J. Microbiol. Biotechnol.

J. Microbiol. Biotechnol. (2016), 26(8), 1428–1438http://dx.doi.org/10.4014/jmb.1603.03074 Research Article jmbReview

Biosorption of Lead(II) by Arthrobacter sp. 25: Process Optimizationand MechanismYu Jin, Xin Wang, Tingting Zang, Yang Hu, Xiaojing Hu, Guangming Ren, Xiuhong Xu, and Juanjuan Qu*

College of Resource and Environment, Northeast Agricultural University, Harbin 150030, P.R. China

Introduction

Heavy metal contamination is a serious global

environmental hazard, since all metals or elements with

metallic characteristics can form toxic compounds. The

presence of these compounds, especially in drinking water,

even at trace levels, may cause anemia, itai-itai, or carcinoma

and other illnesses in humans [7, 9, 17, 24, 27]. Lead (Pb) is

a malleable and heavy post-transition metal generally

applied in building construction, e-manufacturing, mining,

and lead-acid batteries [13, 26, 30, 39]. China is the largest

producer and consumer of lead with an annual output near

135 million tons and annual consumption of 80 million

tons. Outdated technology and equipment and a lack of

effective environmental protection measures by Chinese

industries have increased the lead contamination in the

ecosystem, which may increase the probability of lead

affecting humans through inhalation, ingestion, dermal

contact, or other means. Acute and chronic exposure to

lead can cause anemia, liver and kidney disease, central

and peripheral nervous system damage, stunted growth,

and high blood lead levels [33, 36], especially in young

children; these effects are frequently reported in China [19].

The efficient removal of lead ions from aqueous waste is

essential for environmental safety, but current techniques

such as ionic exchange, membrane separation, and activated

carbon adsorption are inefficient for the treatment of

wastewater containing low concentrations of lead [12].

In China, the threshold limit of lead-containing liquid

from sewage and industrial effluents was set to 1.0 mg/l

(Environmental Quality Standard for Surface Water of China,

GB8978-1996), but wastewater without efficient treatment

procedures cannot achieve the discharge standards. The

biosorption process utilizes various natural materials that

Received: March 31, 2016

Revised: May 4, 2016

Accepted: May 9, 2016

First published online

May 20, 2016

*Corresponding author

Phone: +86-150-4668-5503;

Fax: +86-451-5519-0647;

E-mail: juanjuan4050234@163.com

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2016 by

The Korean Society for Microbiology

and Biotechnology

In the present work, Arthrobacter sp. 25, a lead-tolerant bacterium, was assayed to remove

lead(II) from aqueous solution. The biosorption process was optimized by response surface

methodology (RSM) based on the Box-Behnken design. The relationships between dependent

and independent variables were quantitatively determined by second-order polynomial

equation and 3D response surface plots. The biosorption mechanism was explored by

characterization of the biosorbent before and after biosorption using atomic force microscopy

(AFM), scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction,

and Fourier transform infrared spectroscopy. The results showed that the maximum

adsorption capacity of 9.6 mg/g was obtained at the initial lead ion concentration of

108.79 mg/l, pH value of 5.75, and biosorbent dosage of 9.9 g/l (fresh weight), which was

close to the theoretically expected value of 9.88 mg/g. Arthrobacter sp. 25 is an ellipsoidal-

shaped bacterium covered with extracellular polymeric substances. The biosorption

mechanism involved physical adsorption and microprecipitation as well as ion exchange, and

functional groups such as phosphoryl, hydroxyl, amino, amide, carbonyl, and phosphate

groups played vital roles in adsorption. The results indicate that Arthrobacter sp. 25 may be

potentially used as a biosorbent for low-concentration lead(II) removal from wastewater.

Keywords: Arthrobacter sp., lead(II), biosorption mechanism, optimization of biosorption

process

Biosorption of Lead 1429

August 2016⎪Vol. 26⎪No. 8

exhibit metal sequestering properties, which helps to

decrease the concentration of heavy metal ions from the

ppm to the ppb level [6, 14, 15, 28].

