Rapid catalytic oxidation of As(iii) to As(v) using a: Bacillus spore-2,2,6,6-tetramethylpiperidine-1-oxyl system

Article abstract of DOI:10.1039/c8gc03654c

The oxidation of As(iii) to As(v) is a critical process in the treatment of contaminated water. We found that 95% As(iii) (10 mg L^-1) could be rapidly oxidized to As(v) by a laccase-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) system in 1 h. Based on this finding, we used Bacillus subtilis spores instead of laccase for As(iii) oxidation with the same effect because the former had plenty of CotA-laccase on their surface. The catalytic ability of CotA protein and spores was confirmed by expressing the CotA protein and knocking out the cotA gene from wild-type spores. Both laccase- and spore-TEMPO systems displayed similar oxidation rate constants, Michaelis-Menten constants, and maximal velocities owing to the formation of the oxoammonium cation of TEMPO in the presence of dissolved oxygen. Several other laccase mediators such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic-acid) (ABTS), acetosyringone (AS), 1-hydroxybenzotriazole (HBT), 2-hydroxybutyl acrylate (HBA), violuric acid (VLA), 4-oxo-TEMPO, 4-amino-TEMPO, 4-methoxy-TEMPO, 4-hydroxy-TEMPO benzoate, and 4-hydroxy-TEMPO coupled with spores for As(iii) oxidation were also investigated in detail. The spore-TEMPO system exhibited the highest oxidation efficiency and tolerated the addition of 10 mg L^-1 Al³⁺, Ti⁴⁺, Cu²⁺, K⁺, Fe³⁺, Zn²⁺, Ni²⁺, Mg²⁺, Co²⁺, and Mn²⁺. Both laccase and spores recovered via ultrafiltration and centrifugation, respectively, could be reused for at least five cycles. The developed spore-based system has several advantages including eco-friendliness, ease of operation and storage, low cost, recyclability, sustainability, and without the need for enzyme purification. These findings may have promising implications for developing a new eco-friendly and cost-effective technology for the treatment of arsenic-containing water.

Full text of DOI:10.1039/c8gc03654c

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Rapid catalytic oxidation of As(III) to As(V) by using Bacillus
spore–2,2,6,6-tetramethylpiperidine-1-oxyl system
Yuqing Qin, a, b Fei Peng a, b, Yonggang Hu a, b,
*
Received 00th January 20xx,
Accepted 00th January 20xx
Oxidation of As(III) to As(V) is a critical process in the treatment of contaminated water. We found 95% As(III) (10 mg L–1)
could be rapidly oxidized to As(V) by laccase–2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) system in 1 h. Basing on this
finding, we used Bacillus subtilis spore instead of laccase for As(III) oxidation with the same effect because the former had
plenty of CotA–laccase on their surface. The catalytic ability of CotA protein and spore was confirmed by expressing CotA
protein and knocking out cotA gene from wild-type spore. Both laccase– and spore–TEMPO systems have similar oxidation
rate constants, Michaelis–Menten constant, and maximal velocity owing to the formation of oxoammonium cation of
TEMPO in the presence of dissolved oxygen. Several other laccase mediators such as 2,2-azino-bis(3-ethylbenzothiazoline-
6-sulfonic-acid) (ABTS), acetosyringone (AS), 1-hydroxybenzotriazole (HBT), 2-hydroxybutyl acrylate (HBA), violuric acid
(VLA), 4-oxo-TEMPO, 4-amino-TEMPO, 4-methoxy-TEMPO, 4-hydroxy-TEMPO benzoate, and 4-hydroxy-TEMPO coupled
with spore for As(III) oxidation were also investigated in detail. Spore–TEMPO system exhibited the highest oxidation
efficiency and tolerated the addition of 10 mg L–1 Al3+, Ti4+, Cu2+, K+, Fe3+, Zn2+, Ni2+, Mg2+, Co2+, and Mn2+. Both laccase and
spore recovered via ultrafiltration and centrifugation, respectively, could be reused for at least five cycles. The advantages
of spore-based system include eco-friendliness, easy operation and storage, low cost, recyclability, sustainability, and
without the need for professional training and enzyme purification. These findings may show promising implications in
developing a new eco-friendly and cost-effective technology for the treatment of arsenic-containing water.
