Biotechnology Letters 23: 61–65, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

61

Chromate reduction by Microbacterium liquefaciens immobilised in

polyvinyl alcohol

P. Pattanapipitpaisal, N.L. Brown & L.E. Macaskie∗

School of Biosciences, The University of Birmingham, Edgbaston B15 2TT, UK
∗ Author for correspondence (Fax: +44-121-414-6557; E-mail: L.E. Macaskie@bham.ac.uk)

Received 26 September 2000; Revisions requested 4 October 2000; Revisions received 24 October 2000; Accepted 24 October 2000

Key words: bioremediation of Cr(VI), chromate reduction, immobilised cells, Microbacterium liquefaciens

Abstract
A polyvinyl alcohol-based immobilisation technique has been utilised for entrapping the newly-isolated chromate-
reducing bacterium, Microbacterium liquefaciens MP30. Three immobilisation methods were evaluated: PVA-
nitrate, PVA-borate and PVA-alginate. Chromate reduction was studied in batch and continuous-flow bioreactors,
where the beads maintained integrity during continuous operation. PVA-borate and PVA-alginate cell beads showed
a higher rate and extent of chromate reduction than PVA-nitrate cell beads in batch experiments. With the former
100 µM Cr(VI) was removed within 4 days, while only 40 µM Cr(VI) was removed using the latter, and with no
increase in Cr(VI) removal subsequently. Cell activity was maintained during immobilisation but the rate of Cr(VI)
removal by immobilised cells was only half that of an equivalent mass of free cells. Using PVA-alginate cell beads
in a continuous-flow system, chromate removal was maintained at 90–95% from a 50 µM solution over 20 days
without signs of bead breakdown.

