Biochemical Engineering Journal 36 (2007) 54–58

Biosorption of Cr(III) and Cr(VI) onto the cell surface

of Pseudomonas aeruginosa
So-Young Kang a , Jong-Un Lee b , Kyoung-Woong Kim a,∗
a Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST),
Gwangju 500-712, South Korea
b Department of Civil, Geosystem and Environmental Engineering, Chonnam National University,

Gwangju 500-757, South Korea

Received 6 September 2005; received in revised form 16 May 2006; accepted 12 June 2006

Abstract
Biosorption of the chromium ions Cr(III) and Cr(VI) onto the cell surface of Pseudomonas aeruginosa was investigated. Batch experiments
were conducted with various initial concentrations of chromium ions to obtain the sorption capacity and isotherms. It was found that the sorption
isotherms of P. aeruginosa for Cr(III) were described well by Langmuir isotherm models, while Cr(VI) appeared to fit Freundlich models. The
results of FT-IR analysis suggested that the chromium binding sites on the bacterial cell surface were most likely carboxyl and amine groups. The
bacterial surface of P. aeruginosa seemed to engage in reductive and adsorptive reactions with respect to Cr(VI) biosorption.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Biosorption; Pseudomonas aeruginosa; Chromium; FT-IR spectroscopy; Bioremediation; Wastewater treatment

1. Introduction is known to cause allergic skin reactions and cancer [7]. As a

Toxic heavy metals are frequently contained in wastewaters
produced by many industrial processes, such as those employed
in the electroplating, metal finishing, metallurgical, tannery,
chemical manufacturing, mining, and battery manufacturing
industries [1,2]. The existence of heavy metals in the environ-
ment represents a very significant and long-term environmental
hazard. Even at low concentrations these metals can be toxic to
organisms, including humans. In particular, chromium is a con-
taminant that is a known mutagen, teratogen and carcinogen [3].
Chromium is generally found in electroplating and metal finish-
ing industrial effluents, as well as sewage and wastewater treat-
ment plant discharges [4]. Among the several oxidation states
(di, tri, penta and hexa), trivalent chromium, Cr(III), together
with the hexavalent state, Cr(VI), can be the main forms present
in aquatic environments [5]. Chromate (CrO4 2− ) is the prevalent
species of Cr(VI) in natural aqueous environments, and is the
major pollutant from chromium-related industries [6]. Although
Cr(III) is less toxic than Cr(VI), long-term exposure to Cr(III)
∗ Corresponding author. Tel.: +82 62 970 2442; fax: +82 62 970 2434.
E-mail address: kwkim@gist.ac.kr (K.-W. Kim).
result, the total chromium level in effluent is strictly regulated
in many countries. In the USA, the concentration of chromium
in drinking water has been regulated with a maximum level of
0.1 mg/l for total chromium [8].
The removal of heavy metals from aqueous solutions has
therefore received considerable attention in recent years. How-
ever, the practical application of physicochemical technology
such as chemical precipitation, membrane filtration and ion
exchange is sometimes restricted due to technical or economical
constraints. For example, the ion exchange process is very effec-
tive but requires expensive adsorbent materials [9,10]. The use
of low-cost waste materials as adsorbents of dissolved metal ions
provides economic solutions to this global problem and can be
considered an eco-friendly complementary [11,12]. At present,
emphasis is given to the utilization of biological adsorbents for
the removal and recovery of heavy metal contaminants.
Biomass involving pure microbial strains has shown high
capacities for the selective uptake of metals from dilute metal-
bearing solutions. Several investigations have reported that
Pseudomonas aeruginosa displays efficiency for metal uptake
[13–15]. Chang and Hong [16] found that the amount of mer-
cury adsorbed by a P. aeruginosa biomass sample (180 mg Hg/g
dry cells) was higher than that bound to a cation exchange

