Journal of Industrial and Engineering Chemistry 40 (2016) 118–127

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Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec

Removal of Pb(II) in a packed-bed column by a Klebsiella sp. 3S1 biofilm

supported on porous ceramic Raschig rings
Antonio Jesús Muñoz, Francisco Espı́nola, Encarnación Ruiz *
Department of Chemical, Environmental and Materials Engineering, University of Jaén, 23071 Jaén, Spain

A R T I C L E I N F O A B S T R A C T

Article history: The purpose of this work was to confirm the effectiveness of the bacterium Klebsiella sp. 3S1 for the
Received 18 February 2016 removal of lead ions from aqueous solution in a fixed-bed column. The results provided evidence that
Received in revised form 15 June 2016 the bacterial biofilm formation on the support clearly improved the lead biosorption capability of the
Accepted 17 June 2016
system. FTIR, SEM and TEM techniques provided factual evidence of the role of the bacterial biofilm on
Available online 24 June 2016
lead biosorption and the different mechanisms involved, such as cell surface binding and cytoplasmic
bioaccumulation.
Keywords:
The dynamic behavior of the column in continuous mode was well-established through breakthrough
Lead
Biosorption
curves, with experimental data fitting using different mathematical models, especially the Yang model.
Biofilm Up to 70 mg of lead was retained per gram of microbial biofilm in each cycle without loss of biosorption
Biofilter capacity compared to previous work in batch mode. In addition, the bacteria maintained their
effectiveness when the cells were inactivated after acid desorption cycles. The column was reused four
times after biosorbent regeneration with an elution efficiency of 99% showing that it is appropriate for
industrial applications. The use of a Klebsiella sp. 3S1 biofilm immobilized on a porous support in
commercial biofilters for the removal of heavy metals from wastewater could be a competitive option.
ß 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
reserved.

Introduction an ecofriendly behavior, the regeneration of the biosorbent for

Currently, the pollution caused by heavy metals is an important
issue because of their persistence and toxicity. Lead is non-
biodegradable and can accumulate in living tissues. Thus, it can be
concentrated in the food chain and can be easily absorbed by the
human body. Even a very small amount of lead can cause severe
physiological or neurological damage, and it can also promote
cancer [1]. Therefore, its removal from wastewater prior to
discharge into the environment is necessary [2,3]. The current
European Directive 2008/105/EC [4] establishes a maximum
permissible concentration of 0.0072 mg/L in surface waters.
Various chemical and physicochemical methods for the treat-
ment of wastewater containing lead wastes are known such as
chemical precipitation, electrochemical reduction, ion exchange,
and adsorption. Biosorption has emerged as one of the most
promising techniques. It is generally preferred for the removal of
lead due to its high efficiency, cost effectiveness, easy handling and
the availability of different adsorbents [5–8]. Moreover, it presents
* Corresponding author. Tel.: +34 953212779.
E-mail address: eruiz@ujaen.es (E. Ruiz).
multiple uses is easily performed, and it shows selectivity toward
different metals [9].
The conventional treatments present several limitations that
include excessive usage of chemicals, expensive plant require-
ments and high operational costs [10]. For this reason, the
development of a robust, highly competitive process with good
performance and high efficiency, based on the ability of certain
biological materials to accumulate and eventually transform
metallic species from effluents by physicochemical or metabolic
reactions, is strongly recommended.
In the planktonic state, bacteria have a high surface-area-to-
volume ratio that can provide a large contact interface. This allows
the interaction with metals from the surrounding environment.
Moreover, bacteria usually grow as a biofilm if a suitable support
for cell attachment is present [11]. When bacteria are grouped to
form biofilms, they can produce macromolecules usually called
extracellular polymeric substances (EPS). EPS are metabolic
products of bacteria and are mainly composed of polysaccharides,
proteins, humic substances and uronic acids, which contain several
functional groups such as carboxyl, phosphoric, amine and
hydroxyl groups. They are also associated with the organic matter
present in the effluent to be treated [12,13].

http://dx.doi.org/10.1016/j.jiec.2016.06.012
1226-086X/ß 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127 119