Bacterial biomass shows tremendous potential for use in

heavy metal removal because of its small size, ubiquity,

and applicability to various environments [1, 20, 37].

Polysaccharides, proteins, and lipids on bacterial cell walls

may provide functional groups such as amino, hydroxyl,

carboxyl, and phosphate groups that are able to attract and

bond with metals [25, 28]. Many bacterial species have been

used for the sorption of lead(II). A lead-resistant bacterium,

Enterobacter cloacae strain P2B, was isolated from effluent

discharged by a lead battery manufacturing company and

was shown to resist lead nitrate up to 1.6 mM [29].

Pseudomonas aeruginosa ASU 6a is a promising biosorbent

for the removal of lead(II) and nickel(II) ions from aqueous

solutions [10]. In addition, Bacillus sp. PZ-1 exhibits lead

resistance and has been used as an adsorbent to remove

Pb(II) from aqueous solution even at low temperature [37].

Microbes, including Chlorella vulgaris, Bacillus sp., and yeast,

are used commercially to adsorb or remove heavy metals

by B.V.SORBEX Inc., Visa Tech Ltd., and the US Bureau of

Mines, respectively [48].

Arthrobacter is a genus of bacteria that is commonly

found in soil. Several species in this genus are efficient

in bioremediation. Arthrobacter ps-5 isolated from the

rhizosphere of the maidenhair tree (Ginkgo biloba) produces

extracellular polymeric substances (EPS) that exhibit

high adsorption capacity for copper and lead ions [49].

A. crystallopoietes has been shown to reduce hexavalent

chromium levels in contaminated soil [3]. Additionally, free

and immobilized biomass from pond-isolated Arthrobacter

sp. was successfully utilized for the removal of copper both

in a batch and a continuous system [18].

In this study, a lead-tolerant bacterium, Arthrobacter sp. 25,

was found to show biosorption of low concentrations of

lead ions in aqueous solution. This work aims to optimize

the biosorption process using response surface methodology

(RSM) and determine the removal mechanism of lead(II) by

Arthrobacter sp. 25.

Materials and Methods

Preparation of Biosorbent, Lead(II) Solution, and Batch Biosorption

Experiments

Arthrobacter sp. 25 used in the present study is an aerobic,

elliptical shaped, and gram-negative bacterium with a GenBank

accession number of JQ086764 for its 16S rDNA sequence, which

was isolated from a lead-zinc mine in northeast China [22]. The

free-living cells were collected at the late exponential phase by

centrifugation at 8,000 rpm for 7 min and prepared as biosorbents

after being fully rinsed by phosphate buffer solution and sterile

deionized water. Lead(II) solutions were prepared by dilution of

1 g/l stock solution obtained by dissolving a predetermined quantity

of Pb(NO3)2 (Sinopharm, China) in distilled and deionized water.

Batch biosorption experiments were carried out to investigate

the effects of parameters on lead(II) adsorption in 150 ml flasks in

a thermostatic shaker. After centrifugation and filtration, the

residual lead(II) concentration in the supernatant was determined

on an atomic absorption spectrophotometer (AA-6800; Shimadzu-

GL, Japan). All experiments were conducted in triplicates and the

mean values were calculated. The Q (biosorption rate, %) and the

q (adsorption capacity, mg/g) were determined as Eqs. (1) and

(2), respectively:

(1)

(2)

where C0 and C are the initial and final concentrations of lead(II)

(mg/l). V and M are the volume of solution (ml) and the weight of

adsorbent (g), respectively.

