Enhanced reductive transformation of 2,4-dinitroanisole in a anaerobic system: The key role of zero valent iron

Article abstract of DOI:10.1039/c5ra11197h

Accelerated reduction of typical multi-substituted nitroaromatic compounds (NACs), i.e., 2,4-dinitroanisole (DNAN), was achieved in an anaerobic system coupled with zero valent iron (ZVI), with the underlying role of ZVI in this process elucidated. Both removal of DNAN and formation of its final reductive product 2,4-diaminoanisole (DAAN) were notably improved in the ZVI coupled biosystem. In the ZVI coupled biosystem and biotic control system, complete removal of DNAN could be achieved within 4 h and 20 h, respectively. However, only 28.71 ± 5.06% of DNAN could be removed in the ZVI control system after 20 h. Correspondingly, the formation efficiencies of DAAN in the ZVI coupled biosystem, biotic control system and ZVI control system were 99.66 ± 0.70%, 16.99 ± 1.73% and 0.00 ± 0.00%, respectively. The increased DNAN removal and DAAN formation in the ZVI coupled biosystem was linked to the high accumulation of formate, low oxidation-reduction potential (ORP) and great pH self-buffering capability, which was provided by the addition of ZVI. Compared with the biotic control system, the production of CH₄ was significantly accelerated in the ZVI coupled biosystem, indicating that a favorable environment for methanogens was created at the presence of ZVI. Especially, the ZVI coupled biosystem displayed a more stable performance in terms of DNAN reduction with the coexistence of the competitive electron acceptors, such as nitrate and sulfate. Therefore, the ZVI coupled biosystem could be a promising alternative to the conventional anaerobic reduction process for the removal of multi-substituted NACs from wastewater.

Full text of DOI:10.1039/c5ra11197h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Enhanced reductive transformation of 2,4-
dinitroanisole in a anaerobic system: the key role of
Cite this: RSC Adv., 2015, 5, 75195
zero valent iron†
a
ab
a
a
a
Changjin Ou, Shuai Zhang, Jianguo Liu, Jinyou Shen,* Weiqing Han,
a
a
a
Xiuyun Sun, Jiansheng Li and Lianjun Wang*
Accelerated reduction of typical multi-substituted nitroaromatic compounds (NACs), i.e., 2,4-dinitroanisole
DNAN), was achieved in an anaerobic system coupled with zero valent iron (ZVI), with the underlying role of
(
ZVI in this process elucidated. Both removal of DNAN and formation of its final reductive product 2,4-
diaminoanisole (DAAN) were notably improved in the ZVI coupled biosystem. In the ZVI coupled
biosystem and biotic control system, complete removal of DNAN could be achieved within 4 h and 20 h,
respectively. However, only 28.71 ꢀ 5.06% of DNAN could be removed in the ZVI control system after
20 h. Correspondingly, the formation efficiencies of DAAN in the ZVI coupled biosystem, biotic control
system and ZVI control system were 99.66 ꢀ 0.70%, 16.99 ꢀ 1.73% and 0.00 ꢀ 0.00%, respectively. The
increased DNAN removal and DAAN formation in the ZVI coupled biosystem was linked to the high
accumulation of formate, low oxidation–reduction potential (ORP) and great pH self-buffering capability,
which was provided by the addition of ZVI. Compared with the biotic control system, the production of
4
CH was significantly accelerated in the ZVI coupled biosystem, indicating that a favorable environment
for methanogens was created at the presence of ZVI. Especially, the ZVI coupled biosystem displayed a
more stable performance in terms of DNAN reduction with the coexistence of the competitive electron
acceptors, such as nitrate and sulfate. Therefore, the ZVI coupled biosystem could be a promising
alternative to the conventional anaerobic reduction process for the removal of multi-substituted NACs
from wastewater.
Received 12th June 2015
Accepted 1st September 2015
DOI: 10.1039/c5ra11197h
www.rsc.org/advances
compounds (NACs) harbors a highly electron decient p-electron
1
. Introduction
system, resulting into the difficulty in chemical oxidation or bio-
4
2
,4-Dinitroanisole (DNAN) is an important ingredient in the logical oxidation. Moreover, with the increase of the nitro group
1
production of dyes and insecticides. Recently, DNAN is being number, mineralization of multi-substituted NACs such as DNAN
5
considered as a replacement for sensitive explosives such as 2,4,6- through oxidative pathways becomes more resistant. Thus, there
2
trinitrotoluene (TNT), because of its insensitive properties.
are signicant needs of appropriate methods for the remediation
Compared to TNT, the detonation temperature of DNAN is higher, of the sites contaminated by DNAN.
which is benecial for manufacture, transport and store
Under anaerobic or anoxic conditions, NACs succumb to
processes. However, considering its potential environmental risk, electrophilic attack and can be transformed to their corre-
3
high toxicity, poor biodegradability and wide usage, improper sponding aromatic amines but without cleaving the aromatic
disposal of DNAN containing waste can lead to tremendous ring. Generally, the produced aromatic amines are less toxicity
6
environmental pollution, including water contamination and soil and easier to mineralize than their parent compounds.
problems. Due to the pronounced electron-withdrawing character Nevertheless, due to the highly recalcitrant and toxicological
of the nitro groups on the benzene ring, nitroaromatic nature of NACs, the anaerobic reduction is usually limited by
low degradation rate and poor stability. Therefore, it is impor-
a
tant to improve anaerobic reduction performance to achieve
more effective reduction of NACs such as DNAN.
