Chemosphere 52 (2003) 1523–1529

www.elsevier.com/locate/chemosphere

Reduction of chromate (CrO2
4 ) by an enrichment

consortium and an isolate of marine
sulfate-reducing bacteria
K.H. Cheung a, Ji-Dong Gu a,b,*

a
Department of Ecology & Biodiversity, Laboratory of Environmental Toxicology, The University of Hong Kong,
Pokfulam Road, Hong Kong SAR, PR China
b
The Swire Institute of Marine Science, The University of Hong Kong, Shek O, Cape dÕAguilar, Hong Kong SAR, PR China

Abstract

An enrichment consortium and an isolate (isolate TKW) of sulfate-reducing bacteria (SRB) have been obtained
from metal-contaminated marine sediments of Tokwawan, Hong Kong SAR. These bacteria are capable of reducing
highly toxic and soluble hexavalent chromium (Cr6þ ) enzymatically into less toxic and insoluble trivalent chromium
(Cr3þ ) under anaerobic conditions. The enrichment consortium almost completely (98.5%) reduced 0.6 mM Cr6þ in 168
h and the rate of reduction was 0.5 g (Cr6þ ) g (protein)
1 h
1 . In comparison, with Cr6þ as the sole electron acceptor (as
a surrogate for SO2
4 ), isolate TKW reduced 94.5% of the initially added Cr
6þ
(0.36 mM) in 288 h, with the rate of 0.26 g
(Cr6þ ) g (protein)
1 h
1 . Adsorption by these bacteria was not the major mechanism contributing to the transformation
or removal of Cr6þ . The biomass and Cr3þ in the cultures increased simultaneously with the reduction of Cr6þ . These
indigenous SRB might have potential application in bioremediation of metal contaminated sediments.
Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Hexavalent chromium; Metal reduction; Sole electron acceptor; Marine enrichment consortium; Sulfate-reducing bacteria;
Bioremediation

