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Review
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
Abstract
328
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
335
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
335
1. Introduction
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.
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
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.
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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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].
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
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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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.
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].
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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.
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.
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