According to Percival et al. [14], a biofilm is composed of Ceramic Raschig rings used as support were obtained from
microbial cells immobilized in a matrix of extracellular polymers Guangdong Boyu Group CO., LTD. (China). They are made of kaolin
acting as an independent functional ecosystem that is home- clay with an inner diameter of 5 mm, outer diameter of 11 mm and
ostatically regulated. The capability of the microorganisms to form height of 11 mm. The chemical composition obtained by X-ray
biofilms is considered an adaptive strategy that can increase the fluorescence (XRF) is given in Table 1. The analysis revealed that
chances of survival. The biofilm formation mechanism is very the ceramic Raschig rings support is composed mainly of silica and
complex and is divided into several stages that also involve the alumina (78.5% of the total weight).
expression of genes that are inactive in the planktonic form This support was chosen among several supports that were
[15]. The biofilm composition depends on multiple variables tested because of its good capability to retain bacterial biomass. It
(microbial species, pH, temperature, support type, flow rate, the favors the hydrodynamics of the fixed-bed column, and it also has
type of effluent, etc.), although it can be considered that its principal the advantage of high resistance to acids.
components are water (up to 97%) and EPS that form a stable matrix
in which other macromolecules, proteins, DNA and waste products Batch biosorption assays
are included [16]. Sometimes biofilms are an industrial and sanitary
problem. However, they can be used under controlled conditions to The Raschig rings were placed inside 250-mL Erlenmeyer flasks
remove heavy metals from industrial effluents. This is due to the containing 150 mL distilled water. They were sterilized at 120 8C
polyanionic character of the EPS present in the matrix, which is a for 20 min to release the air inside the pores. Then, they were
significant advantage [17]. It has been found that biofilms are able placed in 100 mL of TSB (30 g/L) medium at 30 8C for 72 h and
to form metallic precipitates as sulfides, phosphates, carbonates, 150 rpm in an incubator inoculated with Klebsiella sp. 3S1 during
oxides and hydroxides. They can also develop mechanisms of ion the exponential phase of the growth curve. After the biofilm
exchange and even incorporate metals into the cytoplasm [18]. formation over the Raschig rings, the support was washed with an
There is great interest in the use of microbial biofilms for electrolyte solution of 0.1 M NaCl.
practical application in biofilters for wastewater treatment. For The biosorption isotherms were obtained at 25 8C. The
this purpose, an inert support is needed to immobilize the bacteria. experiments were performed with 250-mL Erlenmeyer flasks
The use of natural media such as kaolin as a support can be a containing 100 mL of Pb(II) solution and 4.5 g of Raschig rings
suitable option due to their availability and low cost. Kaolin is a 1:1 covered with biofilm. The Pb(II) concentrations tested ranged
aluminum silicate structure with the ideal formula of between 5 and 160 mg/L. Agitation was maintained at a constant
Al2Si2O5(OH)4 [19]. It has been used for the adsorption of heavy rate of 100 rpm until equilibrium was reached (36 h according to
metals by several authors. For example, it has been studied as a soil previous assays). Experiments were repeated without the bacteri-
component adsorbent for different metals such as Cd, Cr, Cu, Ni, Pb um biofilm for comparison purposes, and all of the experimental
and Zn [20,21] or Cd(II) [22]. Kaolin clay has also been reported as a work was conducted in duplicate. The adsorption isotherm models
support for a biofilm of Escherichia coli for the treatment of tested and the manner in which the data were adjusted are
cadmium, iron, nickel and chromium aqueous solutions [19]. described in a previous work [24].
The main focus of this study was the evaluation of the The pH of the metal solution was initially adjusted to 5.0 to
biosorption behavior of a biofilm of Klebsiella sp. 3S1 supported avoid precipitation of lead in the form of metal hydroxides. After
on porous ceramic Raschig rings made of kaolin for the removal of 44 h, the lead solution was filtered through a cellulose filter
lead from aqueous solutions. This strain is a Gram-negative membrane (pore size 0.22 mm). Next, the filtrate was acidified
bacterium isolated from wastewaters [23] that has been investi- with HNO3 and analyzed by absorption atomic spectrometry (AAS)
gated in a previous study as a good lead biosorbent in batch using an AANALYST 800 (Perkin Elmer, Waltham, MA, USA) to
experiments showing a maximum adsorption capacity of determine the final metal concentration. The lead biosorption
140.19 mg/g [24]. In this work, a fixed-bed column was set up to capacity (q, mg metal/g of the dry sorbent) was determined as it is
be used in continuous mode. For comparison purposes, the same explained in previous work [24].
study was carried out with and without the bacterial biofilm.
Adsorption and desorption cycles were also performed to investi- Mercury porosimetry
gate the effect of the biosorbent regeneration. The dynamic
behavior of the column was analyzed by the Thomas, Yoon-Nelson Mercury porosimetry (PoreMaster 60-Quantachrome Instru-
Clark and Yan models. In addition, batch studies were conducted ments, Boynton Beach, FL, USA) was employed to characterize
with the support and the biofilm to study the equilibrium the porosity of Raschig rings by applying various levels of
isotherms, the mechanism involved and the formation of the
bacterial biofilm by the advanced techniques of FTIR, SEM and TEM. Table 1
Chemical composition of Raschig rings by X-ray
fluorescence (XRF).
Materials and methods
Compound Concentration (%)
Microorganism, chemicals and materials SiO2 58.8
Al2O3 19.7
The bacterium was selected from a group of microorganisms K2O 2.61
Fe2O3 1.65
isolated from wastewater treatment plants that exhibited high TiO2 0.353
resistance to a variety of heavy metals [23]. This isolate showed Na2O 0.252
98% homology with that of standard Klebsiella species based on its MgO 0.182
16S-rDNA gene sequence (1476 bp) and was named Klebsiella sp. CaO 0.11
MnO 0.036
3S1 (GenBank accession number HE975030).
Sb2O3 0.028
The synthetic solutions of Pb(II) were prepared by using 0.1 M ZrO2 0.0279
NaCl (Panreac, Barcelona, Spain) as an electrolyte and analytical Rb2O 0.0206
grade salts of Pb(NO3)2 (Panreac). The pH of influent solutions was ZnO 0.013
adjusted to 5 with a pH meter by using 0.1 M NaOH (J.T. Baker, Y2O3 0.00717
SrO 0.003
Center Valley, PA, USA) or 0.1 M HNO3 (Panreac).
120 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127
pressure to a sample (0.6550 g) immersed in mercury and at
20 8C. The pressure required for mercury to intrude into the pore
sample is inversely proportional to the size of the pores. The
analysis was carried out by loading the sample into a
penetrometer, which consists of a sample cup connected to a
metal-clad, precision-bore glass capillary stem. The cup of the
penetrometer and capillary stem are then automatically back-
filled with mercury. As the pressure on the filled penetrometer
increases, mercury penetrates into the pores, beginning with
those pores of largest diameter. The instrument automatically
collects low-pressure measurement over the range of pressures
specified (340 kPa). Then, the penetrometer is moved to the high
pressure chamber, where high-pressure measurements are
taken (410 MPa).
FTIR spectroscopy
The presence of characteristic functional groups on the
ceramic support was evaluated by Fourier transform infrared
spectrophotometry (FTIR) using a VERTEX 70 (Bruker Corpora-
tion, Billerica, MA, USA) spectrophotometer operating in the
range 4000–400 cm1. The rings were washed with distilled
water until the residual substrate was completely removed,
dried at 104.5 8C for 24 h and then ground in an agate mortar
until obtaining a homogeneous powder that was analyzed by
attenuated total reflection (ATR) at a room temperature of
approximately 25 8C.
FE-SEM-EDX analysis
Field emission scanning electron microscopy coupled with
energy dispersive X-ray spectroscopy (FE-SEM-EDX, MERLIN of
Carl Zeiss, Oberkochen, Germany) was used to study the
formation of the bacterial biofilm on the ceramic support. The
superficial disposition of the metal was also investigated with
this technique. The support was surrounded with plastic mesh
to avoid friction, and these were both washed with distilled
water. Two rings were introduced into separate 250-mL
Erlenmeyer flasks with a tryptic soy broth (TSB) (Scharlau,
Barcelona, Spain) culture medium (100 mL) that was then
inoculated with the bacterium. The test remained for 72 h on an
orbital shaker (CERTOMAT IS, Sartorius, Gotinga, Germany) at
150 rpm and 30 8C. Afterwards, the medium was removed and
Fig. 1. Experimental fixed-bed column: (1) feed storage, (2) peristaltic pump with flow regulation, (3) valve, (4) column, (5) sampling vessel, (6) effluent storage.
the rings were washed three times with 100 mL of electrolyte
(0.1 M NaCl). They were kept for 24 h in 200 mL of electrolyte
agitated at 100 rpm and 25 8C. One ring from each flask was
washed with PBS (Sigma, St. Louis, MO, USA) and then fixed
according to the procedure described in previous work [24]. The
second ring was used to test the biosorption of Pb(II) under the
following conditions: 150 mL Pb(II) solution of 50 mg/L,
T = 25 8C, 100 rpm and t = 24 h. After completion of the test,
the rings were treated using the procedure described above.
HR-TEM-EDX analysis
Ultra-high resolution transmission electron microscopy (HR-
TEM) with high-angle annular dark field (HAADF) FEI-TITAN G2
was used to precisely determine the location of the Pb(II) fixed to
the bacteria. First, a seed culture of Klebsiella sp. 3S1 was grown in
TSB medium for 20 h (150 rpm and 30 8C). Then, the biomass was
removed by centrifugation (6000 rpm) and washed with electro-
lyte (0.1 M NaCl and pH 5). The electrolyte was used to prepare the
metal solution. Finally, the biosorption test was started under the
following conditions: V = 50 mL metal solution (100 mg/L), pH 5,
T = 25 8C, 200 rpm, and a biomass concentration of 0.52 g/L. After
completion of the test, the solution was centrifuged at 6000 rpm
for 5 min and then was washed with PBS buffer under identical
conditions in triplicate. All samples were fixed by the procedure
described in previous work [24].
Column experiments
Fig. 1 shows the experimental device that was set up for column
experiments. The dimensions of the glass column were a height of
58 cm (the bed depth) and an inner diameter of 6 cm. When the
column was filled with Raschig rings, a total mass of support of
1490 g and a bed pore volume of 1184 mL were determined. The
formation of the Klebsiella sp. 3S1 biofilm over the support was
performed by pumping the microorganism culture and the
nutrient broth through the bed at a flow rate of 0.7 L/min.
According to previous assays, 1.31 mg biomass/g support was
retained as a biofilm after 72 h, so the total biomass immobilized in
the whole column was estimated at 1.9519 g
Before the start of the biosorption cycles, the bed was washed
out with an electrolyte solution of 0.1 M NaCl at a flow rate of 4 mL/
min and pH 5 for 3 days, the time needed to stabilize the effluent
 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127 121

