Synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate by immobilized cells using amino acid-modified magnetic nanoparticles

Article abstract of DOI:10.1016/j.procbio.2020.07.027

Fe₃O₄-Arg was selected as the optimal carrier due to its high activity recovery of immobilized cells in the preparation of Fe₃O₄-Arg-Cells. The optimal immobilization conditions for the preparation of Fe₃O₄-Arg-Cells were 30 °C, 4 h, pH 7, and 3 g dry yeast. The activity recovery of immobilized cells reached 76.8 percent. For a batch reduction in a shaker in an alternating magnetic field, Fe₃O₄-Arg-Cells were used as a catalyst to gain ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE). For further improvement in reduction productivity, a continuous reduction in the magnetic fluidized bed reactor system (MFBRS) was completed. Under their optimal transformation conditions, it took 24 h for Fe₃O₄-Arg-Cells to complete the conversion of ethyl 4-chloro-3-oxobutanoate (COBE) (0.8553 mol/L) in the shaker and only 8 h for the batch reduction in an alternating magnetic field. Continuous reduction in MFBRS provided new ideas for the efficient production of (R)-CHBE; 1.5882 mol/L (10 mL) of COBE can be completely converted in 6 h. The conversion and enantiomeric excess (e.e.) of (R)-CHBE were 100 percent and above 99.9 percent respectively, in the three reaction systems mentioned above.

Full text of DOI:10.1016/j.procbio.2020.07.027

ProcessBiochemistry99(2020)9–20
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate by immobilized cells
using amino acid-modified magnetic nanoparticles
Yuan Lua, Hongqian Daia, Hanbing Shib, Lan Tanga, Xingyuan Sunb,*, Zhimin Oua,*
a College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, Zhejiang, China
b The Third Affiliated Hospital of Qiqihar Medical College, Qiqihar, Heilongjiang, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fe3O4-Arg was selected as the optimal carrier due to its high activity recovery of immobilized cells in the
preparation of Fe3O4-Arg-Cells. The optimal immobilization conditions for the preparation of Fe3O4-Arg-Cells
were 30 °C, 4 h, pH 7, and 3 g dry yeast. The activity recovery of immobilized cells reached 76.8 %. For a batch
reduction in a shaker in an alternating magnetic field, Fe3O4-Arg-Cells were used as a catalyst to gain ethyl (R)-4-
chloro-3-hydroxybutyrate ((R)-CHBE). For further improvement in reduction productivity, a continuous re-
duction in the magnetic fluidized bed reactor system (MFBRS) was completed. Under their optimal transfor-
mation conditions, it took 24 h for Fe3O4-Arg-Cells to complete the conversion of ethyl 4-chloro-3-oxobutanoate
(COBE) (0.8553 mol/L) in the shaker and only 8 h for the batch reduction in an alternating magnetic field.
Continuous reduction in MFBRS provided new ideas for the efficient production of (R)-CHBE; 1.5882 mol/L
(10 mL) of COBE can be completely converted in 6 h. The conversion and enantiomeric excess (e.e.) of (R)-CHBE
were 100 % and above 99.9 % respectively, in the three reaction systems mentioned above.
Amino acid-modified magnetic nanoparticles
Alternating magnetic field
Asym-metric reduction
Ethyl (R)-4-chloro-3-hydroxybutyrate
Immobilized cells
1. Introduction