Jiangsu Key Laboratory for Chemical Pollution Control and Resources Reuse, School of
Environmental and Biological Engineering, Nanjing University of Science and
Technology, Nanjing, 210094, China. E-mail: shenjinyou@mail.njust.edu.cn;
wanglj@mail.njust.edu.cn; Fax: +86 25 84303965, +86 25 84315941; Tel: +86 25
Zero valent iron (ZVI) is currently attracting wide interest in
the treatment of wastewater and groundwater due to its inex-
pensive, reliable and moderately strong reduction properties.
Some refractory contaminants at oxidative state, such as NACs,
azo dyes and halogenated organic compounds, could be effec-
8
4303965, +86 25 84315941
b
Jiangsu Changhuan Environmental Science Co. LTD, Changzhou 213022, Jiangsu
Province, China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c5ra11197h
7–9
tively reduced in the ZVI process. For the treatment of the
1
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 75195–75203 | 75195

View Article Online
RSC Advances
Paper
wastewater containing these refractory contaminants, ZVI 2-amino-4-nitroanisole (2-A-4-NAN) were purchased from
reduction process is oen used prior to the biological process Bepharm Co. Ltd (Shanghai, China). DAAN was purchased from
4
,10
for reducing toxicity and improving biodegradability.
Ahn Sun Chemical Technology Co. Ltd (Shanghai, China). ZVI
11
et al. reported that the iron pretreatment not only removed powder with analytical purity was purchased from Sinopharm
energetic compounds but also eliminated the toxic effect on Chemical Reagent Co. Ltd (Shanghai, China) and was used
perchlorate reducing bacteria. Oh et al. also showed that the without pretreatment.
ZVI pretreatment transformed recalcitrant hexahydro-1,3,5-
12
trinitro-1,3,5-triazine (RDX) to ring-opening products, i.e., 2.2 Synthetic wastewater and sludge cultivation
formaldehyde, which are more amenable to mineralization by
aerobic bacteria. Therefore, the combined ZVI and biological
The composition of the synthetic wastewater used in this study
ꢁ1
ꢁ1
was as follows: DNAN (50 mg L ), methanol (0.84 mL L ), KH
2
PO
4
process offers bright prospects for the treatment of highly
ꢁ1
ꢁ1
ꢁ1
(
25 mg L ), NH
4
Cl (100 mg L ), MgSO
4
$7H
2
O (200 mg L ), CaCl
2
13
recalcitrant industrial wastewater. In general, ZVI process and
biological process was oen operated in sequence, however,
coupling of ZVI into the biological process could facilitate the
degradation of pollutants in a single reactor, which might take
ꢁ1
ꢁ1
(30 mg L ), and trace element solution (SL-4, 10 mL L ). In order
to give sufficient electron donor for DNAN reduction, methanol
was added excessively. The composition of SL-4 was as described
20
previously by Shen et al.
14
full advantage of both ZVI and biological process. Considering
its reductive property, ZVI is expected to be helpful for creating
an enhanced anaerobic environment which may be benecial to
improve the performance of an anaerobic reactor in wastewater
Anaerobic sludge taken from an anaerobic baffled reactor
treating real NACs containing wastewater was used as the seed
sludge. Before inoculation, the seed sludge was acclimated for
about three months using the synthetic wastewater as the
inuent. Once stable reduction performance of the acclimation
system was achieved, the acclimatized sludge could be used as
the inoculum.
15
treatment. Meanwhile, ZVI corrosion products, especially
alkaline byproducts Fe(OH) or Fe(OH) , can not only act as the
2
3
acid buffers, but also provide another alternative for the
contaminant removal through occulation, adsorption and
16
precipitation.
2.3 Experimental procedure
Given these, attentions have been increasingly paid to the
combined use of ZVI and microbe for enhanced degradation of
In this study, DNAN reduction was performed in batch mode,
which was carried out in a series of 100 mL serum bottles. 80 mL
8,17
18
recalcitrant contaminants from wastewater.
Liu et al.
ꢁ1
synthetic wastewater containing 50 mg L DNAN was added
into each serum bottle. To remove any residual dissolved
oxygen, the synthetic wastewater was purged with nitrogen for
at least 20 min, followed by the addition of the 0.1 g ZVI and 20
mL seed sludge prepurged with nitrogen. The initial DNAN and
MLSS concentrations in each serum bottle were calculated to be
reported that both azo dye decolorization and COD removal
were remarkably improved in an acidogenic reactor packed with
ZVI. At the presence of ZVI, the abundance of methanogens was
signicantly increased and microbial strains responsible for azo
15
dye decolorization were enriched in the anaerobic reactor. Of
even greater importance, the release of H during ZVI corrosion
2
ꢁ
1
ꢁ1
4
0 mg L and 13 g L , respectively. Then, the serum bottles
became an alternative electron donor for hydrogen-consuming
microorganisms, such as methanogenic and denitrifying
were sealed with polytetrauoroethylene/silica plugs and
19
aluminum crimp seals. All serum bottles were incubated on a
bacteria, as well as some reduction related species. Even
though these physicochemical and microbial interactions are
highly important for the overall performance of the coupled
system, systematic investigation on the ZVI coupled anaerobic
reduction system is still limited. In addition, coupling of ZVI
into an anaerobic biological system for the treatment of multi-
substituted NACs containing wastewater has been rarely inves-
tigated, and the underlying role of ZVI in the coupled system
treating NACs containing wastewater is not fully understood.
Therefore, in this study, coupling of ZVI into the anaerobic
system was established with the goal of accelerating the DNAN
removal from wastewater. Specially, the key role of ZVI in the
coupled system was investigated in terms of the intermediate
products, ORP, pH and biogas analysis. The performance of the
ZVI coupled biosystem at the presence of competitive electron
acceptors, such as nitrate and sulfate, was also evaluated.
