SEPARATION SCIENCE AND ENGINEERING

Chinese Journal of Chemical Engineering, 21(11) 1244—1250 (2013)

DOI: 10.1016/S1004-9541(13)60629-1

Fabrication of Poly(γ-glutamic acid)-coated Fe3O4 Magnetic

Nanoparticles and Their Application in Heavy Metal Removal*

CHANG Jing (常菁)1, ZHONG Zhaoxiang (仲兆祥)1,**, XU Hong (徐虹)1,**, YAO Zhong (姚忠)1
and CHEN Rizhi (陈日志)2
1
College of Food Science and Light Industry, Nanjing University of Technology, Nanjing 210009, China
2
State Key Laboratory of Materials-Oriented Chemical Engineering, Jiangsu Key Laboratory of Industrial Water-
Conservation & Emission Reduction, Nanjing University of Technology, Nanjing 210009, China

Abstract In this study, poly(γ-glutamic acid)-coated Fe3O4 magnetic nanoparticles (γ-PGA/Fe3O4 MNPs) were
successfully fabricated using the co-precipitation method. Fe3O4 MNPs were also prepared for comparison. The av-
erage size and specific surface area results reveal that γ-PGA/Fe3O4 MNPs (52.4 nm, 88.41 m2·g−1) have smaller
particle size and larger specific surface area than Fe3O4 MNPs (62.0 nm, 76.83 m2·g−1). The γ-PGA/Fe3O4 MNPs
can remove over 99% of Cr3+, Cu2+ and Pb2+, and over 77% of Ni2+ in deionized water, much higher than γ-PGA
and Fe3O4 MNPs, attributed to the larger specific surface area of γ-PGA/Fe3O4 MNPs. With the solution pH higher
than 6.0, γ-PGA/Fe3O4 MNPs demonstrate better removal activity. The adsorption isotherm of γ-PGA/Fe3O4 MNPs
for Cr3+ fits the Freundlich model well, with the adsorption capacity of 24.60 mg·g−1. γ-PGA/Fe3O4 MNPs are
strongly attracted by permanent magnet, so it is easy to separate them completely from water. With their high effi-
ciency for heavy metal removal and easier separation, γ-PGA/Fe3O4 MNPs have great potential applications in wa-
ter treatment.
Keywords γ-PGA, magnetic nanoparticles, Fe3O4, heavy metal removal

1 INTRODUCTION to surfaces of magnetic nanoparticles for effective

Large volumes of metal-contaminated and slurry
wastewater are generated annually by various agricul-
tural, industrial, and sewage disposal processes [1, 2],
giving great impact on the environment and public
health. Various flocculants, such as inorganic, synthetic
organic polymeric and natural flocculants, are widely
used in industrial processes including wastewater treat-
ment and downstream processing [3]. Aluminum and
iron salts are among the main inorganic flocculants,
but aluminum is toxic to humans, through the food
chain and drinking water, and ferric salt is corrosive
and causes discoloration in water. Organic high-polymer
flocculants are commonly used because they are in-
expensive and highly effective, such as polyacryla-
mide, but several of them are not easily degraded in
nature. It is suggested that monomers derived from
synthetic polymers are harmful to humans [4-6]. Thus
the attention shifts to microbial biopolymer flocculants,
which offer advantages such as non-toxicity, environ-
mental friendliness, and biodegradability [7], and may
be attractive alternative to organic and synthetic poly-
meric flocculants. However, the major drawbacks of
bioflocculants, such as high cost, low removal efficiency,
and large dosage, limit their practical applications.
Recently, magnetic functional nanocomposites
become a research hotspot, owing to its magnetic sepa-
ration feature [8, 9]. Covalently immobilized polymers,
novel molecules or inorganic materials could be grafted
removal of toxic heavy metal ions from wastewater
[10]. For example, chitosan-modified magnetic nano-
composites provide a useful recyclable tool for removal
of heavy metal ions because of the interaction between
chitosan and heavy metal ions [8]. Fe3O4-silica core-
shell microspheres have been used to remove the
heavy metal ions from industrial wastewater [11].
Use of natural or modified microbial biopoly-
mers as flocculants for wastewater treatment becomes
a popular research focus [12]. Poly-γ-glutamic acid
(γ-PGA) is composed of D- and L-glutamic acid units
connected by amide linkages between α-amino and
γ-carboxylic acid groups. It is a water-soluble, anionic,
biodegradable, and edible biopolymer produced by
Bacillus subtilis. Moreover, γ-PGA has diverse poten-
tial applications in food, pharmaceuticals, healthcare,
water treatment, and other related fields [13, 14]. The
biodegradability and high molecular mass characteris-
tics of γ-PGA make it appropriate to be a bioflocculant,
an interest issue in the field of environmental engi-
neering. Moreover, γ-PGA can flocculate a wide range
of organic and inorganic compounds and bind several
metal ions (Ni2+, Cu2+, Mn2+, and Al3+) [15, 16].
γ-PGA displays better flocculation activity com-
bined other compounds as a flocculant [17]. With its
ability of binding metal ions and the magnetic separa-
tion capability of magnetite nanoparticles, the removal
efficiency of heavy metals can be improved. Thus the
present work aims to synthesize γ-PGA-modified

