Biotechnology Letters 22: 829–836, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

829

Reduction of chromate by microorganisms isolated from metal

contaminated sites of Karachi, Pakistan

U. Badar1 , N. Ahmed1 , A.J. Beswick2,∗∗ , P. Pattanapipitpaisal2 & L.E. Macaskie2,∗

1 Centre for Molecular Genetics, University of Karachi, Karachi 75270, Pakistan
2 School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
∗∗ Current address: Health and Safety Laboratories, Broad Lane, Sheffield S3 7HQ, UK
∗ Author for correspondence (Fax: 0044-121-414-5925; E-mail: L.E.Macaskie@bham.ac.uk)

Received 18 November 1999; Revisions requested 30 November 1999; Revisions received 27 March 2000; Accepted 28 March 2000

Key words: bioremediation of Cr(VI), chromate bioreduction, chromate resistance

Abstract
Three bacterial strains, two identified as Pseudomonas stutzeri and one as a strain of cucurbit yellow vine disease
bacterium, isolated from a foundry soil and a tannery, respectively, in Pakistan, were resistant to up to 1 mM
chromate and anaerobically reduced Cr(VI) up to 100 µM. The highest removal was by P. stutzeri CMG463:
88 µmol l−1 (88% of that supplied; specific rate was 3.0 nmol mg−1 protein h−1 ), while 58 and 76 µmol l−1 (58%
and 76%) were removed by P. stutzeri CMG462 and cucurbit yellow vine disease bacterium CMG480, respectively.
These isolates were compared to strains isolated from an uncontaminated coastal site in the UK and designated as
K2 (Pseudomonas synxantha) K3 (Bacillus sp.), and J3 (unidentified Gram-positive strain). Strain K3 was Cr-
sensitive, partially lysed by Cr(VI), but had the highest removal of chromate anaerobically: 92 µmol l−1 (92% of
that supplied) at a specific rate of 71 nmol mg−1 protein h−1 . Analysis of cell sections using transmission electron
microscopy with energy dispersive X-ray analysis showed intracellular chromium in P. stutzeri but the cucurbit
yellow vine disease bacterium and the Bacillus sp. precipitated chromium extracellularly. The isolates from the Cr-
contaminated sites did not remove more Cr(VI), overall, than Cr-unstressed bacteria, but their tolerance to Cr(VI)
is potentially useful for bioremediation, particularly since other studies have shown that the two P. stutzeri strains
can bioaccumulate Cu2+ .

