Journal of Cleaner Production 337 (2022) 130489

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Journal of Cleaner Production

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Iodide ions enhancing sulfamerazine degradation by horseradish

peroxidase/H2O2: Degradation products, degradation mechanism and
toxicity assessment
Jiali Wang a, Zhao Shu a, Zhennan Chen a, Jixin Su a, Chunguang Liu a, b, *
a
School of Environmental Science and Engineering, Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse, China− America CRC for
Environment & Health of Shandong Province, Shandong University, 72# Jimo Binhai Road, Qingdao, Shandong, 266237, PR China
b
Guangdong Provincial Key Laboratory of Environmental Protection and Resources Utilization, PR China

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

Handling Editor: Zhen Leng Biocatalytic treatment using horseradish peroxidase (HRP) is a sustainable technology to remove antibiotics from
wastewater. In order to improve removal efficiency, a HRP/H2O2/I− system has been developed for sulfadiazine
Keywords: (SMR) degradation. The addition of 1.5 mM iodide ion (I− ) significantly increases the degradation efficiency of
Sulfonamides antibiotics SMR from 16.06% to 90.91% by HRP/H2O2, following pseudo-first-order kinetics (r2 = 0.975). Analysis of SMR
Iodine substitution
degradation products by high resolution Q-TOF mass spectrometry shows that SMR degradation mainly involves
Enzymolysis
in iodine substitution, oxidation and polymerization. Iodo-sulfadiazine, as the main intermediates, is gradually
Transformation of pollutants
Bacterial toxicity degraded overtime. The main mechanism of iodide ion promoting SMR degradation is closely related to the
existing forms of iodine. Under the action of HRP/H2O2, iodide ions are converted into iodine and hypoiodic
ions, which are easier to react with SMR by substitution. Molecular simulation shows that iodo-sulfadiazine is
easier to bind HRP than SMR due to its stronger binding energy, thus enhancing SMR degradation. The toxicity of
degradation products to Bacillus subtilis (model bacteria widely existing in the environment) is as follows: final
degradation products < intermediate degradation products < SMR. This study can provide technical support for
developing a feasible and sustainable sulfanilamide-containing wastewater treatment solution, and provide
reference for the fate and risk assessment of sulfonamide antibiotics in the environment.

1. Introduction management technologies becomes a requirement to reduce the

Sulfonamides antibiotics, as a kind of veterinary drug with low price
and good effect, have been widely used in animal husbandry and
aquaculture industries (Jansomboon et al., 2016; Zou et al., 2014). Due
to overuse and inadequate absorption, a large number of antibiotics
enter the environment through animal feces and wastewater discharge
(Conde-Cid et al., 2018; Islas-Espinoza et al., 2012; Padedda et al.,
2017). When they enter the environment, such drugs can induce the
development of bacterial resistance and cross-resistance and increase
hazardous to organisms in the environment and human beings (Llorca
et al., 2014). Sulfonamide antibiotics up to the milligram level have
been detected in feces and soil in Germany, South Korea, China and
Vietnam (Binh et al., 2018; Hannappel et al., 2016; Lee et al., 2019; Yao
et al., 2015). Therefore, the development of effective environmental
ecological and environmental risks of antibiotics.
Currently, there are several methods for antibiotics removal or
degradation, such as adsorption (Meng et al., 2020), biodegradation (Li
et al., 2020; Torresi et al., 2017), photolysis and advanced oxidation
process (Milh et al., 2021; Shad et al., 2018; Yao et al., 2017), but these
methods may have problems such as large energy consumption or high
cost or low efficiency. Enzymes have been used in degradation reactions
as a kind of highly efficient natural biocatalyst (Fernandez-Sanroman
et al., 2020; Primožič et al., 2020; Varga et al., 2019), among which
horseradish peroxidase (HRP) and laccase are widely used (Bilal et al.,
2020; Liu et al., 2021; Maryskova et al., 2021). HRP first reacts with
hydrogen peroxide to form HRPІ and HRPII, which have strong oxidiz­
ability and can oxidize pollutants. Biodegradation of antibiotics with
HRP has been widely studied with the advantages of green

* Corresponding author. School of Environmental Science and Engineering, Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse,
China− America CRC for Environment & Health of Shandong Province, Shandong University, 72# Jimo Binhai Road, Qingdao, Shandong, 266237, PR China.
E-mail address: chunguangliu2013@sdu.edu.cn (C. Liu).