DOI: 10.1039/x0xx00000x
www.rsc.org/
classical ones,12 such as chlorine, ozone, potassium
permanganate, and persulfate. Some catalytic oxidation
systems, such as Fenton reaction,13 metal activated
Introduction
As a serious global problem, arsenic pollution in drinking
water has recently raised public awareness and scientific
concern1 and has affected 150 million people in over 70
countries.2 World Health Organization provisions a guide limit
value in drinking water at 10 g L–1, and the U.S. Environmental
Protection Agency provides a new standard in the range of 2–
20 g L–1.3, 4 In natural water environments, As(III) and As(V) are
the major species, and As(III) is more toxic than As(V).5
Moreover, As(III) is more mobile in natural waters and less
efficiently removed in adsorption/coagulation processes than
As(V) because the former appears predominately in a fully
protonated form (H3AsO3) below pH 9.2, whereas the latter
occurs as a mixture of H2AsO4– and HAsO42– at circumneutral pH
values.6 Therefore, the oxidation of As(III) to As(V) is a critical
process in the treatment of contaminated water.7 Many
oxidants and technologies for the oxidation of As(III) have been
investigated. For example, some radicals, such as hydroxyl
radical,8 superoxide,9, 10 and sulfate radical,11 are considered as
new potential species for As(III) oxidation instead of the
persulfate,14 and TiO2 photocatalytic systems15, 16 have been
applied to As(III) oxidation. Energy such as light11, 15, ultrasonic
waves,17, 18 and electricity19 has been used to enhance As(III)
oxidation. Each of these processes and oxidants has advantages
and disadvantages. For example, chemical oxidants such as
Fenton reagent, hydrogen peroxide, chlorine, and manganese
oxide have high oxidation efficiency but may generate
secondary pollution. Electrochemical peroxidation and
photocatalytic oxidation are environment friendly but have a
low oxidation efficiency and cannot be extensively used in a
natural environment.20, 21 Therefore, developing efficient As(III)
oxidation processes is needed to remove stringent arsenic.
Enzymatic catalysis is a promising green method with
reasonable consumption of energy and reagents for disposing
emerging contaminants because of its high specificity and
efficiency. Peroxidase- and laccase-catalyzed enzymatic
reactions have been successfully used for the removal of several
23
organic pollutants, such as phenols,22,
alcohols,24 and
dichlorophen25, from water. However, the enzymatic reaction
on the transformation of heavy metals such as arsenic has been
much less explored. Mao et al.26 recently suggested to remove
As(III) from water via reactions mediated by laccase with the
presence of oxygen. According to their report, no As(III)
transformation was observed during the first 10 h of reaction.
However, after 10 h, the reaction of As(III) was rapidly
a. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural
University, Wuhan 430070, China
b. College of Life Science and Technology, Huazhong Agricultural University, Wuhan
430070, China.
Email: yongganghu@mail.hzau.edu.cn
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accelerated, and 85.7% removal was achieved at 48 h.
Unfortunately, our findings were different from their repeated
results. In addition, shortcomings, including the non-
recyclability of laccase in the solution and the long reaction
time, still limit its practical application.
Experimental
Materials
View Article Online
DOI: 10.1039/C8GC03654C
B. subtilis was provided by State Key Laboratory of
Agricultural Microbiology, Huazhong Agricultural University
(Wuhan, China). Taq DNA polymerase, restriction
endonucleases and the DNA markers were purchased from
Takara Biotechnology Co., Ltd (Dalian, China). The synthesized
primers were acquired from Tsingke Biotechnology Co., Ltd
(Wuhan, China). Protein markers were obtained from Transgen
Biotechnology Co., Ltd (Beijing, China). Bacterial Genemic DNA
Extraction kit and Plasmid Purification Kit were purchased from
Takara Biotechnology Co., Ltd. (Dalian, China). Sodium arsenite
(NaAsO2) was obtained from Xiya Regent Co., Ltd (Chengdu,
China). Standard stock solution of As(V) was obtained from
National Center of Analysis and Testing for Nonferrous Metals
and Electronic Materials (Beijing, China). Laccase from Trametes
versicolor was purchased from Sigma-Aldrich (St. Louis, MO,
USA). ABTS was purchased from Aladdin Biochemistry
Technology Co., Ltd (Shanghai, China). TEMPO and TEMPO-
derivatives, containing 4-oxo-TEMPO, 4-amino-TEMPO, 4-
methoxy-TEMPO, 4-hydroxy-TEMPO benzoate, and 4-hydroxy-
TEMPO were obtained from TCI Development Co., Ltd (Shanghai,
China). AS, HBT, HBA and VLA used in this work were obtained
from Yuan Ye Biotechnology Co., Ltd (Shanghai, China). The
reagent was diluted and dissolved using ultrapure H2O (18.2 MΩ)
prepared from a model Cascada IX laboratory ultrapure H2O
system from Pall Co., Ltd (Washington, NY, USA).