ꢂ
rotary shaker at 200 rpm and 30 C. At every predetermined
sampling time, one serum bottle was sacriced, and 10 mL of
the solution was ltered through a 0.22 mm membrane for
analysis. The control systems, i.e., the ZVI control system with
the addition of 0.1 g ZVI but without sludge, and the biotic
control system with the addition of 20 mL anaerobic sludge but
without ZVI, were operated according to the same experimental
procedures as the coupled system.
To evaluate the competitive effect of other electron acceptors
on the microbial transformation of DNAN, two common
competing electron acceptors, i.e., nitrate and sulfate, were
added respectively to the batch anaerobic reactors at the
ꢁ1
concentration of 500 mg L . DNAN reduction performance at
the presence of nitrate and sulfate was evaluated.
All experimental runs were performed in triplicate and the
results were reported as an average of the three independent
determinations.
2. Materials and methods
2.1 Chemicals
2.4 Analytical methods
DNAN was a gi from Hubei Dongfang Chemical Co. Ltd in DNAN and its intermediate products were identied and quanti-
Hubei province, China. 2-Nitro-4-aminoanisole (2-N-4-AAN) and ed by high performance liquid chromatography (HPLC) (Waters
75196 | RSC Adv., 2015, 5, 75195–75203
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
2
996, Waters Incorporation, USA). The HPLC analysis was con-
ducted at room temperature using a RP18 column (5 mm, 4.6 ꢃ
50 mm) and a UV-vis detector. The mobile phase was a mixture
of 45% methanol and 55% water pumped at a ow rate of
C ꢁ C ¼ k t
(1)
(2)
0
t
0
2
ln(C
0
/C
t
) ¼ k
1
t
ꢁ1
ꢁ
1
0 t
where C is the initial DNAN concentration (mg L ), C is the
1.00 mL min . The analysis was performed at 254 nm with a
ꢁ
1
ꢂ
DNAN concentration (mg L ) at reaction time t (h), k is the
0
column temperature of 35 C. The analysis of formate ion was
performed on an ion chromatograph (ICS-2100, DIONEX, USA)
using an Ion Pac® As11-HC (4 ꢃ 250 mm) column and a sup-
pressed conductivity detector. The pH and oxidative-reductive
potential (ORP) were measured by a pH meter (FE20K, Mettler-
Toledo instruments, CH) with a redox electrode. At given time
intervals, the volume of biogas produced was measured using a
syringe aer the gas pressure in the headspace was brought to
atmospheric pressure. The composition of biogas was analyzed by
gas chromatography (Agilent 6820, Agilent Technologies, USA)
equipped with a thermal conductivity detector (TCD) using
molecular sieve 5A-60/80 mesh column (ANPEL Laboratory
Technologies Inc., Shanghai, China) as a separation column. N2
was the carrier gas, and the operating temperature of the injection
port, oven, and detector was 150 C, 60 C and 200 C, respec-
tively. Iron concentration in the reactors was determined by
inductively coupled plasma (Optima 7000, PerkinElmer instru-
ments, USA). Scanning electron microscopy coupled with energy
dispersive spectroscopy (SEM-EDS) (Quanta 250FEG, FEI, USA)
was applied to characterize the morphology and chemical
composition of anaerobic sludge aer ZVI treatment.
ꢁ
1
pseudo zero-order rate constant (mg h ), and k is the pseudo
1
ꢁ
1

rst-order rate constant (h ).
3
3
. Results and discussion
.1 Performance of DNAN reduction in the ZVI coupled
biosystem
To verify whether the anaerobic reduction of DNAN could be
enhanced by ZVI, removal of DNAN and formation of its
reduction intermediates in ZVI coupled biosystem, ZVI control
system and biotic control system, were compared. As shown in
Fig. 1a, only 4 h was required for complete DNAN reduction in
the ZVI coupled biosystem, while as long as 20 h was required
for complete DNAN reduction in the biotic control system. The
difference in terms of DNAN removal was signicant, probably
due to the key role of ZVI in DNAN reduction. However, only
ꢂ
ꢂ
ꢂ
2
8.71 ꢀ 5.06% of the total DNAN could be removed in the ZVI
control system aer 20 h, indicating that the ZVI alone could
not serve as an efficient and sufficient electron donor for abiotic
reduction of DNAN, especially at the neutral pH condition
adopted in this study. Therefore, it could be inferred that there
existed some synergistic effects between ZVI and anaerobic
sludge for DNAN reduction.
2.5 Data analysis
DNAN reduction rates in the reduction systems were described
Under anaerobic condition, DNAN could be reductively trans-
by the pseudo zero-order kinetic model (eqn (1)) and pseudo formed into DAAN with 2-amino-4-nitroanisole (2-A-4-NAN) and 2-
2

rst-order kinetic model (eqn (2)), respectively.
nitro-4-aminoanisole (2-N-4-AAN) as the intermediates, which
Fig. 1 Concentration evolution of DNAN and its corresponding reduction intermediates as a function of reduction time (
biosystem, biotic control system, ZVI control system).