pH at 5. Continuous biosorption experiments began by pumping The equilibrium metal uptake, qe (or maximum capacity of the
the metal solutions (50 mg/L) through the bed with a flow rate of column), is defined by Eq. (5) as the total amount of metal sorbed,
4 mL/min and pH 5 at room temperature (22  2 8C). Samples qtotal, per g of sorbent, m, at the end of the total flow time.
(5 mL) were taken at different times and then analyzed for Pb(II)
content by atomic absorption spectrophotometry (AAS). Operation of qtotal
qe ¼ (5)
the column was stopped when the effluent metal concentration m
reached a constant value.
Desorption cycles were carried out by regenerating the biomass The breakthrough time, tr, was defined as the time at which the
with a 0.1 M HCl solution that was pumped through the column metal concentration in the effluent reached 5% of the influent
under the same conditions used in the adsorption cycles. Several value. The bed exhaustion time, ts, was the time at which the metal
samples (5 mL) were taken and analyzed for metal concentration. concentration in the effluent exceeded 95% of the influent value.
Once the Pb(II) retained by the column was eluted, an electrolyte These values were used to evaluate the length of the mass transfer
solution of 0.1 M NaCl was used to wash the bed until the pH zone, Zm, by using Eq. (6) [27].
stabilized at a value of 5. The regenerated bed was employed in the  
next adsorption cycle to investigate the possible reusability of the tr
zm ¼ z 1 (6)
biofilm in multiple operation procedures. The pH of the effluent ts
was measured at predetermined time intervals.
where z is the bed height of the column (cm).
Modeling and analysis of the column The mass transfer zone, Dt (min), can be calculated from the
difference between the column exhaustion time, ts, and column
The fixed-bed performance is described through the break- breakthrough time, tr. The slope of the breakthrough curve from tr
through curve concept. The breakthrough point is the time that the to ts was represented by dc/dt.
adsorbed species are detected in the column effluent at a given The elution curve is equivalent to the breakthrough curve but
concentration, and the breakthrough curve is the shape of a refers instead to the desorption step. It normally has an
concentration–time profile. These are both very important asymmetrical frequency–distribution shape, with an initial rapid
characteristics for determining the operation and the dynamic increase in the released metal concentration followed by a lower
response of a biosorption column because they directly affect the rate of diminution. The elution curves can be described by the
feasibility and economics of the sorption phenomena. The elution efficiency, ED. This parameter was obtained by dividing the
experimental determination of these parameters is strongly metal mass desorbed, qD, by the metal mass bound to the biomass
dependent on the column operating conditions [25]. in the previous adsorption step, qtotal, Eq. (7).
The breakthrough curve shows the dynamic behavior of
adsorption in a fixed-bed column [26]. It usually shows the qD
ED ð%Þ ¼ 100 (7)
normalized concentration as a function of time or volume of qtotal
effluent for a given bed height, where the normalized concentra-
tion is defined as the ratio of effluent metal concentration to inlet where qD is calculated by Eq. (8) from the regeneration curves.
metal concentration (C/C0). The volume of the effluent, Veff (mL),
can be calculated through Eq. (1). Z t
Q
qD ¼ C dt (8)
V eff ¼ Qt total (1) 1000 0