Bacillus spores are a remarkable phase in the life cycle of
Bacillus. These spores can survive for several years and also can
be recycled via fermentation. In this regard, spores expressing
certain enzymes, proteins, or peptides on their surface have
been presented as a stable, simple, and safe new tool for the
biospecific recognition of target analytes, the biocatalytic
production of chemicals, and the delivery of biomolecules with
pharmaceutical relevance.27 The engineered spores have been
used for whole-cell biosensor,28, 29 biocatalysis,30 surface display
of recombinant proteins for bioanalysis and biotechnological
application,31 and as vehicles for vaccines and therapeutic
agents32, 33. Considering the presence of laccase (i.e., CotA
protein) on the surfaces of Bacillus subtilis (B. subtilis) and
Bacillus amyloliquefaciens spores, we successfully used wild-
type bacterial spores as a whole-cell biosensor in antioxidant
capacity34 and phenol assays35. Furthermore, we proposed a
pioneering biological method using inactivated bacterial spores
to prepare functional monodisperse microparticles, which have
been successfully used in immunoassays,36, 37 catalysis,38 pH
sensing,39 DNA analysis,40 and selective enrichment of
phosphorylated proteins41. Therefore, these spores have
advantages, vast potential research value, and application
prospect in different fields.
Methods
In this work, we found for the first time that laccase–TEMPO
(a commercially available and stable N-oxy radical) system could
rapidly oxide As(III) to As(V). Based on this finding, B. subtilis
spores were utilized as a catalysis for As(III) oxidation because
of the presence of CotA protein on the their surface. The effects
of surface CotA protein, and several mediators such as ABTS, AS,
HBT, HBA, VLA, 4-oxo-TEMPO, 4-amino-TEMPO, 4-methoxy-
TEMPO, 4-hydroxy-TEMPO benzoate, and 4-hydroxy-TEMPO on
As(III) oxidation were also investigated in detail. This work may
contribute to the understanding of the main mechanisms of
As(III) oxidation and may aid in the development of a new
biological As(III) decontamination method driven by spores. Our
proposed spore-based method exhibits several advantages,
such as easy operation and storage, low cost, recyclability,
sustainability, and without the need for professional training
and enzyme purification.
Preparation of Spore. B. subtilis was grown in Luria-Bertani (LB)
solid medium supplemented with 0.25 mmol L–1 Cu2+ at 37 °C
for 4 d42. Spores scraped off from the medium were washed
three times with deionized water, and then stored at 4 °C. The
number of spores was determined with bacterial colony
counting method under plating serial dilutions on LB plates43, 44
Activity Assays of Laccase. The activities of laccase, spores and
.
CotA were determined by monitoring the oxidation of ABTS
using
a UV-visible (UV-vis) spectrophotometer (DU780,
Beckman Coulter, USA) at 420 nm (=3.6×104 M–1 cm–1) with 0.5
mM ABTS as the substrate in acetic acid buffer (0.1 mol L–1, pH
4.5) at 37 °C.45 In briefly, 20 L sample and 10 L of 1 mmol L–1
ABTS were added into 170 L, measured at 420 nm. One unit of
laccase activity was defined as the amount of laccase required
to oxidize 1 M of ABTS per min.
As(III) Oxidation by Spore–TEMPO. As(III) oxidation by spore–
TEMPO was conducted with 2 mL centrifuge-tube containing 1
mmol L–1 TEMPO and 7.48×107 CFU mL–1 spores for 10 mg L–1
As(III) at pH 4.5 and incubated at 55 °C for 1 h, terminated by
centrifuge at 12 000 rpm for 2 min, and the supernatant was
detected using molybdenum blue method.46, 47 Briefly, 200 μL
of the withdrawn sample solution was added into 1.8 mL of
mixture solution, containing 100 μL of sulfuric acid (5 mol L–1),
100 μL of molybdate reagent solution (3.51%) and 50 μL of
ascorbic acid (1%), incubating at 60 °C for 15 min. The
concentration of As(V) was measured using UV-vis
spectrophotometer at 840 nm.