ZVI coupled
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 75195–75203 | 75197

View Article Online
RSC Advances
Paper
was conrmed by HPLC analysis (Fig. S1†). In the ZVI coupled Table 1 Constants of pseudo zero-order and pseudo first-order
models for the reduction of the DNAN in three individual systems
biosystem and biotic control system, the maximum accumulation
concentrations of 2-A-4-NAN and 2-N-4-AAN were 9.65 ꢀ 1.09
Pseudo zero-order
kinetics
Pseudo rst-
order kinetics
ꢁ1
ꢁ1
ꢁ1
mg L and 2.40 ꢀ 0.15 mg L , 23.11 ꢀ 0.77 mg L and 3.88 ꢀ
ꢁ
1
0
.22 mg L , respectively, indicating the relatively low accumula-
ꢁ1
2
ꢁ1
2
Experiment condition
k
0
(mg h
)
R
k
1
(h
)
R
tion of reduction intermediates in the ZVI coupled biosystem
ꢁ
1
(
Fig. 1b and c). However, only 3.65 ꢀ 0.62 mg L 2-A-4-NAN
ZVI coupled biosystem
Biotic control system
9.382
1.910
0.625
0.943
0.867
0.990
1.263
0.217
0.018
0.951
0.993
0.988
was detected in the ZVI control system (Fig. 1b). Moreover, the
maximum concentration of nal product DAAN in the ZVI ZVI control system
ꢁ1
coupled biosystem was as high as 27.79 ꢀ 0.69 mg L , which was
ꢁ
1
much higher than 4.74 ꢀ 0.48 mg L in the biotic control system
ꢁ
1
and 0.00 ꢀ 0.00 mg L in the ZVI control system (Fig. 1d).
Correspondingly, the formation efficiencies of DAAN in ZVI mechanisms between biotic system and abiotic system for
coupled biosystem, biotic control system and ZVI control system DNAN removal.
were 99.66 ꢀ 0.70%, 16.99 ꢀ 1.73% and 0.00 ꢀ 0.00%, respec-
For the solid–liquid heterogeneous reaction system, such as
tively. It could be seen that DNAN was only partially reduced in the the ZVI reduction system or the ZVI coupled biosystem, if the
biotic control system, with more 2-A-4-NAN accumulated but less adsorption of contaminants onto the solid surface played a
DAAN produced. These results further indicated that coupling of minor role in the reductive process, the contaminant removal
ZVI into the anaerobic system could accelerate the degradation of oen followed the pseudo zero-order kinetic model, otherwise
DNAN, particularly the formation of its nal reductive product pseudo rst-order kinetic model was more appropriate for the
22,23
DAAN. In addition, accounting for these intermediate species and removal kinetics.
Since no removal of DNAN was observed at
end products gave good mass balance (greater than 85%) for the the initial stage in the ZVI control system (Fig. 1a), the removal
three individual batch systems, suggesting that other reaction of DNAN in ZVI control system could be attributed to the
products were negligible.
reduction by ZVI rather than adsorption by ZVI. As a result, the
pseudo zero-order kinetics model could be applied to the DNAN
23,24
removal process in the ZVI control system.
However, a sharp
3
.2 Reduction pathway of DNAN in the ZVI coupled biosystem
Based on the evolution of intermediate products, it is inter-
esting to note that the reduction of –NO on DNAN was pref-
decrease of DNAN concentration was observed in either ZVI
coupled biosystem or biotic control system within the rst hour
2
(Fig. 1a), probably due to the strong adsorption of DNAN by the
erential at ortho position in the ZVI coupled biosystem,
resulting in the formation of 2-A-4-NAN, which could be
subsequently reduced to DAAN. This phenomenon was in
accordance with the result in the previous study. Olivares et al.
also found that the reduction of the nitro groups on DNAN
sludge inoculated in these two systems, which was conrmed by
the good match between DNAN removal and rst-order kinetic
25
in either ZVI coupled biosystem or biotic control system.
21
As was indicated in Table 1, the pseudo rst-order rate
constant for DNAN removal in the ZVI coupled biosystem was as
under anaerobic condition followed the order of ortho-position
ꢁ1
ꢁ1
high as 1.263 h , which was much higher than 0.217 h in the
biotic control system. This result strongly conrmed that the
removal of DNAN in anaerobic system could be largely
promoted by the addition of ZVI, probably due to the synergistic
interaction between anaerobic microbes and ZVI. However, the
surface adsorption by ZVI was negligible, since the ZVI used in
this study had few surface sites amenable for DNAN adsorption,
as was indicated by the slight removal of DNAN at the early stage
in the ZVI control system.
>
para-position. According to the density function theory
computations analysis, the charge densities of the N atoms at
para-NO and ortho-NO positions of DNAN were 0.211 and
.215, following the order of ortho-position > para-position.
2
2
0
Electron attacks, which were nucleophilic, would occur prefer-
entially at the N atom with more positive charge density.
Therefore, the reduction of DNAN was selectively favored at the
ortho-NO
2
group.
3.3 Kinetics of DNAN reduction in the coupled ZVI
anaerobic system
3.4 The key role of ZVI in the coupled system
Pseudo rst-order and pseudo zero-order kinetic models were
employed to elucidate the DNAN reductive transformation As indicated in previous study, pH is one of the most important
process in the three individual systems (i.e., ZVI coupled bio- parameters affecting the community structure and activity of
14
system, biotic control system and ZVI control system). The rate anaerobic microorganisms. Aer the 20 h reaction, the pH in
2
constant and regression coefficient (R ) relevant to the zero and the biotic control system and ZVI coupled biosystem shied

rst kinetic models were shown in Table 1. It was noteworthy from 7.12 ꢀ 0.01 to 6.35 ꢀ 0.04 and 6.67 ꢀ 0.02 (Table 2),
that the removal of the DNAN in either ZVI coupled biosystem respectively, suggesting that the ZVI coupled biosystem seemed
or biotic control system could be appropriately simulated by the to have a greater pH self-buffering capability than the biotic
pseudo rst-order kinetic model, while the removal of DNAN in control system. This phenomenon could be linked to the ZVI
ZVI control system followed the pseudo zero-order kinetic corrosion in the anaerobic system, which consumed the acidity
model. This result suggested that there existed different produced from the anaerobic acidication process.