where ttotal is the total time (min) and Q is the flow rate of where t is the time until a residual Pb(II) concentration of less than
circulation through the column (mL/min). 0.02 mg/L was found in the effluent.
The area under the breakthrough curve is obtained by Moreover, mathematical modeling is very important when
integrating with respect to time the curve of the plot of adsorbed scaling up fixed-bed columns to larger operations [28]. Table 2
concentration, Cad (mg/L) versus t (min). This area can then be used shows the different models that have been considered in this work
to find the total adsorbed metal quantity (maximum column to study the dynamic behavior of the column.
capacity), qtotal (mg) in the column for a given feed concentration
and flow rate, Eq. (2)
Z ttotal Table 2
Q Mathematical models used to predict the dynamic behavior of
qtotal ¼ C ad dt (2) the column.
1000 0
Model Equation
where ttotal is the total flow time (4000 min), Q is the volumetric
flow rate (4 mL/min), and Cad is the adsorbed metal concentration Thomas [42] C
C0 ¼ h 1 i
1þexp kTh q0 m C 0 t
(C0  C) in mg/L. Q

Yoon–Nelson [43] ln C C
¼ kYN tt kYN
The total amount of metal sent to the column, mtotal (mg), is 0 C
Clark [44]  1=n1
calculated from Eq. (3). C
C0 ¼ 1
1þAexpðrtÞ
Yan [45] C
¼ 1  1 a
C 0 Qt total C0 C 0 Qt
mtotal ¼ (3) 1þ q0 m
1000
where C is the effluent metal concentration (mg/L), C0 is the inlet
The total percent removal of metal, EA (%), from the volume sent metal concentration (mg/L), kTh is the kinetic constant in the
through the column can also be found from the ratio of the total Thomas model (mL/mg min), Q is the flow rate (mL/min), q0 is
adsorbed quantity of metal (qtotal) to the total amount of metal sent the maximum solid-phase concentration of the solute (mg/g), m
to the column (mtotal) as shown in Eq. (4). is the dry weight of biosorbent (g), Veff is the effluent volume
(mL), kYN is the kinetic constant in the Yoon–Nelson model
qtotal (min1), t is the time required for 50% adsorbate breakthrough
EA ð%Þ ¼ 100 (4)
mtotal (min), n is the Freundlich constant, A is the constant in the Clark
model, r is the adsorption rate (mg/L min), a and b are the
modified dose–response model constants.
122 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127

Results and discussion 0.5

Raschig ring characterization 0.4

Support porosity is an important analytical tool for an 0.3

qe, mg/g
understanding of the formation of a biofilm and the mechanisms
of lead removal on the biosorbent surface. Pore sizes are classified 0.2
Support + biofilm
according to the International Union of Pure and Applied
Chemistry (IUPAC) as micropores (diameter < 2 nm), mesopores Support
0.1
(2 nm < diameter < 50 nm), and macropores (diameter > 50 nm).
Fig. 2 shows the support porosity (Raschig rings) separated into
0.0
different ranges. Because pores with a diameter of less than 50 nm
0 50 100 150 200 250 300
constituted only 1.44% of the specific volume of pores, Raschig
Ce, mg/L
rings can be categorized as a macroporous material. This structure
may contribute to its successful use for Klebsiella sp. 3S1 biofilm Fig. 3. The batch biosorption experimental equilibrium data (symbols) and
formation. The total porosity, introduced volume and surface area Langmuir isotherm (lines) for the support alone and support + biofilm.
were 13.1212%, 0.2003 cm3/g and 4.3477 m2/g, respectively.