Scheme 1. Spore–TEMPO and laccase–TEMPO used in the As(III) oxidation.
2 | J. Name., 2012, 00, 1-3
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Cloning, Expression, and Purification of CotA. cotA, encoding B. been effectively used for the oxidation of phenolic compounds
View Article Online
subtilis CotA protein, was amplified by PCR using B. subtilis toward benzylic alcohols and aromatic DaOmI:in10e.s10.5319/ACc8cGoCr0d3in65g4lyC,
chromosomal DNA as the template with the primers cotA-F (5’- laccase-mediator systems are ideal candidates for sustainable
GCTGGATCCATGACACTTGAAAAATTTGTGG-3’) and cotA-R (5’- chemical and oxidoreductive biotechnological applications.
GCTCTCGAGTTATTTATGGGGATCAGTTAT-3’). The PCR con- Among these systems, laccase–TEMPO catalytic system is the
ditions: 7 min at 94 °C, 30 cycles of 30 s at 94 °C, 45 s at 55 °C, 2 most employed due to TEMPO accessibility and high
min at 72 °C, followed by 72 °C for 10 min. The PCR program compatibility with laccases.52
(1542 bp) was digested with XhoI and BamHI, and then cloned
As(III) cannot be converted to As(V) when only laccase or
into pet28a to generate pet28a-cotA recombinant plasmid. TEMPO is present. This conversion reaction was catalyzed and
Finally, the recombinant plasmid was transformed into accelerated in laccase–TEMPO system as detected using
Escherichia. coli (E. coli) Rosetta (DE3) and result was confirmed high-performance
by sequence analysis.
liquid
chromatography–hydride–
generation–atomic fluorescence spectroscopy53 (HPLC–HG–
E. coli Rosetta (DE3) carrying pet28a-cotA plasmid was AFS) (SA-10, Beijing Titan Instruments, China); 97% As(III)
inoculated into LB medium with 50 g L–1 kanamycin and grown conversion to As(V) was achieved in 1 h (Fig. 1a) because the
at 37 °C, 180 rpm until an optical density at 600 nm of 0.5 was substrate of laccase could be effectively explored via the
reached.
Then
0.1
mmol
L–1
isopropyl-β-D- ancillary action of low molecular weight electron transfer
thiogalactopyranoside (IPTG) and 0.25 mmol L–1 CuSO4 were agents (i.e., mediators) in the presence of dissolved oxygen.54
added into the medium and culture conditions were adjusted to The reactions of laccase–TEMPO and laccase–TEMPO–As(III)
25 °C, 140 rpm. The E. coli Rosetta (DE3) was Incubated for a were
characterized by UV-vis spectrophotometer
from
further 5 h until the density at OD600 of E. coli almost was 2.5. Shimadzu Corporation ( Japan) (Fig. 1b). When the TEMPO was
Then switching off the shaking function for microaerobic mixed with laccase, the absorbance at 245 nm decreased,
conditions, the E. coli Rosetta (DE3) was harvested after a whereas that at 300 nm increased, indicating that TEMPO was
further 24 h48, 49. The cells were harvested using a high-speed transferred to its oxoammonium ions via the laccase–oxygen
56
centrifuge (12 000 rpm, 5 min, 4 °C) and resuspended with 10 reaction.55,
In the laccase–TEMPO–As(III) reaction, the
mmol L–1 imidazole binding buffer. Cell was disrupted using Low characteristic spectral features at 245 nm increased, but those
Temperature Ultrahigh Pressure Continuous Flow Cell Disrupter at 300 nm decreased gradually, indicating that the
and the cell debris removed by centrifugation (12 000 rpm, 20 oxoammonium ions were reduced to TEMPO again. The kinetic
min, 4 °C). The CotA protein purification was preformed using a parameters of the laccase–TEMPO-catalyzed As(III) oxidation
Ni-NTA sefinose resin kit. The CotA protein was analyzed by were analyzed (Fig. 1c), and the oxidation reaction reached
SDS-PAGE with 30% polyacrylamide running gel, performed in a equilibrium in 1 h. The oxidation rate constant (k, s–1) was
DYCZ-24DN mini double vertical electrophoresis system.
