Decomposing phenol by the hidden talent of ferromagnetic nanoparticles
Jinbin Zhanga,b, Jie Zhuanga,b, Lizeng Gaoa,b, Yu Zhangc, Ning Guc, Jing Fenga, Dongling Yanga,
Jingdong Zhua, Xiyun Yana,*
aNational Laboratory of Biomacromolecules and Chinese Academy of Sciences, University of Tokyo Joint Laboratory of Structural Virology and Immunology,
Institute of Biophysics, Chinese Academy of Sciences, Mailbox 1, 15 Datun Road, Beijing 100101, China
bGraduate University of Chinese Academy of Sciences, Beijing 100049, China
cState Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
a r t i c l ei n f o
Received 7 April 2008
Received in revised form 21 May 2008
Accepted 22 May 2008
Available online 19 September 2008
a b s t r a c t
Researches on modified Fenton reactions applied in phenol degradation have been focused on reducing
secondary pollution and enhancing catalytic efficiency. Newly developed methods utilizing carriers, such
as Resin and Nafion, to immobilize Fe2+could avoid iron ion leakage. However, the requirement of high
temperature and the limited reaction efficiency still restrained them from broad application. Based on a
recently discovered ‘‘hidden talent” of ferromagnetic nanoparticles (MNPs), we established a MNP-cata-
lyzed phenol removal assay, which could overcome these limitations. Our results showed that the MNPs
removed over 85% phenol from aqueous solution within 3 h even at 16 ?C. The catalytic condition was
extensively optimized among a range of pH, temperature as well as initial concentration of phenol and
H2O2. TOC and GC/MS analysis revealed that about 30% phenol was mineralized while the rest became
small molecular organic acids. Moreover the MNPs were thermo-stable and could be regenerated for
at least five rounds. Thus, our findings open up a wide spectrum of environmental friendly applications
of MNPs showing several attractive features, such as easy preparation, low cost, thermo-stability and
? 2008 Published by Elsevier Ltd.
Phenol and phenolic compounds are the most important repre-
sentative of organic pollutants generated by various industrial pro-
cesses. Since they are highly toxic and suspicious carcinogens,
many processes such as physical adsorption (Nevskaia et al.,
2004), chemical oxidation (Garcia-Molina et al., 2005) and biolog-
ical degradation (Jiang et al., 2006) have been developed to remove
phenol from wastewater. Among these methods, a solution of Fe3+/
Fe2+and H2O2, known as Fenton reagent, is frequently used to drive
the oxidation of organic contaminants (Tarr, 2003). However, the
iron ions and the sludge generated by Fenton reagent required fur-
ther treatments and rose up new disposal problems. To overcome
the drawbacks, various modified Fenton systems have been devel-
oped. For example, immobilized Fenton reactions using Fe3+loaded
on resin, clay or Nafion have been developed (Sabhi and Kiwi,
2001; Catrinescu et al., 2003; Liou et al., 2004; Carriazo et al.,
2005; Liou et al., 2005; Nikolopoulos et al., 2006). In addition, het-
erogeneous Fenton-like systems were made using goethite (Lu
et al., 2002), hematite (Feng et al., 2004), AFe2O4(A = Fe, Ni, Zn)
(Guin et al., 2005) and Fe0/Fe3O4(Moura et al., 2005), which are
usually slow and need additional assistants such as UV and ultra-
sound. Compared with the chemical reagents, enzymatic catalysis
is more active and environmental friendly. For instance, several
commercial peroxidases were investigated for removing phenol
from aqueous solutions (Bodalo et al., 2006). There were also at-
tempts trying to immobilize them on clay and other vectors
(Akhtar and Husain, 2006; Cheng et al., 2006). However, because
of their protein nature, the instability and costliness of the enzyme
made them not feasible in the treatment of wastewater although it
has been reported for almost 30 year (Klibanov et al., 1980). There-
fore, it is important and necessary to find new materials and estab-
lish new methods.
Recently we have found that the ferromagnetic nanoparticles
(MNPs) are intrinsically active catalyst for oxidation reactions sim-
ilar to that found in natural peroxidases (Gao et al., 2007). What is
more, the MNPs have a definite advantage compared with their
protein-based counterparts because they are considerably more
stable over a wide range of temperatures and various levels of acid-
ity. Based on this finding, we studied the potential application of
MNPs in phenol removal and developed a MNP-catalyzed phenol
degradation assay. Our data show that this assay is efficient and
economical. Furthermore, the stability and reusability of MNPs
promised broad applications in (but not restricted in) the treat-
ment of phenolic wastewater.
0045-6535/$ - see front matter ? 2008 Published by Elsevier Ltd.