Received 2013-03-20, accepted 2013-08-23.

* Supported by the National Natural Science Foundation of China (21276124), the Research Project of Natural Science for
Universities Affiliated to Jiangsu Province (10KJB530002), Key Projects in the National Science & Technology Pillar Pro-
gram (2011BAE07B09-3), the Jiangsu Provincial Science and Technology Support Program (BE2011831), and the State High
Technology Research and Development Program of China (2011AA02A201).
** To whom correspondence should be addressed. E-mail: njutzzx@163.com (Z.X. Zhong); xuh@njut.edu.cn (H. Xu)
 Chin. J. Chem. Eng., Vol. 21, No. 11, November 2013
superparamagnetic iron-oxide nanoparticles (Fe3O4)
using the co-precipitation method under non-oxidizing
conditions, examine their capacities and preliminarily
explore the mechanism for heavy metal ion removal.
2 MATERIALS AND METHODS
2.1 Materials
γ-PGA with low molecular mass (~130 kDa) was
the coating material, synthesized from Bacillus sub-
tilis NX-2 through the batch fermentation according to
Yao et al [18]. Cells in the culture broth was separated
by centrifugation, and the supernatant containing
γ-PGA was ultrafiltered by membrane with exclusion
molecular mass of 50-100 kDa to remove salts and
other small molecular compounds. γ-PGA was ob-
tained through the methanol precipitation and lyophi-
lization, and the molecular mass was measured by
gel-permeation chromatography.
For the synthesis of MNPs, analytical grade ferric
chloride (FeCl3·6H2O) and ferrous chloride (FeCl2·4H2O)
were obtained from Sinopharm Chemical Reagent Co.
Ltd. (Shanghai, China). Ammonia solution (ammonium
hydroxide, 28%) was obtained from Nanjing Chemical
Reagent Co. Ltd. (Nanjing, China). Nitrogen gas was
used to create an oxygen-free atmosphere. Ultra-pure
water was obtained from the direct-pure plus water
system (Rephile Bioscience, Ltd.). The corresponding
metal chloride salts were used as metal cation sources.
All chemicals in the study were of analytical grade
and were used without further purification.
2.2 Synthesis of γ-PGA/Fe3O4 MNPs
Both γ-PGA/Fe3O4 MNPs and Fe3O4 MNPs were
synthesized using the co-precipitation method. About
6.2 g of ferric chloride and 4.2 g of ferrous chloride
were dissolved in 100 ml of ultra-pure water. Nitrogen
gas was bubbled into the reactor for 15 min prior to
the reaction to prevent oxidation of Fe2+, and was con-
tinuously bubbled throughout the reaction. The mixed
solution was vigorously stirred using a precision electric
stirrer (JJ-1 100W). The mixture was heated to 85 °C
Figure 1 Schematic diagram of the removal of heavy metals using γ-PGA/Fe3O4 MNPs
1245
through the water bath and 15 ml of ammonia solution
was then added dropwise to co-precipitate the iron
salts. The color of the mixed solution changed from
orange to black, which indicates the nucleation of
Fe3O4. About 50 ml of 20 g·L−1 γ-PGA solution was
subsequently added to the mixture. The mixture was
continuously stirred at 85 °C for 1.5 h for the growth
of Fe3O4 nuclei and coating of γ-PGA on Fe3O4 parti-
cles. The mixture was naturally cooled to room tem-
perature, and the precipitated γ-PGA/Fe3O4 MNPs
were separated from the solution using magnetic de-
cantation with a permanent magnet. The γ-PGA/Fe3O4
MNPs were washed three times with deionized water
and then freeze-dried for characterization. Fe3O4 MNPs
were prepared using the same method but without the
addition of γ-PGA.
2.3 Characterization of MNPs
Scanning electron microscopy (SEM) analysis
was performed using a QUANTA200 microscope (FEI,
Holland) to observe the surface morphology of MNPs.
The samples of Fe3O4 MNPs and γ-PGA/Fe3O4 MNPs
were initially coated with gold using a SCD500 high
vacuum-coating instrument. The SEM pictures were
taken at an accelerating voltage of 15.0 kV. The struc-
tures of Fe3O4 MNPs, γ-PGA/Fe3O4 MNPs and γ-PGA
were analyzed through Fourier transform infrared
(FT-IR) spectroscopy (Nexus 670) in the range of 400
cm−1 to 4000 cm−1 using a Nicolet 6700 spectrometer
(KBr disk, 64 scans, 4 cm−1 resolution). The surface
areas of the samples were measured based on the N2
adsorption isotherms (−196 °C) with a Micromeritics
ASAP 2020 instrument. The average size and size dis-
tribution of nanoparticles were determined by a size
analyzer (MasterSizer 2000, Malvern, UK). γ-PGA/Fe3O4
MNPs dispersed in deionized water were decanted
using a permanent magnet at room temperature.
2.4 Batch adsorption experiments of heavy metals
The schematic diagram of sorption and removal
of heavy metals by γ-PGA/Fe3O4 MNPs is presented
in Fig. 1.
1246 Chin. J. Chem. Eng., Vol. 21, No. 11, November 2013