The Knockout of cotA Gene. Homologous double exchange
was used to knocked out the cotA gene yielding cotA knock-out
strain (Mt-strain) in this work according to our previous works34.
In brief, we amplified the upstream and downstream regions of
calculated using Equation 1 as follows:
푸퐭 = 푸퐞(ퟏ ― 퐞퐱퐩 ( ― 풌풕))
(1)
Where Qt (mg L–1) is the oxidation capacity of As(III) by
target locus using B. subtilis genomic DNA as a template and oxidant at time t (min), and Qe (mg L–1) is the equilibrium
primer pair (F: 5’-CCGCTCGAGCCCGACAAACTTGCCTCT-3’, R: 5’- oxidation capacity. The oxidation kinetics was fitted by a
CGGGGTACCTTAAAAGCTTTTTCCTGGTTTA-3’). The upstream pseudo-first-order model (R2= 0.999) with k = 3.12 s –1
.
and downstream were connected to the pDG780 vector
Michaelis–Menten reaction kinetics was also assessed (Fig.
respectively, and the recombination plasmid was transformed 1d). Michaelis–Menten constant (Km) and maximal velocity
into B. subtilis. Finally, the transformant was screened using (Vmax) were calculated using Equation 2 as follows:
ampicillin and kanamycin resistance. The positive
푽 = (푽퐦퐚퐱 [푺])/(푲풎 +[푺])
transformants showed resistance to kanamycin and
susceptibility to ampicillin and kanamycin. ABTS assay was used
to determine whether the cotA gene was deleted from B.
subtilis genome successfully.
(2)
Where V is the reaction velocity and [S] is the substrate
concentration. Vmax and Km values were calculated using
nonlinear regression and were estimated to be 3.21×10−3 mmol
L–1 min−1 and 0.033 mmol L−1, respectively.
After oxidation reaction, the laccase was collected through
ultrafiltration to remove the buffer solution and ions such as
TEMPO and its oxoammonium ion with arsenic. The laccase still
maintained high activity for As(III) oxidation after five recycles
(Fig. 1e). Previous works indicate that the oxoammonium ion of
TEMPO and arsenic can interact with laccase and lead to the
inactivation of the latter.57, 58 Our results might be due to the
high oxidation efficiency with short reaction time and the low
Results and discussion
As(III) Oxidation by Laccase–TEMPO
Laccases belong to the multi-copper family of oxidases with
four copper ions in two catalytic centers and uses oxygen as an
electron acceptor.50 Laccases are accessible and inexpensive
and can efficiently work in water under mild reaction
conditions. Through coupling with electron transfer mediators,
such as ABTS, benzotriazole, or syringaldehyde, laccase has
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confirmed using HPLC–HG–AFS (Fig. 2d). TheVieswimAritliaclre OUnliVne-
DOI: 10.1039/C8GC03654C
vis spectral characteristic of CotA–TEMPO (Fig. 2e) and laccase–
TEMPO (Fig. 1b) revealed that these As(III) oxidation reactions
have a similar reaction mechanism. The cotA was then knocked
out from the genome of B. subtilis by using homologous double
exchange to yield cotA Mt-strain. Loss of laccase activity was
confirmed by adding mutant spores (Mt-spores) to the ABTS
solution because the solution remained colorless and did not
generate ABTS•+ (Fig. 2f). On the contrary, the colorless ABTS
was also converted into blue-colored ABTS•+ in the presence of
wild-type spore similar to that in the recombinant CotA protein.
These results indicate that the CotA protein induces the spore
to show laccase activity.
Bacterial spores, Mt-spores, Mt-spore–TEMPO, and spore–
TEMPO were respectively mixed with As(III) solution to test
their ability to oxidize As(III) to As(V) (Fig. 2g). As(V) formation
Fig. 1 Oxidation of As(III) by laccase–TEMPO. (a) Chromatogram of As(III) oxidation by
laccase–TEMPO. Conditions: 45 U L–1 laccase, 1 mmol L–1 TEMPO, 10 mg L–1 As(III), pH
4.5, 40 °C,
1 h reactive time. (b) UV-vis monitoring of TEMPO and TEMPO
oxoammonium in the As(III) oxidation reaction by laccase-TEMPO. Conditions: 1 U mL–
laccase, 0.5 mmol L–1 TEMPO, pH 4.5, 40 °C. (c) The kinetic of As(III) oxidation by
1
laccase–TEMPO. Conditions: 45 U L–1 laccase, 1 mmol L–1 TEMPO, 10 mg L–1 As(III), pH
4.5, 40 °C. (d) Michaelis–Menten reaction kinetics of As(III) oxidation by laccase-
TEMPO. Conditions: 45 U L–1 laccase, 1 mmol L–1 TEMPO, pH 4.5, 40 °C, 0-6.667 mg L–1
As(III), 10 min reactive time. (e) The recyclability of laccase in the As(III) oxidation.
