Journal of Environmental Management 146 (2014) 383e399

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Journal of Environmental Management

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Review

Bacterial chromate reductase, a potential enzyme for bioremediation

of hexavalent chromium: A review
Hrudayanath Thatoi a, *, Sasmita Das a, Jigni Mishra a, Bhagwat Prasad Rath a,
Nigamananda Das b
a
Department of Biotechnology, College of Engineering and Technology, Biju Patnaik University of Technology, Techno-Campus, Ghatikia, Bhubaneswar
751003, Odisha, India
b
Department of Chemistry, North Orissa University, Takatpur, Baripada 757003, Odisha, India

a r t i c l e i n f o a b s t r a c t

Article history: Hexavalent chromium is mobile, highly toxic and considered as a priority environmental pollutant.
Received 28 March 2014 Chromate reductases, found in chromium resistant bacteria are known to catalyse the reduction of Cr(VI)
Received in revised form to Cr(III) and have recently received particular attention for their potential use in bioremediation process.
3 July 2014
Different chromate reductases such as ChrR, YieF, NemA and LpDH, have been identified from bacterial
Accepted 10 July 2014
Available online 8 September 2014
sources which are located either in soluble fractions (cytoplasm) or bound to the membrane of the
bacterial cell. The reducing conditions under which these enzymes are functional can either be aerobic or
anaerobic or sometimes both. Enzymatic reduction of Cr(VI) to Cr(III) involves transfer of electrons from
Keywords:
Bioremediation
electron donors like NAD(P)H to Cr(VI) and simultaneous generation of reactive oxygen species (ROS).
Hexavalent chromium Based on the steps involved in electron transfer to Cr(VI) and the subsequent amount of ROS generated,
Chromium resistance two reaction mechanisms, namely, Class I “tight” and Class II “semi tight” have been proposed. The
Chromate reductase present review discusses on the types of chromate reductases found in different bacteria, their mode of
action and potential applications in bioremediation of hexavalent chromium both under free and
immobilize conditions. Besides, techniques used in characterization of the Cr (VI) reduced products were
also discussed.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Further, the rate of degradation of pollutants by microorganisms is

Environmental pollution due to indiscriminate discharge of
hazardous and harmful wastes containing toxic heavy metals at
elevated concentrations from industries and mining sites has been
a growing concern all over the world and therefore, underline the
importance of applying effective treatment methods to reduce the
concentration of heavy metals down to acceptable limit. Among
various approaches, bioremediation using biological agents such as
bacteria, fungi, and their enzyme is one of the attractive and
effective methods for cleaning the environment from toxic pollut-
ants (Ruggaber and Talley, 2006). The microorganisms play an
important role in bioremediation processes which is, however,
limited by several factors. For instance, the microorganisms that are
actively involved in the bioremediation of a specific pollutant might
be inhibited by other pollutants present in the same environment.
* Corresponding author.
E-mail address: hn_thatoi@rediffmail.com (H. Thatoi).
often very slow which limits the feasibility of using them in practice
for bioremediation processes (Whiteley and Lee, 2006). In this
context, the use of sole enzymes isolated from bacterial species is
more advantageous than using whole microorganisms as revealed
from several studies undertaken during last few years (Sutherland
et al., 2004; Pieper et al., 2004). Moreover, the enzymatic bio-
transformations do not generate toxic side products as often found
in the case of chemical and some microbiological processes and
therefore, possess less risk of biological contamination on
ecosystem. Their action is specific to the substrate in comparison to
microorganisms and they are also more mobile than microorgan-
isms because of their smaller size (Gianfreda and Bollag, 2002).
Although the enzymatic treatment processes have tremendous
scope for bioremediation, its practical application often faces with
several challenges in terms low activity, productivity and stability
of the enzyme in addition to sustainability of their application.
Efforts are on in search of potential microbes capable of producing
enzymes that can transform the toxic metal ions to their less/non-
toxic forms under wide range of environmental conditions (e.g. pH,