1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2006.06.005
 S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 54–58
resin (100 mg Hg/g dry resin). Hu et al. [17] reported that P.
aeruginosa strain CSU showed the highest affinity and maximal
capacity for uranium (100 mg U/g dry weight), and that it was
also competitive when compared to commercial cation exchange
resins.
Previous studies on biosorption using microorganisms have
generally focused on the removal of metal ions from aqueous
solutions. However, a few studies were undertaken to interpret
and establish the mechanisms involved in metal ion binding.
Furthermore, the binding sites for chromium have not been
specifically identified.
The objective of the present work was to assess the potential
of P. aeruginosa for the biosorption of chromium. The func-
tional groups involved in chromium biosorption were identified
using FT-IR analysis. These results would contribute to a better
understanding of biosorption phenomena and aid in the devel-
opment of potential biosorbents that possess high capacities for
heavy metal uptake from aqueous environments.
2. Materials and methods
2.1. Preparation of the biomass
P. aeruginosa PAO1 (courtesy of Dr. Beveridge, University
of Guelph, Canada) was used as a biosorbent in these exper-
iments. P. aeruginosa is a bacterium commonly isolated from
various environmental sources such as soil, water and plant sur-
faces [18]. The bacteria were cultured following the procedure
outlined in Kang et al. [19]. In brief, the cells were grown aer-
obically with agitation in a growth medium (TSB; Trypticase
Soy Broth, Difco) at 37 ◦ C and set at 180 rpm in a shaking incu-
bator. After reaching the mid-exponential growth phase of cells
(OD600 nm = 1.0), the cells were harvested by centrifugation at
6000 rpm for 15 min and washed three times with deionized
water. Prepared biomass was used as adsorbent in batch experi-
ments within 1 h under nutrient-absent conditions, and therefore
metabolic processes were minimized or negligible [19,20].
2.2. Surface characterization
To determine the nature of functional groups on the cell
wall for metal sorption, potentiometric titration of an aqueous
cellular suspension and IR spectrum analysis of the solid phase
biomass were performed. For the potentiometric titration, the
biomass was washed three times with deionized water, and the
biomass suspension (3 g dryweight/l) was then potentiometri-
cally titrated with prepared 0.05N NaOH using potentiometric
titrator (Metrohm Ltd., Switzerland). To identify functional
groups using the IR spectrum procedure, the bacteria were
pelleted by centrifugation at 6000 rpm for 15 min and dried
overnight at 50 ◦ C in an oven. One milligram of finely crushed
particles of biomass was encapsulated in 300 mg of KBr. The
infrared spectra of the biomass were recorded in KBr disks
using an FT-IR spectrophotometer (Jasco, Japan) from 900
to 4000 cm−1 under ambient conditions. Infrared spectra of
P. aeruginosa with or without adsorbed chromium ions were
obtained.
55
2.3. Biosorption experiments
Thirty-milliliter solutions of chromium were prepared with
Cr(NO3 )3 ·9H2 O and K2 Cr2 O7 (Sigma–Aldrich Co.) at different
initial concentrations without adjusting pH. A biomass sample
of 75 mg (dry-weight basis) was added separately into the each
flask and the solutions were then agitated in a shaking incubator
at 180 rpm and 25 ◦ C. Five-milliliter samples were obtained and
filtered through a syringe filter (0.2 ␮m pore size) after 10 h
(well above the adsorption equilibrium time of about 5 h [19]),
and dissolved metals in the filtrates were determined. Specific
metal uptake by the cells was then calculated from the initial
and final concentrations of chromium ions.
2.4. Analysis of chromium ions
Total concentrations of chromium in the samples were mea-
sured by an inductively coupled plasma atomic emission spec-
trometer (ICP-AES, Thermo Jarrell Ash, USA). The analyses
of Cr(VI) in aqueous samples were performed using a UV–vis
spectrophotometer (Shimadzu, Japan) at 540 nm after complex-
ation with 1,5-diphenylcarbazide. The analytical method for
chromium is outlined in Standard Methods [21].
3. Results and discussion
3.1. Surface characterization of P. aeruginosa
The bacterial species used in this study was P. aeruginosa,
a gram-negative aerobic species that is commonly found in
near-surface systems [18]. The cell wall of this bacterium is
composed of peptidoglycan and teichoic acids, and possesses
carboxyl, phosphoryl, hydroxyl, and amino functional groups at
the surface [22]. Before a study of the biosorption of metal, an
investigation of the surface characteristics of P. aeruginosa was
required to understand the mechanisms of metal biosorption.
The titration curve of protonated P. aeruginosa shows that the
bacteria provide a substantial buffering capacity that is depen-
dent on the types of functional groups present in the biomass
