Chemical Engineering Journal 204206 (2012) 264271

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Chemical Engineering Journal

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Biosorption of copper(II) from aqueous solutions using volcanic rock

matrix-immobilized Pseudomonas putida cells with surface-displayed
cyanobacterial metallothioneins
Hong Ni a, Zhe Xiong a, Ting Ye b, Zhen Zhang c, Xiaofeng Ma a, Lin Li b,
a
b
c

School of Life Science, Hubei University, Wuhan 430062, China

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Key Laboratory of Subtropical Agricultural Resource and Environment, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China

h i g h l i g h t s
" A new volcanic rock-bacterium-metallothionein triplicate metal sorbent system.
1

" The maximum Cu(II) adsorption capacity was 22.23 mg g

" The system improved biosorption capacity, mass transfer and mechanical strength.
" Repeated adsorption/desorption operations showed high Cu sorption/recovery capacities.
" The regenerable and high-strength nature shows potential for continuous operations.

a r t i c l e

i n f o

Article history:
Received 28 January 2012
Received in revised form 6 May 2012
Accepted 8 May 2012
Available online 18 May 2012
Keywords:
Biosorption
Cu2+
Volcanic rock matrix
Pseudomonas putida
Metallothionein
Immobilization

a b s t r a c t
Biosorption using biomasses has been conrmed by certain studies to be an effective and economical
method in removing heavy metals from wastewaters. However, the usually insufcient physical strength
and regenerability of these biomaterials have restricted their use in continuous or large-scale processes.
The present study investigates the ability of volcanic rock matrix (VRM)-immobilized Pseudomonas putida
cells with surface-displayed metallothioneins to adsorb and recover Cu(II) ions from solutions. The
immobilization conditions of P. putida cells via VRM, as well as the factors affecting Cu(II) biosorption,
were optimized. Adsorption equilibrium was conducted for 60 min. The pseudo-second-order equation
was applicable to all sorption data over the entire time course. The biosorption equilibrium data was
described well by both Langmuir adsorption and Freundlich isotherm models. The maximum Cu(II)
adsorption capacity on VRM-immobilized biosorbent was 22.23 mg g1. The results from Fourier transform infrared spectroscopy indicated that recombinant P. putida cells were mainly responsible for the
biosorption of Cu(II) ions. The engineered system exhibited high capacities of both Cu(II) adsorption
and Cu(II) recovery in ve continuous cycles of adsorption/desorption, which revealed that the system
had high regenerability. Therefore, the VRM-immobilized biosorbent improved both mechanical strength
and performance, showed potential for further large-scale or continuous operations.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Heavy metal pollution in aqueous ecosystems is a serious environmental threat worldwide because these metals are toxic and
nondegradable. Copper ions are commonly found in various industrial efuents and wastewater from electroplating, mining, and metal-processing operations. They are difcult to remove from water
bodies and are ultimately accumulated into living tissues via food
chain [1,2]. Conventional physicochemical methods, such as
Corresponding author. Tel.: +86 27 8728 6952; fax: +86 27 8728 0670.
E-mail address: lilin@mail.hzau.edu.cn (L. Li).
1385-8947/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cej.2012.05.029

chemical precipitation, chemical oxidation and reduction, ion

exchange, electroplating, evaporative recovery, activated carbon
adsorption, and membrane separation processes [13], have been
proven uneconomical or ineffective especially when metal concentration is low [4]. Alternatively, biosorption using biomasses of
bacteria [5,6], fungi [7,8], yeasts [9,10], algae [11,12], and plant
[13,14], has been proven effective and economical in treating
wastewater containing heavy metals. Unfortunately, these biosorption processes have several technical drawbacks, such as the difculty in post-separation of biomass, low adsorption, poor physical
strength of biomaterials, and, particularly, the unavailability of
large-scale or continuous processes [15,16]. Therefore, studies