Introduction fication of Cr(VI)-contaminated solution in batch or

Chromium in wastewaters from modern industries,
such as stainless steel manufacture, leather tanning,
textile manufacture, electroplating and alloy prepa-
ration, is a toxic waste which may be consequently
discharged into the environment. Its hexavalent form
[chromate: Cr(VI)] is highly water-soluble, highly
toxic, mutagenic and probably carcinogenic (Venitt
& Levy 1974, Petrilli & DeFlora 1977); trivalent
chromium, [Cr(III)] is less soluble and less toxic than
Cr(VI) (Cary 1982, Komori et al. 1989). Therefore,
the reduction of Cr(VI) to Cr(III) can be a useful ap-
proach for the removal of chromate from wastewater
and the environment. A wide variety of bacteria have
been reported to be involved in Cr(VI) reduction under
aerobic or anaerobic conditions, e.g., Pseudomonas
fluorescens LB300 (Bopp & Ehrlich 1988), Enter-
obacter cloaceae HO1 (Wang et al. 1989), and Bacil-
lus sp. (Wang & Xiao 1995).
Many groups have investigated the potential of
these and other Cr(VI) reducing bacteria for detoxi-
continuous bioreactors (DeLeo & Ehrlich 1993, Fujie
et al. 1994, Shen & Wang 1994). However, the bio-
logical treatment of Cr(VI)-contaminated wastewater
can be difficult because of Cr(VI) toxicity, which may
cause cell damage and loss of activity. Immobilising
techniques to entrap cells in gel beads can be used in
order to protect the cells. Several support materials
have been reported for cell immobilisation, such as
natural gels (e.g., agar, carrageenan) and synthetic ma-
trices (e.g., polyurethane, polyethylene glycol, poly-
acrylamide). Natural gel matrices are biodegradable
and subject to abrasion (Østgaard et al. 1993, Lee-
nen et al. 1996) whereas synthetic gels have bet-
ter mechanical properties and are not biodegradable.
Polyvinyl alcohol (PVA) is a synthetic gel which is
widely used in cell immobilisation because it is non-
toxic and is very stable (Østgaard et al. 1993, Leenen
et al. 1996). It has also been used for enzyme immo-
bilisation; Imai et al. (1986) entrapped glucoamylase,
invertase, and cellulase into PVA membrane by cross-
linking using UV irradiation and reported that the
62
immobilised enzymes showed good stability on re-
peated usage. Ariga et al. (1987) found that a repeated
freeze thaw technique produced elastic rubber-like gel
beads with high strength. Hashimoto & Furukawa
(1987) developed a new method for immobilization of
activated sludge by using PVA cross-linked with boric
acid. The beads showed high gel strength with no loss
of biological activity. This method was also economic
in comparison with agar or acrylamide gel entrap-
ment methods. Similarly Hanaki et al. (1994) used
this approach to entrap acetate-utilising methanogenic
bacteria, with maintenance of methanogenic activ-
ity for one year. Wu & Wisecarver (1992) entrapped
phenol-degrading Pseudomonas sp. in PVA gel beads
using the PVA-boric acid method with the addition of
calcium alginate to eliminate bead agglomeration. The
cell beads showed high elasticity and phenol was de-
graded in a fluidized bed reactor over 2 weeks. Chang
& Tseng (1998) established a technique using PVA
cross-linked with sodium nitrate for entrapment of Al-
caligenes eutrophus, which showed a high level of
