly boosted [63]. Hence, overexpression of reductase or hydrogenase activities could be an important asset
for strains engineered for metal neutralisation.
4.2. Engineering sulde precipitation
One of the best-known natural metal precipitation
mechanisms is that of sulde production by SRB. SRB
play a crucial role in metal sulde immobilisation in anaerobic sediments that contain high concentrations of metals [8]. By examining bacterial biolms grown in continuous culture, this phenomenon could be attributed to the
deposition of metal suldes at the biolm surface or in the
liquid phase, followed by entrapment of the precipitated
suldes by the exopolymer [67,68]. SRB have been successfully used in the treatment of waters and leachates in
large-scale bioreactors and in pilot laboratory surveys
[69,70]. Mixed sulfate-reducing bacterial consortia are
more eective than pure cultures in the removal of heavy
metals from solution [67,68]. SRB-containing reactors
have also been used in the removal of heavy metals
from soil in a microbial integrated decontamination process [71]. As a rst step, metals were mobilised from the
soil by sulfur-oxidising bacteria. Some 98% of the bioleached metals could then be removed from the euent
by precipitation using an anaerobic reactor containing
SRB [71]. The importance of SRB and iron-reducing bac-

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metals (His or Cys), and a high anity tetrahedral Zn(II)binding site was obtained [48].
Articial selection of peptide variants from libraries that
include millions of random sequences is another powerful
technique for obtaining novel and unpredicted metal chelators. Phage-display technology has been used to select
for hexapeptides with high anities for Cd2 . A polypeptide selected by this method was able to confer increased
survival to toxic concentrations of the metal when expressed in E. coli as a fusion to the OmpA outer membrane protein [49]. Polypeptides able to bind elemental
gold or chromium have been similarly selected in peptide
libraries displayed on E. coli as part of the LamB protein
[50]. A recently described methodology exploits E. coli
mbriae as scaolds for displaying and selecting for peptide variants capable of coordinating zinc ions [51,52].
Serial selection of a peptide library fused to a permissive
site in FimH ^ the adhesin component of type 1 mbriae ^
rendered sequences with anity for Zn2 but without similarity to any known Zn2 -binding protein motifs [52].

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4.3. Engineering phosphate precipitation

In this process, metal precipitation is mediated by the
liberation of inorganic phosphate from organic phosphate
donor molecules. Most work has been carried out with a
Citrobacter sp. strain isolated from metal-polluted soil. It
was discovered that this bacterium could accumulate high
levels of uranium, nickel and zirconium through the formation of highly insoluble metal phosphates, opening the
way for future applications in bioremediation [76,77]. The
precipitation process was catalysed by the crystallisation
of inorganic phosphate liberated via the activity of a periplasmic acid phosphatase (named PhoN), with a role for
phosphate groups within the lipopolysaccharide in crystal
nucleation [76,78]. Although this phenomenon could be
considered an example of metal biosorption, metal phosphate formation by bacterial metabolism plays a pivotal
role. Similarly, Cd immobilisation by Bacillus thuringiensis
DM55 cells is proposed to be phosphate-dependent [79].
The transference of metal phosphate precipitation activ-

FEMSRE 753 22-10-02

ity from Citrobacter species to other bacteria has proven

feasible. Enhanced metal phosphate biomineralisation in
E. coli was achieved by the introduction of phoN from
Citrobacter or of related phosphatases [76].
Another tool to counteract metal or radionuclide contamination is the generation of bacteria with engineered
polyphosphate pathways. This possibility is based on the
apparent relationship between depletion of polyphosphate
reserves in the cell and heavy metal resistance [80]. The
introduction of an inducible polyphosphate kinase into
P. aeruginosa resulted in the accumulation of large quantities of polyphosphate. If the organism was subsequently
exposed to uranyl under phosphate-limiting conditions,
polyphosphate was degraded and inorganic phosphate secreted, removing the uranyl from solution [81].