Introduction the need for effective, economic waste remediation at

Micro-organisms can play an important role in the
detoxification and/or removal of heavy metals from
polluted environments. Chromium is one of the most
toxic and carcinogenic heavy metals. Divalent and
trivalent chromium species are the most stable and
least toxic species, while hexavalent Cr (chromate,
CrO2−4 ) is highly toxic, readily crossing the mem-
branes of eukaryotic and prokaryotic cells (Cervantes
& Silver 1992), causing oxidative cellular damage.
Chromate compounds are widely used, e.g., in leather
tanning, metal finishing, alloy preparation and wood
preservation (Germain & Patterson 1974). Wastes are
often discharged to the environment, especially in
countries which impose inadequate regulatory control;
source is urgent.
Many bacteria, including Pseudomonas sp. (Ishi-
bashi et al. 1990, Horitsu et al. 1987), Aeromonas
(Kvasnikov et al. 1985), E. coli (Ishibashi et al.
1990) and Enterobacter (Wang et al. 1989) can re-
duce Cr(VI) to the less toxic Cr(III), which readily
precipitates as Cr(OH)3 . Cr(VI) reduction and resis-
tance are considered to be independent processes. In
the case of P. fluorescens, a Cr-resistant strain and
a sensitive derivative reduced Cr(VI) equally (Bopp
& Ehrlich 1988). Chromium resistance is usually at-
tributable to a decreased uptake of the metal (Ohtake
et al. 1987), which is of little potential benefit for
development of bioremediation processes. A particu-
830
larly useful characteristic would be the combination
of Cr(VI) resistance with its removal from solution.
The P. stutzeri strains in this study were origi-
nally isolated from a foundry soil on the basis of
their resistance to copper and accumulated Cu2+ (U.
Badar & N. Ahmed, unpublished). Although biocat-
alytic removal of metal cations is well-documented
(e.g., Yong & Macaskie 1997) few studies have in-
vestigated the potential of metal cation-accumulating
bacteria to remediate anionic forms of metals but
many wastes contain more than one metal species
(e.g., copper/chromium/arsenic-based wood preserv-
atives). These strains, together with an isolate from a
chromium-contaminated tannery site, were evaluated
for their ability to reduce and remove Cr(VI) Since
Cr-stress can lead to resistance which may be asso-
ciated with cellular exclusion of Cr(VI) (above) the
potential of the new strains was evaluated in parallel
with 3 strains which had been isolated from a site
uncontaminated with Cr(VI). These had been identi-
fied as potential Cr(VI) reducers from more than 1000
strains screened from samples taken at a coastal lo-
cation in the UK (Tolley 1993, J.A. Finlay, R. Perry,
P. Pattanapipitpaisal & L.E. Macaskie, unpublished
work).
The aim of this study was to compare Cr(VI)
reduction and Cr bioaccumulation, under aerobic
and anaerobic conditions, by strains from metal-
contaminated sites and from an uncontaminated envi-
ronment.
Materials and methods
Bacterial strains, cell growth and chromate resistance
Acetate minimal medium (AMM) contained (g l−1 ):
NH4 Cl 1.0; MgSO4 · 7H2 O, 0.2; FeSO4 · 7H2 O,
0.001; CaCl2 · 2H2 O 0.001; sodium acetate, 5; yeast
extract, 0.5 and K2 HPO4 , 0.5 (to pH 7.0 with
NaOH). The phosphate source was autoclaved sepa-
rately in 10 ml distilled water and added to the bulk
medium when cool. Nutrient broth (Oxoid) was used
as a starter medium. Cultures were routinely shaken
(30 ◦ C) and for chromate reduction tests were inocu-
lated (10% vol/vol inoculum) from AMM pre-growth
cultures (12–16 h) into fresh aerobic shake flasks or
anaerobic serum bottles with butyl rubber stoppers, as
appropriate (see below). Resistance to chromate was
tested on AMM plates supplemented with different
concentrations of sodium chromate. Incubation was at
30 ◦ C and growth was noted after 48 h.
Chromate reduction tests
A single colony was inoculated into nutrient broth
(5 ml) and cultured aerobically (24 h), inoculated
(1 ml) into 45 ml of AMM and grown for 12–16 h
at 30 ◦ C. For aerobic Cr(VI) reduction 10% (vol/vol)
inoculum was put into 45 ml of AMM and shaken
(100 rpm, 30 ◦ C), initially Cr-unsupplemented to initi-
ate exponential growth, and then supplemented with
sodium chromate (to 100 µM) after 3 h. Samples
(1 ml) were taken periodically and frozen at −20 ◦ C.
For anaerobic tests 45 ml of AMM in 50 ml serum
bottles was degassed with O2 free N2 (10–20 min)
via a syringe, inoculated (10% vol/vol inoculum) from
the aerobic pre-growth culture and incubated under
N2 (30 ◦ C). Sodium chromate was added (to 100 µM)
after 3 h and samples were taken as for the aerobic
cultures. For analysis samples (1 ml) were thawed and
the cells removed by centrifugation (room tempera-
ture). The supernatant was retained for assay of Cr(VI)
and the pellet was suspended in 1 ml of isotonic saline
(8.5 g l−1 of NaCl) for assay of bacterial protein.
Assay of Cr(VI) and protein
For determination of Cr(VI) the mixture contained
400 µl of 20 mM MOPS/NaOH buffer (pH 7), 327 µl
distilled water, 33 µl of 3M H2 SO4 , 40 µl of 0.25%
diphenyl carbazide (DPC) solution and culture super-
natant or standard solution (200 µl). The absorbance
was determined immediately at 540 nm. DPC solu-
tion comprised 0.25% w/v diphenylcarbazide in 0.1 M
H2 SO4 in acetone (Urone 1955). The protein assay
was adapted from the bicinchoninic acid method pro-
tein test kit (Sigma) and calibrated against bovine
serum albumin.
Electron microscopy
Bacterial pellets were harvested after 48 h, washed
with distilled water and fixed by immediate resuspen-
sion in 2.5% (v/v) glutaraldehyde in 0.1 M sodium
cacodylate buffer (pH 7.2, 60 min) and then in sec-
ondary fixative (1% osmium tetroxide: 60 min). The
cells were dehydrated in an ethanol series (70, 90,
100, 100, and 100% ethanol: 15 min each), twice with
propylene oxide (15 min) and then in a mixture of
propylene oxide/epoxy resin (1:1; 45 min). Samples
were then embeded in epoxy resin under vacuum in
plastic moulds (20 min) and left to polymerize at at-
mospheric pressure (24 h, 60 ◦ C). Sections (70 nm)
were cut, collected on copper grids and examined by