Optimization of Biosorption Process by Response Surface

Methodology

Plackett-Burman (P-B) design. P-B design was done to screen

out key variables with SPSS 16.0 software. Six main variables

involved in biosorption process were selected, including initial

lead(II) concentration (X1), pH (X2), contact time (X3), rotation rate

(X4), temperature (X5), and biosorbent dosage (X6), where the

ranges of independent variables were determined based on

preliminary single factor experiments (not described in this

article). Each variable was tested at a low level “-1” and a high

level “1”, and three virtual columns (X7, X8, X9) were set to

investigate the experimental error. On the basis of results

obtained by the P-B test, the fitted first-order model is

(3)

where q is the predicted value of adsorption capacity, β0 and βi are

the constant coefficients, Xi is the coded independent variable or

factor, and k is the number of the independent variables.

Steepest ascent experiment. To move rapidly towards the

neighborhood of the optimum response, a steepest ascent

experiment was used to approximate the best response area. The

new units were determined according to the estimated coefficient

ratio from the first-order model. To move away from the first

design center along the path of steepest ascent, the variables were

set to about 16% to 25% higher or lower than the “0” level [40].

Box-Behnken test. With Design Expert 8.0.5 software (State-

Ease Inc., USA), the Box-Behnken test was carried out to ascertain

QC0 C–( )

C0

------------------- 100%×=

qC0 C–M

-------------- V×=

q β0 βiXi

i=1

k

∑+=

1430 Jin et al.

J. Microbiol. Biotechnol.

the optimum adsorption conditions as well as the interaction

between independent variables. The coded levels (-1, 0, 1) were

used to describe the characteristics of the response surface in the

optimum region. The total number of treatment was 2k + 2k + n0,

where k and n0 were the numbers of independent variables and

experiment repetitions, respectively, at the center point. The results

of Box-Behnken test were fitted with a second-order polynomial

equation by multiple regression analysis:

(4)

where q is the predicted value of adsorption capacity, β0 is the

offset term, βi is the ith linear coefficient; βii is the ith quadratic

coefficient; and βij is the ijth interaction coefficient.

Mechanism Analyses

TEM. Transmission electron microscopy (TEM) was used for

morphological observation of the bacterium. A droplet of the

bacterial suspension was placed onto a Formvar film that had

been transferred onto a copper index TEM grid (300 mesh) for

5 min, and the remaining solution was wiped off using a piece of

filter paper. The specimens were then stained by placing a drop of

2% aqueous phosphotungstic acid for 3 min and examined with a

transmission electron microscope (Hitachi H-7650, Japan) [42].

SEM-EDX and AFM. The surface morphology of Arthrobacter

sp. 25 before and after lead(II) biosorption was characterized using

scanning electron microscopy (SEM) (QuANTA200 model; FEI,

USA). The elements were analyzed by an energy dispersive X-ray

analysis system (EDX) (QuANTA200 model; FEI, USA). Prior to

analysis, the samples were coated with a thin layer of gold under

an argon atmosphere to improve electron conductivity and image

quality.

To obtain complementary information, whole cells were also

examined by Nanoscope IIIα atomic force microscopy (AFM)

(Digital Instruments, USA). Both lead-loaded and lead-unloaded

cells were collected by centrifugation, lightly rinse in a sterile

phosphate-buffered saline solution and sterile deionized water,

and then the specimens were transferred to glass slides and

examined by AFM.

FTIR. The surface functional groups of Arthrobacter sp. 25 were

analyzed by Fourier transform infrared spectroscopy (FTIR).

Samples were ground with KBr powder and pressed into pellets

for FTIR measurement in the frequency range of 4,000–400 cm-1

(ALPHA-T, Bruker, German) [38].

XRD. The crystalline structure of the biosorbent was investigated

by X-ray diffraction (XRD) (D/max2200 model; Hitachi, Japan)

equipped with a Cu Kα radiation. The operating conditions were

U = 40 kV and I = 30 mA. The scans were performed between 5°

and 80° with a scan rate of 4°/min and step size of 0.02° at room

temperature.