https://doi.org/10.1016/j.jclepro.2022.130489
Received 7 August 2021; Received in revised form 1 January 2022; Accepted 9 January 2022
Available online 11 January 2022
0959-6526/© 2022 Elsevier Ltd. All rights reserved.
J. Wang et al.
environmental protection and sustainable development (Calcio Gaudino
et al., 2021; de Cazes et al., 2016; Zhao et al., 2017). However, how to
improve the degradation efficiency of pollutants has become the focus of
extensive attention (Gao et al., 2017; Navada and Kulal, 2019). Several
researchers focused on the introduction of mediator (e.g. ABTS) to
enhance the oxidation and made more reaction feasible for the removal
of pollutants (Leng et al., 2020; Varga et al., 2019). At present, most of
the mediators are artificial mediators, such as N-hydroxybenzotriazole,
N-hydroxyacetanilide, and 2-diazo-bis-(3-ethyl-benzodihydrothiazoli­
ne-6-sulfonic acid). Artificial mediators have some disadvantages,
such as high synthesis cost, ecological toxicity, poor stability of in­
termediates, and easy to affect the activity and stability of enzyme,
which affect their further application (Wells et al., 2006). It is necessary
to develop a new and cleaner way to improve the efficiency of
HRP/H2O2.
Recently, the role of iodine in the degradation of organic pollutants
has attracted more and more attention (Deruer et al., 2018; Karade et al.,
2005). The average concentration of iodine in the earth’s crust is
approximately 0.5 mg kg− 1, and in the atmosphere the concentration
ranges from 10 to 20 ng m− 3 (Moreda-Pineiro et al., 2011). It is note­
worthy that iodine promoted the transformation of organic compounds,
but also produced iodinated organics, which may induce potential
ecological risks (Almaraz et al., 2020; Hebert et al., 2010; Liu and Zhang,
2014). For example, iodide ion promoted the degradation of SMR in
water by ultrasonic treatment (Yang, X.-y. et al., 2018). HRP not only
has the ability to catalyze iodide ions to form iodine, but also can oxidize
dechlorination of chlorophenols (Szatkowski et al., 2011). Therefore, we
should not only pay attention to the enhancement of iodide ions on
organic pollutants degradation by HRP/H2O2, but also focus on the
potential ecological risks of the iodized products produced in reaction
process.
The objectives of this study are to (I) determine the effect of iodide
ion on the degradation of SMR by HRP/H2O2, (II) explore the
improvement mechanisms of iodide ions on SMR degradation by HRP/
H2O2, and (III) analyze the ecological risk of degradation products. The
experimental results are expected to develop a green and efficient
technology for SMR degradation, and provide a reference for the anal­
ysis the migration and transformation of SMR in natural environment.
2. Materials and methods
2.1. Chemicals and reagents
Sulfametazine (SMR), potassium iodide (KI) and hydrogen peroxide
(H2O2), sodium hydroxide, hydrochloric acid (37%), disodium
hydrogen phosphate, sodium dihydrogen phosphate, potassium iodate,
potassium hypoiodate, formic acid, methanol, acetonitrile were pur­
chased from Tianjin Kermel Chemical Reagent Co., Ltd. All the reagents
mentioned above are analytically pure. Horseradish peroxidase (HRP)
were purchased from Shanghai Macklin Biochemical Technology Co.,
Ltd. Bacillus subtilis (ATCC 6633) was supplied by Nanjing Lezhi
Biotechnology Co., Ltd.
2.2. Degradation experiment
The degradation experiments of SMR were conducted at 25 ◦ C by
adding SMR, HRP, iodide ion and H2O2 into a beaker (working volume
of 100 mL) in turn. Sample (1 mL) was taken out at different time in­
tervals for the analysis of the concentrations and speciation changes of
SMR and iodide ions. The reactions in the samples were terminated by
adding hydrochloric acid (1 mol/L) before analysis. Timing starts with
the addition of H2O2. In order to determine the optimal removal con­
ditions, the effects of the main experimental parameters (iodide ion,
H2O2 and pH range) on the removal of SMR were analyzed. The con­