75198 | RSC Adv., 2015, 5, 75195–75203
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Furthermore, ZVI corrosion process could create a more
stable and favorable anaerobic environment for microorgan-
15
isms by lowering the ORP. As shown in Fig. 2, the ORP in the
ZVI coupled biosystem approximately ranged from ꢁ128.5 ꢀ
1
2.0 mV to ꢁ265.0 ꢀ 19.8 mV, while it ranged from ꢁ122.5 ꢀ 3.5
mV to ꢁ215.5 ꢀ 20.5 mV in the biotic control system. Lower
ORP value means a better reductive environment, which could
26,27
exert a positive effect on the reduction of nitro group.
Additionally, a sharp decrease of ORP was observed in ZVI
coupled biosystems within the rst 4 h, implying that there was
a substantial depletion of the oxidative compounds in aqueous
solution. Such a phenomenon was well in agreement with the
DNAN reduction in the ZVI coupled biosystem, conrming the
effective reduction of DNAN in the ZVI coupled system.
Fig. 2 Evolution of the ORP during reduction of DNAN as a function of
To further clarify the effect of ZVI on methanol metabolism, time.
the production of the methanol metabolism product, i.e.,
formate, was investigated. As shown in Fig. 3, the production of
formate in ZVI coupled biosystem was signicantly higher than
that in the biotic control system. A previous work showed that
both acidogenesis and activity of fermentative bacteria could be
effectively improved by lowering ORP, which was provided by
28
the addition of ZVI. On the other hand, the ferrous ions from
ZVI corrosion could stimulate the synthesis of key enzymes in
the hydrolysis-acidication process, resulting in the accumu-
29
lation of volatile fatty acids. Considering that formate was an
30
effective electron donor for the reduction process, the
increased production of formate in the ZVI coupled biosystem
could be benecial for the efficient reduction of DNAN.
Generally, methanol as well as the ZVI in anaerobic system
may serve as precursors for the formation of an intermediate H
pool, which could be utilized as the electron donor for the Fig. 3 Concentration evolution of formate in the ZVI coupled bio-
2
19,31
reduction process.
However, no hydrogen was produced in system and biotic control system.
either biotic control system or ZVI coupled biosystem (Fig. 4).
This might be attributed to the slow corrosion rate of ZVI and
high consumption rate of methanol in the anaerobic system. be further converted to methane through methanogenesis
Under anaerobic conditions, the accumulated VFAs could be using ZVI as the direct electron donor, which was necessarily
34
further bioconverted to methane. It was observed that the benecial for the increase of methane production. Further-
cumulative CH
in the ZVI coupled biosystem was about 0.638 ꢀ more, the rapid reduction of DNAN in the ZVI coupled bio-
.017 mmol per mmol methanol, while, it was only 0.017 ꢀ system alleviated the inhibitive effect of DNAN on
.002 mmol per mmol methanol in the biotic control system, methanogens, since DNAN was much more toxic to methano-
4
0
0
6,35
indicating that the methanogenic activity of the sludge could be genic microorganisms than its reduction products.
effectively improved by addition of ZVI. Previous studies have
Additionally, the electronegative anaerobic bacteria could be
shown that an appropriate amount of ferrous ions released easily attached on the surfaces of ZVI due to the static function
from ZVI corrosion could be involved in energy metabolism as a in a mixed anaerobic culture, and a stable ZVI–microbial zoo-
3
2,33
cytochrome and ferredoxin in methylotrophic methanogens.
gloea could be gradually formed, which was benecial for NACs
36
Meanwhile, the CO
2
produced in ZVI coupled biosystem could reduction. The SEM-EDS analysis conrmed the presence of
2+
a
Table 2 Change of pH and Fe concentrations in different systems
Effluent of biotic
Effluent of ZVI
Effluent of ZVI
Parameter
pH
Inuent of wastewater
control system
control system
coupled biosystem
7.12 ꢀ 0.01
6.35 ꢀ 0.04
7.41 ꢀ 0.01
0.06 ꢀ 0.01
6.67 ꢀ 0.02
0.34 ꢀ 0.02
2+
ꢁ1
Fe (mg L
)
n.d.
n.d.
a
n.d. means not detectable.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 75195–75203 | 75199

View Article Online
RSC Advances
Paper
Fig. 5 Effects of nitrate on DNAN reduction as a function of time
(
ZVI coupled biosystem,
biotic control system,
ZVI coupled biosystem with
biotic control system with
nitrate,
nitrate).
In the biotic control system, the inhibitory effect of nitrate on
DNAN removal could be expected considering the much higher
oxidation potential of nitrate compared to DNAN. The standard
Fig. 4 Dynamics of biogas production in the ZVI coupled biosystem
(
a) and biotic control system (b).
ꢁ
ꢁ
electrode potentials for the reduction of NO
3
to NO
2
at
neutral pH were reported to be 0.43 V vs. standard hydrogen
electrode (SHE), while the one-electron reduction of DNAN was
Fe element on the outer layer and inner parts of anaerobic
granules, indicating that ZVI could be a ideal site for the
formation of ZVI–microbial zoogloea complex (Fig. S2†).
Moreover, under anaerobic condition, the ZVI surface area
might be increased by etching and pitting through corrosion,
which was further benecial for mass transfer and reductive
38–40
as low as ꢁ0.40 V.
As a result, nitrate reduction had an
advantage over DNAN reduction in the competition for the
limited electron donor. The ORP increase in the anaerobic
system aer the addition of nitrate was another reason for the
decreased DNAN removal (Fig. S3a†). However, at the presence
of ZVI, the situation was different. The standard electrode
36
transformation of pollutants on it.