http://dx.doi.org/10.1016/j.jenvman.2014.07.014
0301-4797/© 2014 Elsevier Ltd. All rights reserved.
384 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
temperature, presence interring species etc.) for their practical
application in bioremediation processes.
Traditionally, most of the enzymes used in bioremediation
process are confined to bacterial mono- or di-oxygenases, re-
ductases, dehalogenases, cytochrome P450 mono-oxygenases;
enzymes involved in lignin metabolism (such as laccases, lignin
and manganese peroxidases isolated from white-rot fungi) and
bacterial phosphotriesterases (Pieper et al., 2004). The most
representative class of enzyme used in the remediation of polluted
environments are hydrolases, dehalogenases, transferases and ox-
idoreductases; mainly obtained from bacteria, fungi, plants and
microbeeplant associations (Rao et al., 2010). These enzymes are
either intracellular (produced but retained within the originating
microbial cells) or, extracellular (exported outside from the origi-
nating microbial cells) in nature. Enzyme production by microor-
ganisms is usually low in its natural conditions. The production can
be enhanced, although to a limited extent, in laboratory conditions
through optimization of growth parameters. On the otherhand, the
recombinant DNA technology offers a cost effective process for
large scale production of enzymes with enhanced stability and
activity (Alcalde et al., 2006). In fact, the production of enzymes in
industrial scale from the suitable microbial strain is of paramount
importance for wide spread practical applications of enzymatic
bioremediation process. Keeping the above in view, the present
review highlights the production of chromate reductase enzymes
from bacterial sources, their mode of action and prospects of their
applications both under free and immobilized conditions for
bioremediation of one of the toxic environmental pollutants viz.
hexavalent chromium.
2. Chromium toxicity and bioremediation options
The widespread industrial uses of chromium or its compounds
and mining activities result the release of Cr-containing wastes into
the environment that contaminate the soils, sediment and surafe/
ground waters. Although essential for numerous living organisms
in trace quantities, Cr is toxic at elevated levels. As a transition
mental, it exists in different valence states ranging from eII to þVI
with Cr(VI) and Cr(III) being the dominant species in the environ-
ment. Out of two commonly occurring states, Cr(VI) is toxic to
biological systems due to its strong oxidizing potential that
invariably damage the cells (Kotas and Stasicka, 2000). Cr(VI) is
known to harmful to all forms of living systems (Wise et al., 2004)
including microorganisms (Ackerley et al., 2006). Moreover, it is
mutagenic (Puzon et al., 2002), carcinogenic (Codd et al., 2003),
teratogenic (Asmatullah et al., 1998), and has been classified as one
of the priority pollutants by several regulatory agencies including
United States Environmental Protection Agency (USEPA) that pose
greatest threat to humans (Cheung and Gu, 2007). Hexavalent
chromium usually enters the cell via sulphate transporter pathway
and gets reduced to Cr(III) by various enzymatic and non-
enzymatic processes. During this process, the reactive oxygen
species (ROS) are formed that exert deleterious effects on cells by
interacting with protein as well as nucleic acid (Cheung and Gu,
2007). In contrast, trivalent chromium is having much less
toxicity and bioavailabilty (He et al., 2009) as it readily forms
insoluble hydroxide/oxides above pH ~ 5.5. In fact, the biological
cell membranes are nearly impermeable to Cr(III). As such, detox-
ification of Cr(VI) by its reduction to Cr(III) is of great environmental
importance.
Chemical reduction of hexavalent chromium to trivalent form
followed by precipitation is the most common and widely used
methods among others (e.g. electrochemical treatment, reverse
osmosis, adsorption and ion-exchange) employed for its removal
from contaminated bodies. It involves the reduction of Cr(VI) by
many reducing agents such as Fe(0), Fe(II), sulphide, organic C-
based materials etc. Chemical methods usually require high energy
inputs and/or large quantity of chemical reagent. Besides, these
methods are ineffective at lower concentration of Cr(VI) present in
large volume of wastewaters and generate large quantity of toxic
sludges, disposal of which again causes secondary pollution. On the
other hand, biological methods such as microbial detoxification of
Cr(VI) are economical, safe, and sustainable (Shakoori et al., 2000;
Eccles, 1995) and also free from residual pollution problems.
Many bacterial species possess chromate reductase activity, where
the enzyme converts the highly toxic and soluble hexavalent
chromium to less toxic trivalent form having much lower solubility;
thereby reduction by the enzymes affords a means of chromate
bioremediation (Park et al., 2000). In recent years, chromate re-
ductases have raised enormous interest among the researcher
across the globe because of their central role in mediating chro-
mium toxicity and their potential use in bioremediation/bio-
catalysis (Ackerley et al., 2004b). This results in isolation of diverse
chromate reductase bacterial species, characterization and their
use in reduction of Cr(VI) to Cr(III) to develop a relatively envi-
ronment friendly process alternative to the conventional methods.
3. Bacterial resistance to Cr(VI)
The chromosomal resistance in bacteria makes use of strategies
like specific or unspecific Cr(VI) reduction, free radical detoxifying
activities, repair of DNA damage (Morais et al., 2011) and processes
associated with sulphur or iron homeostasis (Ramirez-Diaz et al.,
2008). Many microorganisms have the potential to survive toxic
metal-polluted environments by developing mechanisms to avoid
metal toxicity like, metal efflux, adsorption uptake, DNA methyl-
ation, and metal biotransformation either directly by enzymatic
reduction to less mobile and toxic forms or indirectly through
making complexes with metabolites (such as H2S) (Camargo et al.,
2005; Pei et al., 2009; Soni et al., 2012). Microbial reduction of
Cr(VI) to Cr(III) is particularly important from bioremediation point
of view which can be considered as an additional chromate resis-
tance mechanism (Cervantes et al., 2001). A variety of Cr-resistant
bacteria with high Cr(VI)-reducing potential have been reported
including Pseudomonas, Bacillus, Enterobacter, Deinococcus, Shewa-
nella, Agrobacterium, Escherichia, Thermus and other species
(Ohtake et al., 1987). It has been reported that both chromate
resistant as well as non-resistant strains can reduce chromate but
the growth of later are significantly inhibited at higher concentra-
tions of chromate (Bopp and Ehrlich, 1988). Therefore, the bacterial
property, which is particularly useful for an effective bioremedia-
tion approach, is one that combines high tolerance/resistance with
the ability to reduce Cr(VI) to Cr(III) (Dhal et al., 2013).
Several microorganisms exhibiting Cr(VI)-reducing activities
and resistance have been isolated and identified from chromate-
contaminated environment as well as natural, uncontaminated
ecosystems (Schmieman et al., 1998; Turick et al., 1996; Wang and
Shen, 1995). Microorganisms that have the ability to reduce Cr(VI)
are usually called as chromium reducing bacteria (CRB). Among
CRB, the Gram-positive bacteria are shown to have significant
tolerance to Cr(VI) toxicity at relatively high concentrations,
whereas Gram-negative bacteria are much more sensitive to Cr(VI)
(Coleman, 1988). Microorganisms found in metal contaminated
environment are naturally resistant for such metals. An investiga-
tion carried out by Das et al. (2013) revealed that the bacteria iso-
lated from chromite mine soils are resistant towards Cr(VI) along
with other heavy metals. It is well known that chromate resistance
and reduction are not necessarily interrelated, and not all Cr(VI)-
resistant bacteria can reduce Cr(VI) to Cr(III). Thus, both chro-
mium resistance and reduction are found to be independent
 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
Fig. 1. Mechanisms of chromate resistance in bacterial cells. (A) Mutation in chromosome-encoded sulfate uptake transporters (B) Extracellular reduction of Cr(VI) to Cr(III) (C)
Intracellular reduction of Cr(VI) to Cr(III) by chromate reductases (D) Function of SOS repair system in reducing oxidative stress (E) Efflux of chromate from the cytoplasm. (F) Action
of ROS scavenging enzyme in reduction of oxidative stress.
properties of bacteria (Bopp and Ehrlich, 1988; Silver, 1997). Bac-
terial Cr(VI) reduction can either occur ‘directly’ by the use of en-
zymes or ‘indirectly’, where Cr(VI) reduction is catalysed by the
metabolic end-products such as Fe(II) and HS of iron and
sulphate-reducing bacteria (Hwang et al., 2002). Bacteria employ
different resistance mechanisms to overcome the Cr(VI) toxicity in
the environment which include the reduced uptake of Cr(VI),
extracellular Cr(VI) reduction, reactive oxygen species (ROS)
detoxifying enzymes/intracellular Cr(VI) reduction, DNA repair
enzymes, efflux of Cr(VI) from cell and ROS scavenging are depicted
in Fig. 1(AeF).
(A) Reduced uptake of Cr(VI)
One of the efficient protective systems against the lethal effects
of Cr(VI) is probably associated with reduced uptake of Cr(VI)
similar to sulphate uptake pathway and with sulphur or iron ho-
meostasis. Since chromate ion (CrO2 4 ) has structural resembles
with tetrahedral sulphate ion (SO2 4 ) (Fig. 2), it can easily pass
through cell membranes via SO2 4 transport pathway, with the help
of non specific anionic (SO2 3
4 , PO4 ) carriers (Wenbo et al., 2000).
The transport of chromate is diminished if the chromosome-
encoded sulphate uptake pathway in bacteria is mutated
(Ramirez-Diaz et al., 2008). The microorganisms present in metal
contaminated environment undergo quick mutation to develop
Cr(VI) resistance that leads to reduced Cr(VI) uptake by sulphate
transport pathway. Susceptible organisms can become insensitive
by mutation or by incorporation of the genetic information which
encodes the resistance (Kümmerer, 2004).
Fig. 2. Structural similarity of chromate and sulfate ions.
385
(B) Extracellular Cr(VI) reduction
Extracellular reduction of Cr(VI) to Cr(III) followed by its binding
with functional groups on bacterial cell surface is another resis-
tance mechanism (Ngwenya and Chirwa, 2011). Binding of reduced
Cr(III) to bacterial cell surface helps its easy removal from the
contaminated environment. Peptidoglycan components present in
the cell walls of the bacteria found to be potent binder of Cr(III)
(Hoyle and Beveridge, 1983). It has demonstrated that some species
of bacteria possess adsorptive properties which facilitate the
removal of metal species from aquatic solutions. These properties
are mostly dependent on the distribution of reactive functional
groups like carboxyl, amine, hydroxyl, phosphate and sulfhydryl
groups on the cell wall surface of bacteria (Parmar et al., 2000).
Thus there is no apparent entry of Cr(VI) in the cell when its
reduction occurs extracellularly.
(C) ROS detoxifying enzymes/Intracellular Cr(VI) reduction
During Cr(VI) reduction to Cr(III) a short lived, highly reactive
intermediate Cr(V) radical is generated which redox cycles. Thus,
Cr(V) is oxidized back to Cr(VI), giving its electron to dioxygen and
generating reactive oxygen species, referred as ROS. Generation of
ROS results in oxidative stress in the bacteria. In this process, the
bacterial proteins are also induced by chromate in the defense
against oxidative stress leading to an additional mechanism of
chromate resistance (Ramirez-Diaz et al., 2008). However, the
oxidative stress arising due to the ROS are nullified to a large extent
by detoxifying enzymes like glutathione transferase, superoxide
dismutase (SOD), catalase, etc. (Ackerley et al., 2004b).
(D) DNA repair enzymes.
Protection of bacterial cells by DNA repair enzymes of damaged
DNA caused by Cr(VI) is another defense shield. Cr(VI) enters the
bacterial cell which is readily reduced to Cr(III) by the action of
386 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
Fig. 3. Physical map of the chr locus in O. tritici 5bvl1 strain. The Tn5 insertion in chrA is indicated by the small black vertical arrowhead.
various enzymatic or non-enzymatic activities that leads to gen-
eration of ROS, which in turn exerts deleterious effects on protein
and DNA in the cell. The ROS generated causes damage to DNA, like
base modification, single-strand breaks, double-strand breaks.
Such damages caused to DNA can be repaired by special DNA repair
mechanism like the SOS response enzymes (RecA, RecG, RuvB) (Hu
et al., 2005). For example, in Escherichia coli, Cr(VI) has long been
known to induce the E. coli SOS repair system that protects DNA
from oxidative damage (Llagostera et al., 1986). Similarly DNA
helicases like RecG and RuvB, components of the recombinational
DNA repair system, have shown to participate in the response to
DNA damage caused by chromate in Pseudomonas aeruginosa
(Miranda et al., 2005). Cellular Cr(VI) reduction is the activation
process, producing redox-active intermediates Cr(V/IV) and stable
Cr(III) forming Cr-DNA adducts which is the most abundant form of
DNA damage that causes mutations and chromosomal breaks
(Zhitkovich, 2011).
(E) Efflux of Cr(VI) from cell
Efflux of chromate ions from the cell cytoplasm, mediated by
transporters encoded by specific plasmid-borne genes, is also a
resistance mechanism found in bacteria. Efflux of chromate seems
to be an efficient and widespread mechanism of resistance, which
prevents the accumulation of toxic ions inside the bacterial cells
(Ramirez-Diaz et al., 2008). The best understood chromate-
resistance system is that conferred by P. aeruginosa ChrA protein
belongs to the chromate ion transporter CHR superfamily. ChrA, is a
hydrophobic membrane protein, encoded by plasmids pUM505 of
P. aeruginosa and pMOL28 from Cupriavidus metallidurans (formerly
Alcaligenes eutrophus and Ralstonia metallidurans) (Cervantes et al.,
1990; Nies et al., 1990) that have been demonstrated as involved in
chromate resistance by chromate efflux mechanism (Ramirez-Diaz
et al., 2008). ChrA protein functions as a chemiosmotic pump that
exports chromate from cytoplasm or periplasm to outside driven by
the proton motive force (Alvarez et al., 1999). CHR proteins from
several bacteria have been demonstrated as involved in chromate
resistance by chromate efflux mechanism (Ramirez-Diaz et al.,
2008).
(F) ROS scavenging
Cr(VI) after entering the cell can be reduced to Cr(V). Electron
donors like NAD(P)H or some other organic compounds (like
glucose) donate electrons to Cr(VI), leading to the formation of
relative unstable toxic intermediate Cr(V). Although the chromate
reductases reduce Cr(V) further to Cr(III) by a two-electron transfer
(via “semi-tight” mechanism), sometimes this reaction is not very
rapid. As a result a portion of Cr(V) intermediate is quickly reoxi-
dized to Cr(VI) thereby generating ROS by a Fenton-like reaction.