denitrification activity in a batch system.
A previous investigation (Badar et al. 2000) fo-
cused on the ability of isolates obtained from metal-
contaminated sites to reduce Cr(VI). One isolate, not
described previously and identified as a strain of Mi-
crobacterium liquefaciens, was resistant to Cr(VI) and
also effected bioreduction of CrO2−
4 at the expense of
acetate as the electron donor. The current study fo-
cuses on the ability of this strain to achieve sustained
removal of Cr(VI) in a flow-through immobilised cell
reactor.
Materials and methods
Bacterial strain
The strain used in this study was a chromate-resistant
and chromate-reducing bacterium, Microbacterium
liquefaciens MP30. The strain was isolated from a
Pakistani tannery sample by A.N. Mabbett and identi-
fied using 16S rRNA analysis as described previously
(Badar et al. 2000)
Media
Microbacterium liquefaciens MP30 was grown and
maintained in Luria broth (Oxoid). For chromate
reduction in batch experiments, an acetate-minimal
medium was used. This was made in 2 parts, au-
toclaved separately (121 ◦ C, 15 min), and mixed at
room temperature prior to use. Part 1 consisted of 1 g
NH4 Cl, 0.2 g MgSO4 · 7H2 O, 0.001 g FeSO4 · 7H2 O,
0.001 g CaCl2 · 2H2 O, 5.0 g sodium acetate 3H2 O,
0.5 g yeast extract in 990 ml distilled water (adjusted
to pH 7 with 1.0 M NaOH). Part 2 consisted of 0.5 g
K2 HPO4 in 10 ml distilled water, pH 7. The carrier so-
lution for continuous experiments using immobilised
resting cells comprised 5 g l−1 sodium acetate 3H2 O
in 20 mM MOPS/NaOH buffer, pH 7.
Chemicals
Polyvinyl alcohol (PVA) (2000 degree of polymeriza-
tion, 140% saponification and 86–89 mol% of hy-
drolysation) was obtained from Fluka Ltd. Sodium
alginate (medium viscosity) was obtained from Sigma.
A stock solution of sodium chromate (1 M) was
sterilised by filtration. All other chemicals were of
analytical grade.
Cr(VI) analysis
Chromate-reducing activity was assayed as the de-
crease of chromate with time using the Cr(VI)-
specific colorimetric reagent S-diphenylcarbazide
(DPC). Diphenylcarbazide (0.25%) was prepared in
acetone/H2SO4 to minimize deterioration (Urone
1955) as follows. DPC (0.025 g) was dissolved in
9.67 ml acetone and 330 µl 3 M H2 SO4 was added.
The reaction mixture was set up in an Eppendorf tube
containing the following: 200 µl sample or standard
sodium chromate solution, 400 µl 20 mM MOPS
buffer (pH 7), 33 µl 3 M H2 SO4 , 40 µl 0.25%
diphenylcarbazide, and 327 µl distilled water. Spec-
trophotometric measurements were made immediately
at 540 nm using 1.5 ml cuvettes, 1 cm path length.
Immobilization of cells
Microbacterium liquefaciens MP30 was cultured aer-
obically with shaking at 30 ◦ C for 24 h in Luria
broth. Cells were harvested by centrifugation and re-
suspended in 1 ml Luria broth. Polyvinyl alcohol (1 g)
and sodium alginate (0.16 g) were mixed in 19 ml of
deionized water. The solution was heated to 60 ◦ C to
completely dissolve the PVA. The PVA-alginate solu-
tion was cooled to 35 ◦ C and then mixed thoroughly
with the cell suspension. The final concentration of
the mixture was 5% (w/v) PVA, 0.8% (w/v) sodium
alginate, and 7.5% (w/v) wet weight cells for batch ex-
periments and increased to 10.5% (w/v) for continuous