centrations of iodide were 0, 0.56, 1.12, 1.5, 1.8 and 2.25 mM. The
concentrations of H2O2 were 0, 0.25, 0.5, 1.0, 2.5, 5 mM pH values were
2
Journal of Cleaner Production 337 (2022) 130489
set at 5, 6, 7 and 8 by adjusting with phosphate buffer solution. HRP
activity was determined by purpurogallin production from pyrogallol
under HRP catalysis (Yang, L. et al., 2018a). Purpurogallin shows a
characteristic absorption peak at 430 nm. One U is defined as the
amount of enzyme required to catalyze the oxidation of pyrogallol in 20
s, and is expressed as U/mL.
Samples were filtered through a 0.22 μm water-based membrane,
and then was analyzed by HPLC (high performance liquid chromatog­
raphy, 2030, Shimazu, Japan) to determine the removal of SMR. HPLC
was equipped with a C18 reverse-phase column (4.6 × 150 mm, 5 μm)
and UV detector. Detector temperature and wavelength were set as 30 ◦ C
and 268 nm. Mobile phase consisted of 1% (v/v) formic acid and
acetonitrile (73:27, v/v) at a flow rate of 1 mL/min. IO3− was analyzed
by ion chromatography with AS11-HC analytical column (4 × 250 mm,
Dionex IonPacTM). The mobile phase was 20 mM KOH solution and its
flow rate was 1 mL/min. The suppressor current was 70 mA (ASRS 4 mm
conductivity suppressor), the conductivity suppressor working temper­
ature and the working column temperature were 30 ◦ C and 35 ◦ C,
respectively. The absorbance of I2 and I3− was measured by UV–Vis
spectrophotometer at 460 nm and 350 nm respectively. Each group has
three parallel samples. The formula of removal efficiency is as follows:
[SMR]0 − [SMR]t
Removal(%) = *100%
[SMR]0
where [SMR]0 is the initial concentration of SMR. [SMR]t is the con­
centration of SMR at time t.
2.3. Degradation product analysis
High resolution liquid chromatography Q-TOF mass spectrometry
(LC-ESI-Q-TOF-MS) was used to identify the degradation products of
sulfadiazine. Samples (0.5 mL) were taken at the 4th h and 8th h for
analysis. Firstly, the reaction solution was extracted by SPE column to
remove the high salinity. The High Resolution Q-TOF mass spectrometry
(Bruck impact HD, Germany) equipped with an electrospray ionization
(ESI) was used for identifying degradation intermediates, and was
operated in positive mode, the capillary voltage was set as 3000 V, the
gas flow rate was 4 L min− 1, the capillary energy was 8.0 eV, and the
collision cell RF was 550.0 Vpp. MS data was collected over a mass-to-
charge ratio (m/z) range from 80 to 1300. Elemental Composition
Calculator v1.0 written by Jef Rozenski was used to calculate all possible
combinations of degradation products.
2.4. Interaction of iodide ion, HRP and sulfadiazine
In order to analyze the interaction between iodide ion, SMR and HRP
in the system, ultraviolet spectrophotometry and Molecular Operating
Environment (MOE) were used to analyze the interaction. To observe
the enzyme absorbance changes more clearly the concentration of io­
dide, HRP, SMR and H2O2 were set as 0.6 mM, 2.5 U/mL, 30 mg/L and
10 mM, respectively. The absorbance of HRP, I2 and I3− was measured
by UV–Vis spectrophotometer at 402 nm, 460 nm and 350 nm, respec­
tively. MOE was used to identify preferred binding sites and possible
conformations to further explore the interactions between HRP and SMR
or degradation products. The structures of SMR and degradation product
were built by module builder of MOE, and then the ligand was energy
minimized. The crystal structure of HRP (PDB code: 1H5C) were
downloaded from the RCSB Protein Database (http://www.pdb.org/).
All the water molecules were removed from 1H5C and hydrogen atoms
were added before docking. After optimizing HRP, the ligand (SMR or
iodine substitution products of SMR (I-SMR)) was imported into the
MOE software. Then the docking of HRP and SMR or I-SMR was per­
formed by clicking the button ‘Compute-Simulations-Dock’, which could
obtain a series of binding sites and corresponding binding energy. Based
on the optimal docking result, the most probable binding site of SMR or
J. Wang et al. Journal of Cleaner Production 337 (2022) 130489