ꢁ
ꢁ
potential of NO /NO2 , i.e., 0.43 V, was higher than that of
3
2
+
Fe /Fe, i.e., ꢁ0.44 V. Therefore, the competitive electron
acceptor, i.e., nitrate, could be theoretically reduced by ZVI in
3.5 DNAN reduction at the presence of competitive electron
4
1
ZVI coupled biosystem. As was reported in previous study, at
the presence of nitrate or nitrite, corrosion of iron might be
acceptors
Previous studies suggested that the competitive electron alleviated, especially under neutral or alkaline condition.42,43
acceptors in wastewater, such as nitrate and sulfate, have However, in this ZVI coupled biosystem, slightly acidic condi-
profound impact on the anaerobic reduction of pollutants, such tion was well maintained, probably due to the acidication
as nitrobenzene, pentachloroaniline and 4-chloronitrobenzene, reaction in this anaerobic system. Therefore, corrosion of ZVI
2
6,31,37
etc.
reduction at the presence of these competitive electron tion and DNAN reduction. In addition, ZVI surface area could be
acceptors.
increased by etching and pitting through anaerobic corrosion,
.5.1 Effect of nitrate on DNAN reduction. As shown in which was further benecial for mass transfer and reductive
Therefore, it was essential to investigate the DNAN would make an important contribution for both nitrate reduc-
3
36
Fig. 5, sharp decrease in terms of DNAN removal was observed reduction on it. More importantly, at near-neutral pH condi-
when 500 mg L nitrate was introduced to the biotic control tion, nitrate as a less strong oxidant could oxidize ZVI to form
ꢁ
1
44
system. Only 55.79 ꢀ 1.54% of the total DNAN could be removed the magnetite, overcoming the obstacle from the electron
ꢁ1
aer 20 h at the presence of 500 mg L nitrate, while complete transfer barrier over the corrosion coating, which might be
45
DNAN removal could be observed aer incubation time of 20 h benecial for NACs reduction. Therefore, the ZVI coupled
at the absence of nitrate. Correspondingly, the constant rate (k
biosystem showed excellent performance in terms of DNAN
of DNAN reduction decreased from 0.217 h to 0.024 h due reduction at the presence of nitrate.
to the introduction of nitrate in the biotic control system.
3.5.2 Effect of sulfate on DNAN reduction. It was inter-
However, aer 500 mg L nitrate was introduced, the incuba- esting to observe that DNAN removal was signicantly acceler-
tion time required for the complete DNAN removal in the ZVI ated in the biotic control system at the presence of 500 mg L
coupled biosystem slightly increased from 4 h to 8 h, indicating sulfate (Fig. 6), with the pseudo rst-order rate constant (k ) for
the low competitiveness of nitrate for the electron donor at the DNAN removal increased obviously from 0.217 h to 0.432 h
presence of ZVI.
1
)
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
1
ꢁ
1
ꢁ1
.
75200 | RSC Adv., 2015, 5, 75195–75203
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
In addition, slight increase in terms of DNAN removal was also of sulfate, indicating that a more reductive condition was
observed in the ZVI coupled biosystem aer the introduction of created for DNAN reduction (Fig. S3b†). In terms of the fore-
ꢁ1
5
00 mg L sulfate. These results indicated that the presence of mentioned discussion, it could be concluded that the DNAN
sulfate had no adverse effect on DNAN removal. On the removal was accelerated by addition of sulfate in either ZVI
contrary, DNAN removal could be enhanced to some extent at coupled biosystem or biotic control system.
the presence of sulfate, which was rather different from the
results at the presence of nitrate.
3
.6 Implication of this work
Since the inhibitory effect of sulfate on NACs degradation
26
Compared to the biotic control system and ZVI control system,
both DNAN reduction and DAAN formation were signicantly
improved in the ZVI coupled biosystem. The efficient reduction
of DNAN to DAAN in the ZVI coupled biosystem would result in
a signicant improvement of biodegradability and reduction of
toxicity. More importantly, favorable environment for some
specic microbial species, such as methanogens, could be
created by offsetting the possible pH decline and lowering the
ORP.
had been well recognized, the phenomenon observed in this
study was rather interesting. The reason could be ascribed to
the generation of some reducing agents, e.g., sulphide, which
could be used as the electron donor for DNAN. Similar result
4
6
was also reported by van der Zee et al., where the reduction of
azo dye could be signicantly enhanced at the presence of
sulfate, especially under the anoxic condition. The standard
51
2
4
ꢁ
ꢁ
electrode potential of SO
/HSO
3
was ꢁ0.52 V vs. SHE at
38
neutral pH, which was lower than the one-electron standard
reduction potential of DNAN. Compared with sulfate, DNAN
was more subjected to reduction in this study, as DNAN showed
higher competitiveness for the electron. Besides, another
2
+
What's more, the Fe in the effluent of the ZVI coupled
ꢁ
1
biosystem was generally below 1 mg L , suggesting the slow
rate of ZVI dissolution. The low consumption of iron leads to
easy maintenance and low operating cost. In addition, the low
concentration of ferrous iron was benecial for the growth of
microorganisms.
could be protected from oxygen, with the reduced formation of
iron oxides on the surface. Thus the frequent replacement and
regeneration of ZVI was not required in the ZVI coupled bio-
system. What's more important, under anaerobic condition, ZVI
surface area could be increased by etching and pitting through
corrosion, which was further benecial for mass transfer and
reductive reduction on it.