experiments. Immobilized cell beads were prepared by
three methods:
Method 1: The PVA-alginate cell suspension was
extruded as drops, using a 10 ml disposable plastic
syringe, into 200 ml of solution comprising 50% (w/v)
sodium nitrate and 2% (w/v) CaCl2 · 2H2 O, and im-
mersed for 2 h to form PVA-nitrate cell beads (Chang
& Tseng 1998).
Method 2: PVA-borate beads were prepared by ex-
truding the mixture into 200 ml of saturated boric acid
and 2% (w/v) CaCl2 · 2H2 O solution and immersing
for 24 h (Wu & Wisecarver 1992).
Method 3: PVA-alginate cell beads were prepared
by extruding the mixture into 200 ml of 2% (w/v)
CaCl2 · 2H2 O solution and immersing for 24 h. All
beads were washed 3 times with 200 ml distilled water
and stored in deionized water at 4 ◦ C until further use.
Chromate reducing activity of PVA-immobilized cell
beads in batch culture
Chromate reduction was compared between whole
cells and an equivalent mass of cells in the three
types of cell beads in batch experiments. One milliliter
of cell beads (75 mg wet weight cells ml−1 gel) or
75 mg wet weight cells were suspended in a 12 ml
anaerobic vial with 10 ml of acetate-minimal medium,
containing 100 µM sodium chromate. The vials were
sealed with rubber serum caps, crimped and flushed
thoroughly with O2 -free N2 for 5 min via a needle
prior to Cr(VI) addition. The vials were incubated
at 30 ◦ C with shaking (100 rpm). Samples (200 µl)
were taken at intervals via the needle and the residual
chromate concentration in sample supernatants was
measured using DPC. Control experiments (cell-free
beads) were processed under the same conditions. The
experiments were duplicated on 2 separate occasions
with the data plotted as means ± SEM. Control experi-
ments (cell-free beads, or cells lacking electron donor)
were done in parallel.
Continuous chromate-reduction by PVA-alginate cell
beads
Continuous chromate reduction was carried out in
25 ml flow-through columns containing PVA-alginate
cell beads (15 ml). Sodium chromate solution (50 µM
Cr(VI) in 20 mM MOPS/NaOH buffer pH 7) was fed
continuously at a flow rate of 0.95 ml h−1 from the
bottom of the column at room temperature.
63
Fig. 1. Chromate reduction by free cells and by cells entrapped in
PVA beads prepared by different methods. Immobilised cell beads
were prepared and challenged with Cr(VI) as described in the text
and compared with Cr(VI) reduction by an equivalent mass of free
cells (). Filled symbols: cell-free beads. Open symbols: beads
containing biomass. , : PVA-nitrate beads. N, 4, PVA-borate
beads. , #, PVA-alginate beads.
Results and discussion
Characteristics of the immobilized cell beads and
chromate reduction tests in batch suspension
Microbacterium liquefaciens MP30 was entrapped in
PVA-gel using three methods in order to ascertain
which method was the most suitable for cell immobili-
sation and chromate reduction. After the cell mixtures
were dropped into the solution, spherical gel beads
(3 mm in diameter) were formed without agglom-
eration and exhibited rubber-like properties. This is
probably due to the natural acidic polysaccharide be-
ing rapidly cross-linked with polyvalent metal ions,
here calcium (or alternatively barium, aluminum or
ferric ions or other polyvalent cations), conferring me-
chanical strength (Rosevear et al. 1987). Wu & Wise-
carver (1992) reported that the lowest concentration
of sodium alginate that would prevent bead agglomer-
ation was 0.02%. In the present study the beads were
crosslinked using Ca2+ , and were also prepared using
borate and nitrate since these had been successfully
used in previous denitrification tests (Chang & Tseng
1998).
The time course of chromate reduction with whole
cells and the three types of PVA-cell beads is shown in
Figure 1. Whole cells reduced 100 µM sodium chro-
mate to 2 µM within two days, while an equivalent
biomass in PVA-borate cell beads and PVA-alginate
64
cell beads completely reduced chromate in four days.
Cell-free beads or beads challenged in the absence of
electron donor (acetate) did not reduce Cr(VI). The
chromate reducing activity of PVA-nitrate cell beads
was only 25% of that of free cells in the same pe-
riod and after four days the residual sodium chromate
concentration was constant at 55.6 µM. These results
showed that M. liquefaciens MP30 could be success-
fully entrapped in PVA beads, but that good chromate
reducing activity was observed in PVA-borate cell
beads and PVA-alginate cell beads only, with no im-
provement using borate and with inhibition in the
presence of nitrate. The nitrate could be acting as a
competing electron acceptor; Komori et al. (1989) also
demonstrated that reduction of chromate by Enter-
obacter cloacae HO1 was diminished in the presence
of sodium nitrate. Overall, the chromate-reducing
activity was slower than for whole cells, probably
attributable to diffusional constraints on the electron
donor (acetate) and chromate, which would have af-
fected their entry into the gel. Boric acid treatment
gave no improvement in activity, therefore subsequent
tests used PVA-alginate cell beads only.
Continuous reduction of Cr(VI) using an immobilized
cell reactor
Microbacterium liquefaciens MP30 entrapped in
PVA-alginate beads was packed into columns and
challenged continuously with 50 µM Cr(VI) and ac-
etate in MOPS/NaOH buffer for 20 days. The results
(Figure 2) show that chromate concentration in the
outlet was significantly decreased after startup of the
experiment and was maintained at approximately 90–
95% chromate removal. Cell-free beads, or beads
challenged in the absence of electron donor, did not
remove Cr(VI). No bead breakdown or agglomeration
was observed throughout.
Conclusion
The chromate-reducing bacterium, Microbacterium
liquefaciens MP30 was successfully entrapped in
PVA-gel beads. The rubber-like cell beads maintained
integrity in a flow-through system over 20 days. Chro-
mate removal was observed in batch and continuous
experiments, indicating that the immobilization tech-
nique has little effect on biological activity. Thus,
it is proposed that immobilised cells of M. lique-
faciens MP30 could have future application in the
bioremediation of chromate-contaminated wastewater.
Fig. 2. Continuous removal of Cr(VI) by Microbacterium liquefa-
ciens immobilised in PVA-alginate beads. Beads were prepared and
challenged with Cr(VI) under continuous flow as described in the
text. : Cell-free beads. #: Biomass-supplemented beads.
Acknowledgements
This work was supported by the Government of Thai-
land and by grants from the BBSRC to the LEM and
NLB laboratories. P.P. thanks Drs J. Hobman and S.
Kidd for assistance and useful discussions.
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