(a)
1.0
(b)
1.0

0.8 0.8

(C0-Ct)/C0
0.6 0.6
(C0-Ct)/C0

0.4 0.4

0.2 0.2

0.0 0.0
HRP+H2O2 HRP+I- I-+H2O2 HRP+H2O2 +I- 5 6 7 8
pH

(c)
2.5
I- 0mM
r2=0.97518
(d)
I- 0.56mM 1.00
2
I- 1.12mM r =0.98613
2.0
I- 1.5mM
I- 1.8mM r2=0.93789
0.75
I- 2.25mM
Absorbance of I3-
1.5
ln (Ct/C0)

2.25mM
2 0.50 1.8mM
r =0.9172 1.5mM
1.0
1.12mM
0.56mM
r2=0.94785
0.5 0.25

r2=0.8821
0.0 0.00
20 40 60 80 100 20 40 60 100
t (min) Reaction time (min)

0.20
(e)
0.15
Absorbance of I2

0.10

0.05 2.25mM
1.8mM
1.5mM
1.12mM
0.56mM
0.00
20 40 60 100
Reaction time (min)

Fig. 1. Degradation of sulfadiazine in different systems ([H2O2]0 = 1 mM, [SMR]0 = 10 mg/L, pH = 5, HRP = 0.5 U/mL (a), Effect of pH on degradation of SMR (b),
Effect of iodide concentration on degradation of sulfadiazine (c), Absorbance changes of I3− (d) and I2 (e) ([H2O2]0 = 1 mM, [SMR]0 = 10 mg/L, pH = 5, HRP = 0.5
U/mL, iodide concentration as shown in the figure (c) (d) (e); in the figure(a) (b), [I− ] = 1.5 mM, absorbance of I2 and I3− was measured after three times dilution).

I-SMR on HRP was obtained. The detailed set of site finder module is
according to the previous study (Xu et al., 2018).
2.5. Ecological risk analysis of degradation products
The ecological risk of degradation products was determined by
analyzing the growth profile of Bacillus subtilis (B. subtilis) exposed with
degradation products. Agar drilling method and OD600 of culture me­
dium were used for qualitative and quantitative analysis of bacteria
growth. B. subtilis was cultured to logarithmic phase with LB medium,
and then used for risk assessment of SMR degradation intermediates.
Degradation products of SMR after 4 h and 8 h of degradation were
added into culture medium to determine its ecological risk. The group
with culture medium being added SMR was set as a positive control, and
group with culture medium being added water was set as a negative
control. Before adding to the medium, the degradation products were
first extracted by SPE column (Filier C18, 200 mg/3 mL) to remove
salinity (Heeb et al., 2014), and then eluted with 2 mL methanol solution

3
J. Wang et al.
Fig. 2. Effect of hydrogen peroxide concentration on degradation of SMR in the presence (a) and absence (b) of iodide ion. Conditions: for (a) and (b), [SMR]0 = 10
mg/L, [I− ]0 = 1.5 mM, pH = 5, HRP = 0.5 U/mL; (c) Determination of bromate by ion chromatography. ([SMR]0 = 10 mg/L, pH = 5).
and 13 mL ultra-pure water. Bacterial growth was measured at 12 h, 36
h, 48 h, 72 h and 80 h.
2.6. Statistical analysis
Each experiment included at least two technical replicates and was
repeated three times. All the mean values were calculated from three
independent experiments with duplicate samples.
3. Results and discussion
3.1. SMR degradation and main effect factors
3.1.1. SMR degradation in HRP/H2O2/I− system under different pH
conditions
SMR degradation in different systems are shown in Fig. 1a. The
removal efficiencys of SMR in HRP/H2O2 and H2O2/I− systems are all
less than 10% within 100 min, while SMR in HRP/I− system does not be
degraded with 1.5 mM iodide ions. When I− is added into HRP/H2O2
system, the removal efficiency of SMR increases significantly, and the
removal efficiency reaches 90.91% within 100 min, which is higher than
that by laccase (Fernandez-Sanroman et al., 2020). Conditions of pH also
affect the degradation of SMR in HRP/H2O2/I− system, shown in Fig. 1b.
Within the range of pH = 5–8, the degradation efficiency of SMR de­
creases monotonically with the increase of pH. In fact, the optimal pH of
HRP catalyzing reaction changes with the substrates. For example, the
4
Journal of Cleaner Production 337 (2022) 130489
optimal pH is 4–5 for degradation of pentachlorophenol by HRP/H2O2
(Zhang and Nicell, 2000). Tong et al. (1998) determined that peroxidase
had the highest removal efficiency of phenol and p-chlorophenol at pH
= 9. In this study, the optimal pH for SMR degradation by HRP/H2O2 is 5
in the presence of iodide ions, which is different to the optimum pH of 6
for SMR degradation by HRP/H2O2 without iodide (Yang, L. et al.,
2018b).
3.1.2. Effect of iodide ions concentration on degradation of SMR in the
HRP/H2O2/I− system
The concentration of I− also affects the degradation of SMR, which
follows pseudo-first-order kinetics, as shown in Fig. 1c and Fig. S1.
When conditions are set at 25 ◦ C, pH = 5, HRP = 0.5 U/mL, H2O2 = 1.0
mM, the concentration of I− increases from 0.25 mM to 1.5 mM, the
removal efficiency increases from 70.45% to 90.91% in 100 min.
However, when the concentration of I− increases to 2.25 mM, the
removal efficiency decreases to 55.44%. The decrease of the removal
efficiency of SMR is due to the formation of I3− rather than I2 in the
presence of higher concentration of I− , which is unfavorable the trans­
formation of SMR. As shown in Fig. 1d and e, the absorbance of I3− at
350 nm increases significantly with the increase of initial iodide ions
concentration, but the absorbance of I2 at 460 nm does not increase. The
reaction mechanisms are mainly as equations (1) and (2). It should be
noted that concentrations of I2 and I3− in the system decrease signifi­
cantly over time. Therefore, it can be speculated that I2 reacts with SMR
in the HRP/H2O2/I− system. However, according to Fig. 1e, when the
J. Wang et al. Journal of Cleaner Production 337 (2022) 130489