Recently, for the removal of various contaminants, the
application of ZVI powder, especially the nano zero valent iron
31
compelling evidence hypothesized by Ismail and Pavlostathis,
was that the growth of sulfate reducers was relatively slow when
methanol was used as the energy source. Therefore, the electron
donors used for the reduction of sulfate could be limited, which
was further benecial for DNAN reduction. Meanwhile, the
poisonous effect of sulphide on microorganisms might be
32,33
Moreover, under anaerobic condition, ZVI
15
47
ignored under near-neutral pH condition. More importantly,
the iron oxides and hydroxides on the surface of ZVI could be
eliminated by sulphide, with the formation of mackinawite or
36
48,49
pyrite.
Compared with the iron oxides and hydroxides, the
mackinawite or pyrite was a better promoter of electron transfer
50
to organic pollutants. Unfortunately, the quantication of
sulphide and elemental sulfur during DNAN reduction has been
unsuccessful in this study. This might be due to the low
concentrations of the sulphide and elemental sulfur in the
anaerobic system, which needs further investigation.
(NZVI), has received increasing attention due to their high
surface area and high reactivity. However, coupling of ZVI
powder into an anaerobic system for the treatment of raw
industrial wastewater was limited by far, due to the inherent
weakness of ZVI powder and NZVI, such as poor stability and
easy aggregation. To address these issues, iron shavings may be
a better choice, compared with ZVI powder and NZVI. The
primary reason for this selection was the abundant local supply,
relatively low cost and fairly large surface area. Ma and his co-
workers have undertaken a major research and development
project to investigate the technical and economic feasibility of
iron shavings for the enhance treatment of industrial process
Additionally, the ORP in either biotic control system or ZVI
coupled biosystem was signicantly decreased at the presence
10
wastes, with success achieved. Nowadays, coupling of iron
shaving into the upow anaerobic sludge blanket (UASB) has
been developed in our laboratory for the treatment of high
strength wastewater containing NACs. The interaction between
iron shaving and microorganisms, as well as the dynamic
change of iron surface and microbial population aer long-term
operation, will be investigated in our future study.
4. Conclusions
Fig. 6 Effects of sulfate on DNAN reduction as a function of time
Compared to the biotic control system and the ZVI control
system, both DNAN reduction and DAAN formation were
signicantly improved in the ZVI coupled biosystem. The high
(
ZVI coupled biosystem,
biotic control system,
ZVI coupled biosystem with
biotic control system with
sulfate,
sulfate).
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 75195–75203 | 75201

View Article Online
RSC Advances
Paper
performance of the ZVI coupled biosystem could be attributed 16 X. Xiao, G. P. Sheng, Y. Mu and H. Q. Yu, Water Res., 2013, 47,
to the high accumulation of formate, low ORP and great pH self- 6007–6013.
buffering capability at the presence of ZVI. Compared with the 17 W. Yin, J. Wu, P. Li, G. Lin, X. Wang, B. Zhu and B. Yang,
biotic control system, the survival environment for metha- Chem. Eng. J., 2012, 210, 309–315.
nogens was effectively improved in ZVI coupled biosystem. In 18 Y. Liu, Y. Zhang, Z. Zhao, Y. Li, X. Quan and S. Chen,
addition, the ZVI coupled biosystem showed high efficiency in Bioresour. Technol., 2012, 121, 148–153.
terms of DNAN removal with the coexistence of competitive 19 X. Y. Yu, C. Amrhein, M. A. Deshusses and M. R. Matsumoto,
electron acceptors, such as nitrate and sulfate. The ZVI coupled Environ. Sci. Technol., 2006, 40, 1328–1334.
biosystem could be a promising alternative to the conventional 20 J. Shen, R. He, H. Yu, L. Wang, J. Zhang, X. Sun, J. Li, W. Han
anaerobic reduction process for the removal of recalcitrant and L. Xu, Bioresour. Technol., 2009, 100, 1922–1930.
contaminants from wastewater, especially for the treatment of 21 C. Olivares, J. Liang, L. Abrell, R. Sierra-Alvarez and
wastewater containing multi-substituted NACs.
J. A. Field, Biotechnol. Bioeng., 2013, 110, 1595–1604.
2
2
2
2
2
2
2
2 J. Fan, Y. Guo, J. Wang and M. Fan, J. Hazard. Mater., 2009,
1
66, 904–910.
3 M. Hou, F. Li, X. Liu, X. Wang and H. Wan, J. Hazard. Mater.,
007, 145, 305–314.
Acknowledgements
2
This research is nanced by Innovation Program of Foundation
Product, Major Project of Water Pollution Control and
Management Technology of P. R. China (No. 2012ZX07101-003-
4 L. Liang, W. Sun, X. Guan, Y. Huang, W. Choi, H. Bao, L. Li
and Z. Jiang, Water Res., 2014, 49, 371–380.
5 L. Zhu, H. Lin, J. Qi and X. Xu, Environ. Sci. Pollut. Res., 2013,
001), Zijin Intelligent Program of NJUST (No. 2013-ZJ-02-19) and
2
0, 6119–6127.
6 J. Huang, Y. Wen, N. Ding, Y. Xu and Q. Zhou, Water Res.,
012, 46, 4361–4370.
Research Innovation Grant for Graduate of Jiangsu Common
High School (GXZZ13-0225).
2
7 V. Murali, S.-A. Ong, L.-N. Ho and Y. S. Wong, Bioresour.
Technol., 2013, 143, 104–111.
8 Y. Liu, Y. Zhang, X. Quan, Y. Li, Z. Zhao, X. Meng and
S. Chen, Chem. Eng. J., 2012, 192, 179–185.
References
1
2
3
V. M. Boddu, K. Abburi, A. J. Fredricksen, S. W. Maloney and
R. Damavarapu, Environ. Technol., 2009, 30, 173–181.
W. E. Platten, D. Bailey, M. T. Suidan and S. W. Maloney,
Chemosphere, 2010, 81, 1131–1136.