* Corresponding author. Tel.: +86 10 6488 8583; fax: +86 10 6488 8584.
E-mail address: firstname.lastname@example.org (X. Yan).
Chemosphere 73 (2008) 1524–1528
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2. Materials and methods
Horseradish Peroxidase (HRP), 3,30,5,50-tetramethyl benzidine,
catalase, 5,50-dimethyl-1-pirroline-N-oxide (DMPO) and BSTFA
(N,O-Bis (trimethylsilyl) trifluoro acetamide) + TMCS (Trimethyl-
chlorosilane) were purchased from Sigma–Aldrich Inc (USA).
H2O2 (30% v/v), 4-AAP (4-aminoantipyrine), K3Fe(CN)6, Phenol,
FeSO4,FeCl3? 6 H2O and NH3? H2O were from Beijing Chemical Re-
agents Company (China). All chemicals and reagents used were of
2.2.1. MNP synthesis
MNPs with a diameter of 13 nm were prepared according to the
method of Molday (1984). Briefly, a solution of 10 mM FeCl3and
6 mM FeSO4 were mixed at pH 1.7 under N2 protecting. Then,
ammonia aqueous solution (1.5 M) was dropped into the mixture
solution with violently stirring until the pH of the solution raised
up to 9. The MNPs were washed immediately with water for 5
times and then ethanol for 2 times. Finally, MNPs were either dis-
solved in ethanol (for long time storage) or dried at room temper-
ature under vacuum (for instant usage).
2.2.2. Phenol removal reaction and detection
carried out in a tube with 125 ll H2O (pH 3.0) containing 12 lg
MNPs and 3 mM phenol. For HRP catalysis, the experiments were
performed with 1.5 lg HRP and 3 M phenol in 125 ll 0.1 M Tris-
HCl buffer (pH 8.0). The reactions were initiated by adding H2O2to
a final concentration of 60 mM (for MNPs) or 6 mM (for HRP) for
3 h at room temperature. Then, 125 U ml?1(final concentration)
Catalase was added to stop the reaction and decompose excessive
H2O2. Finally, the reaction was centrifuged at 13000g for 3 min,
and 2 ll supernatant from each reaction was taken for analysis.
Two microliter sample was mixed in a 96-well plate with 50 ll
reaction buffer A (16.7 mM K3Fe(CN)6in 0.25 M NaHCO3) and 50 ll
reaction buffer B (4.18 mM 4-AAP in 0.25 M NaHCO3). After incu-
bation for 5 min, the phenol concentration was measured at
490 nm using a Bio-Rad Microplate Reader 550.
2.2.3. Recycle of MNPs
A phenol removal experiment was performed using 24 lg
MNPs. After each reaction, the MNPs were collected using a
magnet, washed with 200 ll deionized water and sonicated
for 2 min. The regenerated MNPs were applied for the next
2.2.4. Analysis of MNP-catalyzed products
For TOC assay, the MNP-catalyzed products were first diluted
15 times with H2O and then filtrated with a 0.45 lm Millipore filter
before submitted into an analyzer (Analyti kjena AG, Germany).
For GC/MS assay, the sample were first freeze-dried and dis-
solved in 1 ml methylene dichloride, followed by 0.1 ml BSTFA/
TMCS (99:1) for derivatization. After incubating at 60 ?C for
30 min, the sample were dehydrated by Na2SO4and filtrated with
0.45 lm Millipore filter before submitted to an analyzer (Agilent
For electron spin resonance (ESR) assay, 40 ll samples were ta-
ken instantaneously at different time point from phenol removal
reaction and mixed with 10 ll 500 mM DMPO to form DMPO-OH
adduct. The amount of hydroxyl radicals was indicated by ESR sig-
nals using a Bruker model ER 200D spectrometer.
3. Results and discussion
3.1. MNPs catalyze phenol degradation
First, we prepared MNPs using the method of Molday (1984).
The character of the MNPs was studied using TEM and ED photog-
raphy, showing that the MNPs appeared approximately spherical
with an average diameter of 13 nm (Supplemental Figs. 1 and 2).
Their saturation magnetization value was 65.45 emu g?1and coer-
civity value was 20.89 Oe as determined by hysteresis loops (Sup-
plemental Fig. 3) indicating the MNPs with good magnetism
character. The detailed characterization of the MNPs has been pre-
sented elsewhere (Ma et al., 2004).