transmission electron microscopy. For energy disper-
sive X-ray analysis (EDAX) thicker sections (200–
300 nm) were cut and examined by scanning transmis-
sion electron microscopy (JEOL JEM-100CXII) using
a LINK ISIS X-ray analyser to determine elemental
distribution in/on and around the cells.
Identification of isolates
Gram staining was carried out using established meth-
ods (Collins & Patricia 1984). The isolates were iden-
tified using partial 16S rRNA gene analysis. A small
colony of each was resuspended in 0.1 ml of sterile,
de-ionised water (SDW), mixed and heated at 70 ◦ C
(10 min) for cell lysis. Crude lysate (0.2 µl) was
added to SDW (0.0198 ml) and used as a template
in a polymerase chain reaction using the eubacterial
16S targeted PCR primers (pA and pH0 ) as designed
by Edwards et al. (1989). These are known to am-
plify a 1536 base pair (approx. 1.5 kbp) length of
16S rDNA. The reaction mixture for amplification
was as published by Bruce et al. (1992). Cycling
used a MJ thermal cycler (MJ Research Inc., USA)
under tube temperature control and using 30 cycles
of the following program: 94 ◦ C for 40 s, 55 ◦ C
for 1 min, 72 ◦ C for 2 min and a final 10 min ex-
tension at 72 ◦ C. PCR products were cleaned using
Sephacryl S400 columns (Pharmacia, Sweden), and
partially sequenced using 16S sequencing primer 943
reverse (Lane et al. 1985) by Alta Biosciences (Uni-
versity of Birmingham, UK). Sequences (up to 650 bp)
were analysed using ADVANCED BLAST software
to access the EMBL database, Heidelberg, Germany
(Web ref: http://www.ncbi.nlm.nih.gov/cgi-bin/Blast).
Netscape browser interface was used.
Treatment of data
All experiments were done on at least three separate
occasions except anaerobic tests with strain J3 which
were done twice. Data for each time point are pooled
and the mean is given ± standard error of the mean
(SEM).
Results and discussion
Resistance of the polluted site isolates to Cr(VI)
Analysis using atomic absorption spectroscopy and
X-ray fluorescence following acid digestion showed
that the concentrations of metals (Cr, Cu, Pb) in the
831
UK coastal samples were not above normal environ-
mental levels (Tolley 1993); therefore strains isolated
from these had not suffered Cr-stress. The metals-
burden of the foundry and tannery environments was
not measured, but both are known to be heavily con-
taminated. For example a survey of tannery effluents
showed chromium and copper contents ranging from
0.06–7.9 mg l−1 and from 2.8–7.2 mg l−1 respectively
(N. Ahmed, unpublished). Strains CMG462, CMG463
and CMG480 were resistant to 1 mM sodium chro-
mate (growth on plates supplemented with 1 mM
CrO2−
4 ), but K2 and J3 grew only with 100 µM Cr(VI)
and K3 was sensitive to that concentration. It was con-
cluded that the polluted site isolates were Cr-resistant;
the mechanism(s) was not elucidated further.
Identification of isolates
Three isolates were homologous to strains of
Pseudomonas (Table 1), with the highest homology to
P. stutzeri (Pakistan foundry isolates) and P. synxantha
(UK coastal site isolate). The tannery isolate gave a
high homology to an isolate identified only as ‘cucur-
bit yellow vine disease bacterium’, an enterobacterial
plant pathogen (Avila et al. 1998). Strain K3 was a
Gram-positive aerobic sporeformer and was assigned
to the genus Bacillus while J3, also Gram-positive,
was not identified further. All isolates were ∼97%
similar to reference sequences on the EMBL database.
The Pseudomonas spp. were 99% similar to matching
sequences.
Chromate reduction by isolated strains
All strains grew aerobically in 100 µM Cr(VI)-
supplemented minimal medium (doubling time was
generally 4–6 h) but reduced little Cr(VI) (Table 2a).
Anaerobic growth at the expense of Cr(VI) as the elec-
tron acceptor was negligible but Cr(VI) was reduced
and removed (Table 2b). These cultures were essen-
tially resting cell suspensions, permitting determina-
tion of specific rates of Cr(VI) reduction (Table 2).
This was observed only after a lag of 31 h (follow-
ing growth in the aerobic cutures); the rates were
determined from 31–192 h.
Cr(VI) reduction aerobically was low in ac-
cordance with the provision of O2 as the pri-
mary (competing) electron acceptor (Table 2a). The
Pseudomonas spp. CMG462, CMG463 and K3
showed a comparable loss of Cr(VI) after 192 h. With
CMG480 this was small. Strain J3 removed the most
chromate after 192 h aerobically but this was still
832
Table 1. Bacterial strains used in this study.