H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

focusing on cost-effective and innovative technologies for the

removal of heavy metals in aquatic systems are necessary.
Developments in cell immobilization systems for large-scale or
continuous operations have gained increasing attention for their
advantages over freely suspended biomass treatment systems.
These advantages include increased mechanical strength and resistance to chemical constituents, easy separation of cells and efuents,
high biomass loading and minimal clogging under continuous-ow
conditions, and efcient biosorbent regeneration [17]. Among these
immobilization methods, the microbial cell surface display strategy,
which utilizes the ability of surface-immobilized metal-xing motifs or proteins to adsorb and sequester heavy metals from aqueous
solutions, has been suggested as a promising approach due to its
effectiveness, regeneration, and good performance [18].
The surface display of metal-binding proteins on microbial cells
enables fast and goal-oriented binding reaction on cell surface,
thereby promoting reaction rate, eliminating mass transfer limitation, and minimizing the toxicity of heavy metals to living cells.
The regenerative and stable cell platform has been hypothesized
to be particularly suitable in retaining the activity of surfacedisplayed proteins. This hypothesis has been validated by several
previously developed bacterial surface display systems based on
metal-xing peptides or proteins, such as in Escherichia coli
[1922], Pseudomonas putida [23], Magnetospirillum magneticum
[24], Bacillus subtilis [25], and Staphylococcus species [26]. These
studies have demonstrated, in principle, the feasibility of biosorption toward various heavy metals. However, the behavior of these
systems under continuous processes, as well as the mechanisms
involved in the promotion of cell/metal recovery especially in open
water bodies, remain unknown. Therefore, the applications of
immobilized-cell systems with natural or synthetic materials for
cell/biomass attachment or entrapment have gained increasing
interest as a better alternative method for continuous bioprocesses, such as packed bed, uidized bed, and continuous stirred-tank
reactor [2,27].
Volcanic rocks are porous aluminosilicate minerals with crystalline anionic frames and are widely distributed on the Earths surface. Several previous studies have suggested the potential use of
volcanic rock matrix (VRM) as a directly used heavy metal adsorbent because of its properties, such as surface-exposed ionic charge,
porosity, wearability, and large specic surface area [2830]. However, very little information is available on the metal adsorption of
this natural material. Thus far, no effort has been made to develop a
VRM-immobilized bacterial system as a combined biosorbent for
the removal of heavy metals in aqueous solutions. The present
study aims to develop an efcient metal biosorption and recovery
system specic for continuous bioprocess of contaminated water.
P. putida, a highly solvent-tolerant bacterium, is robust and can
grow in highly contaminated habitats. In the present study, P. putida was genetically modied to display two tandem repeats of a
bacterial metal-binding protein (i.e., metallothionein). This protein
has been conrmed to exhibit metal-binding property towards
various metal ions, such as Zn, Cd, and Cu [31]. The engineered P.
putida strain was then immobilized through an economically available VRM material to construct a VRM-cell-metallothionein triplicate biosorbent system. The effect of initial pH, incubation time,
temperature, and initial Cu(II) concentration on the biosorption
capacity of VRM-immobilized P. putida cells was investigated.
The interactions between VRM and P. putida cells, as well as the
biosorbent system and Cu(II), were examined using Scanning
Electron Microscopy (SEM) and Fourier transform infrared (FTIR)
spectroscopy, respectively. After determining the biosorption
kinetics, the biosorption data were then analyzed using the Langmuir adsorption and Freundlich isotherm models. The Cu(II)
adsorption and recovery capacities in continuous ve cycles of
adsorption and desorption performance were investigated.

265

2. Materials and methods

2.1. Bacterial strains, plasmids, and culture conditions
Wild-type P. putida Migula CCTCC (China Center for Type Culture Collection) AB92019 was used as the host strain for the surface display of a cyanobacterial metallothionein [31]. E. coli
DH5a (TaKaRa Bio In.) cells were used to construct the recombinant plasmids. A previous PseudomonasE. coli shuttle expression
vector pYNP (unpublished) containing a constitutively-active promoter (PoprL) and the gene inaK-N (encodes the rst 175 aa of the
ice nucleation protein InaK of Pseudomonas syringae) [32], was used
to construct the recombinant plasmid pYN2S containing the fusion
gene of inaK-N and two tandem-aligned smtA (encodes the metallothionein protein) [31] (inaK-N/smtA-smtA). The recombinant
P. putida-transformed strain containing the plasmid pYN2S was
designated as YN2S.
All strains were generally cultured using the Luria-Bertani (LB)
medium following the standard procedures [33], unless otherwise
specied. Recombinant E. coli cells were cultured in an LB medium
containing 100 lg mL1 ampilillin (Amp) at 37 C, whereas recombinant P. putida strains were grown in an LB medium containing
500 lg mL1 carbenicillin (Cb) at 28 C, as previously described [34].
2.2. Volcanic rock material and P. putida immobilization
Industrial-grade VRM, purchased from Beijing Kangwen Technology Co., Ltd. (China), was used for the present study. Based on
the quality inspection report provided by the manufacturer, VRM
is mainly composed of the following compounds: SiO2 (53.82%),
Al2O3 (16.89%), Fe2O3 (9.08%), CaO (8.36%), Na2O (2.55%), MgO
(2.46%), K2O (2.30%), FeO (1.12%), and TiO2 (0.06%). The VRMs were
ground and ltered using sieves to obtain particles with sizes ranging from 0.45 mm to 0.90 mm. The selected VRMs were immersed
in 0.2 mol L1 HCl for 24 h, and thoroughly washed with a large
amount of deionized distilled water (dd H2O) until a neutral pH
is achieved. Subsequently, the VRMs were desiccated at 110 C
for 4 h and allowed to cool to room temperature until further use.
The overnight culture of P. putida YN2S cells (about 1  1010
cells mL1) was harvested, washed three times with sterile dd
H2O, and diluted to unit cell density (OD600 = 1.5) using phosphate-buffered saline (PBS) at pH 7.0 to immobilize P. putida using
VRMs. Cell density was measured at 600 nm using a UV/Vis spectrophotometer (DU-800 Nucleic Acids/Protein Analyzer, Beckman
Coulter). A total of 25 g of VRMs were added to 100 mL of YN2S cell
suspension in a 250-mL Erlenmeyer ask. The mixed suspension
was then incubated (30 C) with agitation at 60 rpm, unless otherwise stated. The OD600 values of the VRM-free suspensions were
periodically measured to determine the cell immobilization
capacity of VRMs. The immobilization-saturated biosorbents
(immobilization was usually allowed for 24 h) were used for the biosorp- tion of Cu(II) from the prepared Cu(II) solutions. Autoclaved
VRM materials were used to immobilize P. putida YN2S cells at a
time-course under sterile operations to count the number of P. putida live cells after VRM-immobilization. The VRM-immobilized biomasses were vigorously agitated in a large amount of sterile dd H2O.
The cells were then harvested via centrifugation and plated on LB
plates at 30 C for 24 h to facilitate growth. The colony forming units
(CFUs) growing on the LB plates were then counted.
2.3. Biosorption experiments
The experiments of biomass loading, optimized biosorption conditions, and kinetic biosorption were conducted with 20 mg L1,
10 mg L130 mg L1, and 10 mg L140 mg L1 Cu(II) ion solutions,