Results and Discussion

Optimization of Biosorption Process

P-B design. The P-B design to screen key variables is

shown in Table 1. The minimum and maximum values of

the investigated parameters and the regression analysis for

biosorption are listed in Table 2. The order of key variables

that impacted the adsorption characteristics was X2 > X6 >

X1 > X3 > X4 > X5. The pH value (X2) had the largest effect on

the adsorption sites on the cell surface and the chemical

state and distribution of metal ions. In this study, the pH

value showed a positive effect on lead(II) uptake. This

might result from competition between hydrogen ions at

low pH and the weakly acidic nature of the active groups

on the adsorbent, which favors metal adsorption [31].

Biosorbent dosage (X6) and initial lead(II) concentration (X1)

also significantly affected the adsorption process (p < 0.05),

but other variables were not significant (p > 0.05). The first-

order model equation was derived as follows:

q = 5.31 − 0.56X1 + 3.42 X2 + 0.59 X6 (5)

q β0 βiXi

i=1

k

∑ βiiXi2

ii

k

∑ βijXiXj

i j<

k

∑+ + +=

Table 2. Results of Plackett-Burman and regression analysis for

biosorption.

Factors Level

F test Prob>F− 1 + 1

X1 (mg/l) 67 100 14.97 0.0118

X2 4.0 6.0 553.26 <0.0001

X3 (min) 17 25 0.70 0.4405

X4 (rpm) 107 160 0.49 0.5158

X5 (oC) 24 30 0.44 0.5385

X6 (g/l) 6.7 10 16.52 0.0097

Table 1. Plackett-Burman design for screening of key variables.

No.Variables q

X1 X2 X3 X4 X5 X6 X7 X8 X9 (mg/g)

1 1 -1 -1 -1 1 -1 -1 1 1 0.95

2 -1 -1 -1 1 -1 1 1 1 -1 3.56

3 -1 1 1 1 -1 -1 1 -1 1 9.37

4 -1 -1 -1 -1 -1 -1 1 -1 1 1.65

5 1 1 -1 -1 -1 1 1 -1 -1 9.11

6 1 1 1 -1 -1 -1 1 1 -1 7.26

7 -1 1 1 -1 1 1 1 1 1 9.33

8 -1 1 -1 1 1 -1 -1 -1 1 8.37

9 -1 -1 1 -1 1 1 -1 1 1 2.96

10 1 1 -1 1 1 1 1 -1 1 8.96

11 1 -1 1 1 -1 1 -1 -1 -1 1.49

12 1 -1 1 1 1 -1 -1 1 1 0.72

Biosorption of Lead 1431

August 2016⎪Vol. 26⎪No. 8

Steepest ascent experiment. The steepest ascent experiment

is a procedure for moving sequentially along the path of

steepest ascent. Using the steepest ascent experiment, we

determined the following parameters as the response

surface center: initial lead(II) concentration of 100 mg/l,

pH of 6.0, biosorbent dosage of 10 g/l, and q of 8.9 mg/l.

Box-Behnken design of central composite. In order to

test the key variables at different levels of low (−1), high

(+1), and basal (0), a Box-Behnken design of central

composite was employed. According to the experimental

design, a total of 17 runs were required, and the

experimental and predicted q values for each run are

shown in Table 3. The relationship between independent

variables and response values was expressed using the

following second-order polynomial model (Eq. (6)):

qpredicted = 9.61 − 0.61X1 − 0.67X2 + 0.81X6 − 0.63X1X2 +

0.77X1X6 + 0.36X2X6 − 1.39X1 − 0.97X22 − 0.78X6

2 (6)

The regression model was well-fitted and was able to

explain 95.48% of the observed trends in the experiments.

The coefficients R2 and Adj-R2 were 0.9548 and 0.8967,

respectively, which indicated a high correlation between

the experimental and predicted values. The values of

“Prob>F” less than 0.05 (Table 4) indicated the model was

significant and appropriate to describe the process of

lead(II) adsorption [11].