Strain code Identification Origin

CMG462 Pseudomonas stutzeria Foundry soil, Karachi, Pakistanb

CMG463 Pseudomonas stutzeria Foundry soil, Karachi, Pakistanb
CMG480 Cucurbit yellow vine disease bacteriuma Tannery, Karachi, Pakistanc
K2 Pseudomonas synxanthaa Coastal sediment, Southern Englandd
K3 Bacillus sp. Coastal sediment, Southern Englandd
J3 Unidentified Gram-positive Coastal sediment, Southern Englandd
a The closest homology by 16S rRNA sequence analysis (see text).
b Strains isolated by U. Badar by growth on nutrient agar plates supplemented with Cu2+ .
c Strain isolated by F.F. Rizvi by growth on nutrient agar plates supplemented with CrO2− .
4
d Strains isolated by L.E. Macaskie on nutrient agar plates, selected from > 1000 isolates by routine
screening for reductive capability using tetrazolium blue (Tolley 1993) & identified as Cr(VI)-
reducing (liquid culture screening) by J.A. Finlay, R. Perry & P. Pattanapipitpaisal (unpublished
work).

Table 2a. Chromium (VI) reduction by aerobically-incubated cells.

Strain Residual Cr(VI) (µM) Loss of Cr(VI) (µM) Rate (nmol mg−1 protein per h)

CMG462 87 ± 1 14 0.1
CMG463 85 ± 1 15 0.4
CMG480 91 ± 5 9 0.1
K2 93 ± 2 8 0.4
K3 87 ± 1 12 0.1
J3 79 ± 2 23 0.4

The protein content was that following growth, which was complete after 24 h. The high protein con-
tent due to bacterial growth aerobically resulted in a low specific rate of Cr(VI) reduction following
growth.

Table 2b. Chrome(VI)reduction by anaerobically-incubated cells.