Fig. 3. Mass spectrometric analysis of SMR transformation products at 4 h (a) and 8 h (b) in HRP/H2O2/I− system.

concentration of iodine ion is higher than 1.8 mmol in HRP/H2O2/I− H2O2 for HRP catalysis. The additional consumption of H2O2 indicates
system, the removal of SMR is not further enhanced, which is due to the that there are other reactions that consume H2O2 in the HRP/H2O2/I−
formation of iodate from excess iodine ion. system, as shown equations (4) and (5). The reduction of removal effi­
ciency of SMR caused by excessive H2O2 may be due to the following
I3− ⇌ I2 + I− (1) two aspects. On the one hand, excess H2O2 promotes the formation of
I2 + H2O ⇌ HIO + H + I+ −
(2) HRPIII without catalytic activity, leading to the decrease of the removal
efficiency of SMR (James A et al., 1997; Towne et al., 2004). The in­
2I− + H2O2 + 2H+ = I2 + 2H2O (3) crease of H2O2 concentration promotes the generation of iodate in the
solution and results in the reduction of available iodide (Shin et al.,
−
3I2 + 3H2O2 = IO3 + 5I + 6H− +
(4)
2020). Compared with the I− /SMR system, the HRP/H2O2/I− /SMR
+ −
HOI + H2O2 → H + I + H2O + O2 (5) system promotes the oxidation of I− to iodate (shown in Fig. 2c). Also,
excessive H2O2 leads to more iodate production.

3.1.3. Effect of H2O2 concentration on degradation of SMR in the HRP/
H2O2/I− system
Fig. 2a depicts the effect of H2O2 concentration on SMR degradation.
When the concentration of H2O2 increases from 0.25 to 1 mM, the
removal of SMR increases from 16.06% to 90.91% within 100 min, but
decreases to 20.87% with 2.25 mM H2O2. As shown in Fig. 2b, when the
concentration of HRP is 0.5 U/mL, the optimal H2O2 concentration is
0.125 mM. In the HRP/H2O2/I− system, I− react with H2O2 under the
catalysis of HRP to form I2, resulting in the consumption of H2O2. Ac­
cording to equation (3), 0.75 mM of H2O2 is consumed when 1.5 mM I−
is oxidized. In fact, the optimal concentration of H2O2 in the reaction is
1 mM in the HRP/H2O2/I− system. After subtracting the maximum
consumption of H2O2 by iodide ions, the remaining concentration of
H2O2 is 0.25 mM, which is still greater than the optimal concentration of
3.2. Transformation products of SMR by HRP/H2O2/I−
In the process of SMR degradation by HRP/H2O2/I− , the trans­
formation products of SMR change obviously over time. With high res­
olution (above 40,000 to m/z) of mass spectrometry, the main
degradation products of SMR can be separated and detected. Fig. 3a and
b shows the degradation products at 4 and 8 h, respectively. Within the
first 4 h, SMR transformation is dominated by iodine substitution and
sulfamerazine bond breaking. In 4–8 h, the iodine-substituted products
of SMR are fully degraded, and the intermediate products containing
amide and ketone group. The products and their degradation processes
are listed in Fig. 4. Product 1 (C20H22N8O4S2, M/Z = 502.8255) is a
polymerized product of SMR, which is catalyzed by HRP and iodine.
Both HRP and iodine have the ability to catalyze the polymerization of