Batch biosorption
Equilibrium data were obtained experimentally using different
initial concentrations of Pb(II) between 5 and 160 mg/L at 25 8C and
pH 5 (Fig. 3). A chemical equilibrium program (Medusa software)
was used to confirm that at pH 5, all of the lead is in solution
without metal precipitation, so the differences between the Pb(II)
concentration before and after the test can be attributed to the
biosorption process. The results shown in Fig. 6 demonstrated
differences in the biosorption performance between Raschig rings

Fig. 2. Porosimetry study results for the ceramic support (Raschig rings).
 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127 123

Table 3 The maximum biosorption capacity (qm) according to the

Biosorption equilibrium parameters obtained with different isotherm models by
Langmuir model was 0.383 mg/g for the support alone and
support and support + biofilm.
0.415 mg/g for the support plus biofilm.
Support Support + biofilm

Langmuir qm 0.3835 0.4154 FTIR spectroscopic study

b 0.0405 0.0342
r2 0.970 0.969 In the previous stages of the investigation, the characteristic
S(q  qcal)2 0.00125 0.00184
functional groups of the bacterium Klebsiella sp. 3S1 involved in the
Freundlich KF 0.0456 0.0471 biosorption of Pb(II) were identified by FTIR [24]. In this work, the
n 2.5431 2.4486 same analysis was performed to determine the presence of
r2 1.000 0.991
S(q  qcal)2 4.927E6 5.354E4
functional groups in the ceramic support (kaolin). This technique
has already been proven effective for obtaining structural
Sips Ks 0.0457 0.0375 information from different biosorbent materials [29–31]. FTIR
as 0.00135 0.04395
n 2.5581 1.7819
analysis demonstrated that the ceramic support used in this study
r2 1.000 0.995 (kaolin) did not have a high number of functional groups. The
S(q  qcal)2 4.911E6 3.089E4 results showed five peaks at different wavelengths (cm1): 1051
Redlich–Peterson KRP 2.0890 0.04244
(possible elongation Si–O–Si), 795, 777, 694 and 446 (potential
aRP 45.329 0.51985 deflections Si–O). At appropriate pH conditions, these links could
b 0.60859 0.69230 give rise to functional groups such as aluminol (Al–OH) from the
r2 1.000 0.995 octahedral layers and silanol (Si–OH) from the tetrahedral layers of
S(q  qcal)2 4.865E6 2.862E4
the stratified aluminosilicate [32]. Under the experimental
where qm is the maximum biosorption capacity (mg/g), b is the Langmuir conditions employed for the biosorption experiments (pH 5), it
biosorption equilibrium constant (L/mg), KF is the Freundlich characteristic
is likely that these functional groups were partially protonated.
constant related to the biosorption capacity, n is the characteristic constant
related to biosorption intensity, Ks and as are Sips isotherm parameters, KRP, aRP and
This fact could significantly reduce the capacity of the ceramic
b are the Redlich-Peterson’s parameters, b varies between 0 and 1, r2 is the support for the adsorption of metal cations. Fig. 4 shows the
correlation coefficient, S(q  qcal)2 is the sum of the errors squared. spectrum obtained for the ceramic support by the OPUS program.
The spectrum of Klebsiella sp. 3S1 obtained in the previous work
covered with Klebsiella sp. 3S1 biofilm (support + biofilm) and [24] was also included for comparative purposes. It can be
without the bacterium biofilm (the support only). concluded that the presence of the bacterial biofilm provides a
Different isotherm models have been tested, including significant improvement for potential lead adsorption capacity
two-parameter isotherms (Langmuir and Freundlich) and three- over that of the ceramic support alone by increasing the number of
parameter isotherms (Spis and Redlich-Peterson) [24]. The param- binding sites for the metal cations.
eters obtained by non-linear regression are shown in Table 3. The
correlation coefficients (r2) were found to be satisfactory for all Chemical and SEM analysis
predictions, with a value slightly lower in the case of the Langmuir
curve. This observation implies that heterogeneous surface condi- Fig. 5 shows two SEM micrographs and the EDX spectra obtained
tions are predominant, although monolayer biosorption could after the biosorption tests. The first image (Fig. 5A) shows various
coexist. Hence, the overall biosorption of Pb(II) on the biomass is stages of the formation of the bacterial biofilm on the ceramic
complex, involving more than one mechanism. support [33,34]: Primary adhesion, formation of microcolonies,
3281.68