Conditions: 1 U mL–1 laccase, 1 mmol L–1 TEMPO, pH 4.5, 40 °C, 1 h reactive time. The
error bars represent SE of the means. The data represent means SE (n=3).
concentration of TEMPO, which was beneficial to keep the
activity and recycling application of laccase.
As(III) Oxidation by Spore–TEMPO
Bacterial spores have CotA–laccase on their surface and
could survive under environmental challenges (e.g., wet, dry
heat, UV, gamma radiation, and oxidizing agents).59 The use of
spores instead of commercial laccase has several advantages,
including low cost, long life, reproducibility due to their ability
to cycle between the spore and germinating states, and without
the need for professional training and enzyme purification. To
prove this concept, we expressed the recombinant CotA protein
in the cell cultures of E. coli system. The results of SDS-PAGE and
Western blot analysis (Fig. 2a, b) indicated that a specific band
at the molecular weight of 65 kDa belongs to CotA protein, thus
confirming that the CotA protein of B. subtilis was successfully
cloned and expressed. The recombinant protein was then
purified and tested using ABTS assay. The colorless ABTS was
converted into blue colored ABTS•+ in the presence of
recombinant protein (Fig. 2c), thereby confirming that the
recombinant protein has laccase activity. Furthermore, the
ability of the recombinant protein to oxidize As(III) to As(V) was
Fig. 2 The mechanism determination of spore–TEMPO used for As(III) oxidation.
SDS-PAGE analysis (a) and Western blot analysis (b) of CotA protein of B. subtilis,
Lane M, marker; lane1, lane 2, before and after cell extract of IPTG-induced Rosetta
(DE3); (c) ABTS assay used to determinate the laccase activity of CotA. (d)
Chromatogram of As(III) oxidation by CotA-TEMPO. Conditions: 45 U L–1 CotA, 1
mmol L–1 TEMPO, 10 mg L–1 As(III), pH 4.5, 40 °C, 1 h reactive time. (e) UV-vis
monitoring of TEMPO and TEMPO oxoammonium in the reaction of CotA and
TEMPO. Conditions: 1 U mL–1 CotA, 1mmol L–1 TEMPO, pH 4.5, 40 °C. (f) ABTS assay
used to determinate the laccase activity of spore and Mt-spore. (g) Molybdenum
blue method analysis of As(III) oxidation by Mt-spore, Mt-spore–TEMPO, spore, and
spore–TEMPO. CK means only in the presence of As(III). Conditions: 7.48×107 CFU
mL–1 spore or Mt-spore, 1mmol L–1 TEMPO, 10 mg L–1 As(III), pH 4.5, 55 °C, 1 h
reactive time.
was detected using molybdenum blue method after the spores
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Table 1 Comparison of As(III) oxidation by Spore–TEMPO and other oxidants
View Article Online
DOI: 10.1039/C8GC03654C
Oxidants
TiO2/H2O2
UV/ Ti(SO4)2
Qm
Time
(h)
Conditions
Ref.