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H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

respectively, using VRM-free or VRM-immobilized P. putida YN2S

biomass via shake-ask incubation and agitated at 60 rpm (unless
otherwise specied). In all adsorption experiments, the residual
Cu(II) ion contents of the supernatant after biosorption through
VRM, VRM-free biomass, or VRM-immobilized biomass were determined using an atomic absorption spectrophotometer (HITACHI
180-80, Japan). Moreover, residual Cu(II) ion contents were used to
calculate the absolute Cu(II) biosorption capacity or removal efciency of Cu(II).
2.4. Scanning electron microscopy (SEM)
SEM observations of VRMs and VRM-immobilized biosorbent
were conducted using a JSM-6390/LV SEM (NTC, Japan). The dried
VRMs (with and without P. putida immobilization) were ground to
obtain powdered samples. A small amount of the prepared sample
was xed in 2.5% glutaraldehyde for 12 h, dehydrated using a series of gradient ethanol solutions, vacuum dried, plated in a sputter
coater for gold coating, and then observed under SEM following the
instructions provided by the manufacturer.

Biosorption experiments were conducted by varying the initial

Cu(II) ion concentration from 10 mg L1 to 40 mg L1. A total of
25 g of VRM-immobilized P. putida biomass biosorbent (wet
weight) was added to the asks containing 100 mL of Cu(II) solution. The adsorptions were performed using optimized conditions
(i.e., at pH 6, 30 C, and with 60 rpm shaking for 30 min). The residual Cu(II) ion contents of the supernatants were measured after the
adsorption reaction equilibrium was reached. The equilibrium of
Cu(II) adsorption capacity (qe, mg g1) was plotted, and adsorption
isotherm curves were established. Adsorption capacity was calculated using the following equation:

C i  C e
M

2.9. Data analysis

All data presented are the averages of at least three assays. Statistical analysis was carried out using SPSS 17.0 statistical software. P < 0.05 was considered statistically signicant.

2.5. Kinetics and isotherm analysis

qe

Adsorptions were performed following the procedures described

in Section 2.3. The biosorbents were harvested via centrifugation
after adsorption for 1 h, and were subjected to immediate desorption experiments by immersing into a solution with a pH of 2.0 and
agitated at 60 rpm for 30 min. The biosorbents were again harvested and washed three times with a large amount of sterile dd
H2O. Adsorption/desorption experiments were again performed
at cycle 2. A total of ve adsorption/desorption cycles were performed using the same batch of biosorbent. The live cells after each
adsorption/desorption cycle were counted following the procedures described in Section 2.2. Cu(II) adsorption and desorption
capacities were recorded by measuring the residual or redissolved
Cu(II) contents of corresponding solutions using the method described in Section 2.3. The relative adsorption efciency (setting
adsorption capacity at cycle 1 as 100%) and Cu(II) recovery efciency (the percentage of redissolved Cu in adsorbed Cu in the
same cycle) of each cycle were calculated.