(a) (b)
Figure 2 SEM images of Fe3O4 MNPs (a) and γ-PGA coated Fe3O4 MNPs (b)

γ-PGA/Fe3O4 MNPs were mixed with a 100 ml
solution containing Cr3+, Cu2+, Pb2+ or Ni2+ in the 250
ml stopper conical flasks to explore their capacity in
removing heavy metals from aqueous solutions.
Without special mentioning, the heavy metal ion con-
centration of the solution was 50 mg·L−1. The stopper
conical flask was agitated at 200 r·min−1 in an orbital
shaker at 30 °C for 2 h. The adsorbent was separated
with a permanent magnet and the solution was col-
lected for metal concentration measurement. The
metal concentrations of Cr3+, Cu2+, Pb2+ and Ni2+ were
measured using an inductively coupled plasma-atomic
emission spectrometer (ICP Optima 7000DV, Perki-
nElmer, USA). The heavy metal sorption efficiencies
of γ-PGA and Fe3O4 MNPs were examined via the
same manner. For γ-PGA, the adsorbent was separated
with a membrane with the pore size of 0.22 μm. The
removal efficiency of heavy metals is determined by
the following equation:
C −C
removal efficiency = 0 × 100% (1)
C0
where C0 is the initial heavy metal concentration and
C is the final concentration of the solution.
concentrations (mg·L−1), V is the volume of Cr3+ solu-
tion (L), and W is the mass of absorbate (g).
3 RESULTS AND DISCUSSION
3.1 Characterization
Figure 2 shows the SEM images of Fe3O4 MNPs
and γ-PGA/Fe3O4 MNPs. Both of MNPs are roughly
spherical and tend to aggregate together because of
their high surface energy and adhesion. Fig. 3 shows
that the average size of Fe3O4 MNPs is 62.0 nm and
that of γ-PGA/Fe3O4 MNPs is 52.4 nm. The surface
area of Fe3O4 MNPs is 76.83 m2·g−1 and that of
γ-PGA/Fe3O4 MNPs is 88.41 m2·g−1. γ-PGA/Fe3O4
MNPs present smaller particle size and larger specific
surface area, since one layer of biopolymer on the
surface of Fe3O4 MNP nuclei prohibits further growth
[19] and aggregation due to the steric and electrostatic
repulsion effects [20].