The model was appropriate to predict the lead(II) removal

efficiency as a function of the variable factors, including

initial lead(II) concentration, pH, and biosorbent dosage,

based on statistical analysis. However, this model alone

does not physically explain the biosorption phenomena.

Optimal conditions of lead(II) biosorption. The hierarchical

linear model (Eq. (6)) obtained in this study was used to

represent the response surface. The biosorption process

was optimized using the response surface and contour

plots. The adsorption capacities exhibited at different pH,

biosorbent dosage, and initial lead(II) concentrations are

presented in Figs. 1A, 1B, and 1C. The contour plots show

the effects on adsorption capacity of varying two factors

when the other factors were maintained at a basal level.

The pH value can affect the protonation of functional

groups on the biosobent as well as the metal chemistry [34].

The effects of pH on the lead(II) adsorption capacity of

Arthrobacter sp. 25 are shown in Figs. 1A and 1C. As the pH

value of the solution increased from 5.0 to 5.7, the

adsorption capacity increased from 9.3 to 9.9. When the pH

value was higher than 5.7, the insoluble metal hydroxides

were precipitated and the adsorption capacity decreased.

Similar results were observed for lead(II) removal using

dried cells of Lactobacillius bulgaricus and shells of Pistacia

vera L. [40, 51]. The cell surface of bacteria contains complex

polysaccharides, proteins, and lipids that can provide

amino, carboxyl, and sulfate groups [2]. At low pH, cell

surface ligands can protonate, which impedes interaction

with metal cations owing to the repulsive force. As higher

pH values, more ligands such as amino, phosphate, and

carboxyl groups become exposed, and these metal-binding

functional groups are able to attract more metal ions.

Table 3. Results from Box-Behnken design for lead(II) uptake

(mg/g).

No.Factor 1

X1

Factor2

X2

Factor 3

X6

qexperimental qPredicted

1 0 +1 -1 8.22 7.82

2 +1 +1 0 8.32 8.36

3 0 -1 +1 7.53 7.94

4 0 0 0 9.46 9.61

5 -1 0 +1 5.63 5.26

6 -1 +1 0 5.44 6.02

7 0 0 0 9.37 9.61

8 +1 0 +1 8.24 8.41

9 0 0 0 9.69 9.61

10 0 0 0 9.81 9.61

11 -1 0 -1 8.17 8.01

12 +1 -1 0 9.54 8.97

13 +1 0 -1 7.72 8.09

14 0 -1 -1 7.68 7.89

15 0 +1 +1 5.54 5.34

16 0 0 0 9.71 9.61

17 -1 -1 0 8.12 8.08

Table 4. Box-Behnken analysis of adsorption capacity.

FactorCoefficient

estimate

Standard

errorF Value Prob>F

Intercept 9.61 0.21 16.43 0.0006

X1 -0.61 0.17 13.46 0.0080

X2 -0.67 0.17 16.28 0.0050

X6 0.81 0.17 23.75 0.0018

X1X2 -0.63 0.23 7.28 0.0307

X1X6 0.77 0.23 10.67 0.0138

X2X6 0.36 0.23 2.40 0.1649

X12 -1.39 0.23 36.99 0.0005

X22 -0.97 0.23 18.17 0.0037

X62 -0.78 0.23 11.65 0.0112

1432 Jin et al.

J. Microbiol. Biotechnol.

However, as pH further increased, lead(II) interacted with

the oxygen or hydroxyl ions resulting in oxide or hydroxide

precipitation, subsequently hindering the adsorption process

[43].

Biosorbent dosage showed a significant influence on the

biosorption process at a specified initial concentration. The

effect of biomass dosage on lead(II) removal is depicted in

Figs. 1A and 1B. The biosorption capacity increased as

biomass dosage increased from 7.5 to 10 g/l, but at higher

dosages, the adsorption capacity decreased greatly. This

trend could be due to the partial aggregation of biomass at

higher biomass concentrations, decreasing the effective

surface area available for biosorption [23]. When the

concentration of adsorbents and initial lead(II) were

equivalent, the adsorption capacity reached a maximum.