During this process hydroxyl radicals ( OH) are formed in the mi-
crobial cells (Shi and Dalal, 1994) as depicted in the equation below:
Cr(V) þ H2O2 / Cr(VI) þ OH þ OH


During the reduction process, molecular oxygen is reduced to O2
radicals, which generates H2O2 via dismutation. Hexavalent chro-

mium reacts with H2O2 to generate OH radicals via a Fanton like
reaction. This mechanism is similar to the oxidation of Fe(II) with

H2O2 in the Fenton reaction as the production of OH from Fe(II) via
Fenton reaction is facilitated greatly by the formation of Fe(II)
complexes that have vacant sites for H2O2 coordination.
4. Genetic mechanism of Cr(VI) resistance and tolerance
The bacterial species, able to grow in the toxic conditions
prevalent in Cr(VI) contaminated environment, are generally
assumed to be tolerant/resistant to chromium (Viti and
Giovannetti, 2001). The terms resistance and tolerance are often
used interchangeably although their significance is different. Ac-
cording to Gadd (1992), the resistance is “the ability of a microor-
ganism to survive toxic effects of metal exposure by means of a
detoxification mechanism produced in direct response to the metal
species concerned” while tolerance defined as “the ability of a
microorganism to survive metal toxicity by means of intrinsic
properties and or environmental modification of toxicity”.
High Cr(VI) resistance and a high Cr(VI) reduction capability
makes a bacterial strain suitable candidate for effective remediation
purposes (Christ et al., 2012). The capability of Cr(VI)-reduction is
effected by an additional chromosome or plasmid resistance
mechanism that represents a potentially useful detoxification
process for several bacteria (Pattanapipitpaisal et al., 2001;
Cervantes et al., 2001). It has been reported that genes for Cr(VI)
reduction can either be plasmid borne as observed in the cases with
several Pseudomonas species (Bopp and Ehrlich, 1988; Bopp et al.,
1983) or located on the chromosomal DNA as found in several
Bacilli and Enterobacteriaceae (Li and Krumholz, 2007).
Several chromosomal genes have been found to be responsible
for imparting resistance to Cr(VI) in bacteria. For example, the
chrR gene located on the chromosome of P. aeruginosa conferred
resistance to chromate (Aguilar-Barajas et al., 2008). Morais et al.
(2011) reported that Ochrobactrum tritici contains several chro-
mate resistance genes namely chrB, chrA, chrC and chrF located in
the chromosomal DNA (Fig. 3) of which chrB and chrA genes were
essential for establishment of high resistance in chromium-
sensitive bacteria. The genetic studies also showed that ruvB
gene of O. tritici is related to chromate resistance (Morais et al.,
2011). In yet another bacterium, Ralstonia metallidurans (Fig. 4),
the chromate resistance determinant chr2 (comprising genes
chrB2, chrA2 and chrF2) was reported to be present on the chro-
mosome (Jhunke et al., 2002). Apart from chromosome, bacterial
plasmids also contain genes which are resistance to many toxic
metals and metalloids (Gadd, 2010) and encode systems devoted
to protect bacterial cells from the oxidative stress caused by
chromate. Resistance systems related to plasmid genes are
encoded membrane transporters, which mediate the efflux of
chromate ions across the cytoplasmic membrane. This mecha-
nism has been studied in detail with P. aeruginosa. In this bac-
terium, the chromate transporter chrA functions as a
 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399 387

Fig. 4. AeC. Genes involved in chromate resistance in Ralstonia metallidurans CH34. AeC. Regions on megaplasmid pMOL28 (A) or the bacterial chromosome (B, C) involved in
chromate resistance.

chemiosmotic pump that extrudes chromate using the proton
motive force (Alvarez et al., 1999). Genetic analyses of chromate
resistant P. aeruginosa (Cervantes et al., 1990) and A. eutrophus
(Nies et al., 1990) have shown that reduced accumulation of
Cr(VI) by bacteria is plasmid-mediated. Genes for a hydrophobic
polypeptide, chrA, were identified in chromate resistance plas-
mids of both P. aeruginosa and A. eutrophus. The polypeptide chrA
is postulated to be responsible for the outward membrane
translocation of chromate anions (Cervantes and Silver, 1992). In
another instance, plasmid pMOL28 from R. metallidurans, was
found to encode the chrA chromate efflux pump, in addition to
chrC and chrE proteins that seem to be involved in chromate
resistance (Jhunke et al., 2002). The chromate resistant strain of
Pseudomonas fluorescens (LB300), isolated from the Hudson River,
was found to be 40e200 fold more resistant to chromate than
were either Pseudomonas taken from other environments or
other bacteria taken from the same environment. Plasmids
mediated chromate resistance has also been reported in the cases

Fig. 5. Comparison of genetic determinants of chromate resistance and chromate reduction between (a) Bacillus cereus SJ1 and (b) Bacillus thuringiensis strain 97-27 (He et al., 2010).
388 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399

Fig. 6. Pathways of anaerobic chromate reduction by bacteria.

of Pseudomonas putida (Friello and Chakrabarty, 1980) and
Streptococcus lactis (Efstathiou and McKay, 1977).
In some cases, the sequence elements carried on the plasmid
DNA are transposable across species. Branco et al. (2008) identified
a transposon based chrBACF operon as the key determinant for high
chromate tolerance of Ochrobactrum tritici strain 5bvl1 (Fig. 3). The
activation of this operon was highly selective and provided resis-
tance principally through efficient extrusion of chromate. For
instance, the plasmid pLHB1 from P. fluorescens LB300 carrying the
Cr(VI) reducing genes could be transferred to E. coli to produce
E. coli ATCC 33456 by conjugation (Shen and Wang, 1993). Zou et al.
(2013) reported that the gene encoding thioredoxin oxidoreductase
located on a mre operon in Desulfovibrio desulphuricans G20, was
also involved in reduction of Cr(VI).
The regulation of Cr(VI) reduction in an operon structure was
also observed in Bacillus cereus SJ1 and Bacillus thuringiensis strain
97-27. The Cr(VI) reduction genes were found to be upward regu-
lated by the promoter chrI which in turn regulated the Cr(VI)
resistance gene chrA1 and arsenic resistance genes arsR and arsB
(He et al., 2010) (Fig. 5). In B. cereus SJ1 putative chromate transport
operon, chrlA1 and two additional chrA genes encoding putative
chromate transporters imparted resistance to chromate. ChrA1 and

Fig. 7. Mechanisms of enzymatic Cr(VI) reduction under aerobic (left) and anaerobic (right) conditions. (MR: membrane bound chromate reductase; SR: soluble chromate
reductase). Dotted lines depicted the exceptional reduction cases.
 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
chrl were found to be inducible genes. Azoreductase genes azoR and
four nitroreductase genes nitR were also found to be involved in
chromate reduction of B. cereus SJ1 species (He et al., 2010).
5. Mechanisms of enzymatic Cr(VI) reduction
Microorganisms can remove a number of metallic and metalloid
species from the environment or waste streams by reducing them
to a lower oxidation state (Lovely, 1995). Infact most of the toxic
compounds, especially heavy metals, preferably follow the reduc-
tion pathway instead of oxidative one by native microbes (with a
few exceptions) as their reduced forms are comparatively less toxic.
In case of metals, the higher oxidation states are always more toxic
(10e100 times) than lower oxidation state. The microbes have the
ability to reduce almost all metallic/metalloid species in higher
oxidation state and the microbial reduction of Cr(VI) to Cr(III) forms
the most widely studied example of metal bioremediation.
A wide diversity of microorganisms is known to have evolved
biochemical pathways for reducing toxic compounds by both aer-
obic and anaerobic processes. Aerobic bioremediation involves
microbial reactions that require oxygen to go forward. The bacteria
use a carbon substrate as the electron donor and oxygen as the
electron acceptor. Aerobic reduction is a cometabolic process where
microbes do not gain energy or carbon from degrading a contam-
inant, instead, the contaminant is degraded via a side reaction (EPA,
2006). On the other hand, anaerobic bioremediation involves mi-
crobial reactions occurring in the absence of oxygen. It encom-
passes many processes, including fermentation, reductive
dechlorination, methanogenesis, and sulphate- and nitrate
reducing conditions depending on the contaminant. In anaerobic
metabolism, sulphate, nitrate, carbon dioxide, oxidized materials,
or organic compounds may replace oxygen as the electron acceptor.
Many of the microorganisms that catalyse redox reactions use the
metals or metalloids as terminal electron acceptors in anaerobic
respiration. Such microorganisms, known as dissimilatory metal-
reducing bacteria, are diverse both phylogenetically (Lonergan
et al., 1996) and physiologically (Lovley et al., 1997). Among the
different groups microorganisms, Cr(VI) reduction was investigated
in large number of bacterial species. The mechanisms through
which bacterial strains reduce Cr(VI) to Cr(III) are variable and
species specific dependant (McLean et al., 2000).
Microbial Cr(VI) reduction takes place in two different pro-
cesses. Cr(VI) reduction is found to be cometabolic (not partici-
pating in energy conservation) in aerobic condition where as it is
predominantly dissimilatory under anaerobic conditions (Ishibashi
et al., 1990).
5.1. Anaerobic Cr(VI) reduction
Anaerobic reduction by bacteria is usually associated with
membrane bound reductases like flavin reductases, cytochromes
and hydrogenases that can be part of electron transport system and
use chromate as the terminal electron acceptor. Microbial Cr(VI)
reduction was first reported by Romaneko and Korenkov (1975)
using an anaerobic bacterium, Pseudomonas dechromaticans, iso-
lated from sewage sludge. This Gram-negative, motile, facultative
anaerobe bacterium reported to reduce Cr(VI) under anaerobic
conditions using Cr(VI) as a terminal electron acceptor. Later a
facultative anaerobic bacterium, Enterobacter cloacae, isolated from
industrial wastewater, was also found to use chromate/dichromate
as terminal electron acceptor during the Cr(VI) reduction (Wang
et al., 1989, 1991) in the periplasmic space by membrane bound
hydrogenase or reduced cytochrome (Fig. 6).
Membrane-associated chromate reductase activity was first
observed in E. cloacae HO1 where the insoluble form of reduced
389
chromate precipitates was seen on the cell surface (Wang et al.,
1989; Fuji et al., 1990). In this case, the Cr(VI) reduction was sub-
stantially inhibited by the presence of oxygen. Studies with
E. cloacae HO1 have implicated the respiratory chain in the transfer
of reducing equivalents to Cr(VI) through cytochrome c (Fig. 7). The
chromate reductase activity of Shewanella putrefaciens MR-1 was
found to be associated with the cytoplasmic membrane of anaer-
obically grown cells (Myers et al., 2000) where formate and NADH
served as electron donors for the reductase. In case of P. putida,
unlike S. putrefaciens, NADPH served as an electron donor for the
chromate reductase (Park et al., 2000). Anaerobic Cr(VI) reduction
involving membrane-associated reductase has also been reported
in some chromate-reducing bacteria which utilized H2 as electron
donor and Cr(VI) as an electron acceptor in the electron transport
chain (Lovley and Phillips, 1994; Quilntana et al., 2001). There are,
however, exceptions in which soluble enzymes have been found to
mediate the process of Cr(VI) reduction under anaerobic conditions
(Barrera-Díaz et al., 2012). For instance, Cr(VI) reduction by soluble
cytochrome c3, isolated from anaerobic bacterium Desulfovibrio
vulgaris, has been reported (Lovley and Phillips, 1994).
In Shewanella, Enterobacter and sulphate reducing bacteria the
terminal electron acceptors like nitrate and sulphate are found to
be replaced by chromate (Chardin et al., 2003; Myers et al., 2000).
Besides these, a number of inorganic species including O 2
2 , SO4 ,
NO 2 , NO
3 , Fe(III) and Cr(VI) can also act as terminal electron ac-
ceptors during bacterial respiration. In the absence of oxygen,
Cr(VI) acts as a terminal electron acceptor in the respiratory chain
for a large array of electron donors, including carbohydrates, pro-
teins, fats, hydrogen, NAD(P)H and endogenous electron reserves
(Cheung and Gu, 2007).
The overall bio-reduction of Cr(VI) and precipitation of Cr(III) is
illustrated in Eqs. (1) and (2). Under anaerobic conditions with
glucose as an electron donor, microbial Cr(VI) reduction is related
to Eq. (3) (Singh et al., 2011).
þ 
CrO2 3þ
4 (aq) þ 8H (aq) þ 3e / Cr (aq) þ 4H2O (1)
Cr3þ(aq) þ 4H2O / Cr(OH)3(s) þ 3Hþ(aq) þ H2O (2)