(mg L–1)
60
22.5
0.198
4
4
0.35
8
30 mg L–1 As(III), 0.5 g L–1 TiO2,
2 mmol L–1 H2O2, pH 2.5
0.2 mg L–1 As(III), 5 mg L–1
Ti(SO4)2, pH 5
15
61
Resin-supported nanosized zero-valent
iron
5 mg L–1 As(III), 500 mg L–1 nZVI,
140 rpm, pH 6.5, 25 °C
62
63
64
Immobilized bacteria
0.92
0.1
1
1 mg L–1 As(III), 1.6 × 1010 cell,
260 mg L–1 NH4-N, pH 7, 20 °C
Wolffia/ phyllosphere
168
24
0.1 mg L–1 As(III), 14 h light
every day, pH 5.5, 25 °C
bacteria
Gammaproteobacterial/ graphite
electrode
0.42
0.46
0.2
15 mg L–1 As(III), polarized graphite electrode, 22-24 °C
11
65
Fe(III)/ sulfite
Microbial fuel cells
Spore–TEMPO
0.5
168
1
0.5 mg L–1 As(III), 0.1 mmol L–1 Fe(III) and Na2SO3, visible light,
pH 6
0.2 mg L–1 As(III), power density of MFCs was 752.6  17 mW
m–2, 22  2 °C
9.5
10 mg L–1 As(III), 7.48 × 107 CFU mL–1 spore, 1 mmol L–1 TEMPO,
pH 4.5, 55 °C
This work
were removed from the reaction system via centrifugation. This
detection method is cheaper and more convenient than HPLC–
HG–AFS because the formation of As(V) could be observed with
the naked eye. Only the color of spore–TEMPO–As(III) system
turned blue, indicating that only wild-type spores can catalyze
TEMPO and oxidize As(III) to As(V) due to the presence of CotA–
laccase on their surface.
The oxidation of 10 mg L–1 As(III) was used as a model to
evaluated spore–TEMPO– As(III) catalysis reaction system in the
following reaction conditions: pH in the range of 3–9 and
temperature in the range of 40 °C–70 °C (Fig. 3). This system
exhibited high oxidation efficiency within a wide range of pH
and temperature because CotA–laccase is an intrinsically highly
thermostable and pH-stable enzyme.66 The maximal oxidation
efficiency of As(III) was obtained at pH 4.5 and 55 °C. This
developed method was significantly superior over many other
systems using different materials for As(III) oxidation (Table 1).
The ability of several laccase-mediators such as ABTS, AS,
HBA, HBT, VLA, 4-oxo-TEMPO, 4-amino-TEMPO, 4-methoxy-
TEMPO, 4-hydroxy-TEMPO benzoate, and 4-hydroxy-TEMPO to
oxidize As(III) was investigated and compared with that of
TEMPO by adding them into the spore–As(III) solution. These
mediators with different chemical structures (Fig. 4) represent
the following three different reaction mechanisms underlying
laccase catalytic reaction: via an ionic oxidation that is
suggested for TEMPO and TEMPO derives,24 via electron
transfer that is suggested for ABTS,67 or via a hydrogen atom
transfer that is suggested for AS, HBA, HBT, and VLA, which
Fig. 3 The effect of pH (a) and temperature (b) in As(III) oxidation by spore–
TEMPO. Conditions: 7.48×107 CFU mL–1 spore, 1 mmol L–1 TEMPO, 10 mg L–1
As(III), 1 h reactive time. The error bars represent SE of the means. The data
represent means SE (n=3).
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
68, 69
requires substrates with relatively weak C-H bonds50,
.
Table 2 Oxidation of As(III) using spore–mediators system
View Article Online
Approximately 0.418 mL min–1 O2 was also added into the
spore–As(III) solution to evaluate its effect for As(III) oxidation.
As shown in Table 2, except for CotA–laccase, the redox
potentials of O2 and laccase-mediators are higher than that of
As(V). After 1 h, 95%, 34%, 32%, 4.8%, 25%, and 20% of As(III)
was oxidized to As(V) by using TEMPO, 4-methoxy-TEMPO, 4-
hydroxy-TEMPO, 4-oxo-TEMPO, 4-amino-TEMPO, and 4-
hydroxy-TEMPO benzoate, respectively. However, no
transformation of As(III) was observed when excessive O2, AS,
HBA, HBT, and VLA were used for 10 h. ABTS exhibited low
transformation rate (10%) during 10 h. These results indicate
that the mechanism underlying the reaction between mediator
and substrate is the key for As(III) oxidation.
Furthermore, the transformation efficiency of TEMPO, 4-
methoxy-TEMPO, 4-hydroxy-TEMPO, 4-oxo-TEMPO, 4-amino-
TEMPO, and 4-hydroxy-TEMPO benzoate increased to 100%,
95%, 95%, 8.9%, 89%, and 46%, respectively, in 10 h, indicating
that the conversion rate of As(III) could be improved by
prolonging the reaction time. The difference of TEMPO and
TEMPO derives for As(III) oxidation is due to the different
stability of oxoammonium cations under acidic condition70 and
redox potential71. That is, the high redox potential of TEMPO-
analogous compounds compared with CotA–laccase leads to
slow oxoammonium cation formation, which may require a long
time to oxidize As(III) to As(V).72 Therefore, TEMPO with the
lowest redox potential and stable oxoammonium cation has the
highest oxidation efficiency.