1. Introduction (Hedgecott, 1994). As the application of chromium is

Chromium (Cr) is an essential trace metal for living
organisms; however, its high toxicity, mutagenicity and
carcinogenicity render it hazardous at very low con-
centration (Venitt and Levy, 1974; EPA, 1998; McLean
et al., 2000; Cheung and Gu, 2002). Medical research
has further confirmed that hexavalent chromium (Cr6þ )
exposure is associated with increased human lung cancer
risk (Gibb et al., 2000). Accordingly, chromium and its
compounds are placed on the priority list of toxic chem-
icals of many countries including USA, UK and Canada
*
Corresponding author. Tel.: +852-2299-0605; fax: +852-
2517-6082.
E-mail address: jdgu@hkucc.hku.hk (J.-D. Gu).
0045-6535/03/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0045-6535(03)00491-0
extensive in various industries like chrome-plating, wood
preservation and alloy (especially important for the
resistance of corrosion in stainless steel) formation,
chromium-associated pollution is of increasing concern
nowadays (Shakoori et al., 2000; Gu and Cheung, 2001;
Ryan et al., 2002). Apart from traditional physico-
chemical treatments, a number of biological assays using
microorganisms have been studied and developed to
remedy chromium-contaminated water. The two major
processes being investigated are adsorption of metals on
biological materials (i.e. biosorption) including cells of
microorganisms and plants, and dissimilatory reduction
of metal ions from higher valent state to lower one (i.e.
biotransformation) through enzymatic reaction or indi-
rectly with metabolite produced (Lovley, 1993; Wang
and Shen, 1995; Tobin and Roux, 1998; Lee et al., 2000;
1524 K.H. Cheung, J.-D. Gu / Chemosphere 52 (2003) 1523–1529
Valls et al., 2000). As a transition metal, chromium ex-
ists in a wide range of oxidation states from )2 to +6,
while the dominant species in nature are hexavalent
chromium (Cr6þ ) and trivalent chromium (Cr3þ ) (Fukai,
1967). Cr6þ is about 100 times more toxic, and soluble
than Cr3þ . Reduction of Cr6þ to Cr3þ will not only relief
the toxicity of chromium acting on living organisms, but
also help precipitating chromium out at neutral pH
(mainly as Cr(OH)3 ) for further physical removal, and
this has been considered as an economical alternative for
wastewater treatment (Yamamoto et al., 1993; Shen and
Wang, 1995; Ganguli and Tripathi, 2002).
Since the first report of chromium reduction by
bacteria published in the late 1970s (Romanenko and
KorenÕkov, 1977), a number of bacterial species have
been isolated and shown to be capable of reducing Cr.
Species in the genus of Pseudomonas, Escherichia, Ba-
cillus, Enterobacter, and sulfate-reducing bacteria (SRB)
like Desulfovibrio, Desulfomicrobium and Desulfotomac-
ulum are being studied most extensively (Wang et al.,
1989; Ishibashi et al., 1990; Wang and Shen, 1995;
Lovley and Phillips, 1994; Tebo and Obraztsova, 1998;
Tucker et al., 1998; McLean and Beveridge, 2001; Mi-
chel et al., 2001; Ganguli and Tripathi, 2002). Besides,
other species of bacteria have also been investigated for
their Cr reducing property, such as Shewanella alga
(Guha et al., 2001), Microbacterium sp. MP30 (Pat-
tanapipitpaisal et al., 2001), Agrobacterium radiobacter
(Llovera et al., 1993), Pyrobaculum islandicum (Kashefi
and Lovley, 2000), Deinococcus radiodurans (Fredrick-
son et al., 2000) and Pantoea agglomerans (Francis et al.,
2000). Although a number of Cr reducing bacteria have
been isolated and reported, most of them were derived
from industrial effluent and groundwater (i.e. freshwater
origin). In this study, a consortium of SRB has been
enriched, with subsequent isolation, from metals con-
taminated marine sediments and studied for their Cr
reducing property, using chromate (CrO2
4 ) as the sole
electron acceptor.
2. Materials and methods
2.1. Source, enrichment and isolation of bacteria
Marine sediment slurry was collected with a Ponar
dredge (Wildco Instrument, Michigan) at Tokwawan, a
metals-polluted bay area in Hong Kong SAR (EPD,
1999). The sediment slurry was transferred immediately
to a 0.5 l Nalgene bottle until it was completely filled,
then the bottle was sealed tightly to prevent the direct
contact with atmospheric oxygen.
Enrichment of chromium-resistant bacteria was car-
ried out immediately after transporting the sample into
laboratory. 5 ml sediment slurry was transferred into a
serum bottle containing 50 ml sterilized culture medium
amended with approximately 1.0 mM Cr6þ (autoclaved
at 121 °C, 2 atm for 15 min). The medium was modified
from Postgate medium C (Postgate, 1984) and com-
posed of (in g l
1 of distilled water): sodium lactate 1.2,
sodium citrate 0.3, yeast extract 0.1, Na2 SO4 4.5,
CaCl2  2H2 O 0.06, NH4 Cl 1.0, KH2 PO4 0.5, MgSO4 
7H2 O 2.0, FeSO4  7H2 O 0.5, disodium ethylendiamine-
tetraacetate (EDTA) 0.3, and K2 CrO4 0.2. Resazurin
(1.0 lg ml
1 ) was added as a redox indicator to show any
potential contamination in the medium by molecular
oxygen. Na2 S  9H2 O (0.25 g l
1 ) was added to reduce the
trace amount of O2 remaining in the medium after
autoclaving, EDTA was added as chelator to enhance
metal solubility to bacteria in the medium (Lovley,
1997). pH was adjusted to 7.2 ± 0.2, and the headspace
of serum bottle was filled with pure N2 gas, then capped
with tert-butyl rubber stopper and crimp sealed. The
above processes were conducted inside an Anaerobic
Chamber (Coy Laboratory Products, Michigan). The
inoculated serum bottles were then put into a rotary
shaker (150 rpm) at 30 °C in the dark. After 1 week of
incubation, 5 ml enrichment culture was taken and
transferred into 50 ml freshly made culture medium
amended with approximately 1.0 mM Cr6þ according to
the procedures described above, and using this process
the second enrichment culture was established. Follow-
ing another week of incubation of the second enrich-
ment transfer culture, the third enrichment culture was
initiated in similar manner.
Hundred microlitre of the third enrichment culture
was transferred onto agar plates with composition as
described above plus 1.5% agar, and spread evenly with
sterilized glass beads. After 1 week of incubation an-
aerobically in the dark at 30 °C, individual colony was
selected for streaking on agar slant in Belch tube, and
the inoculated slant was incubated under identical con-
ditions. Colonies with uniform colony morphology were
observed in each agar slant, individual colony was
picked and inoculated to freshly prepared culture me-
dium, and it was serving as the pure isolate culture
thereafter. All procedures above were carried out asep-
tically and inside the Anaerobic Chamber (Coy Labo-
ratory Products) (Hademan et al., 1977).
2.2. Chromate reduction (with sulfate) by the enrichment
consortium
Five millilitre of the third enrichment culture was
transferred into 50 ml fresh culture medium as described
above and containing 0.6 mM Cr6þ , the inoculated
cultures were incubated on a rotary shaker (150 rpm) in
the dark at 30 °C. 1 ml sample was withdrawn using pre-
sterilized syringe and needle at regular time interval, and
stored at 4 °C for analyses of Cr6þ and total amount of
proteins. A set of uninoculated medium was prepared
 K.H. Cheung, J.-D. Gu / Chemosphere 52 (2003) 1523–1529 1525