2.5 Kinetics and isotherm of adsorption for Cr3+

In order to study the relationship between the

adsorbed amount and time for Cr3+, 100 ml of Cr3+
solution in 50 mg·L−1 concentration was contacted
with about 0.05 g γ-PGA/Fe3O4 MNPs in a shaker at
room temperature.
To determine the effect of Cr3+ concentration on
the absorption capacity of γ-PGA/Fe3O4 MNPs, dif- Figure 3 Particle size distribution of Fe3O4 MNPs and
ferent concentrations of Cr3+ (10 mg·L−1 to 100 γ-PGA/Fe3O4 MNPs
mg·L−1) were used, and the pH value of solution was
adjusted to 6.0 with 0.1 mol·L−1 HCl or 0.1 mol·L−1 Figure 4 shows the FT-IR spectra of the samples.
NaOH. The adsorption amount of Cr3+ per unit mass For the Fe3O4 MNPs [Fig. 4 (a)], the peak at 590.19
of absorbate (mg·g−1) is calculated as follows cm−1 is related to the vibration of Fe O functional
( C0 − Ce )V group and fits well with the characteristic peak of
qe = (2) Fe3O4 [19, 21]. Three characteristic peaks in the γ-PGA
W IR spectrum [Fig. 4 (b)] correspond to the C O
where C0 and Ce are the initial and equilibrium Cr3+ stretching of free carboxylic acids at 1636.58 cm−1,
 Chin. J. Chem. Eng., Vol. 21, No. 11, November 2013 1247

(a) (b)

(c)
Figure 4 FT-IR spectra of Fe3O4 MNPs (a), γ-PGA (b) and γ-PGA/Fe3O4 MNPs (c)

Figure 5 Schematic diagram of formation mechanism for γ-PGA coating on Fe3O4

asymmetric COO¯ stretching at 1593.08 cm−1, and
symmetric COO¯ stretching at 1402.55 cm−1 [22]. The
four peaks at 1632.95, 1560.33, 1404.01, and 582.71
cm−1 in the γ-PGA/Fe3O4 MNPs IR spectrum [Fig. 4
(c)] consist of those of Fe3O4 MNPs and γ-PGA, indi-
cating that PGA is coated on the surface of Fe3O4
MNPs. However, the formation mechanism of γ-PGA
coating on Fe3O4 in the reactions is unclear. Magnetite
nanoparticles prepared by co-precipitation have exten-
sive hydroxyl groups on the surface because of contact
with the aqueous phase. Liu et al. [23] consider that
oleic acid forms a coating layer on the surface of
Fe3O4 MNPs through “esterification” or electrostatic
interaction between carboxylic acid groups of oleic
acid and hydroxyl groups of Fe3O4 MNPs. Thus, we
suggest that the carboxylic acid groups of γ-PGA and
hydroxyl groups of Fe3O4 MNPs form ester bonds or
carboxylic acid groups of γ-PGA and hydroxyl groups
of Fe3O4 MNPs interacte through electrostatic interac-
tion, as shown in Fig. 5.
Figure 6 (a) shows that γ-PGA/Fe3O4 MNPs dis-
perse in deionized water uniformly. Cheng et al. [20]
have reported that a surfactant coating on the surface
of Fe3O4 MNPs make nanoparticles disperse excel-
lently. γ-PGA generates self-exclusion through steric
and electrostatic repulsion effects because it is a linear
1248 Chin. J. Chem. Eng., Vol. 21, No. 11, November 2013