C6H12O6 þ 8CrO2
4 (aq) þ 14H2O / 8Cr(OH)3(s) þ 10OH (aq)
þ 6HCO(aq) (3)
Sulphate and iron reducing bacteria (SRB and IRB) are impor-
tant members of anaerobic microbial communities with economic,
environmental and biotechnological interest. Cr(VI) reduction by
biogenic iron(II) and sulphides generated by IRB and SRB is ~100
times faster than that by CRB alone. SRB produce H2S, which serve
as a Cr(VI) reductant. Anaerobic bacteria mainly use Cr(VI) as ter-
minal electron acceptor or reduce Cr(VI) in the periplasmic space
by hydrogenase or reduced cytochrome. For example sulphate
reducing bacteria like D. vulgaris and Desulfovibrio fructosovorans,
the involvement of cyt c3 and NieFe dehydrogenase in Cr(VI)
reduction has been reported by Chardin et al. (2003) and Michel
et al. (2001). Chardin et al. (2003) have reported that among
[Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the
genera Desulfovibrio and Desulfomicrobium, [Fe] hydrogenase from
D. vulgaris strain Hildenborough showed reduction of Cr(VI) with
highest rate. In a previous study with SBR, Desulfotomaculum
reducens MI-1, Cr(VI) acted as the sole electron acceptor for its
reduction (Tebo and Obraztsova, 1998). Some other anaerobes
capable of reducing Cr(VI) to Cr(III) are Microbacterium sp. MP30
(Pattanapipitpaisal et al., 2001), Geobacter metallireducens (Lovley
et al., 1993), Pantoea agglomerans SP1 (Francis et al., 2000). Agro-
bacterium radiobacter EPS-916 (Llovera et al., 1993), etc. Even
bacteria tolerant to radiations like Deinococcus radiodurans R1 are
390 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
quite efficient in reducing Cr(VI) anaerobically (Fredrickson et al.,
2000).
Microorganisms are also capable of indirect Cr(VI) reduction by
a biotic-abiotic coupling. A wide range of bacteria are known to
couple the oxidation of organic compounds and H2 for reduction of
Fe(III) and SO2
4 to Fe(II) and H2S, respectively under oxygen stress
conditions (Nevin and Lovley, 2002). Reduction of Cr(VI) with
bacterially produced H2S followed by precipitation of reduced
Cr(III) is an important mechanism in sulphate-rich soil environ-
ments when anaerobic conditions prevail (Losi et al., 1994; Pettine
et al., 1994). The H2S produced by sulphate respiring bacteria in
anaerobic systems diffuses out into the medium and reduces Cr(VI)
(Kamaludeen et al., 2003).
5.2. Aerobic Cr(VI) reduction
Aerobic Cr(VI) reduction is generally associated with soluble
proteins and requires NAD(P)H as an electron donor (Shen and
Wang, 1993) (Fig. 7). These soluble proteins are localized as cyto-
solic proteins as demonstrated by Puzon et al. (2002) in E. coli.
Apart from this, the Cr(VI)-reducing activity in aerobes like Pseu-
domonas ambigua, P. putida, E. coli (Shen and Wang, 1993) and Ba-
cillus coagulans (Philip et al., 1998) have been found to be present in
the soluble fraction of the cells (Ishibashi et al., 1990). Garbisu et al.
(1998) have reported that a Gram-positive aerobe, Bacillus subtilis
reduced hexavalent chromium by using cell free extracts of the
bacterium. In case of Gram-negative bacteria, several Pseudomonas
have also been investigated to assess their Cr(VI) reduction abilities.
The ability of an aerobic bacterium such as P. aeruginosa to reduce
Cr(VI) has been extensively studied (Bopp et al., 1983; Ohtake et al.,
1987). Other aerobes involved in Cr(VI) reduction are A. eutrophus,
Bacillus sp., E. coli sp ATCC 33456, Shewanella alga BrY-MT, etc.
(Guha et al., 2001; Camargo et al., 2003). Besides, a number of
bacteria belonging to genera Bacillus, Ochrobactrum, Pseudomonas,
Escherichia and Paracoccus have also been reported for their Cr(VI)
reduction abilities under aerobic conditions (Hussein et al., 2005;
Sultan and Hasnain, 2005; Das et al., 2014).
The NADH was also the preferred electron donor for the
reduction of chromate by the soluble enzyme present in cytoplasm
of Pseudomonas sp. CRB5 (McLean and Beveridge, 2001). Previous
studies also showed that NADH/NADPH served as electron donors
for Cr(VI) reduction by the soluble enzymes present in P. ambigua
(Suzuki et al., 1992) and P. putida (Ishibashi et al., 1990). Another
Gram-positive aerobe belonging to Vigribacillus sp., isolated from
saline environment, with the ability to reduce Cr(VI) at higher
concentrations in salt medium has been reported recently by
Mishra et al. (2012). As exceptions, P. maltophilia O-2 and Bacillus
megaterium TKW3 were found to utilize membrane-associated re-
ductases for Cr(VI) reduction, in spite of being aerobes (Cheung and
Gu, 2007).
In presence of oxygen, the enzymes from aerobes responsible
for reducing Cr(VI) to Cr(III) require NAD(P)H. Two reaction steps
have been suggested to be involved in the reduction reactions; first
Cr(VI) accepts one electron from one molecule of NADH to gener-
ates Cr(V) as an intermediate (Eq. (4)) and then Cr(V) accepts two
electrons to form Cr(III) (Eq. (5)) (Singh et al., 2011).
Cr6þ þ e / Cr5þ (4)
Cr5þ þ 2e / Cr3þ (5)
The ability to reduce Cr(VI) by aerobic bacteria such as
P. aeruginosa (Bopp et al., 1983; Ohtake et al., 1987) has been
extensively studied and found the resistance to Cr(VI) is propor-
tional to the increased efflux of the heavy metal by cell membrane.
Similar behaviour was also observed with P. synxantha (McLean
et al., 2000). In contrast, several species like P. fluorescens showed
that the Cr(VI) resistance is not depended on the capacity of their
reduction of heavy metal. In Pseudomonas sp. CRB5, NADH was the
preferred electron donor for the reduction of chromate by the
soluble enzyme contained in cytoplasm (McLean and Beveridge,
2001).
Soluble enzyme that can catalyse the reduction of Cr(VI) to
Cr(III), can detoxify its environment. Once reduced, Cr(III) usually
binds to the electronegatively charged functional groups on the
bacterial cell surface, which serve as nucleation sites for further
precipitation. Cr(III) also undergoes complexation with cell enve-
lope and capsule exopolymer which in turn inhibits the metals
from entering the cytoplasm. By employing this process, chromate
can be effectively detoxified and removed from Cr(VI) enriched
medium (McLean and Beveridge, 2001).
5.3. Both aerobic and anaerobic reduction
Some bacteria are capable of reducing chromate under both
aerobic and anaerobic conditions, for example, P. fluorescens LB300,
isolated from chromium-contaminated sediments, was capable of
reducing Cr(VI) to Cr(III) both aerobically and anaerobically (Bopp
and Ehrlich, 1988). Under anaerobic condition P. fluorescens LB300
utilized acetate as an electron donor for chromate reduction while
the microorganism used a variety of electron donors for chromate
reduction under aerobic conditions (Bopp and Ehrlich, 1988). A
number of other chromate-resistant bacteria including Achromo-
bacter sp. (Ma et al., 2007), E. coli, P. ambigua (McLean and
Beveridge, 2001), P. putida (Barak et al., 2006), E. cloacae (Wang
et al., 1989), Providencia sp. (Thacker et al., 2006), Brucella sp.
(Thacker et al., 2007) and Bacillus sp. (Liu et al., 2006) have also
shown the Cr(VI) reduction ability under both aerobic and anaer-
obic conditions although their rate of reduction varied widely. For
instance, P. ambigua G-1 and P. putida PRS2000 reduced Cr(VI)
under both these conditions with higher reduction rates whereas
faster reduction rate of Cr(VI) under aerobic conditions than
anaerobic conditions has been reported in the case of E. coli ATCC
33456 (Shen and Wang, 1993).
6. Biological Cr(VI) reduction pathways
Several components of protoplasm of bacterial cells such as
NADH (NAD(P)H in some species), flavoproteins and other hem-
eproteins (Ackerley et al., 2004a) readily reduce Cr(VI) to Cr(III).
Bacterial reduction of Cr(VI) can occur directly through enzymatic
activity (Lovely and Coates, 1997; Losi et al., 1994) or indirectly
through non-enzymatic pathways by producing compounds such
as glutathione, cystein etc. that can reduce Cr(VI) (Fendorf and Li,
1996) (Fig. 8).
6.1. Non-enzymatic Cr(VI) reduction
In non-enzymatic process Cr(VI) reduce to Cr(III) in presence of
different chemical compounds, produced in the bacterial metabolic
process. For instance, it is well known that Fe(II) and HS, the
anaerobic metabolic end products of iron and sulphate-reducing
bacteria, can reduce hexavalent chromium. The most powerful
non-enzymatic chromate reductants could be ascorbic acid, gluta-
thione (GSH), cysteine, hydrogen peroxide (H2O2) for microbial
cells, and ascorbate for higher organisms (Poljsak et al., 2010).
Reduction of Cr(VI) may also take place by chemical reactions
associated with compounds present in intra/extra cellular com-
pounds such as amino acids, nucleotides, sugars, vitamins, organic
acids or glutathione (Dhal et al., 2013).
 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399 391