Strain Residual Cr(VI) (µM) Loss of Cr(VI) (µM) Rate (nmol mg−1 protein per h)

CMG462 42 ± 3 58 3.0
CMG463 12 ± 3 88 2.6
CMG480 24 ± 7 76 4.3
K2 21 ± 2 79 5.6
K3a 8±7 92 71.5
J3 28 73 3.2

Negligible growth occurred anaerobically in the absence of added electron acceptor (other than that
present in the yeast extract).
a Strain K3 gave very poor growth anaerobically before Cr(VI) addition. The protein content was low,
hence the specific rate of Cr(VI) reduction was high.

less than 25% of that provided (Table 2a). In contrast
Cr(VI) reduction was apparent in all strains anaero-
bically; the best strains were Pseudomonas stutzeri
CMG 463 and Bacillus sp. K3 in terms of total Cr(VI)
removed (88% and 92%, respectively). K3 gave the
best overall rate, largely attributable to a low pro-
tein content associated with very poor growth in the
3 h prior to Cr(VI) addition. Strain K2 (P. synxan-
tha) removed Cr(VI) comparably to the two P. stutzeri
strains. Comparison of strains of the same genus
(Pseudomonas spp.) suggested that pre-exposure to
metal contamination and enrichment for resistance
to Cr(VI) did not promote reduction of Cr(VI), in
accordance with other studies in the literature.
 833

Fig. 1. Transmission electron microscopy of sections of Cr(VI) challenged cells withdrawn after 48 h and prepared as described in Materials
and methods. (a, c, e, g): Controls (No Cr(VI)). (b, d, f, h): Cr(VI)-challenged cells. (a, b): Pseudomonas stutzeri GMG462. (c, d): Pseudomonas
stutzeri CMG463. Pseudomonas synxantha K2 is not shown but was similar to (a, b). (e, f): Cucurbit yellow vine disease bacterium, CMG480.
(g, h): Bacillus sp. K3. Bars are 500 nm.
834

Fig. 2. EDAX analysis of electron opaque areas. (a, c): P. stutzeri CMG463, (b, d, f): Cucurbit yellow vine disease bacterium. (e): resin matrix
around the cells (blank). (a, b): Cell edge regions. (c, d): regions inside cells. In (c) this corresponds to an electron opaque inclusion body as
visible in Figure 1d. In (f) this corresponds to the electron opaque extracellular particles as shown in Figure 1f.

Electron microscopy and analysis by EDAX
A study of the three Pseudomonas strains (CMG462
and 463, and K2) suggested two mechanisms of chro-
mate resistance and accumulation. P. stutzeri CMG462
and P. synxantha K2 showed some electron opaque
material at the outer cell surface (Figure 1b) but the lo-
calised concentration of this was below the sensitivity
of the EDAX technique. P. stutzeri CMG 463 showed
occasional intracellular deposits of electron opaque
material (Figure 1d) which gave a positive result for
Cr and also for P by EDAX (Figure 2c). Analysis
of the cell surface (Figure 2a) and background resin
around the cells (Figure 2e) gave no detectable Cr.
Detection of intracellular Cr suggests that the anionic
CrO2−4 can enter the cell but for a waste remediation
process a cell surface-localized deposit is preferable
because it may be possible to remove this and conserve
the biomass for re-use. Two strains showed extracellu-
lar electron opaque deposits. In accordance with the
toxicity tests (above) Bacillus sp. K3 showed lysis
of some cells (Figure 1h). The observation that the
inhibited culture reduced Cr(VI) similarly to the Cr-
tolerant cultures (Table 2) suggested that dead cells or
cell-lysates probably retained Cr(VI)-reductive ability.
A similar phenomenon was seen with strain CMG480
(Figure 1f). Analysis of the extracellular material of
CMG480 by EDAX clearly showed co-deposition of
Cr and P (Figure 2f) which were not visible inside
the cells (Figure 2d), at the cell surface (Figure 2b)