2958.30
2925.30
2854.37

1637.61
1531.23
1452.70
1395.68

1232.97

1062.60
967.14
913.57
859.52
780.08

515.07
0.20

Klebsiella sp. 3S1

0.00
Absorbance Units

4000 3500 3000 2500 2000 1500 1000 500

1051.46

795.27
777.23
693.75
560.36
445.91
0.20

Ceramic support
0.00

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

Fig. 4. Comparative FTIR spectra of the ceramic support and the Klebsiella sp. 3S1 obtained in the previous work [24].
124 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127
Fig. 5. Scanning electron micrographs (SEM) and energy dispersive X-ray (EDX) spectra obtained after Pb(II) biosorption. (A) Micrograph that shows various stages of the
formation of the bacterial biofilm on the ceramic support. (B) Micrograph that shows the presence of the retained metal after the biosorption test. (C) EDX spectrum in zone
1 of B. (D) EDX spectrum in zone 2 of B.
their maturation and the subsequent formation of the matrix
exopolysaccharides (EPS). In the second image (Fig. 5B), the
presence of retained metal is observed after the biosorption test.
EDX spectra (Fig. 5C and D) clearly show that Pb(II) is mainly fixed in
the biofilm. This could be attributed to the fact that the EPS matrix
produced by Gram-negative bacteria usually consists of polyanionic
or neutral polysaccharides (uronic acids). This anionic character
could allow the binding of the biofilm with divalent cations
[17,18]. Therefore, the presence of the Klebsiella sp. 3S1 biofilm
gives the ceramic support a capability that it did not have before.
Chemical and TEM analysis
The micrographs and EDX spectra obtained by this technique
(Fig. 6) show that the bacterium Klebsiella sp. 3S1, in their
planktonic state, is able to retain the Pb(II) ions. This process occurs
both at the surface level as well as with the incorporation of metal
within the cytoplasm. Fig. 6B and C shows elemental maps for
phosphorus and lead, where the location of metal is highlighted,
and the relationship between both elements [35,36]. There is
evidence that some prokaryotes have acidocalcisomes, organelles
rich in polyphosphates (Poly P) and pyrophosphates (PPi). These
organelles can sequester cations of Pb, Cd, Ni, Cu, etc. [37,38]. This
accumulation of heavy metals stimulates the activity of the
enzyme exopolyphosphatase (PPX), releasing orthophosphate (Pi)
from PPi. In this way, and thanks to the presence of the proton
pump in the membrane of the organelle, the phosphate and metal
complexes can be expelled to the exterior. Zone 2 of Fig. 6A could
indicate the presence of one of the mentioned organelles. Some
authors have reported the existence of bacteria with only one
acidocalcisome with a polar position [36,39].
Fig. 6D shows the EDX spectrum in zone 1 of Fig. 6A. In this
figure, the presence of the metal on the cell surface is confirmed,
possibly precipitated as a phosphate compound [40]. This fact is in
accordance with previous findings [24] where the functional
groups potentially responsible for the surface adsorption of metal
cations by Klebsiella sp. 3S1 were identified. Fig. 6E shows the EDX
spectrum in zone 2 of Fig. 6A. In this case, it can be seen that the
metal is also incorporated in the bacterial cytoplasm. This ability
has been demonstrated in other bacteria [40]. These images
provide evidence of the ability of Klebsiella sp. 3S1 to remove Pb(II)
from aqueous solutions. This fact, together with the previously
verified capability of biofilm formation, constitutes an important
advantage for potential practical applications in the treatment of
contaminated wastewater.
Column studies
Five Pb(II) adsorption–desorption cycles were completed in a
continuous sequence. Fig. 7 shows the breakthrough curves
obtained for the different adsorption cycles, one for the Raschig
rings without the biofilm (blank) and four for the Raschig rings
covered with biofilm. It can be observed that from the first cycle,
the curves are practically identical and different from the curve
corresponding to the blank. The column operating parameters are
presented in Table 4. The breakthrough time (tr) was found to
remain practically constant during the four sorption cycles with
only a small decrease in the second cycle due to a behavior
anomaly. However, this difference was not observed in the rest of
the parameters. Moreover, as was expected, the length of the mass
transfer zone (Dt), which is described by the difference between
the exhaustion and breakthrough times, did not change consider-
ably during the process. This implies that the overall sorption zone
remains approximately constant during the cycles. A different
behavior for the breakthrough time as the cycles progressed can be
observed in the literature for metal sorption–desorption in the
column [25,41].
It is remarkable that the metal uptake (q values in Table 4)
resulted in a considerable increase with the presence of biofilm.
Thus, when comparing the blank with the third biosorption cycle,
the total adsorbed metal quantity (qtotal) increased from 89.9 mg to
226.0 mg (150%). This is due only to the 1.9519 g of the biofilm.
Thus, the amount of metal adsorbed per g of biofilm is important
(69.7 mg/g). This biosorption capacity is in the range of that
obtained by Klebsiella sp. 3S1 in its planktonic state [24] with qe
values between 67 and 153 mg/g under different conditions in
batch experiments. This implies that no significant loss of total lead
biosorption capacity is produced when the bacteria is used as a
biofilm immobilized on an inert support in continuous mode, even
if the time operation contact is significantly lower. Another
positive finding was the fact that the Pb(II) uptake capacity
remained practically constant for the four cycles, with a slight
 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127 125

Fig. 6. Transmission electron micrographs (TEM) and energy dispersive X-ray (EDX) spectra obtained for a thin section of Klebsiella sp. 3S1 after Pb(II) biosorption. (A)
Micrograph that shows the location of fixed lead. (B) Elemental map for phosphorous. (C) Elemental map for lead. (D) EDX spectrum in zone 1 of (A), corresponding to surface
biosorption of lead. (E) EDX spectrum in zone 2 of (A), corresponding to the intracellular accumulation of lead.