(Fig. 1). The biomass titration results revealed that there are at
least two main functional groups on the surface of bacteria. To
evaluate the properties of functional groups, we can consider a
chemical model incorporating the reactions between the groups
of bacteria and the protons in the solution. The reactions and
equilibrium constants (Ka , Kb ) of certain functional groups may
be defined as follows:
[A− ][H+ ]
AH ↔ A− + H+ , Ka = (1)
[AH]
[B− ][H+ ]
BH ↔ B− + H+ , Kb = (2)
[BH]
where [A− ], [B− ] and [AH], [BH] represent the concentration of
deprotonated and protonated surface species, respectively, and
[H+ ] represents the activity of protons in solution. We solved
the equilibrium constants of each surface functional group using
the surface equilibrium program FITEQL 4.0 [23]. The negative
56 S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 54–58
Fig. 1. Biomass potentiometric titration: P. aeruginosa was washed by distilled
ionized water, and then potentiometrically titrated with 0.05N NaOH. Symbols
are experimental data and the curve is the prediction of the model.
logarithm of the equilibrium constants pKa and pKb were found
to be 5.2 and 9.5, respectively, which correspond to carboxyl
(4 < pKa < 6) and hydroxyl (9 < pKa < 11) groups [24,25].
An FT-IR analysis was carried out to confirm the type of
functional groups. As shown in Fig. 2, the FT-IR spectrum of P.
aeruginosa displays a number of absorption peaks, indicating
the complex nature of the biomass examined. The broad absorp-
tion peak around 3430 cm−1 is indicative of the existence of
–OH and –NH stretching, thus showing the presence of hydroxyl
and amine groups on the bacteria. The band at 2938 cm−1 can be
assigned to the –CH stretch. The absorption bands at 1660 cm−1
(mainly C O stretch) and 1551 cm−1 (mainly –NH stretch) can
be attributed to the amide I and amide II bands of amide bond
due to the protein peptide bond. The moderately strong bands
at 1080 cm−1 could be assigned to the –CN stretching vibration
of the protein fractions. It was clear that the carboxylate ions
gave rise to two bands: C O stretch at 1468 and 1414 cm−1 .
A band at about 1246 cm−1 , representing –SO3 stretching, was
observed in the FT-IR spectrum of P. aeruginosa.
Fig. 2. IR spectrum in solid phase of the lyophilized P. aeruginosa in KBr disk.
Fig. 3. Biosorption isotherms of Cr(III) onto P. aeruginosa. The biomass was
contacted with metal solution for 10 h at 25 ◦ C and 180 rpm in shaking incubator.
The line was produced by using the MINEQL+.
3.2. Biosorption isotherms of chromium
Experimental biosorption isotherms of chromium ions were
obtained to evaluate sorption capacity and understand the pattern
of chromium biosorption by P. aeruginosa.
Sorption experiments of Cr(III) were carried out at a vari-
ety of initial concentrations, and revealed that the amount of
specific Cr(III) uptake increased in response to augmentation
of the initial metal concentration of each metal for the range
0–5 mmol/l of equilibrium concentration (Fig. 3). However, the
amount of Cr(III) adsorbed onto the cells remained constant
beyond 150 ␮mol/l of equilibrium concentration and was unaf-
fected by increases in the initial input metal concentration, with
the sorption profile showing a clear plateau. Sorption isotherms
displaying such a pattern indicated that Cr(III) uptake by P.
aeruginosa cells reached equilibrium, with the mechanisms of
uptake being saturated. The isotherms derived from these exper-
iments fit the Langmuir isotherm model to a reasonable degree.
The linearized forms of Langmuir adsorption isotherms were
used to evaluate the sorption data and are represented as follows
[26]:
1 1 1 1
= + (3)
Γ Γmax Kads Γmax [A]
where Γ is the amount of adsorbed metal ion per mass of
adsorbent (␮mol/g) and Γ max is the maximum adsorption capac-
ity of metal ion (␮mol/g); A is the equilibrium concentration
of metal ions in solution (␮mol/l) and Kads the equilibrium
adsorption constant (l/␮mol). Linear transformation of the plots
in Fig. 3 using the Langmuir model showed that the maxi-
mum amount of Cr(III) adsorbed by P. aeruginosa cells was
136 ␮mol/g of dry biomass weight and that Kads was equivalent
to 1.1 × 10−6 l/␮mol.
The biosorption of Cr(VI) ions from aqueous solution by P.
aeruginosa was also studied in a batch system (Fig. 4), with
results showing that biosorption of Cr(VI) by P. aeruginosa
includes two processes. The first process is the reduction of
Cr(VI) to Cr(III) by reductive functional groups according to
 S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 54–58
Fig. 4. Biosorption isotherms of Cr(VI) onto P. aeruginosa. The biomass was
contacted with metal solution for 10 h at 25 ◦ C and 180 rpm in shaking incubator.
The lines were produced by using the MINEQL+.
the following reaction:
Cr2 O7 2− + 16e− + 14H+ ↔ 2Cr 3+ + 7H2 O
A number of previous experimental studies of bacterial Cr(VI)
reduction reported the enzymatic reduction as a product of
