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Marine Pollution Bulletin 49 (2004) 974–977

Accumulation of hexavalent chromium by anexopolysaccharide producing marine Enterobacter cloaceae

Anita Iyer, Kalpana Mody *, Bhavanath Jha

Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

Abstract

An exopolysaccharide producing Enterobacter cloaceae (AK-I-MB-71a) was tested for its Cr (VI) tolerance. This isolate was not

only resistant to this heavy metal but also showed enhanced growth and exopolysaccharide production in the presence of Cr (VI) at

25, 50 and 100 ppm concentrations. XRF analysis of both the biomass as well as the exopolysaccharide revealed that a sum total of

about 60–70% chromium was accumulated by this bacterium. This indicated that this organism could prove to be a potential can-

didate in the field of bioremediation with respect to chromium removal.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Bacteria; Bioremediation; Enterobacter cloaceae; Exopolysaccharide; Hexavalent chromium; Marine

1. Introduction

Chromium (Cr) is an essential trace metal for livingorganisms, but, its high toxicity, mutagenicity and car-

cinogenicity render it hazardous at very low concentra-

tion (Cheung and Gu, 2003). As the application of

chromium is extensive in various industries like

chrome-plating, wood preservation and alloy formation,

chromium–associated pollution is of increasing concern.

Apart from traditional physicochemical treatments, a

number of biological assays using microorganisms havebeen studied and developed to remedy chromium con-

taminated water. The two major processes being investi-

gated are adsorption of metals on biological materials

(i.e. biosorption) including cells of microorganisms and

plants (Davis et al., 2003), and dissimilatory reduction

of metal ions from higher valent state to lower one

(i.e. biotransformation) through enzymatic reaction or

indirectly with metabolite produced (Lee et al., 2000).

0025-326X/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.marpolbul.2004.06.023

* Corresponding author. Tel.: +91 278 256 7760; fax: +91 278 256

6970.

E-mail address: salt@csir.res.in (K. Mody).

Gadd (1988) and Brierley (1990) have described many

ways in which bacteria, fungi and algae can take up

toxic metal ions. Heavy metal ions can be entrappedin the cellular structure and subsequently biosorbed

onto the binding sites present in the cellular structure.

This method of uptake is independent of the biological

metabolic cycle and is known as biosorption or passive

uptake. The heavy metal can also pass into the cell

across the cell membrane through the cell metabolic

cycle. This mode of metal uptake is referred as active up-

take. The metal uptake by both active and passivemodes can be termed as bioaccumulation.

As a transition metal, chromium exists in a wide

range of oxidation states from �2 to +6, while the dom-

inant species in nature are hexavalent chromium Cr (VI)

and trivalent chromium Cr (III). Cr (VI) is about hun-

dred times more toxic and soluble than Cr (III). Reduc-

tion of Cr (VI) to Cr (III) will not only reduce the

toxicity of chromium acting on living organisms but alsohelp in precipitating chromium out at neutral pH for

further physical removal.

A number of Cr reducing bacteria have been studied.

These include Pseudomonas, Escherichia and Bacil-

lus and a number of sulfate reducing bacteria like

A. Iyer et al. / Marine Pollution Bulletin 49 (2004) 974–977 975

Desulfovibrio, Desulfomicrobium and Desulfotomaculum

(Cheung and Gu, 2003). Besides, other species of bacte-

ria have also been investigated for their chromium

reducing property such as Shewanella alga (Guha

et al., 2001) and Pyrobaculum islandicum (Kashefi and

Lovley, 2000).Metal chelation by unicellular algae has been re-

ported by Kaplan et al. (1987). Biosorption of heavy

metals by brown algae is also known since a long time.

This includes sorption of heavy metals like Cd+2, Cu+2,

Zn+2, Pb+2, Cr+3 and Hg+2 by a number of cell wall con-

stituents such as alginate and fucoidan. Most of the re-

search in this area has been carried out on marine and

soil algae (Davis et al., 2003).Extracellular polysaccharides produced by several

classes of algae as well as bacteria. The need for eco-

nomical, effective and safe methods for the removal of

heavy metals from wastewater has directed attention

to extracellular polysaccharide (EPS) produced from

algae, bacteria, fungi and yeast. Few reports are availa-

ble on metal chelation using exopolysaccharides from

bacteria. The adsorption of heavy metals by EPS isnon-metabolic, energy independent and can be caused

by interaction between metal cations and negative

charge of acidic functional groups of EPS. Here the

EPSs are recommended as surface-active agents for hea-

vy metal removal because of their extensive capacity.