5
J. Wang et al.
Fig. 4. Proposed degradation products and decomposition pathway of SMR in HRP/H2O2/I− system (The blue pathway represents the products degraded for 4 h, and
the black pathway represents the products degraded for 8 h).
phenolic organic compounds (Enríquez-Medrano et al., 2013). Product 2
(C10H10IN4O2S, M/Z = 377.0477) has a apparently iodine isotope
peak, which suggests the production of iodine-substituted product of
SMR. According to the relative semi quantification of mass spectrom­
etry, iodine-substituted product of SMR is one of the main trans­
formation products within 4 h. Although the substitution reaction of
pyrimidine is difficult, the halogenation reaction is relatively easy.
Because the electron cloud density of pyrimidine at position 5 is the
lowest, the reaction is most likely to take place at this position. Dong
et al. (2019) also confirmed the substitution reaction of chloration at the
pyrimidine 5 position of sulfadiazine. After 8 h, the sharp decrease of
iodine-substituted products is due to the subsequent σ-breaking reac­
tion, which results in the removal of bound iodine. In addition, HRP has
a certain dehalogenation effect. Therefore, the presence of HRP in the
system can help to degrade iodine-substituted products. Product 3
(C10H11N4, M/Z = 187.0982) is the result of the rearrangement of SMR
in the form of Smiles-type. Sulfonamide group is a strong
electron-withdrawing group, and N of pyrimidine is in possession of a
high-density electron cloud. After the C–S bond breaks, electrons
transfer from N of pyrimidine to C next to sulfonamide group to form a
new carbon nitrogen bond with the nitrogen at the 3-position of py­
rimidine ring, making Smile rearrangement occur eventually. The
rearrangement products were also detected in the degradation of SMR
by MnO2/HRP/O3 systerm (Gao et al., 2012; Yang, L. et al., 2018a).
Product 4 (C4H9N3, M/Z = 98.9846) and product 5 (C6H7NO3S, M/Z
= 172.9766) are produced by the sulfanilamide bond break of SMR, and
the C– – N addition reaction takes place before product 4. Zhang et al.
(2017) also reported a similar addition reaction of pyrimidine during
chlorine disinfection. Product 5 lost an amino group and formed product
6 (C6H6O3S, M/Z = 158.0026).
6
Journal of Cleaner Production 337 (2022) 130489
3.3. Mechanisms of SMR transformation in HRP/H2O2/I− system
3.3.1. Contribution of each component in HRP/H2O2/I− system to SMR
transformation
In order to further analyze the contribution of each component in
HRP/H2O2/I− system to SMR transformation, effect of I2 formed in
HRP/H2O2/I− system was analyzed with H2O2/I2 system as a control
group. As shown in Fig. 5a and b, I2 exhibits obvious transformation
ability to SMR. As shown in equations (1) and (2), I− can convert into I2
and further convert into HIO in the weak acid condition, which has
higher oxidability than that of I2. Therefore, the transformation of SMR
involves substitution and oxidation in the presence of iodide ions.
However, when I2 and H2O2 coexist, the degradation efficiency of SMR
decreases, which is because although H2O2 can oxidize I− and I2 to I2 and
IO3− , respectively (equation (3) and (4)). The constants of the oxidation
reaction is much lower than that of H2O2 reducing HIO to I− (equation
(5) and Table S1). Therefore, the transformation of SMR was reduced.
Furthermore, IO3− is difficult to degrade phenolic compounds (Shin
et al., 2020). However, when HRP is added to H2O2/I− systems, the
removal efficiency of SMR is greatly improved. HRP can react with H2O2
to produce HRPІ/HRPII radicals (equation (6)), which are believed to
play a major role in the process of degradation (equation (8)). Moreover,
HRP rapidly catalyzes I− to produce I2 (Dunford, 1972) (equation (7)),
and then significantly enhances the oxidative degradation and haloge­
nation of SMR (Butler and Sandy, 2009) (equation (9)). On the other
hand, when excess hydrogen peroxide, HRP competes with iodide sub­
stances to consume H2O2, which promotes the degradation of SMR by I2.
HRP + H2O2 →HRP І/ HRPII (6)
−
HRP I + 2I → I2 + HRP (7)
SMR + HRP І/ HRPII→ HRPII/ HRP + SMR− Products
(8)
SMR + I2 → SMRI (9)
SMR + HIO → SMR − Products
(10)
J. Wang et al. Journal of Cleaner Production 337 (2022) 130489