Fig. 8. A schematic diagram of bacterial Cr(VI) reduction (direct and indirect).

6.1.1. Enzymatic Cr(VI) reduction enzymes are extracellular in nature. A couple of examples are
The enzymes from different organisms, ranging from bacteria to available in support of this observation. Priester et al. (2006) re-
mammals, take part in the reduction of hexavalent chromium. As ported that the chromate reductases originating in the cytoplasm
far as mammalian sources are concerned, the enzymes like alde- reduced Cr(VI) extracellularly in P. putida. Cheung and Gu (2007)
hyde oxidases, cytochrome p450 and Dt-diaphorase are involved in reported that extracellular enzymes (cytosolic proteins) are solu-
Cr(VI) reduction. However, microbial enzymes are most important ble chromate reductases such as flavin reductases, nitrate re-
for reduction of Cr(VI) from environmental point of view. ductases, flavin proteins and ferrireductases. Wang et al. (1991)
Patra et al. (2010) reported the role of several enzymes involved observed that bacteria with membrane bound reductases can also
in Cr(VI) reduction in bacteria. Several oxidoreductases (Fig. 9), reduce Cr(VI) to Cr(III) by extracellular processes, by using electron-
with otherwise different metabolic functions that have been re- shuttling compounds coupled to membrane reduction. Most of
ported to catalyse Cr(VI) reduction in bacteria, include nitro- these enzymes are produced inductively i.e., they are produced in
reductase (Kwak et al., 2003), iron reductase, quinone reductases
(Gonzalez et al., 2005), hydrogenases (Chardin et al., 2003), flavin
reductases (Ackerley et al., 2004a) as well as NAD(P)H dependant
reductases (Puzon et al., 2002). The type of bacterial chromate re-
ductases depends on the nature of bacteria carrying out the
reduction reaction, i.e., aerobic or anaerobic. Bacterial chromate
reductases are either localized in the membrane fraction (Wang
et al., 1991; Cheung et al., 2006) or in the cytosolic fractions
(Suzuki et al., 1992; Park et al., 2000; Bae et al., 2005) of the
chromate reducing bacteria. The enzymatic reduction of Cr(VI) in
bacteria is accomplished in different ways. Soluble reductases can
participate either in extracellular or intracellular reduction of Cr
(VI) (Elangovan et al., 2010; Das et al., 2014) whereas, membrane
bound reductase reduces by extracellular means (Wang et al., 1991).

6.1.2. Extracellular
In some cases, the Cr(VI) reducing enzymes, produced by bac-
terial cells, are exported into the media to reduce Cr(VI). These Fig. 9. Oxidoreductase enzymes found in bacteria that catalyse Cr(VI) reduction.
392 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
Table 1
Involvement of chromate reductases in one and two electron reduction mechanism.
Mechanism employed for chromate reduction
One electron Reduction of Cr(VI) to Cr(III) occurs via Cr(V) intermediate.
reducers A continuous shuttle between Cr(VI) and Cr(V) forms hap-
pens with Cr(V) transferring 1 e- to O2, generating ROS
(superoxide, O2-2 ) in a Fenton-like reaction (Shi and Dalal,
1994; Barak et al., 2006)
-
Two electron Transfer of 3 e to Cr(VI) results in its direct reduction to
reducers Cr(III).
1 e is transferred to O2, forming ROS (superoxide, O2 2 ).
No Cr(V) intermediate is involved hence no redox cycle
occurs.
presence of Cr(VI) in the solution and are therefore highly regulated
(Cheung and Gu, 2007). This regulation is controlled by regulatory
elements (e.g., starvation promoter) that allow massive synthesis
under the unfavourable in situ environment. Smith and Gadd
(2000) established the extracellular Cr(VI) reduction pathways in
sulphate reducing bacteria through a mass balance in which 90% of
the reduced Cr was detected in the supernatant.
6.1.3. Intracellular
In intracellular processes, Cr(VI) is reduced in the cytosol us-
ing cytoplasmic soluble reductase enzymes. These enzymes play
an intermediate role between associated biological electron do-
nors. The electron donors involved in an intracellular Cr(VI)
reduction are NADH and NADPH. Puzon et al. (2005) suggested
that Cr(VI) was reduced intra-cellularly in the cytoplasm by a
bacterial enzyme, using NADH as the reductant. Streptomyces
sp. MC1, isolated from sugar cane, has been shown to reduce
Cr(VI) intracellularly (Polti et al., 2011). Many bacteria are also
known to participate in the intracellular reduction of Cr(VI).
Some examples of Gram negative bacteria that reduce Cr(VI)
intracellularly are P. aeruginosa, Pseudomonas sp. CRB5, E. coli
ATCC 33456, Rhodobacter sphaeroides, Alcaligenes, Enterobacter
and P. fluorescens LB300. A Gram positive bacteria B. subtilis was
reported to carry out intracellular reduction of Cr(VI) by Ohtake
et al. (1990).
7. Explanation of one and two electron Cr(VI) reduction
mechanism
During Cr(VI) reduction, the bacterial cells are subjected to
oxidative stress due to simultaneous generation of ROS (Cheung
et al., 2006). The oxidative stress affects viability of cells and the
efficiency of Cr(VI) reduction. On the basis of the magnitude of
oxidative stress generated, the enzymes reducing hexavalent
chromium can be categorized as either one electron reducers or
two electron reducers (Table 1).
In one electron reduction, Cr(VI) gets reduced to form the highly
unstable Cr(V) intermediate. Cr(V) can get oxidized back to Cr(VI) in
a redox cycle, giving its electrons to molecular oxygen and thus,
generating a large amount of ROS. The one electron reducers being
flavin dependent enzymes follow the reaction as given below:
FMN (Ox.) þ e þ Hþ / FMNHþ (Sq.)
Flavin nucleotide (FMN) is a tight, sometimes covalently bound
and are a part of flavoproteins. The oxidized flavin nucleotide
(FMN(Ox.)) accepts one electron yeilding semiquinone (a stable
free radical) form (FMNH(Sq.)).
Some of the identified chromate reducing enzymes belonging
to one electron reducers are lipoyl dehydrogenase (LpDH) from
Clostridium kluyveri, cytochrome c, glutathione reductase and
Examples Effect on cells
- LpDH Highly detrimental to bacterial cells
- Cytochrome c due to more ROS generation.
- Glutathione reductase Chromate reducing efficiency of the
- Ferridoxin-NADP oxidoreductase bacteria declines.
- NfsA (E. coli)
- ChrR (Ps. putida) Much lesser ROS generated than
- YieF (E. coli) one electron reducer, hence less
toxic to bacterial cells
ferridoxin-NAD (Shi and Dalal, 1990; Ackerley et al., 2004b). The
physiological roles of these enzymes are to catalyse energetic or
biosynthetic reactions (Barak et al., 2006).
On the other hand, two electron reduction process of Cr(VI) to
Cr(III) proceeds without forming Cr(V) intermediate. As a result
much lesser amount of ROS is generated during this process than
that of one electron reduction. Two electron reducers being NAD(P)
dependent enzymes, are characterized by the transfer of hydride
ion (H, the equivalent of a proton and two electrons) in a reaction
as follows:
NADþ þ 2e þ 2Hþ / NADH þ Hþ
NADPþ þ 2e þ 2Hþ / NADPH þ Hþ
Some of the chromate reducing enzymes behaving as two
electron reducers include ChrR from P. putida, YieF and NfsA from
E. coli (Barak et al., 2006). The varying one and two electron
reducing ability of different enzymes have been experimentally
verified. Matin (2006) conducted an experiment to categorise two
chromate reducing enzymes viz., LpDH and YieF as either one or
two electron reducer. The in vitro study, using pure lipoyl dehy-
drogenase (LpDH) by ‘redox balance’ method, revealed the con-
sumption of only 24% of electrons for chromate reduction while
remaining 70% electrons for ROS generation. The results indicated
LpDH as the one electron reducer. In contrast, an identical test with
YieF showed the use of only 25% of electrons for ROS generation
while the remaining electrons for Cr(VI) reduction indicating
YieF as the two electron reducer. Ackerley et al. (2004b) proposed
a broad classification of enzymes carrying out Cr(VI) reduction
i.e. Class I or Class II reductases enzymes based on sequence
homologies.
7.1. Class I chromate reductase
Two of the most commonly studied Class I enzymes include
ChrR and YieF (Ackerley et al., 2004b). YieF dimer brings this Cr (VI)
reduction in one step without redox cycling, so that ROS generation
is minimal where as the ChrR dimer appears to reduce chromate by
a combination of two and one electron reduction steps, generating
more ROS than YieF. The transfer of two electrons from the enzyme
can either occur simultaneously (“tight”; as with YieF) or non-
simultaneously (“semi-tight”; as with ChrR).
YieF, a dimeric flavoprotein, reduces chromate to Cr(III), isolated
from E. coli (Park et al., 2002; Ackerley et al., 2004b). The YieF
enzyme is unique as it direct reduces of Cr(VI) to Cr(III) through a
four-electron transfer, in which three electrons are consumed in
reducing Cr(VI) and fourth electron is transferred to oxygen. Since
the quantity of ROS generated by YieF in Cr(VI) reduction is mini-
mal, it is regarded as a more effective reductase than ChrR for Cr(VI)
reduction (Park et al., 2002). In contrast to ChrR, YieF did not show
 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399 393