following strictly the same procedures and served as 2.4. Scanning electron microscopy
control. All procedures were conducted aseptically
under anaerobic conditions in duplicate. One millilitre enrichment culture was filtered onto a
The concentration of Cr6þ in samples were quantified 0.2 lm-pore-size polycarbonate membrane filter (Gelman
by the colorimetric diphenylcarbazide (DPC) method Science, Ann Arbor, Michigan). It was then prepared
modified from Lovley and Phillips (1994). Subsamples for scanning electron microscopy (SEM) examination
(0.3 ml) were pipetted to test tube containing 9.7 ml 0.2 following procedures of fixation in 2% glutaraldehyde
NH2 SO4 . Then 25 ll H3 PO4 was added, followed by 0.5 then 1% OsO4 . Thereafter, it was dehydrated in a series
ml DPC reagent (0.025 g 1,5-diphenylcarbazide dis- of ethanol solutions, critical-point dried in liquid CO2 ,
solved in 10 ml acetone). After 5 min of reaction, the and coated with gold–palladium as described by Gu and
absorbance of sample was measured with UV-1201V Cheung (2001). The prepared samples were then ob-
Spectrophotometer (Shimadzu, Kyoto) at wavelength served under a Leica Cambridge S440 scanning electron
540 nm (i.e. A540 ). microscope (Cambrigde, UK).
For the measurement of bacterial growth, Bradford
assay (Daniels et al., 1994) was adopted to quantify the
total amount of proteins in culture samples instead of 3. Results
directly measuring the optical density (OD). This is be-
cause copious production of black metal sulfide by the 3.1. Enrichment culture and isolate TKW
bacteria will obstruct the accuracy of directly using OD.
0.2 ml subsamples were added to 2.5 ml Coomassie An active enrichment culture of SRB was obtained
Brilliant Blue reagent (100 mg Coomassie Brilliant Blue from Tokwawan marine sediments (after three enrich-
G250; 50 ml 95% ethanol; 100 ml 85% H3 PO4 ; 1 l dis- ment transfers) with Cr6þ as the major electron acceptor
tilled water). After reacting for 5 min, A595 of the sample and lactate as the major electron donor under sulfate
was measured with Spectrophotometer (Shimadzu). reducing conditions (Fig. 1). The morphological ap-
Concentrations of standard protein (Albumin) showed pearance of the bacteria is characteristic of SRBs. The
linear relationship between 2.5 and 30 lg ml
1 . activity of SRB was indicated by the intense production
of ferrous sulfide (FeS, black precipitates formed when
H2 S reacts with Fe2þ in the culture medium) resulting
2.3. Chromate reduction (without sulfate) and adsorption from the reduction of sulfate (SO2
4 ), and biomass in-
by isolate TKW crease as reflected by the total amount of proteins in the
enrichment culture (Fig. 2a). When the precipitates were
To investigate whether the isolate (designated as allowed to settle, the originally yellow aliquot became
isolate TKW) can utilize K2 CrO4 as the sole electron colorless, indicating that Cr6þ (yellow in color) has been
acceptor in the medium, 5 ml of the isolate TKW culture transformed in the medium (colorless). On the spread
was transferred into 50 ml fresh culture medium, which plates of the third enrichment culture, a number of
contained no sulfate (SO2
6þ
4 ) but 0.36 mM Cr . The colonies with uniform colony morphology were ob-
medium was composed of (in g l
1 of distilled water): served. A pure isolate, designated as isolate TKW, was
sodium lactate 1.2, sodium citrate 0.3, yeast extract 0.1,
CaCl2  2H2 O 0.06, NH4 Cl 1.0, KH2 PO4 0.5, disodium
EDTA 0.3, and K2 CrO4 0.07. The inoculated medium
was incubated at 30 °C with shaking (150 rpm) in the
dark. 1 ml sample was taken and stored at 4 °C for
analyses of Cr6þ and total amount of proteins as de-
scribed above. Another set of culture medium contain-
ing neither SO2
2