29 X. Meng, Y. Zhang, Q. Li and X. Quan, Biochem. Eng. J., 2013,
73, 80–85.
30 X. Quan, X. Zhang and H. Xu, Water Res., 2015, 78, 74–83.
N. N. Perreault, D. Manno, A. Halasz, S. Thiboutot, 31 Z. Z. Ismail and S. G. Pavlostathis, Biodegradation, 2010, 21,
G. Ampleman and J. Hawari, Biodegradation, 2012, 23, 287–
95.
J. Y. Shen, C. J. Ou, Z. Y. Zhou, J. Chen, K. X. Fang, X. Y. Sun,
43–57.
2
32 C. F. Shen, N. Kosaric and R. Blaszczyk, Water Res., 1993, 27,
25–33.
4
J. S. Li, L. Zhou and L. J. Wang, J. Hazard. Mater., 2013, 260, 33 G. Zhen, X. Lu, Y. Y. Li, Y. Liu and Y. Zhao, Chem. Eng. J.,
93–1000.
2015, 263, 461–470.
S. Susarla, Y. Yonezawa and S. Masunaga, Water Res., 1998, 34 S. Karri, R. Sierra-Alvarez and J. A. Field, Biotechnol. Bioeng.,
2, 639–648.
2005, 92, 810–819.
B. A. Donlon, E. Razo-Flores, J. A. Field and G. Lettinga, Appl. 35 D. O. Tas and S. G. Pavlostathis, Environ. Sci. Technol., 2005,
Environ. Microbiol., 1995, 61, 3889–3893. 39, 8264–8272.
J. Singh, S. Comfort and P. Shea, J. Environ. Qual., 1998, 27, 36 W. Zhang, L. Chen, H. Chen and S. Q. Xia, J. Hazard. Mater.,
240–1245. 2007, 143, 57–64.
B. T. Oh, C. L. Just and P. J. J. Alvarez, Environ. Sci. Technol., 37 J. F. Devlin and K. O. Allin, Environ. Sci. Technol., 2005, 39,
001, 35, 4341–4346.
1868–1874.
C. B. Wang and W. X. Zhang, Environ. Sci. Technol., 1997, 31, 38 R. K. Thauer, K. Jungermann and K. Decker, Bacteriol. Rev.,
154–2156.
1977, 41, 100–180.
0 L. M. Ma and W. X. Zhang, Environ. Sci. Technol., 2008, 42, 39 P. Clauwaert, K. Rabaey, P. Aelterman, L. de Schamphelaire,
9
5
6
7
8
9
3
1
2
2
1
1
1
1
1
1
5
384–5389.
1 S. C. Ahn, D. K. Cha, B. J. Kim and S.-Y. Oh, J. Hazard. Mater.,
011, 192, 909–914.
2 S. Y. Oh, P. C. Chiu, B. J. Kim and D. K. Cha, Water Res., 2005,
9, 5027–5032.
T. H. Ham, P. Boeckx, N. Boon and W. Verstraete, Environ.
Sci. Technol., 2007, 41, 3354–3360.
40 M. Uchimiya, L. Gorb, O. Isayev, M. M. Qasim and
J. Leszczynski, Environ. Pollut., 2010, 158, 3048–3053.
41 H. Hwang, D. G. Kim and H. S. Shin, J. Hazard. Mater., 2011,
185, 1513–1521.
2
3
3 J. Shen, Z. Zhou, C. Ou, X. Sun, J. Li, W. Han, L. Zhou and
L. Wang, J. Environ. Sci., 2012, 24, 1900–1907.
4 W. W. Li, Y. Zhang, J. B. Zhao, Y. L. Yang, R. J. Zeng, H. Q. Liu
and Y. J. Feng, Bioresour. Technol., 2013, 149, 38–43.
5 Y. B. Zhang, Y. W. Liu, Y. W. Jing, Z. Q. Zhao and X. Quan, J.
Environ. Sci., 2012, 24, 720–727.
42 M. J. Alowitz and M. M. Scherer, Environ. Sci. Technol., 2002,
36, 299–306.
43 K. Ritter, M. S. Odziemkowski, R. Simpgraga, R. W. Gillham
and D. E. Irish, J. Contam. Hydrol., 2003, 65, 121–136.
75202 | RSC Adv., 2015, 5, 75195–75203
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
4
4
4
4 T. Suzuki, M. Moribe, Y. Oyama and M. Niinae, Chem. Eng. J., 48 A. J. Pyzik and S. E. Sommer, Geochim. Cosmochim. Acta,
012, 183, 271–277. 1981, 45, 687–698.
5 Y. H. Huang and T. C. Zhang, Water Res., 2006, 40, 3075– 49 L. A. Hoferkamp and E. J. Weber, Environ. Sci. Technol., 2006,
082. 40, 2206–2212.
6 F. P. van der Zee, I. A. E. Bisschops, V. Blanchard, 50 M. Elsner, R. P. Schwarzenbach and S. B. Haderlein, Environ.
2
3
R. H. M. Bouwman, G. Lettinga and J. A. Field, Water Res.,
003, 37, 3098–3109.
7 M. A. Reis, J. S. Almeida, P. C. Lemos and M. J. Carrondo,
Sci. Technol., 2004, 38, 799–807.
2
51 J. Hawari, F. Monteil-Rivera, N. N. Perreault, A. Halasz,
L. Paquet, Z. Radovic-Hrapovic, S. Deschamps, S. Thiboutot
and G. Ampleman, Chemosphere, 2015, 119, 16–23.
4
Biotechnol. Bioeng., 1992, 40, 593–600.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 75195–75203 | 75203