where Ci denotes the initial Cu(II) ion concentration (mg g1), Ce denotes the equilibrium Cu(II) concentration (mg L1), and M denotes
the mass concentration amount of rock particle-immobilized cells
or cell-free rock particles.
2.6. Flow cytometry analysis
Recombinant P. putida YN2S cells were harvested, washed with
PBS (pH 7.2), and then incubated in PBS containing polyclonal
InaQ-N antiserum [35]. The subsequent procedures were performed as previously described [34]. For each experiment, up to
50,000 cells were measured for Cy5 uorescence using a single
laser system at an excitation wavelength of 635 nm and emission
wavelength of 670 nm.
2.7. Fourier transform infrared (FTIR) spectroscopy
The FTIR spectra of VRM-immobilized P. putida biosorbents before and after Cu(II) adsorption (for 24 h in 20 mg L1 initial copper
ion concentration) were analyzed using an FTIR spectrometer
(Spectrum One, Perkin-Elmer). All infrared spectra were recorded
within the 4004000 cm1 range. Sample preparation was generally performed as previously described [36].
2.8. Repeated adsorption/desorption operations in shake-ask trials
Each sample containing 25 g of VRM-immobilized biosorbent
(with saturated P. putida YN2S cells) was added in aqueous solutions containing Cu(II) at initial concentrations of 100 mg L1.

3. Results and discussion

3.1. Construction P. putida cell surface display system
The N-terminal domain of ice nucleation protein InaK from P.
syringae has been conrmed efcient in surface-immobilization
of target proteins [32]. The truncated InaK variant, with only N-terminal domain (InaK-N), was used as anchoring motif to construct a
surface projection system to detect cyanobacterial metallothionein
(SmtA) on the surface of P. putida AB92019. The recombinant plasmid pYN2S containing fusion gene with inaK-N and two tandem
repeats of smtA was used to express fusion protein InaK-N/
SmtA-SmtA during the entire growth phase of P. putida AB92019
cells, which was obtained from an E. coliPseudomonas dual-active
promoter PoprL (Fig. 1a).
Surface localization of fusion protein InaK-N/SmtA-SmtA on
P. putida YN2S cells was veried via ow cytometry analysis. Cy5
uorescence was detected for P. putida YN2S intact cells, and the
control strain P. putida AB92019 incubated with anti-InaQ-N antibody (InaK-N is identical with InaQ-N in the aa sequence) followed
by Cy5-conjugated secondary antibody. The mean percentage of
Cy5-uorescent P. putida YN2S and recombinant P. putida
AB92019 cells were 48.5% and 0.8%, respectively. These values
clearly indicated the surface immobilization of fusion protein in
recombinant P. putida YN2S cells, expressing the InaK-N/SmtASmtA fusion protein (Fig. 1b).
The absorbability of Cu(II) ions by engineered P. putida YN2S
strain was determined in shake-ask trials. The results showed
that the net adsorption capacity of P. putida YN2S strain was significantly increased by 35.5 nmol mg1 dry cells compared with that
of the recipient strain. This result indicated that the surface-displayed metallothionein SmtA was functional in host cells.
3.2. Preparation of VRM-immobilized biosorbent via immobilization of
recombinant P. putida YN2S
The capacity of VRMs to immobilize P. putida YN2S cells was
determined by monitoring the cell densities of VRM-free supernatants during incubation. The cell density of VRM-free suspension
after VRM immobilization showed a continuously decreasing pattern over the time course (0270 min) (Fig. 2), indicating that

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H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

(a)

525 bp

352 bp

inaK-N

smtA

NcoI

smtA

BamHI

NcoI

EcoRI

3.33, respectively), and temperatures (20, 30, and 40 C) in shakeask incubation was performed to increase the capacity of VRM
in immobilizing P. putida YN2S cells (Table 1). Table 2 shows that
the four factors exhibited an effect on the VRM-initiated immobilization of P. putida YN2S in the following order: temperature >
pH > initial cell concentration > shaking speed. The optimized factors correspond to the combination D2A2C1B1, indicating that
the optimized treatment conditions are as follows: temperature
of 30 C, pH value of 7, shaking speed of 60 rpm, and the largest
amount of initial cell concentration.
SEM was performed to examine the immobilization proles between VRM and P. putida cells (Fig. 3). The porous and rugged
structures of VRMs (Fig. 3b), as well as its ion-charged surfaces,
caused the immobilization of P. putida cells in VRMs, which constitute the immobilized P. putida biomass (Fig. 3c).

pYN2S
(5763 bp)

inaK-N BamHIgfp
EcoRI

PoprL

pYNP

rep

5614 bp
Amp

(b)

48.5%

0.8%
(i)

3.3. Cu(II) biosorption by immobilized biosorbent

(ii)

Fig. 1. Map of the recombinant plasmid pYN2S (a) and ow cytometric analysis of
P. putida YN2S cells (b). In (a), PoprL, constitutive promoter in P. putida; Amp,
ampicillin resistance gene; rep, plasmid replicon; inaK-N, N-terminal domain of
inaK; gfp, green uorescence protein gene; and smtA, cyanobacterial metallothionein protein gene. In (b), (i) P. putida YN2S; (ii) P. putida AB92019 (negative control).