4 nor CrO4 (i.e. no electron acceptor)
was inoculated, together with a set of uninoculated
medium, serving as controls. All procedures were con-
ducted aseptically and anaerobically in duplicate.
To study whether adsorption of Cr by isolate TKW
was significant, 5 ml of the isolate-TKW culture was
heat-treated (autoclaved) prior to inoculation into 50 ml
culture medium (with SO2
4 ) containing 0.75 mM Cr ,
6þ

and incubated under identical incubation conditions as

described above. 1 ml sample was withdrawn periodi- Fig. 1. A scanning electron micrograph of the enrichment
cally and analyzed for the concentration of Cr6þ by the culture from Tokwawan marine sediments showing a consor-
DPC method. tium of sulfate-reducing bacteria (SRB) with precipitates.
1526 K.H. Cheung, J.-D. Gu / Chemosphere 52 (2003) 1523–1529

3.3. Reduction of chromate as the sole electron acceptor

Similar to the reduction of Cr6þ under culturing

condition with SO2
4 , 94.5% of the initially added Cr
6þ
µ

were reduced by isolate TKW, while the time required

was substantially longer (288 h) with CrO2
4 as the sole
electron acceptor (Fig. 3a). At an initial concentration of
0.36 mM Cr6þ , the rate of Cr reduction was 0.26 g (Cr6þ )
g (protein)
1 h
1 , about half slower than that with SO2

4 ,
and with residual concentration of 0.015 mM Cr6þ (Fig.
3a). The total amount of proteins did not change sig-
nificantly at the beginning until 144 h of incubation, and
(a)
then it increases as Cr6þ being continuously reduced
(Fig. 3b). No growth was observed in the culture with-
out any electron acceptor, i.e. neither SO2
4 nor CrO4
2

6þ
(data not shown); no significant reduction of Cr (Fig.
3a) and increases in the total amount of proteins (Fig.
3b) were observed in the uninoculated control.

3.4. Adsorption of chromium

Heat-treated bacterial cells of isolate TKW were in-

oculated into culture medium amended with 0.75 mM
Cr6þ . Unlike the biologically active cultures, the heat-
(b) treated one removed only approximately 30% of Cr6þ

Fig. 2. (a) Total amount of proteins measured over time as

Cr6þ being reduced by the enrichment culture with normal
culture medium (with SO2
4 ) and no increase of protein in the
uninoculated controls. (b) Cr6þ reduction by the enrichment
culture with normal culture medium (with SO2
4 ) and no change
in the uninoculated controls over time. Arrow indicating the
first appearance of black precipitates.

obtained from one of these colonies and was used for

further investigation.