or in the extracellular resin matrix (Figure 2e). Ca and
Fe were co-deposited with Cr and P (Figure 2f); the
precipitate was probably a mixed metal phosphate. A
similar result was obtained by analysis of extracellular
precipitate of strain K3 (not shown). Strain J3 ap-
peared to remove Cr(VI) aerobically as well as anaer-
obically (Table 2) but was no better than P. stutzeri
CMG463, overall, in terms of total Cr removal, and
was not considered further.
Conclusions and wider implications
Substantial reduction of Cr(VI) occurred only anaer-
obically but growth at the expense of Cr(VI) as the
electron acceptor was not observed. Many bacterial
strains reduce chromate anaerobically or aerobically
but not both (Germain & Patterson 1974). Strain J3
could be an exception but the total Cr(VI) reduced was
low aerobically, and anaerobically this strain was not
exceptional. Strain K3 showed cellular damage and
sensitivity to 100 µM Cr(VI) and would probably be
insufficiently robust for industrial use.
The strains isolated from the metal contaminated
sites were the most tolerant to Cr(VI) and the P.
stutzeri removed Cr(VI) comparably to P. synxantha
(strain K2), which had not been previously metal-
stressed. The high Cr(VI) resistance of these isolates
makes them attractive for Cr-waste remediation, no-
taby because reduced Cr(VI) uptake was reported pre-
viously in a Cr-resistant strain of P. fluorescens (Bopp
& Ehrlich 1988).
Strain CMG480 is noteworthy. Analysis of the ex-
tracellular precipitate identified by EDAX suggests
a method of Cr deposition via reduction of Cr(VI)
to Cr(III) followed by precipitation of CrPO4 at the
expense of phosphate provided in the medium. It is
possible that CrO2−4 is taken up, reduced and effluxed
for precipitation exocellularly. Extracellular precipi-
tate was seen in all Cr(VI)-cultures of CMG 480 (3
experiments).
Strain CMG480 was virtually identical to cucur-
bit yellow vine disease bacterium over 308 bp of the
16S rRNA gene including variable regions. It could
be argued that this provides insufficient data for a
definitive identification. However the 308 bp frag-
ment was from the middle region of the 16S rRNA
gene which contains a key hypervariable region which
provides a substantial amount of discriminatory infor-
mation about the bacterium. In addition there are less
variable regions which also contribute to the identifi-
835
cation; such an approach has been used previously in
the identification of pathogenic isolates (Beswick et al.
1999).
The potential phytopathogenicity of the strain may
preclude its widespread industrial use. The reason for
its occurrence in a tannery environment is conjectural.
However as tannins and similar polyphenolic com-
pounds are important secondary metabolites of many
plants, a tolerance to relatively high concentrations
of such compounds would be a likely prerequisite for
a phytopathogenic bacterium; high concentrations of
such compounds building up from bark used in tradi-
tional tanning processes could impose a selection on
the environmental microflora towards species tolerant
of these plant products. It seems possible that this
putative plant pathogenic strain has evolved a mech-
anism(s) of resistance to free radical toxicity which, in
the present case, may be associated with the oxidative
effects of Cr(VI).
Industrial workplaces in Pakistan provide novel
enrichment environments for the natural selection of
novel strains of metal resistant bacteria because there
is little regulatory control over industrial emissions
and environmental burdens can be severe. The strains
described in this study, in addition to being capa-
ble of Cr(VI) reduction to an extent comparable to
good isolates from a natural environment, have other
potentially useful properties and numerous plasmids
(U. Badar & N. Ahmed, unpublished) that will be
reported elsewhere. The conclusion from this study
is that while the new strains may not have the high-
est rates of Cr(VI) reduction as compared to the best
strains reported in the literature, they combine Cr(VI)
resistance with its reduction and also have the potential
for removal of metal cations.
Acknowledgements
The authors acknowledge, with thanks, the support of
the British Council in support of U.B. P.P. was sup-
ported by the Government of Thailand and A.B. was
supported by grants to Prof N.L. Brown (University of
Birmingham, from BBSRC, EU and PHLS). They also
acknowledge, with gratitude, useful discussions with
the late Dr R.B. Pearce in aspects of plant pathology.
836
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