VRMs can efciently adsorb P. putida YN2S cells, thereby immobilizing the added cells. Live cells of VRM-immobilized biomasses in
the same time course were counted to evaluate the inuence of
VRM-immobilization on cell viability. The cell counts increased
when cell densities of the supernatants immobilized by VRMs decreased (Fig. 2). This result suggested that VRM-immobilization did
not cause remarkable loss in cell life. The saturated immobilization
capacity of VRM was 1.35  109 cells g1 VRM.
An orthogonal test at four factors/three levels with regard to the
effects of pH values (4.0, 7.0, and 10.0), shaking speeds (60, 120, and
160 rpm), initial cell concentrations (1.7  108, 3.3  108, and
1.0  109 cells mL1, which correspond to OD600 of 0.57, 1.10, and
VRM + P. putida cells
VRM-immobilized alive cells
P. putida cells (control)

Table 1
L9-orthogonal test of P. putida YN2S immobilization by VRM.

1.5

1.5

1.2

1.2

0.9

0.9
0.6
0.6

-1

0.3
0.3
0.0
0

50

100

150

200

3.3.2. Optimization studies on biosorption conditions

The Cu(II) biosorption capacity of VRM-immobilized biosorbent
was determined under shake-ask incubation conditions. Fig. 5
shows that immobilized biosorbent exhibited a remarkable effect
on Cu(II) adsorption, whereas cell-free VRM material exhibited
only limited Cu(II) biosorption capacity. These results indicated
that the immobilized P. putida YN2S cells with surface-projected
metallothioneins mainly contributed to the biosorption of Cu(II)
ions.
Several previous studies on biomass-mediated heavy metal biosorption have revealed that pH, temperature, adsorption time, and
initial metal concentration can signicantly modulate biosorption
capacity [1,2,38]. An orthogonal trial at four factors (pH value,

1.8

CFUs (10 cells L )

Cell density of supernate (OD600)

1.8

3.3.1. Effect of different biomass loadings on Cu(II) biosorption

Diverse biomass loading affects metal biosorption capacity. Certain previous studies reported that an increase in biomass loading
increased metal biosorption [2]. However, several other studies reported that the increase in metal biosorption can lead to a decrease
in metal uptake [37]. Hence, the effect of different VRM-immobilized biomasses on Cu(II) removal efciency was veried using biosorption experiments. Fig. 4 shows that Cu(II) removal efciency
generally increased when biomass was increased from 42.2%
(1.2  108 cells L1, 30 min) to 94.5% (3.9  108 cells L1, 30 min)
in treating a solution containing 20 mg L1 Cu(II) ions. However,
Cu removal efciency did not increase as the biomass was further
increased from 3.9  108 cells L1 to 5.5  108 cells L1. These results indicated that VRM-immobilized biomass exhibited a certain
threshold to efciently remove Cu(II).

250

Immobilization time (min)

Fig. 2. Time-course effect of VRM on the immobilization of P. putida YN2S cells and
live cell counts after immobilization onto VRM.

Run

Factorsa
A

1
2
3
4
5
6
7
8
9

1
1
1
2
2
2
3
3
3

1
2
3
1
2
3
1
2
3

1
2
3
2
3
1
3
1
2

1
2
3
3
1
2
2
3
1

4.0
4.0
4.0
7.0
7.0
7.0
10.0
10.0
10.0

60
120
160
60
120
160
60
120
160

3.33
1.10
0.57
1.10
0.57
3.33
0.57
3.33
1.10

20
30
40
40
20
30
30
40
20

yib(%)

Levels

0.014
0.023
0.005
0.024
0.002
0.049
0.015
0.000
0.007

a
A: pH value; B: shaking speed (rpm); C: the P. putida YN2S concentration
(OD600); D: temperature (C).
b
Net OD600 value before and after immobilization by VRM, which is calculated by
the equation: yi = Initial OD600 of supernate residual OD600 of supernate after VRM
immobilization.

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H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

Table 2
Signicance analysis of the factors in the L9-orthogonal test of the P. putida YN2S
immobilization.
Aa
Mean

B
Mean

C
Mean

D
Mean

1
2
3
Rjb
Rank

1.41
2.34
0.27
2.07
2

1.76
1.13

0.63
4

2.10
1.34
0.35
1.75
3

0.17
2.89
0.96
2.72
1

Cu(II) removal efficiency (%)

Levels

100

A: pH value; B: shaking speed (rpm); C: biomass concentration; D: temperature

(C).
b
Range of the corresponding yi values of each factor.