Figs. 1B and 1C show the effect of initial lead(II)

concentration on the biosorption capacity of Arthrobacter

sp. 25. The adsorption capacity increased gradually when

the lead(II) concentration increased from 80 to 109 mg/l,

and then decreased as the concentration was additionally

increased. At lower metal ion concentrations, the ratio

of the initial moles of metal ion to the available surface

area was low; subsequently, the fractional sorption was

independent of the initial concentration. However, at

higher concentrations, the sites available for sorption were

fewer relative to the moles of metal ions; thus, the removal

of metal ion was strongly dependent upon the initial

concentration [45].

These results showed that the optimal conditions for

lead(II) biosorption by Arthrobacter sp. 25 (free-living cells)

from aqueous solution were an initial lead(II) concentration

of 108.79 mg/l, pH of 5.75, and a biosorbent dose of 9.9 g/l.

The predicted maximum theoretical adsorption capacity of

lead(II) was 9.88 mg/g, as indicated in the response surface

and contour plots (Fig. 1).

Model validation test. Under optimum conditions, the

observed adsorption capacity was 9.6 mg/g, close to the

theoretical maximum value of 9.88 mg/g, for a 97.2%

prediction accuracy. This level of accuracy proved the

feasibility of RSM in the optimization of the adsorption

conditions.

Other bacteria have been used for lead biosorption and

exhibited adsorption capacities in the range of 1.29-

110 mg/g under different operational conditions (Table 5).

This large range of efficacy can be attributed to differences

in surface structure, functional groups, available surface

area, and the conditions of the cells (living or lyophilized/

heat-dried cells) [4, 21, 32, 41]. In this study, free-living

cells were used as biosorbent and exhibited an adsorption

Fig. 1. 3D surface plots of sorption capacity versus two

independent factors: (A) pH and biosorbent dosage, (B) initial

lead(II) concentration and biosorbent dosage, and (C) pH and

initial lead(II) concentration.

Biosorption of Lead 1433

August 2016⎪Vol. 26⎪No. 8

capacity of 9.6 mg/g (using a wet/dry ratio of 8.42:1). The

capacity could theoretically reach 83.2 mg/g with dried

cells, and a direct comparison between these different

results is not valid.

Mechanism Analyses

TEM. The cells of Arthrobacter sp. 25 were ellipsoidal in

shape with an average length of 1.25 ± 0.2 μm and width of

0.83 ± 0.2 μm, as shown in Fig. 2. A thick layer of EPS

extended up to 0.5 μm from the cell membrane and

uniformly covered the cell. Both cell walls and the EPS

layer provided abundant sites for the binding of metal ions

[42].

SEM-EDX and AFM analyses. SEM images of Arthrobacter

sp. 25 with and without lead(II) loading are shown in Fig. 3

(A, B). Lead(II)-loaded cells differed in morphology from

the unloaded ones. For the samples containing the unloaded

cells, the majority of cells remained intact, smooth, and

closely connected with one another, providing a large

surface area for biosorption (Fig. 3A). In the case of the

loaded samples, the matrix layers of the cell wall appeared

to shrink and stick. Naik et al. [29] observed that cells of

Enterobacter cloacae strain P2B shrunk significantly after

exposure to lead nitrate, which may be consistent with our

observations. The changes in cellular morphology and size

may result from mechanical force and reciprocation between

surface-active components and metallic ions [20]. Therefore,

this structural change was attributed to the strong

crosslinking of metal (lead) and negatively charged chemical

groups on the cell wall polymers.