3.2. Chromate reduction in the presence of sulfate

(a)
Reduction of Cr6þ (0.6 mM) by the enrichment
consortium took place after a brief lag period (12 h).
Then, the rate of reduction of Cr6þ was rapid, at ap-
proximately 0.5 g (Cr6þ ) g (protein)
1 h
1 until 168 h,
where no further reduction of Cr6þ occurred afterwards.
µ

98.5% of Cr6þ was reduced during the experiment pe-

riod, while 0.009 mM Cr6þ (residual concentration) re-
mained unreduced even after 350 h of incubation (Fig.
2b). It should also be pointed out that appearance of
black precipitates was observable only after Cr6þ was
consumed (after 199 h). At the same time period, the
uninoculated control did not show significant change of (b)
Cr6þ concentration, nor production of black precipi- Fig. 3. (a) Cr6þ reduction by isolate TKW with CrO2
4 as the
tates, over the entire duration of this incubation (Fig. sole electron acceptor (no SO2
in culture medium) and the
4
2b). The total amount of proteins (Fig. 2a) and Cr3þ uninoculated controls over time. (b) Total amount of proteins
(data not shown) were increasing simultaneously as Cr6þ measured over time as Cr6þ being reduced by isolate TKW with
was reduced. CrO2
2

4 as sole electron acceptor (no SO4 in culture medium).
 K.H. Cheung, J.-D. Gu / Chemosphere 52 (2003) 1523–1529
Fig. 4. Cr6þ removal (mainly abiotic adsorption) by heat-
treated bacterial cells of isolate TKW.
even after 264 h of incubation (Fig. 4). Adsorption of Cr
by isolate TKW was not the major mechanism respon-
sible for Cr removal by this bacterium.
4. Discussion
The level of Cr that can be reduced by both marine
SRB consortium and the isolate TKW was relatively
high as the common reduction range of Cr6þ by bacteria
(with nearly complete reduction) was below 0.5 mM
(Ishibashi et al., 1990; Shen and Wang, 1993, 1994;
Lovley and Phillips, 1994; Michel et al., 2001; Pat-
tanapipitpaisal et al., 2001; Ganguli and Tripathi, 2002),
while the concentration of Cr that can almost be com-
pletely reduced in this study was 0.6 mM Cr6þ . As the
culture medium being used was at neutral pH, together
with a chelator (EDTA) being added to enhance the
solubility of metal ions (Lovley, 1997), not much
chemical precipitation of Cr as Cr(OH)3 ) is expected,
and Cr6þ being added is presumably available to the
microorganisms. Both the enrichment consortium and
isolate TKW experienced a brief lag period at the initial
stage as they need to be acclimatized to the culturing
environment amended with high concentration of Cr6þ
before they started to actively metabolize, this was
demonstrated by the almost constant concentration of
Cr6þ and total amount of proteins at the beginning of
incubation. Both enrichment consortium and isolate
TKW, instead of other physico-chemical means, were
responsible for the reduction of chromium as elucidated
by the negligible amount of Cr6þ being reduced in the
uninoculated controls. Moreover, the negative relation-
ship between the total amount of proteins and Cr3þ in
the medium with Cr6þ , i.e. the total amount of proteins
and Cr3þ increased simultaneously when Cr6þ was being
reduced, further indicates that the bacteria were re-
sponsible for the reduction of Cr, and the end-product
of Cr reduction was Cr3þ as described in previous find-
1527
ings (Wang et al., 1989; Llovera et al., 1993; Shen and
Wang, 1993, 1994; Lovley and Phillips, 1994). Never-
theless, it should be noted that Cr reduction does
not necessarily accompany with bacterial growth. Pat-
tanapipitpaisal et al. (2001) reported that the Micro-
bacterium sp. they isolated was capable of reducing Cr
anaerobically without measurable growth.
The study of Cr reducing bacteria has drawn more
attention after Romanenko and KorenÕkov successfully
isolated the first Cr reducing bacteria in 1977 (Roman-
enko and KorenÕkov, 1977). Since then, a number of Cr
reducing bacteria have been isolated and investigated,
including aerobes, facultative anaerobes and SRB. For
instance, a consortium of SRB enriched from electro-
plating sludge (Fude et al., 1994), Desulfotomaculum
reducens (Tebo and Obraztsova, 1998), Desulfovibrio
vulgaris strain Hildenborough (Lovley and Phillips,
1994; Michel et al., 2001), Desulfovibrio desulfuricans
(Tucker et al., 1998; Michel et al., 2001), Desulfomicro-
bium norvegicum, Desulfovibrio gigas, and Desulfomi-
crobium escambiense were investigated for Cr reduction
(Michel et al., 2001). This consortium of SRB and the
isolate TKW are unique in that they can grow with
CrO2
4 as the sole electron acceptor, which served as a
surrogate for SO2