3.3 Removal activity

Figure 8 shows the removal efficiency of Cr3+

(a) (b)
Figure 6 γ-PGA/Fe3O4 MNPs dispersed in water (a) and
attracted by a permanent magnet (b)
polymer with a large free carboxyl group, so its
coating on the surface of Fe3O4 MNPs results in good
dispersion characteristics. Fig. 6 (b) shows that all
γ-PGA/Fe3O4 MNPs are strongly attracted by a per-
manent magnet in a magnetic decantation. The floc-
culants and metal γ-PGA/Fe3O4 MNPs can be easily
separated from the solution in wastewater treatment.
3.2 Heavy metal removal by γ-PGA/Fe3O4 MNPs
with γ-PGA/Fe3O4 MNPs, Fe3O4 MNPs, and γ-PGA.
As the amount of γ-PGA/Fe3O4 MNPs and Fe3O4
MNPs increased from 0.4 g·L−1 to 1.4 g·L−1, the re-
moval efficiency increased from 59.54% to 99.24%
and from 15.31% to 69.47%, respectively. With the
addition of γ-PGA in the range of 50 mg·L−1 to 400
mg·L−1, the removal efficiency increased from 8.74%
to 75.80%, attributed to more binding sites. The re-
sults indicate that γ-PGA/Fe3O4 MNPs have better
heavy metal removal efficiency than Fe3O4 MNPs and
γ-PGA. With γ-PGA, the heavy metal cations ab-
sorbed are separated by membrane and those unab-
sorbed pass through the membrane, lowering heavy
metal removal activity. Fe3O4 MNPs have been effec-
tively applied in the removal of Cr3+ from wastewater
because of their high surface area and high surface
energy that could adsorb metal ions on its surface.
Jing et al. [25] reported that Fe3O4 MNPs has the abil-
ity to remove metals in natural waters. γ-PGA/Fe3O4
MNPs overcome the drawbacks of γ-PGA and have
higher surface area than Fe3O4 MNPs, presenting bet-
ter heavy metal removal activity.

Figure 7 shows the effects of dosage of

γ-PGA/Fe3O4 MNPs on the heavy metal removal effi-
ciency with 100 ml of 50 mg·L−1 Cr3+, Cu2+, Ni2+,
Pb2+ solutions. The removal efficiency increased from
33.79% to 77.17% for Ni2+, 46.31% to 99.63% for
Cu2+, 59.54% to 99.24% for Cr3+, and 99.57% to
99.63% for Pb2+ with the increase of dosage. The re-
moval activity order of γ-PGA/Fe3O4 MNPs is Pb2+>
Cr3+> Cu2+>Ni2+. According to the hard soft acid base
principle proposed by Pearson [24], hard acids prefer
to bind with a hard base through ionic interactions and
soft acids prefer a soft base through covalent interac- (a)
tions. Thus these heavy metals interact with carboxy-
late anions and amide groups of γ-PGA. Pb2+ has a
better binding capacity with γ-PGA/Fe3O4 MNPs,
because it has higher molar mass and less amount of
charge than Cr3+.

(b)
Figure 8 Removal efficiency of heavy metals with γ-PGA/
Fe3O4 MNPs and Fe3O4 MNPs (a) and γ-PGA (b)