Next, we mixed these MNPs with hydrogen peroxide and phe-
nol together in water solution to find out whether the peroxi-
dase-like activity of MNPs could catalyze the decomposing of
phenol. As shown in Fig. 1a, the MNPs dramatically degraded phe-
nol in the reaction and about 60% phenol was removed within the
first 60 min. The maximal phenol removal efficiency of MNPs was
higher than 85% after 180 min. In contrast, little change in phenol
concentration was observed in control reactions without H2O2. In
addition, the absorption of phenol by MNPs was neglectable even
with double dose (Fig. 1b). These data demonstrate that it is
MNP catalysis but not unspecific absorption that results in phenol
Fig. 1. The MNPs catalyzed phenol removal with little non-specific absorption. (a) Phenol concentration decreased as MNP catalysis went on and (b) absorption of phenol by
MNPs was neglectable.
J. Zhang et al./Chemosphere 73 (2008) 1524–1528
3.2. MNP-catalyzed phenol removal depend on H2O2and phenol
To test whether the catalytic activity of the MNPs is, like the
conventional enzyme-HRP, dependent on the concentration of
H2O2, we performed phenol removal experiments using a range
of H2O2concentrations from 0.3 to 240 mM. Reactions were initi-
ated by adding either 12 lg MNPs or 1.5 lg HRP. The results
showed that both MNPs and HRP removed phenol effectively, but
their dependence on H2O2was largely different. To reach the max-
imal level of phenol removal, the MNPs required 60 mM H2O2,
which is one order of magnitude higher than HRP (6 mM). We be-
lieve that is because of their different catalyzing mechanism. As
essentially iron oxides, MNPs might catalyze phenol removal
through a chemical catalysis mechanism in which the Fe2+/Fe3+
on the surface of Fe3O4particles might play as a Fenton-like re-
agent whereas HRP catalyzed the reaction in an enzymatic way.
In addition, we found that further increasing of H2O2concentration
would result in inhibition of both HRP and MNP catalysis. The HRP
catalysis was sharply suppressed when H2O2concentration was
more than 10 mM (Fig. 2a), while the MNP catalysis was inhibited
when H2O2increased to 480 mM (Supplemental Fig. 4).
To detect whether the phenol concentration also affects the
MNP catalysis, we used a range of initial phenol concentrations
from 0.038 to 13.76 mM. As shown in Fig. 2b, MNP and HRP catal-
ysis were both decreasing with the increase of phenol concentra-
tion. More than 90% phenol was removed by either MNPs or HRP
when the initial phenol concentration was around 2 mM. However,
when phenol concentration increased to 12 mM, the MNPs showed
80% efficiency while the HRP remained less than 30%. Moreover,
further study showed that the optimal H2O2/phenol ratio for HRP
catalysis varied from 0.5 to 2.0 which is consistent with the previ-
ous report (Klibanov et al., 1983; Liu et al., 2002). However, the
optimal H2O2/phenol ratio for MNPs ranged from 2 to 160, which
is much broader than HRP. This indicates that the MNPs are better
than HRP in handling large amount of pollutants.
3.3. MNP-catalyzed phenol removal depend on pH but not
In order to find optimal condition of MNPs in phenol removal,
we set up a range of values of pH from 1 to 9.5 and temperatures
from 4 to 90 ?C. The results showed that the MNP-catalyzed phenol
removal was dependent on pH but not temperature. The optimal
pH for MNP catalysis was restricted around 3. When the pH was
lower than 2 or higher than 4, the MNP catalysis was sharply de-
creased (Fig. 3a). This observation is consistent with the typical
Fenton process which is carried on at pH 3.0 (Edwards et al.,
1992). It might be due to the catalytic hydration shell forms differ-
ent complexes according to the pH range. In more acidic solutions,
Fig. 2. Phenol removal efficiency of HRP and MNPs depend on the initial concentrations of both H2O2(a) and phenol (b). The reactions were taken at pH 3 for MNPs and pH 8
Fig. 3. Phenol removal efficiency of MNPs depends on pH (a) rather than temperature (b).
J. Zhang et al./Chemosphere 73 (2008) 1524–1528
shell water; at more basic pH-values, the OH groups might replace
the H2O groups. Both of the effects are detrimental to the Fe-cata-
lytic activity of decomposing H2O2to generate hydroxyl radicals.
Surprisingly, unlike typical Fenton processes which usually re-
quire high temperature from 40 to 80 ?C (Catrinescu et al., 2003;
Liou et al., 2005), MNPs effectively catalyzed phenol degradation
under temperatures varied from 16 to 95 ?C (Fig. 3b). Over 85%
phenol removal efficiency could still be achieved even at 16 ?C.