The EDX analysis showed that the elemental composition

of strain 25 was significantly changed after lead(II)

adsorption (Figs. 4A, 4B; Table 6). The atomic percentage of

element Pb (At %) was 6.31, C (At %) decreased from 22.47

to 8.23, and O and P increased respectively from 34.04 to

36.61 and 6.19 to 12.34 after lead(II) uptake. These results

indicated that lead(II) could covalently bond with C-, O-,

and P-containing functional groups. The functional groups

(carboxylate, hydroxyl, amino, and phosphate) on the

bacterial cell surface were previously found to be essential

for lead(II) adsorption [25, 28, 44, 50]. The adsorption of

lead(II) by strain 25 occurred primarily on the cell surface.

The atomic percentage of Na and K decreased from 2.94 to

1.82 and 1.20 to 0.60, respectively, after the cells were

mixed with lead(II), indicating the possible exchange of Pb

with Na and K on the cell surface [8, 47].

Table 5. Uptake capacity of lead by different bioadsorbents.

Biosorbent qmax/mg/g References

Geobacillus thermodenitrificans 1.29 [4]

Staphylococcus saprophyticus 100 [21]

Pseudomonas aeruginosa PU21 110 [41]

Hot spring consortium of microbes 74.4 [41]

Bacillus cereus 36.7 [32]

Alcaligenes BAPb.1 66.7 This study

Fig. 2. TEM image of Arthrobacter sp. 25.

Fig. 3. SEM images of Arthrobacter sp. 25 before (A) and after (B) lead(II) uptake.

1434 Jin et al.

J. Microbiol. Biotechnol.

To evaluate the changes of surface roughness, depth, and

width in response to lead(II) adsorption, the two-dimensional

and its corresponding three-dimensional topographic AFM

images of the strain 25 cell were compared and analyzed as

shown in Fig. 5. Before adsorption, the cells were elliptical

in shape with an average size of 2-3 μm length and 1 μm

width (Fig. 5A). This was longer than the length measured

by SEM, suggesting the layer of extracellular polymeric

substances may contribute to the increased thickness of the

cell surface. The cell surface was relatively smooth with a

RMS (root mean square) of 91.7 nm and Ra (roughness

average) of 20.4 nm (data not shown). After lead(II)

adsorption, the cell appeared to be coated with a soft,

compressible material. The RMS and Ra values, respectively,

increased to 160.0 nm and 21.9 nm. Obviously protuberant

components were unevenly distributed on the cell surface,

and stretched out upon retraction of the tip to hundreds of

nanometers in length (Fig. 5B), possibly resulting from the

bonding of lead(II) with the EPS polysaccharide, protein,

and amide [52]. The results indicated that lead(II) adsorption

by strain 25 included processes of surface adsorption and

micro-precipitation. The irregular shape and topography of

the cell resulted from the interactions between lead(II) and

the surface of the biosorbent. These observations are

consistent with the TEM and SEM analyses. Moreover, the

morphological changes after lead(II) adsorption were

partly attributed to different interaction forces between the

cell and substrate surfaces [5].

XRD analyses. XRD analyses before and after lead(II)

adsortpion are shown in Fig. 6. There was a wide diffraction

peak at the incident-beam 2θ of 14°-30°, which was formed

by polysaccharide and other organic ingredients. The

shape of this peak indicated that the original absorbents

had an amorphous structure. The XRD pattern of the

lead(II)-loaded biomass was distinct and complex with

some peaks narrowing and two new peaks emerging at

26.52° and 30.26°, indicating the deposition of lead hydroxides

and lead carbonates in the form of crystallized lead [53].

According to Jade 5.0 software analysis, the new peaks

were generated by the integration of lead(II) with P=O and

–OH. Together with the EDX analysis, it was deduced that

phosphoryl and hydroxyl were the main cellular surface

functional groups for lead(II) biosorption.