variation in the second one. This diminution in the second cycle
could be attributed to the effect of the prior acid desorption step.
Otherwise, the slope of the breakthrough curves (dC/dt) may be
used to characterize them. Table 4 shows the obtained values,
which experienced a small decrease.
Different mathematical models have been developed to predict
the dynamic behavior of the column, such as the Thomas [42],
1
0.8
0.6
C/Co
Yoon–Nelson [43], Clark [44] and Yan [45] models (Table 2).
Among these, the Thomas model is the most widely employed in
the literature. The Yoon–Nelson model is a more recent model that
has been used to predict breakthrough curves, but it is
mathematically similar to the Thomas model. Moreover, Yan
et al. [45] proposed a modified dose–response model that
successfully describes the column kinetics of metal biosorption
onto immobilized Mucor rouxii. The successful design of a column
biosorption process requires prediction of breakthrough curves
and maximum biosorbent uptake capacity. Table 5 summarizes the
model parameters obtained during the different cycles, which have
been determined by nonlinear regression. Fig. 8 allows the
comparison between the theoretical curves and the experimental
data in the case of the fourth biosorption cycle. It can be observed
that all four models very closely represent the experimental
breakthrough curves. However, the best fit for the retention of

Blank
Pb(II) was obtained with the Yan model.
Cycle 1
0.4 The Thomas model assumes Langmuir adsorption–desorption
Cycle 2
kinetics, that no axial dispersion is derived from the adsorption
Cycle 3
Cycle 4
and that the rate-driving force obeys second-order reversible
0.2 reaction kinetics. In this work, the maximum uptake capacity (q0)
obtained from the Thomas model (Table 5) is quite similar to the
experimental uptake values (qe) that are shown in
0 Table 4. Moreover, the results obtained for the rate constant (kTh)
0 1000 2000 3000 4000 5000 6000 7000 are similar to those obtained by various other authors [46–48].
t, min Yoon and Nelson [43] have developed a relatively simple model
addressing the adsorption and breakthrough of adsorbate vapors
Fig. 7. Multiple breakthrough curves obtained in column experiments for lead
biosorption by the support alone (blank) and the support + biofilm (cycles) at
or gases with respect to activated charcoal. This model is based on
constant flow rate (4 mL/min). C is the Pb(II) concentration at a time t, C0 is the the assumption that the rate of decrease in the probability of
initial Pb(II) concentration (50 mg/L). adsorption for each molecule is proportional to the probability of
126 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127

Table 4
Breakthrough parameters for 5 sorption–desorption cycles, using a fixed-bed column for the removal of lead (50 mg/L) by Raschig rings without biofilm (blank) and covered
with biofilm.

Cycle qtotal (mg) qe (mg/g) tr (min) ts (min) dC/dt (mg/L min) EA (%) zm (cm)

Blank 89.9 0.0603 18 2000 0.0224 11.4 57.48

1 201.8 0.1353 136 3366 0.0142 24.7 55.66
2 165.6 0.1110 71 3253 0.0143 20.5 56.73
3 226.0 0.1515 200 3575 0.0131 28.6 54.75
4 204.1 0.1368 142 3800 0.0117 26.8 55.83

where qtotal is the total adsorbed quantity of metal in the column (mg), qe is the total amount of metal adsorbed per g of sorbent (mg/g), tr is the breakthrough time, the time at
which metal concentration in the effluent reached 5% of the influent value (min), ts is the exhaustion time, the time at which metal concentration in the effluent exceeded 95%
of the influent value (min), dC/dt is the slope of the breakthrough curve from tr and te (mg/L min), EA is the total removal percent of metal, zm is the length of the mass transfer
zone (cm)

Table 5
Breakthrough curves parameters at different cycles for the mathematical models tested.

Cycle 1 Cycle 2 Cycle 3 Cycle 4

Thomas kTh (mL/mg min) 0.0505 0.0459 0.0516 0.0484

q0 (mg/g) 0.1151 0.0850 0.1326 0.1195
r2 0.974 0.973 0.986 0.984
S(q  qcal)2 0.155 0.105 0.074 0.092

Yoon–Nelson kYN (min1) 0.00258 0.00232 0.00255 0.00231

ts (min) 842 627 1000 935
r2 0.974 0.973 0.986 0.984
S(q  qcal)2 0.155 0.105 0.074 0.092

Clark n 1.000177 1.000158 1.000078 1.000245

A 0.000511 0.000315 0.000267 0.000686
r (mg/L min) 0.001829 0.001808 0.01711 0.001626
r2 0.988 0.987 0.996 0.994
S(q  qcal)2 0.070 0.050 0.024 0.032

Yan a 1.8332 1.5401 2.0409 1.7919

q0 (mg/g) 0.0925 0.0652 0.1120 0.0939
r2 0.997 0.993 0.996 0.997
S(q  qcal)2 0.019 0.025 0.020 0.019

where: kTh is the kinetic constant in the Thomas model (mL/mg min), q0 is the maximum solid-phase concentration of the solute (mg/g), r2 is the correlation coefficient,
S(q  qcal)2 is the sum of the errors squared, kYN is the kinetic constant of Yoon-Nelson model (min1), ts is the time required to absorb 50% metal depending on the
breakthrough curve (min), n is the Freundlich constant, A is the constant in the Clark model, r is the rate of adsorption (mg/L min), a is the constant in the Yan model.