5. Enzymatic transformation of metals and metalloids

Bacteria exhibit a number of enzymatic activities that
transform certain metal species through oxidation, reduction, methylation and alkylation. Apart from the enzymatic transformations that lead to metal precipitation
and immobilisation (see above), other biological reactions
can be applied to bioremediation because they generate
less poisonous metal species. Mercury and arsenic are
the elements for which these reactions have been best
studied.
5.1. Hg
Resistance to mercury is considered a paradigm of metal
detoxication by enzymatic transformation to a less noxious species. The mechanism of bacterial resistance to
Hg2 is its reduction by mercuric reductase (the product
of the merA gene) to the less toxic and volatile Hg0 species
[82,83]. Natural isolates often show broad-spectrum mercury determinants, encoding the capacity to degrade organomercurials ^ such as the highly poisonous methylmercury ^ to Hg2 , which is subsequently transformed to Hg0
[84]. The reductase activity provides a means of mercury
removal by mobilisation of the metal to the atmosphere as
an alternative to metal immobilisation strategies. The prociency of natural mercury-tolerant isolates of bacteria in
mercury volatilisation has been assayed under dierent
conditions and experimental systems [85^88]. For instance,
a P. putida strain removed over 90% of the metal from a
40 mg l31 solution in 24 h [89]. The merA reductase activity has been eectively introduced into diverse engineered strains. An E. coli variant containing simultaneously the merA and glutathione S-transferase genes was able
to withstand high mercury concentrations (30 mg l31 ) and
to reduce to Hg0 some of the metal present in the solution
[90]. Deinococcus radiodurans, the most radiation-resistant
organism known, was recently modied to express merA
cloned from E. coli BL308. The strain was instrumental in

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teria such as Shewanella and Geobacter species in the immobilisation of heavy metals in natural sediments lays the
basis for their use in remediation processes in situ [8,72].
As their genomic sequences become available (see http://
www.jgi.doe.gov or http://www.ncbi.nlm.nih.gov for most
recent updates), the genetic and biochemical bases of their
activities on metals will be revealed.
However, the fact that even low levels (20^200 WM) of
free Cd(II), Zn(II) or Ni(II) ions are toxic to SRB such as
Desulfovibrio may limit their use [67]. A Klebsiella planticola strain able to thrive in high Cd2 concentrations and
precipitate signicant amounts of cadmium sulde could
be an alternative to SRB for metal immobilisation in anaerobic conditions [73]. Another alternative for improving
sulde-dependent metal removal might be the transference
of the dissimilatory sulfate reduction pathway to environmental bacteria. The rst attempt in this direction was the
expression of the thiosulfate reductase gene from Salmonella enterica serovar typhimurium in E. coli. E. coli DH5K
strains harbouring this enzyme produced signicantly
more sulde than the control strains under both aerobic
and anaerobic conditions. Moreover, one of the recombinant strains removed 98% of the available cadmium (from
up to 200 mM solutions) under anaerobiosis [74].
An additional prospect of metal precipitation based on
sulfur cycling by bacteria is the metabolic engineering of
sulfate reduction to make the process eective under aerobic conditions. In one study, serine acetyltransferase and
cysteine desulfhydrase were overexpressed in E. coli so
that cysteine was overproduced and subsequently converted to sulde [75]. The resulting strain was eective in
aerobically precipitating cadmium as cadmium sulde,
which was deposited on the cell surface. This bacterium
may provide a feasible substitute to anaerobic SRB metal
decontamination.

M. Valls, V. de Lorenzo / FEMS Microbiology Reviews 26 (2002) 327^338

the decontamination of radioactive waste, since it could

grow in the presence of both radiation and ionic mercury,
eectively volatilising the metal [91]. A combined method
of chemical leaching and subsequent volatilisation of mercury by bacteria has been developed that removed about
70% of mercury from polluted Minamata Bay sediments
[92]. Nevertheless, the volatilisation of mercury remains a
matter of controversy which arose after the mer system
was introduced into plants designed for phytoremediation
[93]. The mobilisation of mercury could solve a local problem, but there is public concern that this might eventually
contribute to global atmospheric pollution.
5.2. As