1.0 further degradation. This can also explain that the iodine-substituted
(a) I2 0.75mM products of SMR produced in the first 4 h are almost completely
I2 0.75mM+H2O2 0.25mM degraded in the 8th h. Therefore, one of the main pathways for I− to
0.8 I2 0.75mM+HRP 1U/mL +H2O20.25mM promote SMR degradation by HRP/H2O2 may be that SMR first forms
substitution products with iodine, and then is further degraded.
On the other hand, the enhancement of HRP stability by I− is another
SMR Removal

0.6 factor to promote the degradation of SMR. Fig. 6c shows that the binding
site of SMR to HRP is in the active pocket of HRP, which promotes the
degradation of SMR and affects the structure and activity of HRP.
0.4 Compared with SMR, the structure of HRP becomes more stable in the
presence of I− . As shown in the Fig. 6d, I− induces the red shift and
enhancement of Fe-His absorption peak of HRP peak at 404 nm, which
0.2 means that the addition of I− enhances the stability of the HRP (Dipak K.
Bhattacharyya, 1993; Enzo Laurenti et al., 2000). Therefore, iodide
promotes the degradation of SMR by enhancing the structural stability
0.0 of HRP.
20 40 60 80
t (min) 3.4. Ecological risk of SMR transformation products
1.0
(b) I- 1.5mM+HRP 0.5U/mL+H2O2 1mM In order to determine the ecological risk of SMR transformation
A: HRP 1U/mL+H2O2 0.25mM products produced from HRP/H2O2/I− system, bacteria toxicity exper­
0.8 B: I2 0.75mM iments were conducted with the exposure of SMR transformation
A+B products. Bacillus subtilis, as a beneficial bacteria, is widely present in
various environment, and monitoring its growth can reflect the quality
SMR Removal

0.6 of environment. In this study, Bacillus subtilis growth indicates the

environmental risk of SMR degradation byproducts. As shown in Fig. 7a,
compared with the positive control group with SMR, the toxicity of SMR
0.4 transformation products on bacteria growth weakens. Moreover, with
the increase of HRP/H2O2/I− treatment time, the inhibition of SMR
transformation products on bacteria growth futher obviously relieves.
0.2 The plate culture method (Fig. 7b) also verifies that the toxicity of SMR
degradation products to bacteria is less than that of SMR, and the
toxicity of SMR transformation products after 8 h treatment is less than
0.0 that of SMR transformation products after 4 h treatment. This may be
20 40 60 80 because the active structure in SMR has been destroyed (Nunes et al.,
t (min) 2020). Hebert et al. and Liu et al. proved that halogenated organic
compounds (halomethane, halophenol) have certain biological toxicity
Fig. 5. Contribution of each component in (a) HRP/H2O2/I2 or (b) HRP/H2O2/
(Hebert et al., 2010; Liu and Zhang, 2014). However, the iodized
I− system to SMR transformation ([SMR]0 = 10 mg/L, pH = 5, HRP = 0.5 U/
products of SMR are gradually degraded overtime, resulting in the
mL, [H2O2]0 = 1 mM).
obvious reduction of the toxicity of SMR degradation products to bac­
teria. Therefore, the ecological risk of SMR degradation products by
HRP/H2O2/I− is low.
SMRI + HRPII→ SMR− Products
(11)