adsorption and the probability of adsorbate breakthrough on the
adsorbent. As observed in Table 2, t is the time required for 50%
adsorbate breakthrough (min), and kYN is the rate constant
(min1). The results obtained in this work for these parameters
were similar to those obtained by Quintelas et al. [10] working
with columns containing a biofilm supported on granular activated
carbon.
1.00
0.80
0.60
C/Co
Clark [44] defined a new simulation of breakthrough curves.
This model combines the Freundlich equation and the mass
transfer concept. The values of A and r in the Clark equation were
determined using by non-linear regression analysis and are also
shown in Table 5.
The Yan model, as was mentioned earlier, described with great
precision the tendency of the experimental curves (Fig. 8).
However, the q0 values obtained with this model (Table 4) were
slightly lower than the experimental values in the present work.
Concerning the pH of the effluent solution, a decreasing
tendency was detected as the Pb(II) was adsorbed from the initial
value of approximately 5 to a constant value of approximately
4.5 when the bed was saturated. As the metal solution contacts the
biomass, an exchange between the Pb(II) adsorbed and protons
released from the sorbent takes place, and therefore, a pH decrease
can be observed. When the bed is exhausted, no more protons are

Cycle 4
liberated and then the pH tends toward a constant value.
0.40 Thomas and Yoon-Nelson On the other hand, desorption cycles were performed in order to
regenerate the biosorbent. Thus, an elution step was carried out
Clark after each adsorption cycle after the column bed was saturated. The
0.20 effective operation of the next adsorption cycle was clearly
Yan influenced by the efficiency of the preceding desorption. After
each elution operation, the column was washed with an electrolyte
0.00 solution to eliminate the rest of the acid in the bed until a pH value
0 1000 2000 3000 4000 5000 6000 between 4.5 and 5 was achieved in the effluent solution. The most
t, min
important parameter that defines the elution performance is the
Fig. 8. Comparison between the experimental data obtained at cycle 4 and the elution efficiency, ED (Eq. (7)). In this work, the ED value was
breakthrough curves predicted with different mathematical models. approximately 99%. The tendency of the pH during the regeneration
 A.J. Muñoz et al. / Journal of Industrial and Engineering Chemistry 40 (2016) 118–127
steps showed a rapid decrease in the first minutes, followed by a
slow and continuous decrease toward a constant value (approxi-
mately 1.1). The situation was opposite to that observed in the
adsorption cycles. The protons of the eluent were exchanged with
the metal retained, which produces an initial rapid decrease in the
pH value up to the point of maximum Pb(II) concentration released
in the effluent. As occurs in adsorption cycles, this decreasing
tendency in pH supports the idea of ion exchange as the
predominant mechanism in metal biosorption [49].
The column system tested was able to operate for a period of
50 h for each sorption cycle maintaining a Pb(II) effluent
concentration lower than 0.02 mg/L (from a 50 g/L initial Pb(II)
concentration) for at least 2 h in the four cycles performed.
Furthermore, the metal desorption with HCl solution was fast, with
a maximum Pb(II) concentration peak obtained around an elapsed
time of 5 min or less and a residual metal concentration lower than
0.9 mg/L after 1 h of elution for all of the cycles studied. After the
acid treatment, the biofilm was able to support an uninterrupted
use for at least 28 days subjected to continuous Pb(II) sorption–
desorption cycles in the fixed-bed column with no apparent
diminution of its biosorption performance.
Conclusions
In this work, the effectiveness of a Klebsiella sp. 3S1 biofilm to
bioadsorb Pb(II) ions has been proved. Both batch and continuous
column experiments clearly demonstrated that the bacterial biofilm
significantly improved the adsorption properties of the support.
FTIR spectra showed that Klebsiella sp. 3S1 provides extra binding
sites for the retention of the metal cations. SEM studies also revealed
that the bacteria gave the support additional adsorption properties
that could be attributed to the EPS matrix produced by the
microorganism. Different stages in the biofilm formation have been
corroborated by the SEM micrographs. The biosorption equilibrium
data better fit the Freundlich model, which implies heterogeneous
surface conditions. TEM analysis revealed that several mechanisms
could be involved in the lead biosorption process because the metal
was detected on the cell surface and also in the cytoplasm.
The mathematical modeling of the breakthrough curves
obtained during the column experiments showed a good
correspondence with the different well-established column
models that were assessed. The presence of the biofilm resulted
in an important improvement in the metal uptake with up to a
150% increase in the lead biosorption capacity of the porous
support. Four biosorption-desorption cycles were carried out in a
continuous sequential manner without a significant loss in the
effectiveness of lead retention. The desorption efficiency was close
to 100%. The results obtained supported that the developed packed
bed column biosorption process with a Klebsiella sp. 3S1 biofilm
has a high potential for use in industrial applications for the
removal of lead from wastewater. Future research could be
conducted to test higher-porosity supports and a maximal amount
of immobilized Klebsiella sp. 3S1 biofilm to achieve a robust, large-
scale process for heavy-metal biosorption.
127
Acknowledgement
The authors acknowledge the support of the Spanish Agency for
International Development Cooperation (Project Ref. A/018600/08).
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