Table 2
Some Cr(VI)-reducing bacteria, along with the type of enzymes that they make use of to reduce Cr(VI) to Cr(III).

Sl No. Enzyme Type of enzyme Class/type Organism Mol wt (kDa) Structure Reference

1 ChrR Quinone reductase I E. coli Eswaramoorthy et al., 2012

2 Gh-ChrR Chromate reductase I Gluconoacetobacter hansenii Tetramer Jin et al., 2012
3 ChrR Flavoenzyme I Ps. Putida 50 Dimer Park et al., 2000
4 ChrA Flavoprotein family of reductases I Ps. aeruginosa, Cupriavidus Diaz-Perez et al., 2007
metallidurans (plasmid)
5 Chr NADH-dependent I R. sphaeroides 42 Monomer Nepple et al., 2000
6 YieF FMN or NAD(P)H dependent I E. coli 50 Dimer Ackerley et al., 2004b
chromate reductase
7 OYE enzyme Flavin oxidoreductase I S. carlsbergensis 45 Monomer Saito et al., 1991
8 Frp Flavin reductase P I Vibrio harveyi Zenno et al., 1998
9 NemA Chromate reductase I E. coli Robins et al., 2013
10 AzoR Azoreductase I E. coli Robins et al., 2013
11 Chromate NAD(P)H-dependent I Bacillus sp RE Elangovan et al., 2006
reductase
12 NfsA Nitroreductase (flavoprotein) II E. coli 50 Dimer Ackerley et al., 2004b
13 NfsB Nitroreductase II Vibrio harveyi 50 Monomer Kwak et al., 2003
14 _ NADPH dependent Thermus scotoductus 72 Dimer Opperman et al., 2008
15 YcnD FMN reductase II B.subtilis Morokutti et al., 2005
16 Suzuki enzyme NAD(P)H dep chromate reductase II Ps. Ambigua 65 Dimer Suzuki et al., 1992
17 EcdA Soluble chromate reductase II Ps. Putida Park et al., 2000
18 YdgL Nitroreductase Bacillus subtilis Minton et al., 2004 [patent]
19 KefF Nitroreductase II E.coli Prosser et al., 2010
20 chrBAC Efflux transporter Shewanella strain ANA-3 Aguilar-Barajas et al., 2008
21 25 kDa protein Cell free extract (inducible) Bacillus sp JDM-2-1 Zahoor and Rehman, 2009
22 FRase I FMN reductase II Vibrio (or Photobacterium) fischeri Zenno et al., 1994