90
80
70
60
50
40
1.2
2.4
3.9
5.5

30
20
10
10

20

30

40

50

60

-1

10 cells L
8
-1
10 cells L
8
-1
10 cells L
8
-1
10 cells L

70

80

90

Adsorption time (min)

Fig. 4. Effect of different P. putida YN2S biomass loadings on the Cu2+ removal
efciency.

1.0

-1

qe (mg g )

0.8

0.6

0.4

0.2
VRM-immobilized biomass
Cell-free VRM

0.0
15

30

45

60

75

Adsorption time (min)

Fig. 5. Effects of VRM-immobilized biomass on the biosorption capacity of Cu2+
ions.

effects on VRM-immobilized biosorbent (Table 3). The results

showed that all four factors affect Cu(II) removal efciency at
different degrees. pH and temperature had more signicant effect
on Cu(II) biosorption compared with other factors (Table 4). Moreover, the result was in good agreement with those of a previous
study on Cu(II) adsorption using the same metallothionein protein
[31]. The ANOVA result (Table 5) showed that the P values of the
four factors were all less than 0.01, indicating that the effect of four
factors on Cu(II) biosorption is very signicant. An optimized
immobilization condition for Cu(II) biosorption can be dened as
follows: pH value of 6, temperature of 30 C, initial copper ion concentration of 20 mg L1, and adsorption time of 60 min.

3.4. Kinetics of biosorption

Fig. 3. Representative SEM micrograph for P. putida YN2S cells (a), VRM (b), and
VRM-immobilized biosorbent (c).

adsorption time, temperature, and initial Cu2+ concentration) and

three levels (pH values: 6, 7, and 8; adsorption times: 15, 30, and
60 min; temperatures: 20, 30, and 40 C; and Cu2+: 10, 20, and
30 mg L1) was used to test the effect of saturated VRM-immobilized biosorbent (1.35  109 cells mL1) prepared using optimized
procedures and to investigate whether these factors have similar

Except for the initial Cu2+ concentrations, optimum biosorption

conditions (pH 6.0, 30 C, and 60 min for saturated adsorption) were
applied to varying Cu2+ concentrations, namely, 1040 mg L1. The
time-course patterns of Cu biosorption using VRM-immobilized biosorbent were determined to identify biosorption kinetics (Fig. 6).
The results showed that the biosorption of Cu2+ ions was rapidly
conducted for approximately 90% of the adsorption equilibrium
capacity within the rst 30 min, and maintained an increasing trend
until 60 min, when nal adsorption equilibrium was reached. Under

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H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

Table 3
L9-orthogonal test of Cu2+ removal efciency by the VRM-immobilized biosorbent.

1
2
3
4
5
6
7
8
9

1
1
1
2
2
2
3
3
3

1
2
3
1
2
3
1
2
3

1
2
3
2
3
1
3
1
2

1
2
3
3
1
2
2
3
1

6
6
6
7
7
7
8
8
8

15
30
60
15
30
60
15
30
60

20
30
40
30
40
20
40
20
30

10
20
30
30
10
20
20
30
10

Levels

Cu2 + removal efciency (%)

1.25

31.6
45.1
42.0
34.9
42.6
33.9
27.5
17.3
31.5

1.00
1

Factorsa

qe (mg L )

Run

10 mg L
1
20 mg L
1
30 mg L
1
40 mg L

0.75

0.50

0.25

A: pH value; B: adsorption time (min); C: temperature (C); D: initial Cu2+

concentration (mg L1).

0.00
0
Table 4
Signicance analysis of the factors in the L9-orthogonal test of Cu2+ removal efciency
by VRM-immobilized biosorbent.
Levels

Aa
Mean

B
Mean

C
Mean

D
Mean

1
2
3
Rj
Rank

39.56
37.12
25.45
14.11
1

31.32
34.48
35.99
4.67
3

27.59
37.33
37.20
9.74
2

35.22
35.48
31.42
4.06
4

A: pH value; B: adsorption time (min); C: temperature (C); D: initial

Cu2+concentration (mg L1).

10

20

30

40

50

60

70

80

90

Adsorption time (min)

Fig. 6. Biosorption equilibrium curves for VRM-immobilized biomass on different
initial Cu2+ ion concentrations.

Table 6
Fitting parameters of the pseudo-second order kinetics.
C0 (mg L1)

k2 (g mg1 min1)

qe (mg g1)

40
30
20
10

0.2824
0.3603
0.2964
0.3995

1.2314
0.9253
0.6379
0.3299

Table 5
Analysis of variance for selected factorial model.