FEMSRE 753 22-10-02

6. Towards in situ bioremediation strategies

Current research interests are moving towards the application of in situ strategies in an attempt to avoid the
costs and pollution dispersal problems caused by transportation of contaminated soils or waters to treatment
plants. The rst and best-established approach for in situ
detoxication of metal-polluted euents is their circulation through a bacterial, algal and higher-plant consortium. In one example, the so-called Meander system, metals were removed from the water column with greater than
99% eciency [69]. Precipitation, biosorption and particulate entrapment seem to be the underlying mechanisms of
detoxication, with a major role for SRB in the sediment
[104]. However, the usefulness of articial wetlands for
metal removal is unpredictable, and the nal fate of the
metals is a question of concern, since long-term applications could lead to secondary contamination of the environment [105].
More recently, a novel approach for the in situ immobilisation of heavy metals in polluted soils was described
[30]. In this system, the metal-tolerant bacterium R. metallidurans CH34 was modied by expressing mouse MT on
its surface to promote metal biosorption. When this recombinant bacterium was introduced into contaminated
peat soil, it was able to immobilise cadmium in situ, protecting plants from the noxious eects of the metal (Fig. 3).
Since the toxic metal seemed to undergo a degree of precipitation around the bacterial cells [30], metal immobilisation in soil using bacteria (and the corresponding decrease in bioavailability) could have a longer-term eect,
not unlike addition of chemical amendments, such as zeolite, beringite and hydroxyapatite [106^108]. Microorganisms could therefore be employed to immobilise metals in
moderately polluted elds, thus allowing their use in agriculture [30]. It has also been shown that inoculation of
metal-resistant bacteria into soils protected the indigenous
bacterial community from the eects of heavy metals
[109]. Bioaugmentation is therefore under development
as an instrument for the in situ protection of micro- and
macrobiota from metal toxicity.
Still related to the improvement of in situ technologies,
some eort is currently being devoted to obtaining bacteria better adapted to the actual conditions in which
biotreatments are to be performed. Since heavy metals
usually coexist in wastes with other kinds of contaminants
such as organic pollutants, the introduction of several
detoxifying functions into a single organism is being attempted. Recent reports include the characterisation of a
microbial consortium that could couple the oxidation of
phenol to Cr(VI) reduction [110], the study of an enriched
microbial population able to degrade metal^EDTA complexes [111], and the development of a strain that can
detoxify both mercury and toluene in radioactive wastes
[91]. Recent advances in the engineering of radiationresistant microorganisms, such as D. radiodurans, could

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Microbial transformations of metalloids (e.g. the methylation of arsenic, selenium and tellurium) have been reported in environments such as anaerobic sewage sludge
[94,95]. Several bacterial species have been shown to methylate arsenic compounds to volatile dimethyl- or trimethylarsine, although the process is better characterised in
fungi [96,97]. In methanogenic bacteria, methylation of
inorganic arsenic under anaerobic conditions is coupled
to the methane biosynthetic pathway. The process proceeds by reduction of arsenate to arsenite followed by
methylation to dimethylarsine [97]. Most volatile metal
compounds exhibit higher toxicity than their inorganic
counterparts since organic derivatives are lipophilic and
thus more biologically active. Exceptions are arsenic and
selenium, where the inorganic non-methylated forms are
more toxic than the methylated derivatives [95]. Arsenic
volatilisation may thus be used as a mechanism for metal
detoxication, although greater knowledge of its molecular basis may be required before it can be fully exploited.
As an alternative to arsenic methylation, accelerating
the oxidation of As(III) to the more readily adsorbed species As(V) may also result in increased immobilisation and
attenuated toxicity [98,99]. The oxidation of arsenite to
arsenate in nature is predominantly a microbially driven
process. Chemical oxidation is slow under most environmental conditions. For instance, As(III) was oxidised by
Thermus sp. at a rate approximately 100-fold greater than
abiotic rates [100]. The arsenite-oxidising bacteria that
have been isolated are often heterotrophic, such as Alcaligenes faecalis [101], but a small number of aerobic chemolithoautotrophic microbes have been found to derive
metabolic energy from the oxidation of As(III) [98,99].
Some of these strains exhibit high tolerance to As (III)
and other heavy metals such as cadmium, and are good
candidates for arsenic mitigation in heavily polluted sites
[102]. Finally, As(V) forms insoluble suldes upon exposure to H2 S more readily than As(III). Microbial oxidation in this context is a useful way of precipitating As
from solution when combined with a separate step of exposure to SRB [103].

333

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M. Valls, V. de Lorenzo / FEMS Microbiology Reviews 26 (2002) 327^338

7.1. Strain improvement

open the way for metal bioremediation in radioactive

wastes [112]. Knowledge of the microbial processes that
occur at high temperature may be applied to the decontamination of metal-polluted waters under such conditions. It was recently noted that Pyrobaculum islandicum,
a hyperthermophilic bacterium, had the ability to reduce
U(VI), Cr(VI), Co(III), and Tc(VII) using hydrogen as
the electron donor [113]. This enzymatically catalysed
metal reduction could account for the formation of
uranium deposits at around 100C in hydrothermal
environments. Finally, further study of anaerobic respiration may result in novel strategies for the in situ treatment
of organic and metal contaminants in the subsurface
[55,114].

7. Future perspectives
Imminent developments in the eld include the further
genetic improvement of strains and the adaptation of existing methodologies to large-scale and in situ decontamination processes.