3.4 Effects of pH on heavy metal removal

Figure 7 Effect of the amount of γ-PGA/Fe3O4 MNPs on
the removal efficiency of heavy metals
2+ −1 3+ −1 2+ −1
■ Cu (50 mg·L ); ● Cr (50 mg·L ); ▲ Ni (50 mg·L ); pH value is an important parameter in an adsorption
−1
2+
▼ Pb (50 mg·L ) system because it involves the adsorption mechanism.
 Chin. J. Chem. Eng., Vol. 21, No. 11, November 2013
With 100 ml solution in 50 mg·L−1 of Cr3+ and 0.8 g
of γ-PGA/Fe3O4 MNPs, Fig. 9 shows that the removal
rate of Cr3+ increases remarkably from 7.19% to
99.66% as pH increases from 2 to 6. The pH value is
important because it changes the conformation of mo-
lecular structure and influences the stability and
speciation of metals [26]. The average pKa value of
native γ-PGA is 3.6 [1]. Since the initial pH of 2.0 is
lower than the pKa value of γ-PGA, the carboxyl
group of γ-PGA remains in its non-ionized form. In
addition, the R-helix conformation of γ-PGA is more
compact at low pH, which could cause the formation
of intermolecular hydrogen bonds in γ-PGA [14, 27].
All these prevent binding with γ-PGA. Poor adsorp-
tion of Cr3+ occurs at pH 2.0 when the conformation
of γ-PGA changes from R-helix to a random coil, and
when the carboxyl groups are ionized at pH 4.0>3.6.
Therefore, the removal efficiency increases rapidly as
the pH value increases from 2.0 to 4.0.
Figure 9 Effect of pH on heavy metal removal efficiency
with γ-PGA/Fe3O4 MNPs
3.5 Equilibrium adsorption isotherm
Figure 10 shows the absorption amount with time
Figure 10 Adsorption of Cr3+ with γ-PGA/Fe3O4 MNPs
(Cr3+ solution: 100 ml, 50 mg·L−1; γ-PGA/Fe3O4 MNPs: 0.05 g)
Table 1 Langmuir and Freundlich isotherm constants
Langmuir isotherm
B qm
γ-PGA-Fe3O4 MNPs 0.00997 162.6
1249
for Cr3+. About 120 min is required to reach the ad-
sorption equilibrium, which is the basis for choosing
the adsorption time for other experiments.
Adsorption isotherms are obtained by varying the
initial concentration of Cr3+. The experimental data are
analyzed using the Langmuir and Freundlich equations.
The Langmuir isotherm model has the following form [28]
bq C
qe = m e (3)
1 + bCe
where Ce is the equilibrium concentration of metals in
the solution (mg·L−1), qm is the amount of adsorbate
forming a complete monolayer coverage (mg·g−1), and
b is a constant relevant to the heat of adsorption.
The Freundlich isotherm model has the following
form [28]
qe = K FCen (4)
where KF (mg) represents the adsorption capacity
when the metal equilibrium concentration is equal to 1
and n represents the degree of dependence of adsorp-
tion with equilibrium concentration.
The adsorption isotherm of γ-PGA/Fe3O4 MNPs
for Cr3+ is shown in Fig. 11. The constants and corre-
lation coefficients R2 obtained through non-linear fit-
ting are listed in Table 1. The adsorption isotherm is
well represented by the Freundlich model. It is diffi-
cult to directly compare the magnetite nanoparticles
with other adsorbents, because of different experi-
mental conditions. Generally, the adsorption capacity
could be represented by the value of Freundlich pa-
rameters KF, and some values of KF by various ad-
sorbents in literature [29-33] are summarized in Table 2.
Compared with other adsorbents, the γ-PGA/Fe3O4
MNPs developed in this study are among those with
good adsorption capacities.
Figure 11 Adsorption isotherm for Cr3+ with γ-PGA/Fe3O4
MNPs at pH 6.0 (initial concentration range: 10-100 mg·L−1
for Cr3+; γ-PGA/Fe3O4 MNPs dose: 2 g·L−1; temperature: 30 °C;
contact time: 2 h)
Freundlich isotherm
2
R KF 1/n R2
0.9901 24.60 0.408 0.9004
1250 Chin. J. Chem. Eng., Vol. 21, No. 11, November 2013
Table 2 Comparison between the adsorbent in this study
with other adsorbents in literature
Adsorbents KF/mg·g−1 References
γ-PGA/Fe3O4 MNPs 24.60 this study
maghemite 19.2 [29]
diatomite 11.55 [30]
activated carbon 15.47 [31]
beech sawdust 16.13 [32]
mesoporous magnetic γ-Fe2O3 15.6 [33]
4 CONCLUSIONS
In this study, we successfully synthesized
γ-PGA/Fe3O4 MNPs via the co-precipitation method.
γ-PGA/Fe3O4 MNPs display excellent removal activ-
ity for Cr3+, Cu2+, Pb2+, and Ni2+, with better removal
activity than γ-PGA and Fe3O4 MNPs. The removal
activity of γ-PGA/Fe3O4 MNPs also depends on the
pH value of the heavy metal solution, exhibiting the
best removal activity at pH of 6.0. The adsorption
isotherm of γ-PGA/Fe3O4 MNPs for Cr3+ fits well
with the Freundlich model, and its adsorption capacity
(KF) is 24.60 mg·g−1. The results indicate that the
γ-PGA/Fe3O4 MNPs have great potential for use in
water treatment.
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