Biosorption of Pb, Cu, Zn has been reported by a novel

acidic polysaccharide produced from Bacillus firmus

(Salehizadeh and Shojaosadati, 2003). Similarly, a cellbound polysaccharide produced by the marine bacte-

rium Zoogloea spp. is also known to act as an adsorbent

of metal ions like cadmium, ferrous, lead and chromium

(Kong et al., 1998).

Today better cost effective and consistent alternatives

to reduce the concentration of such toxic compounds

are being explored to meet legislative standards. This re-

search was carried out to explore the potential of an exo-polysaccharide producing bacterium, E. cloaceae to

accumulate chromium ions.

2. Materials and methods

The exopolysaccharide producing marine bacterium

was isolated from a marine sediment sample from thewest coast of India and identified as E. cloaceae using

the API system for identification of bacteria. E. cloa-

ceae was grown for 16 h in Seawater medium (Matsuda

et al., 1992) and used as an inoculum (approximately

108 cellsml�1). One millilitre of inoculum was added

to 250 ml of Seawater medium containing different con-

centrations of Cr (VI), i.e., 25, 50 and 100 ppm, as

potassium dichromate. Each of the sets was preparedin duplicates. One set of medium without Cr (VI)

was also inoculated and kept as control. After inocula-

tion the flasks were incubated at room temperature for

80 h.

The bacterial cells were pelleted out of the medium by

centrifuging the culture broth at 10,000 rpm for 20 min.

The effect of chromium on biomass production (and in

that way tolerance of the culture to Cr) was recordedin terms of dry weight of the cells. Exopolysaccharide

production was also recorded for each set. The tradi-

tional method of alcohol (isopropanol) precipitation

of the supernatant was used for the recovery of the

exopolysaccharide. The precipitates thus obtained were

subjected to air-drying and dry weight of the exopoly-

saccharide was measured. The chromium content in

the bacterial biomass as well as in the exopolysaccharideproduced by it was measured using XRF spectrometry

(Model: Bruker AXS Spectrometer S4 Pioneer XRF).

It was ascertained that all the samples subjected to

XRF analysis were moisture free.

3. Results and discussion

After 80 h of incubation, luxuriant growth of E. clo-

aceae was observed in all the flasks (control as well as

those containing Cr (IV)). As seen in Table 1, an in-

crease in concentration of chromium, in fact, caused

an increase in biomass production (which can be seen

by the increase in dry weight of the cell pellets). A sim-

ilar pattern was also observed in case of the exopolysac-

charide production. This indicated that the culture wasnot only tolerant to hexavalent chromium, but its pres-

ence stimulated exopolysaccharide production. It was

also observed that the stimulating effect was increased

with an increase in chromium concentration.

The XRF data revealed the presence of chromium in

cell pellet as well as in the exopolysaccharide. This indi-

cated that pellet as well as exopolysaccharide could

accumulate chromium.In case of cell pellet, it was observed that an increase

in chromium concentration did not influence the per-

centage chelation. This remained almost constant i.e.,

approximately 26%. Whereas in the case of exopolysac-

charide an increase in chelation from 37% to 49% was

observed as the concentration of chromium was in-

creased from 25 to 100 ppm. Thus the cumulative metal

chelation by the cell pellet as well as by exopolysaccha-ride was 63%, 68% and 75% in the presence of 25, 50 and

100 ppm of chromium, respectively. Chromium was not

detectable, both in the cell pellet and exopolysaccharide

of the control sample.

Tolerance and accumulation of hexavalent chromium

by two marine seaweed associated fungi is reported by

Vala et al. (2004) who observed that both the isolates

could accumulate more than 25% of the chromium sup-plied. Removal of metal ions from aqueous solution by

a polysaccharide producing Bacillus firmus was reported

Table

1

Dry

weightandchromium

contentin

E.cloaceae

Serialno.

Crconcentration(ppm)

E.cloaceae

Cellpellet

EPS

Totalchelation(%

)

Dry

weight(g)

Crcontent(m

gg�1)

Crchelation(%

)Dry

weight(g)

Crcontent(m

gg�1)

Crchelation(%

)

10(C

ontrol)

0.3655

ND

a0

0.9739

ND

a0

0

225

0.4924

2.6

25.62

1.1045

1.7

37.55

63

350

0.7189

3.6

25.88

1.1526

3.7

42.64

68

4100

1.133

4.6

26.00

1.1816

8.3

49.04

75

aND––non-detectable.