FEMSRE 753 22-10-02

7.2. Process improvement

To render metal biotechnologies attractive for use, they
must be adapted to specic contamination problems.
Treatment of mixed wastes containing both metals and
organic contaminants and the development of in situ
methodologies are some of the main goals (see above).
In this context, the use of live bacteria in reactors for
the treatment of metals from contaminated soil is a concept receiving renewed interest [15,71]. A recent approach
is based on the observation that some bacteria, such as R.
metallidurans CH34, can solubilise metals in soil via the
production of siderophores and adsorb them into their
biomass. The addition of R. metallidurans CH34 to a
sandy soil contaminated with metals, followed by removal
of the bacterium by a water otation and occulation process, reduced 6^7-fold the cadmium, lead and zinc concentrations [15]. This study shows that well-characterised systems can be used in dierent ways, opening up future
possibilities.
Biosurfactants ^ surface active chemical species, frequently polymers produced by bacteria or fungi ^ can
also assist in the solubilisation and desorption of metals
from polluted soils or sediments. These molecules are competitive in ex situ treatments, particularly because they
have excellent surface properties and are biodegradable
and less poisonous than synthetic surfactants [117].
Rhamnolipids produced by P. aeruginosa have been extensively studied and are produced commercially [118]. Engi-

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Fig. 3. In situ approach for metal immobilisation in soil using designed

bacteria [28]. Three-month-old Nicotiana benthamiana plants grown in a
Cd-polluted sterile soil (1), in the same soil inoculated with wild-type R.
metallidurans (2), or with a genetically engineered R. metallidurans strain
displaying mouse MT on the cell surface (3). Metal toxicity in the
plants is manifested as reduced growth and pigment deciency (chlorosis) in leaves. Protection to metal toxicity is caused by microbially driven metal immobilisation in the soil. Notice the vigour of the plant when
the modied bacterial strain with increased metal-binding capacity is
present. A lesser protective eect is provided by the wild-type strain.

The impact of heavy metals on biological processes has

led to the emergence of a variety of resistance mechanisms
(see above). Interestingly, it is precisely the study of these
adaptations to metal tolerance that is providing the tools
to carry out bioremediation [115,116]. The study of newly
isolated heavy metal-resistant bacteria may contribute to
progress in this direction. For instance, little is currently
known about the transformation of metals by archaea,
which often colonise extreme environments and may
have advantageous activities. The discovery that Fe(III)reducing archaea can reduce and eciently precipitate
gold is indicative of what might still be discovered [57].
Moreover, the exploration of novel metal-binding molecules, such as designed phytochelatin analogues [34,47]
and selected polypeptides with new anities, may enable
further improvement of metal biosorption. Access may
also be gained to new, enticing activities through genomic
and proteomic approaches. In this regard, the availability of the genome sequences for some of the strains that
inhabit niches polluted with heavy metals (for instance, the
reference strain R. metallidurans CH34, http://www.jgi.
doe.gov/JGI_microbial/html/ralstonia/ralston_mainpage.
html) will prove very advantageous for future research
eorts.

M. Valls, V. de Lorenzo / FEMS Microbiology Reviews 26 (2002) 327^338

8. Concluding remarks
The examples reviewed above indicate that remediation
technologies using microorganisms are feasible alternatives to the physical cleansing of soils or the concentration
of metals in polluted waters by physical or chemical
means. The advantages of biological approaches include
a higher specicity than the physical and chemical methods, their suitability to in situ methodologies (e.g. the
avoidance of high energy or toxic chemical addition),
and the potential for improvement by genetic engineering.
Molecular approaches may enable the design of biomass
with specic metal-binding properties through the expression of metal-chelating proteins and peptides, the improvement of metal precipitation processes and the introduction of metal transformation activities in robust
environmental strains. In spite of this, large-scale biological applications are still rare. This is due to the reluctance
of the market to embrace new technologies, especially
those involving recombinant microorganisms, but also to
the inherent diculty of reproducing these processes at
large scale. The fact that the bacterially mediated precipitation of metal in the form of suldes as well as the
use of biosurfactants are the objects of large-scale commercial development holds the promise that other biotechnologies could be used in the eld of metal decontamination.

Acknowledgements
Work in the authors laboratory is supported by EU
contracts QLK3-CT2000-00170 and QLK3-CT199900041, by the Spanish CICYT grant BIO98-0808 and by
the Plan de Grupos Estrategicos de la Comunidad Autonoma de Madrid.

FEMSRE 753 22-10-02

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