4 , and only a few microorganisms have
been reported to bear this characteristic (Lovley, 1993;
Tebo and Obraztsova, 1998).
In the metabolism of SRB, sulfate (SO2
4 ) is normally
reduced to sulfide (S2
), and bacterially produced hy-
drogen sulfide (H2 S) is capable of reducing Cr6þ in
marine environment. This process is regarded as the
major route of Cr reduction by SRB (Smillie et al., 1981).
Besides D. vulgaris (Hildenborough) ATCC 29579, which
has been found to reduce Cr6þ enzymatically, other Cr
reducing SRB were found to reduce Cr6þ indirectly
with H2 S being generated (Ehrlich, 2002). The reduc-
tion of Cr6þ in our marine SRB should, however, be
an enzymatic process instead of precipitation with S2

because they are capable of reducing Cr6þ , which served
as the surrogate electron acceptor, without SO2
4 in the
culture medium. Presumably, no S2
would be pro-
duced and thus impossible for S2
to precipitate Cr6þ
out.
Anaerobic bacterial reduction of heavy metals has
potential advantages over traditional physico-chemical
means, like no costly chemical additives or aeration is
needed, only mild culturing conditions (e.g. neutral pH
and moderate temperature) are required, and the sludge
production is minimized by using anaerobes. Thus, the
operating cost of adopting bioremediation would be
lowered (Ohtake and Silver, 1994; Wang, 2000).
Conclusively, a consortium of marine SRB has been
enriched and isolated (isolate TKW) from metals-
polluted sediment slurry. They are resistant to high con-
centration of Cr6þ , can reduce Cr6þ biologically as the
sole electron acceptor. These properties of the bacteria
1528 K.H. Cheung, J.-D. Gu / Chemosphere 52 (2003) 1523–1529
make them potentially useful for the treatment of in-
dustrial effluent and in situ bioremediation for chro-
mium contaminated sediment and water.
Acknowledgements
This research was supported by the CRCG Initiation
Programme of The University of Hong Kong. We thank
technical assistance of the Electron Microscopy Unit,
The University of Hong Kong and Jessie Lai of this
laboratory.
References
Cheung, K.H., Gu, J.-D., 2002. Bacterial color response to
hexavalent chromium, Cr6þ . The Journal of Microbiology
40, 234–236.
Daniels, L., Hanson, R.S., Phillips, J.A., 1994. Chemical
analysis. In: Gerhardt, P., Murray, R.G.E., Wood, W.A.,
Krieg, N.R. (Eds.), Methods for General and Molecular
Bacteriology. American Society for Microbiology, Wash-
ington DC, pp. 512–554.
Ehrlich, H.L., 2002. Geomicrobiology, 4th ed. Marcel Dekker,
New York. pp. 768.
Environmental Protection Agency (EPA) 1998. Toxicological
Review of Hexavalent Chromium. US Environmental
Protection Agency, Washington DC.
Environmental Protection Department (EPD) 1999. Marine
Water Quality in Hong Kong in 1999. HK Environmental
Protection Department, Hong Kong SAR.
Francis, C.A., Obraztsova, A.Y., Tebo, B.M., 2000. Dissimi-
latory metal reduction by the facultative anaerobe Pantoea
agglomerans SP1. Applied and Environmental Microbio-
logy 66, 543–548.
Fredrickson, J.K., Kostandarithes, H.M., Li, S.W., Plymale,
A.E., Daly, M.J., 2000. Reduction of Fe(III), Cr(VI), U(VI),
and Tc(VII) by Deinococcus radiodurans R1. Applied and
Environmental Microbiology 66, 2006–2011.