DOI: 10.1039/C8GC03654C
As(III) oxidation (%)
Redox
potential
(vs NHE)
0.5673
2.4273
1 h
–
–
10 h
–
–
As(V)
O2
TEMPO
95
34
32
4.8
25
20
100
95
95
8.9
89
46
0.75, 0.7470
0.8072
4-methoxy-TEMPO
4-hydroxy-TEMPO
4-oxo-TEMPO
4-amino-TEMPO
4-hydroxy-TEMPO
benzoate
0.8072
0.7872
0.8972
1.0074
ABTS
AS
< 1
–
10
–
0.69 and 1.1075
0.6076
HBA
–
–
0.9077
HBT
–
–
1.0975
VLA
–
–
0.9275
CotA from B. subtilis
–
–
0.4678
4-methoxy-TEMPO and 4-hydroxy-TEMPO have moderately
oxidation efficiency, though their redox potentials are higher
than that of TEMPO possibly because their oxoammonium
cations can stabilize under the reaction condition. Moreover,
both of them have similar oxidation efficiency owing to their
same redox potential. The oxidation efficiency of 4-hydroxy-
TEMPO benzoate was lower than those of TEMPO, 4-methoxy-
TEMPO, and 4-hydroxy-TEMPO owing to its highest redox
potential. The low oxidation efficiency of 4-amino-TEMPO
might be mainly due to its unstable oxoammonium cations at
pH 4.5 and its high redox potentials. Among TEMPO-analogous
compounds, 4-oxo-TEMPO has the lowest oxidation efficiency
because its oxoammonium cations decomposed irreversibly
into nonradical species at pH above 3.5.70
The kinetics and Michaelis–Menten reaction kinetics of As(III)
oxidation by spore–TEMPO system were also investigated (Fig.
5a, b). The oxidation reaction achieved equilibrium in 1 h and
was fitted by a pseudo-first-order model (R2= 0.995). The k
value was 2.46 s–1. A Km of 0.04 mmol L–1 and a Vmax of 3.10×10–3
mmol L–1 min–1 were also obtained. These parameters are
similar to those of commercial laccase, which further confirmed
that spores and laccase both have similar catalysis mechanism.
The effects of adding inorganic metal ions (e.g., Al3+, Ti4+,
Cu2+, K+, Fe3+, Zn2+, Ni2+, Mg2+, Co2+, and Mn2+) with the same
concentration for 10 mg L–1 As(III) oxidation were investigated
(Fig. 5c). Except for Mn2+, these tested metal ions did not affect
As(III) oxidation. The oxidation efficiency of As(III) slightly
decreased in the presence of Mn2+ possibly because a part of
spore–laccase was consumed to oxidize Mn2+ to Mn3+.79, 80
These results indicate that the spore–TEMPO system for As(III)
oxidation has preferable tolerance to metal ions.
After each use, the spores were collected through
centrifugation and washed twice for the next cycle of oxidation.
The As(III) oxidation efficiency did not significantly decreased
after fifteen cycles (Fig. 5d). This finding is similar to that
obtained by using commercial laccase and strongly suggests
Fig. 4 The chemical structure of mediators used in this work.
6 | J. Name., 2012, 00, 1-3
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ARTICLE
This work was supported by the National Natural Science
View Article Online
Foundation of China (grant 2167505D7O),I: 1t0h.e103N9/aCt8ioGnCa0l36K54eCy
Research and Development Program of China (grant
2016YFD0500900), and the Fundamental Research Funds for
the Central Universities (grant 2662018PY054).
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In this work, we revealed for the first time that laccase–
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DOI: 10.1039/C8GC03654C
Rapid catalytic oxidation of As(III) to As(V) by using Bacillus
spore–2,2,6,6-tetramethylpiperidine-1-oxyl system
Yuqing Qin, a, b Fei Peng a, b, Yonggang Hu a, b,
*
a State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural
University, Wuhan 430070, China
b College of Life Science and Technology, Huazhong Agricultural University, Wuhan
430070, China
Email: yongganghu@mail.hzau.edu.cn
A rapid and green oxidation of As(III) to As(V) was proposed using Bacillus Spore–
TEMPO system.