Levels

Squares

df

Mean
square

F valuea

P value

pH
Adsorption time
Temperature
Initial concentration
of Cu2+
Error

682.59
68.61
374.99
61.87

2
2
2
2

341.30
34.31
187.50
30.94

604.6565
60.77657
332.1762
54.8061

5.9  1010
5.0  106
6.4  109
7.5  106

5.08

0.56

Langmuir model assumes a monolayer sorption of a solute from

a liquid solution, which can be expressed as follows:

Q max K s C e
1 K s Ce

or presented in linear form as follows:

** symptomizes signicant difference.

optimized conditions, VRM-immobilized biosorbent obtained a

maximum Cu(II) adsorption capacity of 22.23 mg g1 in 60 min.
The pseudo second-order equation was used to model the sorption data over the time course. The pseudo-second-order Lagergren
equation based on the adsorption equilibrium capacity is generally
expressed as follows [2]:

dqt
k2 qe  qt 2
dt

Qe

where k2 is the second-order biosorption rate constant (g mg1

min1) and qe and qt are the amounts of metal sorbed per unit
weight of the sorbent at equilibrium and time t, respectively
(mg g1).
The time-course sorption prole was tted using Eq. (2). The results showed that the pseudo-second order model (R2 = 0.9902
0.9915) was able to describe the sorption kinetics better (Table 6).
The equilibrium adsorption capacity qe increased when the initial
concentration of copper ions was increased; however, k2 did not signicantly change.
3.5. Biosorption isotherms
The biosorption isotherm data of copper ions onto VRM-immobilized biomass at different initial concentrations of copper ions
were analyzed using the Langmuir and Freundlich equations. The

1
1
1

Q e Q max K s Q max C e

where Qe is the equilibrium of heavy metal ion concentration on the

biosorbent (mg g1), Qmax is the maximum biosorption capacity of
the sorbent (mg g1), Ce is the liquid phase equilibrium concentration of heavy metal ions (mg L1), and Ks is the saturation constant
(mg L1).
The Freundlich model is based upon sorption on a heterogeneous surface, which can be expressed as:

Q e K f C e1=n

or in linear form as follows:

In Q e In K f 1=nIn C e

where Qe and Ce are the same parameters indicated above, and Kf

and n are the Freundlich constants denoting the adsorption capacity
and adsorption intensity, respectively.
Based on the linearized Langmuir and Freundlich plots (Fig. 7),
Qmax, Ks, and the Freundlich constants (Kf and n) can be calculated
as follows: Qmax = 22.23, Ks = 0.0386, Kf = 0.8164, and n = 1.0618.
The results showed that the obtained experimental data described
well with both the Langmuir adsorption model and Freundlich isotherm model (R2 > 0.99).

270

H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

Measured values
Langmuir isotherm model (a)
Freundlich isotherm model (b)

1.6

1.5899

1.2323

0.9091

0.6492

881

1534
2

a: y = 1.165x + 0.045; R = 0.9988

0.4
0.3553

b: y = 0.9418x - 0.2028; R = 0.9952

0.0
0.6

1451 1235
1050
1392

2963
2926

0.8

0.9

1.2

1.5
1

1.8

2.1

2+

Ce (mg L Cu )

3298

Transmittance (%)

qe (mg g )

1.2

VRM (control)
VRM + P. putida YN2S
2+
VRM + P. putida YN2S + Cu
P. putida YN2S
2+
P. putida YN2S + Cu

1658

2968
2924

1546

3296

3294

1655
2968
2925

1545
1655 1399

2925

The regeneration and reusability of the biosorbent can denitely confer desirable potential for continuous biosorption processes [2]. Cyanobacterial metallothionein has been revealed to
have Cu2+-dissociation characteristics under low pH conditions
(as low as pH 2.35 for half dissociation) [31]. Thus, Cu adsorption
and desorption efciency of VRM-immobilized biosorbent in a continuously repeated adsorption and desorption operation was further examined. Fig. 9 shows that the system retained relatively
high Cu(II) adsorption and Cu(II) recovery although both Cu(II)
adsorption and desorption capacities of VRM-immobilized biomass
exhibited a decreasing prole at cycle 1 to cycle 5. This result was
further veried by higher adsorption efciencies of 94.2%, 88.1%,
83.1%, and 74.9% at cycles 25 (the cycle 1 was set as 100%),
respectively. Moreover, the result exhibited comparable Cu(II)
recovery efciencies of 98.1%, 96.7%, 95.3%, 93.1%, and 91.2% at cycles 15, respectively. In addition, no signicant decline of P. putida
1
For interpretation of color in Figs. 1, 2, 48, the reader is referred to the web
version of this article.

1048

578
578

578

1048

3.6. FTIR spectra analysis

3.7. Cu adsorption and desorption in continuously repeated

adsorption/desorption cycles

747

1645
3294

1033

4000

3600

3200

2800

2400

2000

1600

1200

800

400

Wavenumber (cm )
Fig. 8. FTIR spectra for P. putida YN2S biomass and VRM-immobilized P. putida
YN2S biomass before and after Cu2+ biosorption.