semiquinone flavoprotein generation during chromate reduction
and consumed only some 25% of NADH electrons to molecular
oxygen in ROS generation. The ChrR and YieF homologs were
shown to contain the characteristic signature of the NADH-dh2
family of protein, which consists of bacterial and eukaryotic NAD(P)
H oxidoreductases (Li and Krumholz, 2007). He et al. (2010)
introduced another chromate reductase namely ChrA protein, iso-
lated from B. cereus SJ1 and B. thuringiensis strain, which belong to
class I family.
N-ethylmaleimide reductase (NemA), isolated from E. coli, is a
member of the “old yellow enzyme” (Class I) family of flavopro-
teins and catalyses the reduction of N-ethylmaleimide (NEM) to
N-ethylsuccinimide (Miura et al., 1997). NemA is able to accept a
variety of substrates, including chromate (Robins et al., 2013) and
catalyses chromate reduction through the addition of one or two
electrons from the cofactors NADH/or NADPH (Roldan et al.,
2008).
7.2. Class II chromate reductase
The Class II charomate reductases, which possess nitroreductase
activity bear no homology to the class I enzyme but are homolo-
gous to chromate reductase purified from P. ambigua (Park et al.,
2002). Two other members of the class II family, namely NfsA
protein of E. coli and the ChfN protein of B. subtilis (Park et al., 2002).
NsfA reduces nitrocompounds and quinone by an obligatory two
electron transfer (Zenno et al., 1996; Ackerley et al., 2004b). Thus
NfsA, like ChrR is semitight chromate reducer (Ackerley et al.,
2004b). The Class II enzymes reduce quinones and nitro-
compounds effectively, but vary in their ability to transform chro-
mate (Park et al., 2002). The NfsA protein has received wide
attention because of its ability to detoxify nitrocompounds. This
protein possesses therapeutic properties and ability to activate
prodrugs used in cancer chemotherapy (Carroll et al., 2002). Park
et al. (2002) reported another enzyme of Class II family ChfN pro-
tein of B. subtilis, in electrophoretically pure state, to possess
chromate reductase activity (accession numbers 3225092). Kwak
et al. (2003) reported that the nitroreductases are efficient in
chromate reduction.
8. Identification of Cr(VI) reductases from bacteria
A number of researchers have isolated and identified chromate
reductase enzymes (Table 2) from different bacterial sources. The
chromate reductase that play a role in reducing Cr(VI) belong to
several classes, viz. quinone reductases (in E. coli), nitroreductases (in
Vibrio sp.), NADPH-dependent enzymes (in Bacillus sp.), etc. ChrR, a
quinone reductase enzyme belongs to the flavodoxin superfamily
and isolated from E. coli, able to reduce Cr(VI) (Eswaramoorthy et al.,
2012). Jin et al. (2012) reported that a putative chromate reductase
viz. Gh-ChrR, isolated from the bacterium Gluconoacetobacter han-
senii, reduces Cr(VI) to Cr(III) in presence of NAD(P)H. ChrA,
belonging to NADPH dependent flavoprotein family of reductase and
isolated from P. aeruginosa/Cupriavidus metallidurans (formerly
Alcaligenes eutrophus), was also found responsible for resistance and
reduction of Cr(VI) (Ramirez-Diaz et al., 2008). ChrA is transporter
enzyme responsible for chromate resistance trough chromate efflux
mechanism. A soluble oxidoreductase (NemA), isolated from E. coli,
can able to reduce Cr(VI) by using NADH as a cofactor (Robins et al.,
2013). A NADH-dependent oxidoreductase (YcnD), isolated from a
Gramepositive bacterium B. subtilis, has been reported to reduce
chromate along with nitroorganic compounds and series of azo dyes
(Morokutti et al., 2005). Another NAD(P)H dependent chromate
reductase purified from P. putida viz. Suzuki enzyme with molecular
weight 65,000 Da efficiently reduced Cr(VI) at relatively higher
temperature and pH (Suzuki et al., 1992). A soluble chromate
reductase EcdA, isolated from P. putida by Park et al. (2000) was also
found to reduce chromate at higher temperature and pH. Other
chromate reductase enzymes having chromate reduction ability
include OYE enzyme from Thermus scotoductus (Saito et al.,1991), Frp
flavin reductase from E. coli (Zenno et al., 1998), FerB from Paracoccus
denitrificans (Sedlacek and Kucera, 2010) and Azo reductase from
Bacillus sp. OY1-2 (Maier et al., 2004).
9. Progress in Cr(VI) reduction by free and immobilized
enzymes
Elangovan et al. (2010) studied the Cr(VI) reduction by cell free
extract (CFE) from Arthrobacter rhombi-RE in presence of NADH.
394 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
The specific Cr(VI) reductase activity of the CFE of this strain at
30  C and a pH of 6.0 was found to be 0.46 nM Cr(VI) reduction/mg
protein/min. Addition of NADH enhanced the chromate reductase
activity to 0.77 nM Cr(VI) reduced/mg protein/min. In this case, the
supernatant obtained after harvesting the cells (extracellular en-
zymes) showed less chromium reductase activity (0.13 nM Cr(VI)
reduction/mg protein/min) as compared to CFE (Elangovan et al.,
2010). This clearly showed that the Cr(VI) reduction was associ-
ated with the soluble fraction of the cells and not with extracellular
enzymes, which corroborates the earlier observations with E. coli
(Shen and Wang, 1993), Desulfovibrio vulgaris (Lovley and Phillips,
1994), P. putida (Ishibashi et al., 1990) and Bacillus QC1-2 (Campos
et al., 1995). Cr(VI) reductase activity of the bacteria were studied
by measuring the decrease in Cr(VI) per unit of enzyme per minute
(Elangovan et al., 2010). In a recent study, Rath et al. (2014) reported
high extracellular chromate reductases activity (3.67 Uml1) under
optimized set of conditions from Bacillus amyloliquefaciens isolated
from chromite mine soil of Sukinda, India. The chromate reductase
activity of CFE of another strain, Arthrobacter sp. SUK 1201 has been
studied by Dey and Paul (2013) which found to be a constituent in
nature and reduced Cr(VI) with decreasing efficiency at increased
Cr(VI) concentration. Previously, the in vitro reduction of Cr(VI) was
studied using cell-free extracts from a Cr(VI) reducing Bacillus fir-
mus KUCr1 strain. Chromium reductases activity of this bacterium
was found to be constitutive and its activity was observed both in
soluble cell fractions and membrane cell fractions (Sau et al., 2010).
Experiments on Cr(VI) reduction by cell free extract revealed
that additional electron donors like NADH and NAD(P)H have
accelerated the Cr(VI) reductase activity of CFE of Bacillus sphaericus
AND 303 (Pal and Paul, 2004). Fractionation and assay of CFE of
Bacillus ES 29 revealed that a soluble NADH dependent enzyme was
responsible for the reduction of Cr(VI) (Camargo et al., 2003). Philip
et al. (1998) reported that the residual organic matter present in the
CFE of Bacillus coagulans might have acted as the electron donor for
Cr(VI) reduction. A soluble Cr(VI) reductase present in E. coli ATCC
33456 was responsible for Cr(VI) reduction under both aerobic and
anaerobic conditions (Shen and Wang, 1993). The enzyme was
purified and characterized from the cytoplasm from this bacterium
which is significantly inhibited by the presence of other heavy
metal ions like Ag2þ, Cd2þ, Hg2þ and Zn2þ (Bae et al., 2005).
Apart from use of free enzymes, immobilized enzymes were also
used in bioremediation programme. The immobilized enzymes and
proteins have been extensively used in antibiotic production, drug
metabolism, food industry, biodiesel production and bioremedia-
tion (Khan and Alzohairy, 2010). It has been also reported that the
immobilized enzymes are much better suited for ex situ bioreme-
diation (Russell et al., 2003). Immobilized enzymes have long life-
spans, much easier to handle and separate from the products
(Kandelbauer et al., 2004). Thus, enzyme immobilization facilitates
their reuse in bioremediation. It has also been reported that
immobilization makes the enzyme more resistant to temperature,
pH and substrate concentration swings giving it a longer lifetime
and higher productivity per active unit (Russell et al., 2003;
Gianfreda and Rao, 2004; Kandelbauer et al., 2004).
Several natural and synthetic support materials such as agar,
alginate, carrageenan, cellulose, and its derivatives like collagen,
gelatin, polyacrylamide, polyester, polystyrene, and polyurethane
have been used for cell immobilization (Munjal and Sawhney,
2002). With the advancement in enzyme immobilization technol-
ogy, it is anticipated that the application of immobilized Cr(VI)
reductase could be a promising approach for bioremediation of
Cr(VI)-contaminated wastewater over a wide range of environ-
mental conditions. The effect of different matrices on Cr(VI)
reduction by CFE-immobilized Arthrobacter rhombi-RE was inves-
tigated by Elangovan et al. (2006). They observed that the activity of
enzyme immobilized on alginate beads and Zeolite resin was
higher than gelatin gel, polyacrylamide gel, and PVA. Applications
of immobilized enzymes in chromium(VI) reduction have been
sparsely studied. However, there is a great scope of application of
enzyme immobilization technology for Cr(VI) bioremediation pro-
gramme which needs to be explored.
10. Techniques used for characterization of Cr(VI) reduced
product
Hexavalent chromium is a strong oxidant and its chemical
reduction by biomolecules results Cr(III) usually via Cr(V)/Cr(IV)
intermediates which precipitates as hydroxide, Cr(OH)3, at pH  7.0
(Garcia et al., 2006). In biological systems such as microbial
reduction, the reduction of Cr(VI) to Cr(III) is rather complex and
the nature of the reduced product is mostly dependent on the type
of bacterial strains, reaction parameters (pH, media composition,
presence of electron donor or complexing agents etc.) and reaction
environment (aerobic/anaerobic). Once reduced by microbial pro-
cesses, the Cr(III) may either bind to the functional groups of bac-
terial cell or form stable nucleation sites for further precipitation of
Cr(III) mineral phases (McLean et al., 2000). It is well known that
bacteria are excellent nucleation sites for fine-grained mineral
formation due to their high surface area-to-volume ratio and the
presence of electronegative functional group such as carboxyl,
phosphoryl and hydroxyl groups. A proper knowledge of final
reduced product and identification of reduction intermediate is,
therefore, essential to develop bioremediation process for practical
use. With the advent of sophistication in analytical techniques, a
wide variety of spectroscopic, microscopic, X-ray diffraction etc.
methods have been used to identify the bioreduced Cr(III) species
during last two decades and the important observations are briefly
described in this section.
It is widely believed that reduction to Cr(III) is the final pathway
step in the microbial Cr(VI) reduction chain because bacterial cells
become encrusted with Cr-rich precipitates (Fude et al., 1994). A
TEM/EDS study of Cr(VI) reduced species associated with bacterial
cell of a Gram-negative bacterium, resembling P. synxantha, showed
that the bacterial cell surfaces were uniformly covered with bound
Cr(III) (with surface functional groups) and amorphous precipitates
(McLean et al., 2000). In a study conducted later, McLean and
Beveridge (2001) reported the formation of fine-grained Cr(III)
precipitates, mostly chromium hydroxide mineral phases such as
Cr(OH)3 by Pseudomonas after Cr(VI) reduction. This result was also
supported by TEM-EDX and SAED analyses which revealed that the
surfaces of the bacterial cells were uniformly stained with bound
Cr(III) and amorphous precipitates. SEM-EDAX study also revealed
the extracellular reduction of Cr(VI) by Leucobacter sp. encrusted
with amorphous Cr(OH)3 on cell surface (Zhu et al., 2008).
In a recent study (Srivastava and Thakur, 2007) on reduction of
biosorbed chromate to Cr(III) by Acinetobactor sp. and its micro-
scopic analyses by SEM-EDS and TEM revealed Cr(III) precipitation
with hydroxyl or carboxyl group in side the bacterial cell (intra-