In Fig. 5b, the sum of the removal efficiencys for SMR by I2 and HRP/
H2O2, respectively, is less than that of HRP/H2O2/I− , which indicates
that iodide ion and HRP play a synergistic role in the degradation of
SMR. This may be due to the enhancement of SMR degradation by iodide
acting as the mediator of enzymatic in the degradation system. On the
other hand, according to the analysis of degradation products, the sub­
stitution product of SMR (equation (10)) may be easier to bind to HRP
(equation (11)) to enhance the removal of SMR.
3.3.2. Mechanisms of I− enhancing the degradation of SMR by HRP/H2O2
In order to further clarify the mechanism of I− promoting the
degradation of SMR by HRP/H2O2, MOE was used to simulate the
docking site, binding energy and interaction mechanism of HRP with
SMR and its iodized products. Fig. 6a and b shows the most stable
binding model of HRP and SMR or iodine-substituted products of SMR.
Both SMR and its iodine-substituted products are bound to the Hem 350
residue near the active site of HRP by ionic bonds (as shown in Table 1).
The binding energy between SMR and HRP is − 13.5381 kcal/mol, while
the binding energy between iodine-substituted products of SMR and
HRP is − 15.5507 kcal/mol. According to the theory that the lower the
binding energy, the more likely the reaction occurs. HRP is more likely
to react with iodine-substituted products of SMR, thus promoting their
4. Conclusions
Iodide ions promote SMR degradation by HRP/H2O2 at a less than or
equal to 1.8 mmol but inhibit at a high level of over 1.8 mM, and the
maximum removal efficiency of SMR is 90.91% in HRP/H2O2/I− system
within 100 min due to the synergistic effect of H2O2/I− (7.07%) and
HRP/H2O2 (5.13%). The main mechanisms of iodide ion promoting SMR
degradation are that HRP/H2O2 can not only degrade SMR directly by
sulfonamide bond breaking, polymerization and rearrangement, but
also catalyze iodide ion to form iodine element, which is easier to react
with SMR by substitution and i-cleavage. The substitution products of
SMR are more likely to bind to HRP, thus further enhancing SMR
degradation. The degradation products of SMR after HRP/H2O2/I−
treatment for 8 h shows a negligible bacteria toxicity. This is the first
report that I− can accelerate SMR degradation by HRP, which provides
more basis for further application of HRP to removal sulfonamides
antibiotics.
CRediT authorship contribution statement
Jiali Wang: Experiment, Writing – original draft, Data curation,
Investigation, Methodology, Formal analysis. Zhao Shu: Investigation,
Methodology. Zhennan Chen: Investigation, Methodology. Jixin Su:

7
J. Wang et al. Journal of Cleaner Production 337 (2022) 130489

Fig. 6. Bind interaction of SMR to HRP related amino acid residues (a); Bind interaction of iodine substitution products of SMR to HRP amino acid residues (b); 3D
docking simulation diagram of SMR and HRP, the binding interaction of HRP’s heme center to SMR, red model stick model represents heme, blue model stick model
represents 1-,8-methyl, yellow ball-and-stick model represents SMR (c); Effect of iodide ion and sulfadiazine on the absorbance of HRP. [I− ]0 = 0.6 M, [SMR]0 = 30
mg/L, [H2O2] 0 = 10 mM (d); Bind interaction of heme center to amino acid residues in HRP (e).

Table 1
The bonds simulated by using MOE.
Ligand Receptor Residue Type Binding Energy Distance

SMRI O 5227 NH 600 ARG 38 H-acc − 15.550 kcal/mol 2.52 Å

N 5238 Fe 4698 HEM 350 ionic 2.31 Å
SMR H 5235 O 2614 LYS 174 H-don − 13.538 kcal/mol 2.40 Å
N 5240 Fe 4698 HEM 350 ionic 2.31 Å

8
J. Wang et al.
Fig. 7. Ecological risk of SMR degradation products.
Formal analysis, Writing –- review & editing. Chunguang Liu: Super­
vision, experiment design, Formal analysis, and, Writing – original draft.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This study has been supported by National Natural Science Foun­
dation of China (42077391), Shandong Provincial Natural Science
Foundation, China (ZR2019BB049), Guangdong Foundation for Pro­
gram of Science and Technology Research (2020B1212060053). We
would like to thank Zhifeng Li and Jingyao Qu from State Key laboratory
of Microbial Technology of Shandong University for help and guidance
in test analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jclepro.2022.130489.
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