976 A. Iyer et al. / Marine Pollution Bulletin 49 (2004) 974–977

by Salehizadeh and Shojaosadati (2003) who observed

the influence of pH, metal concentration and polysac-

charide concentration on metal uptake. Utilization of

a cell bound polysaccharide produced by a marine bac-

terium, Zoogloea sp. for metal adsorption was studied

by Kong et al. (1998). Chelating properties of extracellu-lar polysaccharides from a unicellular alga, Chlorella sp.

was reported by Kaplan et al. (1987). They observed

that composition of polysaccharide, especially the pres-

ence of uronic acid, influenced the metal uptake.

In the present investigation, the exopolysaccharide

from E. cloaceae contains high uronic acid and sulfate

(unpublished data) which may be responsible for its

metal chelation property. This culture could grow luxu-riantly at 25–100 ppm chromium concentration which

indicated that it possessed high tolerance to various con-

centration of chromium. At 100 ppm chromium, this

organism could successfully chelate approximately 75%

of chromium which is of great significance. High toler-

ance and high chelating ability of this culture make it

a potential candidate as a heavy metal scavenger with re-

spect to chromium. Though the mechanism of chro-mium chelation is not studied, this type of chelation

can be classified under biosorption as mentioned by

Valls and de Lorenzo (2002). According to them, carb-

oxylic and sulfate groups present in acidic exopolysac-

charide works as a non-specific ion exchange material

which may render chelating property. However, the

non-specific nature of this particular exopolysaccharide

can be established by only after conducting similar typeof studies using other metals.

Acknowledgments

The authors thank Dr. P.K. Ghosh, Director, CSM-

CRI, for providing facility and encouragement. The firstauthor also thanks CSIR for providing financial support

in the form of a fellowship.

References

Brierley, C.L., 1990. Bioremediation of metal-contaminated surface

and ground water. Geomicrobiology Journal 8, 499–503.

Cheung, K.H., Gu, J.D., 2003. Reduction of chromate (CrO4�2) by an

enrichment consortium and an isolate of marine sulfate-reducing

bacteria. Chemosphere 52, 1523–1529.

Davis, T.A., Volesky, B., Mucci, A., 2003. A review of the biochem-

istry of heavy metal biosorption by brown algae. Water Research

37, 4311–4330.

Gadd, G.M., 1988. Accumulation of metal by microorganisms and

algae. In: Rehm, H. (Ed.), Biotechnology: a complete treatise, vol.

6B, Special Microbial Process, vol. 4. Verlagsgesellschaft, Wein-

heim, VCH, pp. 401–403.

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.

A. Iyer et al. / Marine Pollution Bulletin 49 (2004) 974–977 977

Kaplan, D., Christiaen, D., Arad, S., 1987. Chelating properties of

extracellular polysaccharides from Chlorella spp. Applied and

Environmental Microbiology 53 (12), 2953–2956.

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 56, 2268–2270.

Kong, J.Y., Lee, H.W., Hong, J.W., Kang, Y.S., Kim, J.D., Chang,

M.W., Bae, S.K., 1998. Utilization of a cell-bound polysaccharide

produced by the marine bacterium Zoogloea sp.: New biomaterial

for metal adsorption and enzyme immobilization. Journal of

Marine Biotechnology 6, 99–103.

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 and Biotechnology 54, 445–448.

Matsuda, M., Worawattanamateekul, W., Okutani, K., 1992. Simul-

taneous production of muco- and sulfated polysaccharides

by marine Pseudomonas. Nippon Susian Gakkaishi 58 (9), 1735–

1741.

Salehizadeh, H., Shojaosadati, S.A., 2003. Removal of metal ions from

aqueous solution by polysaccharide produced form Bacillus firmus.

Water Research 37, 4231–4235.

Vala, A.K., Anand, N., Bhatt, P.N., Joshi, H.V., 2004. Tolerance and

accumulation of hexavalent chromium by two seaweed associated

aspergilli. Marine Pollution Bulletin 48 (9–10), 983–985.

Valls, M., de Lorenzo, V., 2002. Exploiting the genetic and biochem-

ical capacities of bacteria for the remediation of heavy metal

pollution. FEMS Microbiology Reviews 26, 327–338.