cellular precipitation). Intracellular reduction of Cr(VI) to Cr(III) has
also been reported in the case of bacterial species A. haemolyticus
(Pei et al., 2009). Analyses of reduced species associated with
bacterial cell by FT-IR, FESEM-EDX and TEM indicated the forma-
tion of intracellular Cr(III) in complex form with carboxyl, hydroxyl
and amide groups of bacteria. Absence any precipitate on the cell
wall region and reductase test further supported the intracellular
reduction of Cr(VI) transported into the cell. Cheung et al. (2006)
also reported aggregation of reduced Cr(III) deposited along the
intracellular membrane region for Bacillus megaterium TKW3
incubated with Cr(VI).
 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
On the other hand, the studies on Cr(VI) detoxification by a
sulphate reducing bacteria, Desulfovibrio vulgaris, revealed the
accumulation of Cr(III) precipitate on the cell surface or at a dis-
tance from cell (Goulhen et al., 2006). Identification of mineral
phases in these precipitate by high angle dark filed (HAADF)
coupled with electron energy loss spectra (EELS) indicated the
aggregate of nanometric basic unit which are amorphous in nature.
EELS fine structure analysis further showed the presence of Ca, Cr
and P in the mineral precipitate on the bacterial cell or distant from
the cell with tentative composition Ca0.34Cr0.7735(PO4).
Precipitation of reduced Cr(III) on the outer surface of Shewa-
nella oneidensis only has also been suggested by Daulton et al.
(2002) as there was no electron-dense precipitate in the inner re-
gion of the bacterial cell. SAED image showed that the grains were
predominantly amorphous due to high degree of hydration. Bac-
terial cells having thickness of 30e40 nm with distinct cell line
indicated saturation of cell boundary with absorbed element of
heavy mass i.e., Cr.
In bacterial Cr(VI) reduction it is commonly seen the reduced
Cr(III) species is present both in soluble and insoluble forms. Often a
significant amount of reduced Cr(III) is present in the supernatant
in soluble form. A basic understating regarding immobilisation
reduced Cr(III) in different bacterial species and its nature inter-
action with the bacterial cell is, therefore, essential for bioremedi-
ation of Cr(VI) in real system. In view of above, Li et al. (2008) have
made several studies on Cr(VI) reduction with high Cr(VI) tolerant
bacteria isolated from Cr-contaminated site, Ochrobactrum
anthropi, in varying culture media and in presence aminoacids as
coordinating ligands. XPS/EPR, FT-IR and SEM/AFM investigations
showed the accumulation of most of reduced Cr(III) on the cell
surface indicating the chemical interaction between functional
groups (eCOOH and eNH2) on the cell surface and Cr(III) (Cheng
et al., 2009). In addition, the precipitation of intracellular Cr(III)
was also evident from thin-section TEM analysis of bacterial cell.
The immobilization efficiency of Cr(III) was found media dependent
showing up to 95% immobilization by the bacteria in TriseHCl
buffer medium as against only 70% in LB medium. In a later study
(Cheng et al., 2010), the X-ray absorption fine structure (XAFS)
analysis on the cell debris provide further evidence of coordination
of reduced Cr(III) on the surfaces via carboxyl- and amido-
functional groups. However, in presence low molecular weight
aminoacids or their derivatives, most of the immobilized Cr(III) on
cell surface leached into supernatant indicating their stronger co-
ordination ability than the cell surface functional groups. Based on
their observation, a practical strategy has also been proposed for
effective immobilization of reduced Cr(III) species and possible
application in real system.
In a recent study of Cr(VI) reduction by B. amyloliquefaciens in LB
medium from our group also showed immobilization of more than
60% of reduced Cr(III) on bacterial cell which was very similar to
those reported in case of Cr(VI) reduction by Ochrobactrum anthropi
and P. synxartha (McLean et al., 2000). In an another study, rela-
tively lower amount of immobilisation of reduced Cr(III) (35%) on
the bacterial cell was found in the case Cr(VI) reduction by Vig-
ribacillus sp. in LB medium (Mishra et al., 2012). On the other hand,
the Cr(VI) reduction with Arthrobacteria and Bacillus, revealed the
presence of almost all the reduced Cr(III) in the supernatants as
soluble Cr(III) end products studies (Megharaj et al., 2003).
Several other studies also reported about distribution of reduced
Cr(III) species. For instance, Cr(VI) reduction by bacterial strains
(e.g., Pseudomonas sp. G1DM21 and P. putida) led to the production
of soluble Cr(III) end products and not Cr(OH)3 precipitate (Smith
and Gadd, 2000). Indirect evidence of solubility Cr(III), due to
complex formation, was obtained from the fact that the solubility of
Cr(III) species significantly enhanced by strong interaction of Cr(III)
395
Fig. 10. A schematic diagram of Cr(VI) biomass interaction; Cr(VI) initially binds with
the functional groups of the biomass and then reduced to Cr(III).
with microbial exudates (e.g., exopolymeric substance (EPS), cit-
rate, alginate) in natural systems (Priester et al., 2006).
FT-IR is one most extensively used techniques to identify the
functional groups present in different bacterial cells and their co-
ordination with reduced Cr(III) species (Bueno et al., 2008;
Lameiras et al., 2008; Quintelas et al., 2008; Das and Guha, 2007).
FTIR analysis of P. aeruginosa by Chatterjee et al. (2011) revealed in
the involvement of two major functional groups of cell wall
carboxyl and amino groups on the bacterial surface are responsible
for binding with the reduced Cr(III) (Fig. 10). The shift of peak po-
sition in the FT-IR spectra of reduced species associated with Ba-
cillus sp. cell indicated Cr(III) coordination with surface functional
group (Dhal et al., 2010).
A number of studies from our group have also been devoted to
assess the fate of bioreduced products of Cr(VI) using a variety of
techniques. In one of the recent studies (Mishra et al., 2012), the
TEM-SAED analyses of Vigribacillus sp. cell associated with reduced
product showed the accumulation of reduced Cr(III) species, either
as Cr(OH)3 precipitate in nanometric size or immobilized on the
bacterial cell surface, both on cell envelop and inside the cell sur-
face. The XRD pattern without any characteristic peaks of Cr(OH)3
indicated its amorphous nature or formed in small amount which
did not produce any observable diffraction peak in the XRD pattern.
A similar result has also been obtained with the reduction of Cr(VI)
by Bacillus sp. in LB medium (Dhal et al., 2010) or by Bacillus
amyloliquefaciens in M9 medium. Interestingly, the characteristic
peaks of crystalline Cr(III) phosphate was observed in the XRD
pattern when the reduction was carried out using Bacillus sp. in M9
media containing phosphate anion in the media (Dhal et al., 2010).
Thus the media composition greatly affects the overall distribution
and nature of Cr(III) species.
UVeVis is another simple but versatile technique used for an-
alyses of different types bio-based samples in solid state. Analyses
of diffuse reflectance spectra (UVeVis-DRS) provide valuable in-
formation in respect of oxidation state of reduced species. For
instance, UVeVis-DRS of dried mass (Vigribacillus sp. cell associated
with reduced species) in the reduction of Cr(VI) by Vigribacillus sp.
showed the characteristic peaks of Cr(III) species at ~600, 420 and
280 nm corresponding to spin allowed, Laporte forbidden transi-
tions from the fundamental state 4T2g (F) / 4T2g (F), 4T2g (F) / 4T1g
(F) and 4T2g (F) / 4T1g (P), respectively indicating the formation of
Cr(III) from Cr(VI) (Mishra et al., 2012). A similar observation was
also made in the case of Cr(VI) reduction with Bacillus amylolique-
faciens (Das et al., 2014). Distribution of total chromium content of
the reduced product in B. amyloliquefaciens was estimated by
Atomic Absorption Spectroscopic analysis. Formation of Cr(III) in
this case was also confirmed from the characteristic peak (at
~600 cm1) of Cr(III) in the Raman spectra (Das et al., 2014). Further
analyses by TEM-EDX and SAED image showed the presence of
electron dense particle within the bacterial cell indicating intra-
cellular deposition of amorphous Cr(III) species in nanometric
396 H. Thatoi et al. / Journal of Environmental Management 146 (2014) 383e399
range. Intracellular bioreduction of chromate was also reported in
the case of its reduction Shewanella oneidensis on the basis of
Raman spectra coupled with TEM image (Ravindranath et al., 2011).
11. Potential of chromate reductase in anticancer therapy
Recent studies have suggested that enzymes efficient in
reducing chromate, with some modifications can also play a role in
cancer chemotherapy (Patterson et al., 2003; Grove et al., 2003;
Barak et al., 2006). The most elaborate instance of such an enzyme
is Y6 enzyme. It is a modified version of the E. coli YieF, that results
due to the technique of error-prone PCR. Y6 can act as a ‘prodrug’,
that means, it is non-toxic in native form but highly toxic when
reduced. These drugs kill by generating DNA adducts and can target
both growing and non-growing tumour cells (Patterson et al.,
2003). Error prone PCR was carried out using the E. coli yieF gene
and the evolved genes were screened for improved chromate
reductase activity. Among the enzymes showing superior chromate
reductase activity, Y6 showed improved activity for prodrug
reduction when tested with HeLa cells (Barak et al., 2006). A class of
enzymes that has already been well studied in gene-delivered
enzyme prodrug therapy is bacterial nitroreductases, such as,
NfsA and NfsB from E. coli (Grove et al., 2003; Weedon et al., 2000).
Y6 has been found to kill HeLa cells with 5-fold greater efficiency
than NfsA. Thus such modified chromate reductases can certainly
provide new potentialities into cancer chemotherapy (Barak et al.,
2006). The efficiency of bacterial bioremediation can be increased
in many ways. One of the approaches is to promote the growth of
the specific bacterial species that reduces Cr(VI), by providing
specific nutrients for it, while outcompeting the growth of other
species (Anderson et al., 2003).
12. Conclusions
The enzymes that play a role in reducing Cr(VI) have been re-
ported from different microorganisms are belong to several classes,
like quinone reductases, nitroreductases, NADPH-dependent etc.
which vary in their ability to transform chromate and follow
different pathways. Several bacteria reduce Cr(VI) through mem-
brane bound reductases such as flavin reductase, cytochromes and
hydrogenases. These enzymes can be part of electron transport
system and use chromate as the terminal electron acceptor. Bac-
terial strains of various genera also possess soluble chromate
reductase activity in cytosol. The chromate reductases use NAD(P)H
as electron donors to reduce Cr(VI). Compared to membrane bound
chromate reductases, soluble reductases are suitable for develop-
ment of biocatalyst for bioremediation since those are more
amenable to protein engineering to suit environmental conditions
of contaminated sites. Though soluble chromate reductases have
been reported from numerous bacteria, only a few of them have
been purified and characterized. In rare cases, genes responsible for
chromate reductase production have been identified. Cloning of the
genes may allow enhanced production of large quantities of pure
using modern molecular approach for efficient chromate reduction.
Unlike free enzymes, enzymes can be immobilized onto a carrier
and can be used for a longer period in bioremediation programme.
Apart from these, the enzymes that are efficient in reducing chro-
mate, can also play a role in cancer chemotherapy and has gener-
ated interest for exploitation of the enzyme for pharmaceutical
application.
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