Biosorption
Desorption

1.5

1.2
2+

qe (mg g Cu )

0.9

-1

The FTIR spectroscopic analysis of P. putida YN2S cells and VRMimmobilized biosorbent before and after Cu(II) biosorption was performed to verify which component of the sorbent (represented by
corresponding band groups) is involved in Cu(II) binding. Fig. 8
shows that the spectra displayed peaks in the 33002800 cm1 region, attributed to the alkyl groups like CH3, CH2, and CH [36]. Specically, the peaks at 2924, 2925, 2926, 2963, 2968, 3292, and
3296 cm1 represent the CH stretching vibrations belonging to P.
putida cells, but not to the VRM. The peaks at 1544 and 1545 cm1
represent NH stretching vibration, and the peak at 1534 cm1 is
the NH stretching vibration peak from P. putida YN2S cells after
Cu(II) biosorption. Therefore, the variance of the wavenumber is
the consequence of Cu(II) binding to NH band groups (Fig. 6, indicated by the arrow on the purple curve). Furthermore, the corresponding peaks of both VRM-immobilized biosorbent (red1 curve
in Fig. 8) and VRM-immobilized biosorbent after Cu(II) binding
(brown curve in Fig. 8) are 1545 and 1537 cm1, respectively. This
result indicated that P. putida cells with surface-displayed metallothioneins may have signicant role in Cu(II) binding. The peak
associated with Cu(II) binding is 1534 cm1, and not 1537 cm1.
This result is due to the superposition at the corresponding wave
number position with that of the VRM peak.

742

1537
1655

3293

Fig. 7. Langmuir isotherm plots of P. putida YN2S immobilized on VRM.

1309
1450 1239
1401
1049

0.6

0.3

0.0
1

2+

Cu adsorption/desorption cycles
Fig. 9. Cu2+ adsorption capacity and Cu2+ recovery capacity at continuously
repeated adsorption and desorption cycles.

live cell count from VRM-immobilized biosorbents was found

when the biosorbents were treated at pH 2.0 for 30 min after 5-cycle operations (data not shown). This result reected the strong
viability of P. putida cells under such treatments, thereby strongly
suggesting the good regeneration efcacy and potential use of the
engineered system in a continuous Cu(II) adsorption and desorption operation.
Numerous studies have documented the removal of heavy metals from wastewater using naturally occurring or recombinant
microorganisms or other biomasses (for reviews, see [15,17,27]).
Several previously described systems exhibited higher levels of
Cu adsorption with maximum adsorption capacities at 34.1 mg g1
[3], 48.4 mg g1 [39], and 71.2 mg g1 [40]. Unfortunately, these
systems have to be validated in large-scale or continuous processes
where a high physical strength of biomaterials and feasible regeneration are often required. Although the absolute Cu adsorption
capacity of the current system is not very high due to insufcient
immobilization efciency, the signicant physical strength and
regenerative characteristics, as well as intrinsic features, such as

H. Ni et al. / Chemical Engineering Journal 204206 (2012) 264271

the absence of mass transfer limitation, high adsorption using surface-immobilized metal-binding protein, and easy desorption and
metal recovery procedures, indicated the potential use of this system in a continuous Cu process or other metal bioadsorption processes. On the other hand, modifying the pore sizes and surface ion
charges is necessary to allow a more efcient and more coordinated immobilization of engineered cells. The development of
capacity-promoted VRM-P. putida cell-metallothionein systems
is now one of our primary research goals.
4. Conclusions
The present study demonstrated, for the rst time, that VRMimmobilized P. putida cells using surface-projected cyanobacterial
metallothioneins can remove copper ions from aqueous solutions.
The adsorption equilibrium was conducted for 60 min under optimized conditions. The maximum adsorption capacity was
22.23 mg g1 for Cu(II). The results from the FTIR analysis indicated
that engineered P. putida cells with surface-projected metallothioneins exhibited a signicant role in Cu(II) biosorption. The engineered system exhibited substantial activities without
signicantly decreasing both Cu(II) adsorption capacity and Cu(II)
recovery efcacy in ve continuous cycles of adsorption/desorption
operations, indicating the favorable performance and regenerability
of the system. Therefore, the VRM material efciently immobilized
P. putida cells and improved the physical strength of the sorbent.
This system can be especially valuable for further large-scale or continuous biosorption processes.
Acknowledgments
The authors are grateful to Dr. Yu Peng, and Dr. Yun Chen for
their technical help. The present study was supported by grants
from the National Natural Science Foundation of China (Grant
Nos. 31070111 and 40830527) and the Fundamental Research
Funds for the Central Universities (Program No. 2012MBDX011),
and was partially supported by a Grant from the State Key Laboratory of Agricultural Microbiology (Grant No. AMLKF201002).
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