Fude, L., Harris, B., Urrutia, M.M., Beveridge, T.J., 1994.
Reduction of Cr(VI) by a consortium of sulfate-reducing
bacteria (SRB III). Applied and Environmental Microbio-
logy 60, 1525–1531.
Fukai, R., 1967. Valency state of chromium in seawater. Nature
(London) 213, 901.
Ganguli, A., Tripathi, A.K., 2002. Bioremediation of toxic
chromium from electroplating effluent by chromate-reduc-
ing Pseudomonas aeruginosa A2Chr in two bioreactors.
Applied Microbiology & Biotechnology 58, 416–420.
Gibb, H.J., Lees, P.S.J., Pinsky, P.F., Rooney, B.C., 2000.
Lung cancer among workers in chromium chemical pro-
duction. American Journal of Industrial Medicine 38, 115–
126.
Gu, J.-D., Cheung, K.H., 2001. Phenotypic expression of
Vogesella indigofera upon exposure to hexavalent chro-
mium, Cr6þ . World Journal of Microbiology & Biotechno-
logy 17, 475–480.
Guha, H., Jayachandran, K., Maurrasse, F., 2001. Kinetics of
chromium (VI) reduction by a type strain Shewanella alga
under different growth conditions. Environmental Pollution
115, 209–218.
Hademan, L.V., Cato, E.P., Moore, W.E.C., 1977. Anaerobic
Laboratory Manual. 4th ed., Anaerobe Laboratory, Vir-
ginia Polytechnic Institute and State University, Blacks-
burg, Virginia. pp. 156.
Hedgecott, S., 1994. Prioritization and standards for hazardous
chemicals. In: Calow, P. (Ed.), Handbook of Ecotoxicology.
Blackwell Scientific Publications, Oxford, pp. 378–382.
Ishibashi, Y., Cervantes, C., Silver, S., 1990. Chromium
reduction in Pseudomonas putida. Applied and Environ-
mental Microbiology 56, 2268–2270.
Kashefi, K., Lovley, D.R., 2000. Reduction of Fe(III), Mn(IV),
and toxic metals at 100 °C by Pyrobaculum islandicum.
Applied and Environmental Microbiology 66, 1050–1056.
Lee, D.-C., Park, C.-J., Yang, J.-E., Jeong, Y.-H., Rhee, H.-I.,
2000. Screening of hexavalent chromium biosorbent from
marine algae. Applied Microbiology & Biotechnology 54,
445–448.
Llovera, S., Bonet, R., Simon-Pujol, M.D., Congregado, F.,
1993. Chromate reduction by resting cells of Agrobacterium
radiobacter EPS-916. Applied and Environmental Micro-
biology 59, 3516–3518.
Lovley, D.R., 1993. Dissimilatory metal reduction. Annual
Review of Microbiology 47, 263–290.
Lovley, D.R., 1997. Microbial Fe(III) reduction in subsurface
environments. FEMS Microbiology Reviews 20, 305–313.
Lovley, D.R., Phillips, E.J.P., 1994. Reduction of chromate by
Desulfovibrio vulgaris and its c3 cytochrome. Applied and
Environmental Microbiology 60, 726–728.
McLean, J., Beveridge, T.J., 2001. Chromate reduction by a
Pseudomonad isolated from a site contaminated with
chromated copper arsenate. Applied and Environmental
Microbiology 67, 1076–1084.
McLean, J.S., Beveridge, T.J., Phipps, D., 2000. Isolation and
characterization of a chromium-reducing bacterium from a
chromated copper arsenate-contaminated site. Environmen-
tal Microbiology 2, 611–619.
Michel, C., Brugna, M., Aubert, C., Bernadac, A., Bruschi, M.,
2001. Enzymatic reduction of chromate: comparative stud-
ies using sulfate-reducing bacteria. Applied Microbiology &
Biotechnology 55, 95–100.
Ohtake, H., Silver, S., 1994. Bacterial detoxification of toxic
chromate. In: Chaudhry, G.R. (Ed.), Biological Degrada-
tion and Bioremediation of Toxic Chemicals. Chapman &