FTIR analyses. FITR was used to identify the biomass

functional groups involved in the adsorbing process, which

is important for elucidation of the surface-bonding

mechanism. The FTIR spectrum with and without the

lead(II) revealed significant differences in the absorption

peaks of functional groups (Figs. 7A, 7B). The FTIR spectrum

showed that the peaks of hydroxyl and amine groups shifted

from 3,296.61 to 3,307.13 cm-1, indicating the formation of

Fig. 4. Energy dispersive X-ray analyses of Arthrobacter sp. 25

before (A) and after (B) lead(II) uptake.

Table 6. Atomic ratio analyses of Arthrobacter sp. 25 before (A)

and after (B) lead(II) uptake.

Before biosorption After biosorption

Element At. % Element At. %

CK 22.47 CK 08.23

OK 34.04 OK 36.61

PK 06.19 PK 12.34

NaK 02.94 NaK 01.82

KK 01.20 KK 00.60

PbK — PbK 06.31

Matrix ZAF Matrix ZAF

Biosorption of Lead 1435

August 2016⎪Vol. 26⎪No. 8

more -OH and -NH by complexation; an anti-symmetric

stretching peak of -CH shifting from 2,927.06 to 2,929.03 cm-1

by interaction with lead(II); a symmetric stretching peak of

C=O shifting from 1,652.48 to 1,652.59 cm-1; an enhancement

of an amide peak between 1,535 to 1,560 cm-1; a stretching

vibration peak of C-O-C shifting from 1,234.47 to 1,231.30 cm-1;

a stretching peak of C-OH located at around 1,000 to

1,100 cm-1 now migrated from 1,066.11 to 1,055.33 cm-1; and

the reduced transmittance of the phosphate peak at

953.35 cm-1 indicated that the presence of metals caused

less stretching. NH- stretching was found to be responsible

for copper(II) and lead(II) bonding, and the C-O bond played

an important role in lead(II) sorption [34]. Additionally, a

report by Gupta and Rastogi [16] found that the presence of

amino, carboxyl, hydroxyl, and carbonyl groups on the

surface of the green alga Spirogyra was responsible for the

bonding of lead [45]. These phenomena further supported

the conclusion that available hydroxyl, amino, amide, carbonyl,

and phosphate functional groups on the Arthrobacter sp. 25

cell surface were associated with lead(II) biosorption.

In conclusion, the optimum conditions for lead(II)

adsorption by Arthrobacter sp. 25 were an initial lead ion

concentration of 108.79 mg/l, pH 5.75, and biosorbent

dosage of 9.9 g/l. Under these conditions, the maximum

adsorption capacity was 9.6 mg/g.

The experimental results showed that the lead-resistant

strain Arthrobacter sp. 25 can be effectively used for the

removal of lead ions from wastewater at low concentrations.

The complexity of the lead(II) removal mechanism was also

confirmed.

The cells of Arthrobacter sp. 25 are ellipsoidal shaped and

covered with EPS. The mechanism of biosorption of lead(II)

involves surface phenomena such as physical adsorption

Fig. 5. AFM images of Arthrobacter sp. 25 before (A) and after (B) contacting with lead(II).

Each group of images includes two-dimensional topographic images of the cell surface (left); sectional analysis determining the depth of cell

(middle); and three-dimensional topographic images of the cell surface (right). The lead(II) concentration is 100 mg/l.

Fig. 6. X-ray powder diffraction analyses of Arthrobacter sp. 25

before (A) and after (B) lead(II) uptake.

1436 Jin et al.

J. Microbiol. Biotechnol.

and microprecipitation as well as ion exchange. Phosphoryl,

hydroxyl, amino, amide, carbonyl, and phosphate functional

groups were all engaged in lead(II) adsorption.

This lead-resistant bacterial strain may be utilized as a

potential biotechnological agent for the bioremediation of

lead-contaminated water. This work may provide some new

insights into heavy metal removal in real-life bioprocesses

and contribute to the application of functional Arthrobacter

species.

Acknowledgments

This research is supported by the Educational Commission

of Heilongjiang Province, China (12541043).

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