These data support that MNP catalysis is pH but not temperature
shell protonation will result in releasing hydration
3.4. MNPs are thermo-stable
As inorganic nanomaterial, MNPs are expected to be more sta-
ble than the enzyme-HRP which is a protein and easy to be dena-
tured. To examine the stability of MNPs, we first treated the HRP
and MNPs at a range of temperatures from 5 to 90 ?C for 3 h, and
then the heat-treated catalysts were applied for phenol removal
experiment under the optimal conditions. As shown in Fig. 4, the
MNPs appeared very good thermo-stability. Even after treated at
90 ?C for 3 h, the MNPs still remained more than 80% activity in
phenol removal. In contrast, the enzymatic activity of HRP dramat-
ically decreased after treatment at temperatures greater than
60 ?C. These data suggest that MNPs are thermo-stable, which
can be easily stored at temperature from 0 to 90 ?C.
3.5. MNPs are reusable
The catalytic property coupled with magnetically driven cap-
ture techniques endowed the MNPs a unique advantage in waste-
water treatment. To examine the reusability of MNPs, we repeated
phenol removal experiments for 5 times using the same MNPs
regenerated simply by sonication and washing with deionized
water. After 5 rounds of recycle, the MNPs still remained almost
100% catalytic activity (Fig. 5). The combination of catalytic activity
and magnetic recovery of MNPs on the one hand prevents second-
ary contamination caused by MNP leakage, on the other hand fur-
ther lower the cost per treatment. Considering both the cost of
preparation ($2.5 kg?1) and the reusability of MNPs, it would be
very cheap when applied in wastewater treatment.
Fig. 4. Comparing the thermo-stability of MNPs and HRP. Both catalysts were first
treated at temperatures indicated in the figure for 3 h and then applied for phenol
removal experiment at the optimal condition.
Fig. 5. Reusability of the MNPs in phenol removals.
Fig. 6. TOC removal efficiency of MNPs. MNPs catalyzed reactions were stopped at
different time points when samples were analyzed for both TOC and phenol
Fig. 7. ESR spectrum detected in phenol removal reaction with MNPs (a) or not (b). (c) ESR signal of DMPO-OH radical observed in MNP catalysis at different time points.
J. Zhang et al./Chemosphere 73 (2008) 1524–1528
3.6. Analysis of phenol degradation product
In order to figure out whether all the phenol was mineralized
during the MNP catalysis, we further tested the TOC values of reac-
tions carried on for different times. The results showed that 30% of
phenol was mineralized by MNPs after 3 h reaction (Fig. 6). The
rest of reaction products were identified by GC/MS to be small
molecular organic acids, including lactic acid, tartaric acid, oxalic
acid, succinic acid, maleic acid and 1,2-propylene glycol. These
data suggest that MNPs degraded phenol partially by cleaving phe-
nyl to form small molecular organic acids.
Furthermore, we used ESR assay to detect whether any hydro-
xyl radicals were produced during MNP catalysis. The four-line sig-
nal with a peak height ratio of 1:2:2:1 showed in the results is
matched with the pattern of the typical DMPO-OH adduct
(Fig. 7a) while in the contrast no signal was observed in control
reactions (without MNPs, Fig. 7b), indicating the presence of hy-
droxyl radicals in the products. Moreover, although the phenol oxi-
dation reaction was almost stopped after 3 h, the intensity of
DMPO-OH radical was still about one third of that when the reac-
tion started (Fig. 7c). This implies that the MNPs could drive the
generation of hydroxyl radicals constantly which is in consistent
with the reusability. Taken together, we speculate the mechanism
of MNPs in phenol removal is that the MNPs catalyzed H2O2to gen-
erate hydroxyl radicals, which in turn attacked the phenyl and
break down it into small molecular organic acids.
Our results demonstrated that MNPs can be used as a promising
catalyst to remove phenolic pollutants from aqueous solutions.
Over 85% phenol could be removed at optimal reaction condition.
There are 4 significant advantages of using MNPs in wastewater
treatment: (1) The MNPs are cheap and easy to prepare, which
make them more feasible than other effective but expensive re-
agents or processes. (2) As being composed of inorganic materials,
MNPs are robust and thermo-stable. (3) With the chemical essen-
tial of Fe3O4, MNPs are nontoxic and environmental friendly. (4)
The MNPs could be recycled and regenerated. These attractive fea-
tures endowed them broad applications in various processes such
as phenolic wastewater treatment.
This work is partly supported by grants from the NSFC founda-
tion (Nos. 90406020 and 30672436), the Knowledge of Innovation
Program of the Chinese Academy of Sciences (Kjcx2.yw.nano02,
kjcx2-yw-r-121 and kjcx2-yw-m02), the Chinese Ministry of Sci-
ences (2006CB933204, 2006DFB32010 and 2006CB910901), the
Chinese 863 Project (2006AA02A245), and Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan.
Appendix A. Supplementary material
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