Hall, London, pp. 403–415.
Pattanapipitpaisal, P., Brown, N.L., Macaskie, L.E., 2001.
Chromate reduction and 16S rRNA identification of bac-
teria isolated from a Cr(VI)-contaminated site. Applied
Microbiology & Biotechnology 57, 257–261.
Postgate, J.R., 1984. The Sulphate-Reducing Bacteria, 2nd ed.
Cambridge University Press, Cambridge. pp. 208.
Romanenko, V.I., KorenÕkov, V.N., 1977. A pure culture of
bacteria utilizing chromates and bichromates as hydrogen
acceptors in growth under anaerobic conditions. Mikro-
biologiya 46, 414–417.
Ryan, M.P., Williams, D.E., Chater, R.J., Hutton, B.M.,
McPhail, D.S., 2002. Why stainless steel corrodes. Nature
(London) 415, 770–774.
Shakoori, A.R., Makhdoom, M., Haq, R.U., 2000. Hexava-
lent chromium reduction by a dichromate-resistant Gram-
 K.H. Cheung, J.-D. Gu / Chemosphere 52 (2003) 1523–1529
positive bacterium isolated from effluents of tanneries.
Applied Microbiology & Biotechnology 53, 348–351.
Shen, H., Wang, Y.-T., 1993. Characterization of enzymatic
reduction of hexavalent chromium by Escherichia coli
ATCC 33456. Applied and Environmental Microbiology
59, 3771–3777.
Shen, H., Wang, Y.-T., 1994. Biological reduction of chromium
by E. coli. Journal of Environmental Engineering 120, 560–
572.
Shen, H., Wang, Y.-T., 1995. Simultaneous chromium reduc-
tion and phenol degradation in a coculture of Escherichia
coli ATCC 33456 and Pseudomonas putida DMP-1. Applied
and Environmental Microbiology 61, 2754–2758.
Smillie, R.H., Hunter, K., Loutit, M., 1981. Reduction of
chromium(VI) by bacterially produced hydrogen sulphide in
a marine environment. Water Research 15, 1351–1354.
Tebo, B.M., Obraztsova, A.Y., 1998. Sulfate-reducing bacte-
rium grows with Cr(VI), U(VI), Mn(IV), and Fe(III) as
electron acceptors. FEMS Microbiology Letter 162, 193–
198.
Tobin, J.M., Roux, J.C., 1998. Mucor biosorbent for chromium
removal from tanning effluent. Water Research 32, 1407–
1416.
Tucker, M.D., Barton, L.L., Thomson, B.M., 1998. Reduction
of Cr, Mo, Se and U by Desulfovibrio desulfuricans
1529
immobilized in polyacrylamide gels. Journal of Industrial
Microbiology & Biotechnology 20, 13–19.
Valls, M., Atrian, S., de Lorenzo, V., Fernandez, L.A., 2000.
Engineering a mouse metallothionein on the cell surface of
Ralstonia eutropha CH34 for immobilization of heavy
metals in soil. Nature Biotechnology 18, 661–665.
Venitt, S., Levy, L.S., 1974. Mutagenicity of chromates in
bacteria and its relevance to chromate carcinogenesis.
Nature (London) 250, 493–495.
Wang, Y.-T., 2000. Microbial reduction of chromate. In:
Lovley, D.R. (Ed.), Environmental Microbe–Metal Inter-
actions. American Society of Microbiology, Washington,
DC, pp. 225–235.
Wang, Y.-T., Shen, H., 1995. Bacterial reduction of hexavalent
chromium. Journal of Industrial Microbiology 14, 159–
163.
Wang, P.-C., Mori, T., Komori, K., Sasatsu, M., Toda, K.,
Ohtake, H., 1989. Isolation and characterization of an
Enterobacter cloacae strain that reduces hexavalent chro-
mium under anaerobic conditions. Applied and Environ-
mental Microbiology 55, 1665–1669.
Yamamoto, K., Kato, J., Yano, T., Ohtake, H., 1993. Kinetics
and modeling of hexavalent chromium reduction in Ente-
robacter cloacae. Biotechnology and Bioengineering 41,
129–133.