metabolic activity [27,28], and most of these focused on the
requirement of external electron donors for reduction to occur
[29,30]. However, Fein et al. [31] suggested that the Cr(VI)
reduction is not dependent on cell metabolism and that some
component of the cell wall serves as the electron donor for the
reduction reaction. We have also investigated the reduction of
Cr(VI) to Cr(III) in the absence of externally supplied electron
donors.
In the second process, chromium ions are removed from
wastewater using the adsorptive functional groups of P. aerug-
inosa. The adsorptive property is due to the electrostatic inter-
action between the charged surfaces of bacteria and chromium
ions. The experimental sorption isotherms of Cr(VI) are rep-
resented by the Freundlich sorption isotherm in Fig. 4. The
linearized form of Freundlich is represented by the following
equation [26]:
log Γ = log m + n log[A]
where m represents the Freundlich constant and n is the measure
of the nonlinearity involved. Values of m and n were, respec-
tively, found to be 80.8 and 1.03 as the total adsorbed chromium
ions; 38.6 and 1.02 as the adsorbed Cr(III) in Cr(VI) biosorp-
tion to P. aeruginosa. The difference of concentration between
total and hexavalent chromium was taken as the concentration of
trivalent chromium. These results show that the bacterial func-
tional groups of P. aeruginosa can act as reductive and adsorptive
sites in metal biosorption.
3.3. FT-IR spectra of chromium-loaded P. aeruginosa
To confirm the difference between functional groups in rela-
tion to biosorption of Cr(III) and Cr(VI), the FT-IR study was
57
Fig. 5. FT-IR spectra of P. aeruginosa prepared in KBr disks: (a) pristine; (b)
(4) Cr(III)-loaded; (c) Cr(VI)-loaded bacteria.
carried out using chromium-loaded P. aeruginosa. The absorp-
tion spectrum of chromium-loaded biomass (at pH 5) was
compared with that of pristine biomass. The chromium-loaded
biomass was washed, dried and powdered after biosorption of
chromium ions under the same conditions used in the preparation
of pristine biomass. A change of absorption bands can be seen
when comparing the FT-IR spectra of pristine and chromium-
loaded biomass (Fig. 5).
Fig. 5(b) shows the changes in the spectrum of the biomass
after sorption of Cr(III) by P. aeruginosa. An interesting phe-
nomenon was the sharp decrease in the band intensity at
1414 cm−1 corresponding to C O stretching after metal bind-
ing. On the basis of the change of the band, it was reasonable
to assume that the peak value suggested the chelating (biden-
tate) character of the Cr(III) biosorption onto carboxyl groups
[32]. The structure of the metal bound to carboxyl ligands on
the bacteria is likely to take the following form [33]:
(5)
In the case of Cr(VI)-loaded bacteria, the spectral analysis
of P. aeruginosa before and after metal binding indicated that
–NH is involved in Cr(VI) biosorption (Fig. 5(c)). There is a
substantial decrease in the absorption intensity of –NH bands
at 1660 and 1551 cm−1 . The broad overlapping range for N–H
and O–H stretching in the range 3200–3600 cm−1 also presents
some changes, but it is difficult to determine the group that causes
the shift. These amino groups are protonated at pH 3 [34] and
the negatively charged chromate ions become electrostatically
attracted to the positively charged amines of the biomass cell
wall. Similar to Cr(III)-loaded bacteria, the characteristic peak
58 S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 54–58
of C O stretching at 1414 cm−1 decreased and indicated Cr(III)
binding after reduction of Cr(VI) to Cr(III).
4. Conclusions
This study shows that P. aeruginosa can be applied to
chromium-contaminated wastewater. The sorption of chromium
ions by P. aeruginosa was modeled well by the Langmuir and
Freundlich sorption isotherms. The data of potentiometric titra-
tion indicated the presence of two major functional groups on the
cell wall, corresponding to pKa values of 5.2 and 9.5. FT-IR spec-
trometry showed bindings of chromium ions were dominated by
complexation to the carboxyl and amine groups on the biomass
surface. In the case of Cr(VI) biosorption of P. aeruginosa, the
reduction and adsorption of Cr(VI) occurred coincidently in an
abiotic process. This phenomenon is environmentally significant
because most bacteria in the subsurface exist in nutrient-poor
or -absent conditions under natural conditions [31]. This study
shows the potential for the use of P. aeruginosa for chromium
recovery in various water and wastewater treatment applications,
and highlights the efficacy of using biological agents for the
remediation of polluted aqueous environments.
Acknowledgements
This research was supported by the Gwangju Institute of
Science and Technology (GIST) Research Fund and National
Research Laboratory Project (Arsenic Geoenvironment Lab.) to
K.-W. Kim.
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