coli CCZU-A13 [15], E.coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) [16],
and a novel alcohol dehydrogenase from Lactobacillus curieae S1L19
(R)-CHBE, an optically active alcohol, is a key intermediate for the
production of chiral drugs [1,2], including L-carnitine [3], (−) mac-
rolactin A, (R)-γ-amino-β-hydroxybutyric acid (GABOB) [4] and (R)-4-
hydroxy-pyrrolidone [4,5]. Although various biocatalysts have been
found to give (S)-CHBE, the opposite enantiomer of (R)-CHBE, with
excellent enantioselectivity and high yields [6–9]. However, few bio-
catalysts that could synthesize (R)-CHBE with high enantioselectivity
have been reported. Several biocatalysts are known to convert ethyl 4-
Chloro-3-oxobutanoate (COBE) to (R)-CHBE including gox2036 from
Gluconobacter oxydans [10], AKRs from Sporobolomyces salmonicolor
[11], LEK from Lodderomyces elongisporus [12], a reductase from Ba-
cillus sp. ECU0013 [13] and Burkholderia gladioli [14]. (R)-CHBE (100 %
e.e.) was produced by Gox2036 obtained from G. oxydans with > 99 %
conversion and 96.9 % yield (968.6 mg/L) [10]. LEAKR50 obtained
from S. salmonicolor could completely transform 20 mM COBE to (R)-
CHBE (98 % e.e.) [11]. The molar conversion yield of COBE was 78.0 %
using LEK from L. elongisporus, and the e.e. value of (R)-CHBE was > 99
% [12]. Thus, great efforts have been made to explore more robust
biocatalyst, select appropriate reaction medium and immobilize the
enzyme and whole-cell to increase the reuse times of catalyst.
[17] were adopted in the asymmetric reduction of COBE to synthesize
(R)-CHBE for improving catalytic efficiency and stereoselectivity. An
appropriate reaction medium is beneficial to prepare (R)-CHBE effi-
ciently. Traditional aqueous phase [16], organic phase, water/oil
system [18], and an ionic liquid [Bmim]PF6-hydrolyzate media [15]
were also reported in the biotransformation of COBE to obtain (R)-
CHBE. The conversion is low in the aqueous phase because the enzyme
and whole cell have substantial catalytic activity in water and because
several organic substrates are difficult to dissolve in water. A biocata-
lyst in the organic phase or water/oil system is used to enhance the
reaction because the organic substrate readily dissolves in the organic
phase, and certain enzymes and whole cells still retain activity in or-
ganic solvents or at the oil-water interface [19]. The addition of the
ionic liquid and auxiliary reagent contribute to enhancement of the cell
permeability and increase of mass transfer rate and conversion [15].
Meanwhile, immobilization of the enzyme and whole cell has the ad-
vantage of reusing the biocatalyst, which improves production effi-
ciency.
In recent years, Fe3O4 magnetic nanoparticles (MNPs) are dramatic
materials showing great performance in the immobilization of bioca-
talysts [20–24]. Compared with other nanoparticles, such as terracotta
Much work has been done in finding a more robust biocatalyst. E.
⁎ Corresponding authors.
E-mail addresses: 708900599@qq.com (X. Sun), oozzmm@163.com (Z. Ou).
https://doi.org/10.1016/j.procbio.2020.07.027
Received 21 April 2020; Received in revised form 11 July 2020; Accepted 30 July 2020
Availableonline26August2020
1359-5113/©2020ElsevierLtd.Allrightsreserved.

Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
beads, coconut bract, corncob, Ca-alginate beads, and straw-alginate
beads, Fe3O4 MNPs have unique properties, including the smaller core
size, larger surface area for functionalization, higher coercive force,
stronger surface adsorption ability, better suspension property, super-
paramagnetic behavior, and low toxicity. Fe3O4 MNPs have the re-
markable characteristic of superparamagnetism, which is essential and
critical for reaction in the magnetic field. Moreover, Fe3O4 MNPs can be
easily separated and effectively recycled under an external magnetic
field. Numerous varieties of cells have been successfully modified by
MNPs to construct immobilized cells [25–28]. Immobilized cells are
economically available biocatalysts widely applied in continuous bio-
processes currently. MNPs can be modified by biomolecules capable of
selectively interacting with surface groups of cells to form functiona-
lized MNPs, which could efficiently capture the cells. However, many
such biomolecules are large molecules that would decrease the amount
anchored onto the MNPs surface and therefore affect the cell im-
mobilization efficiency. Jin et al. [29] used three strong positive
charged amino acids, arginine (Arg), lysine (Lys) and poly-L-lysine
(PLL) to modify MNPs through a simple and economical two-step
transformation process (TST) for bacteria capture and removal. Mod-
ification of Fe3O4 nanoparticles with amino acids greatly enhanced the
bacteria capture efficiency and kinetics towards both Gram-positive B.
subtilis and Gram-negative E. coli. For both bacterial cells, the three
amino acid-functionalized Fe3O4 particles (Fe3O4-AA) types could
capture more than 97 % of the bacterial cells in 20 min. Cell capture by
Fe3O4-AA is governed by energy interactions between cells and nano-
particle surface. These interactions mainly include van der Waals forces
and electrostatic double-layer repulsion. In addition, hydrophobic in-
teraction and hydrogen bonding might be involved [29].
2. Materials and method
2.1. Materials
Ferric chloride hexahydrate (FeCl3•6H2O), ferrous sulfate heptahy-
drate (FeSO4•7H2O), L-arginine powder (98 %), L-lysine powder (99
%), L-glutamate powder (99 %) and L-aspartic acid powder (98 %) were
purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Ethyl
4-chloro-3-oxobutanoate (99 %) standard was purchased from
Bangcheng Chemical Co., Ltd. Shanghai, China. (S)-CHBE and (R)-
CHBE standard were purchased from Aladdin reagent Co., Ltd.
Shanghai, China. S. cerevisiae CGMCC No. 3361 was preserved in China
General Microbiological Culture Collection Center.
2.2. Analytical methods
The activity recovery of immobilized cells was analyzed by spec-
trophotometer. The cell solution without Fe3O4-AA carrier was used as
a reference solution. The ultraviolet absorption (A0) at a wavelength of
260 nm of the cell solution diluted twenty times was used as the initial
concentration. The absorption of the supernatant liquid after im-
mobilization was taken as the Ai. The activity recovery of immobilized
cells (AD) was calculated by Eq. (1):
A0 Ai
A0
AD =
× 100%
(1)
The conversion and enantiomeric excess of (R)-CHBE were analyzed
using a Shimadzu GC-2014 gas chromatograph. The conversion was
defined as the ratio of the converted substrate concentration to the
initial substrate concentration. The enantiomer excess of CHBE was
detected by the derivatization method. At the end of reduction, the
catalyst was recovered by an external magnetic field. 4 mL of ethyl
acetate was added in a stoppered test tube containing 11 mL of the
supernatant. 1 mL of ethyl acetate extract was adopted in a glass bottle
for the derivatization reaction (natural volatile solvent at room tem-
perature, then 2 drops of acetic anhydride and 2 drops of pyridine were
added in a fume hood and placed in a boiling water bath for 1 h). After
slightly cooling, 1 mL of ethyl acetate was used to dilute the above
sample. The sample was analyzed by GC equipped with a Agilent
CP7502 J&W CP-CHirasil-Dex CB chiral column (Machery-Nagel; 25
m × 0.25 mm × 0.25 mm). Injector, column and FID temperature were
250, 110 and 250 ℃. H2 pressure: 83 kPa. Split ratio: 1:15. Retention
time of COBE, (S)-CHBE and (R)-CHBE were 10.107, 20.035 and
21.000 min. Eqs. (2) and (3) evaluated the conversion (X) and en-
antiomeric excess of CHBE.
In this study, Saccharomyces cerevisiae CGMCC No. 3361, which is
highly capable of asymmetric reduction of COBE, was selected. Amino
acids are desirable chemicals as ligands anchored with MNPs. Two
positively charged polar amino acids (arginine, lysine) and two nega-
tively charged polar amino acids (glutamate, aspartic acid) were used in
the modification of MNPs. A facile and inexpensive process was applied
in the preparation of four types of Fe3O4-AA. Fe3O4-AA-Cells were
prepared by the efficient immobilization of S. cerevisiae CGMCC No.
3361 on Fe3O4-AA. (R)-CHBE was synthesized by asymmetric reduction
of COBE with Fe3O4-AA-Cells in a shaker in an alternating magnetic
field. Both carbonyl reductase and NADPH were present in cells.
Carbonyl reductase catalyzed the asymmetric reduction of COBE to (R)-
CHBE. NADPH served as the hydrogen donor for reduction. The me-
chanism of asymmetric reduction of COBE is shown in Fig. 1.
P × Ms
X (%) =
× 100%
Q × Mp
(2)
Ms and Mp are the molecular weight of the substrate and the pro-
duct. P and Q stand for the mass of the product at the end of the re-
action and the initial mass of the substrate.
CR CS
eep =
× 100%
CR + CS
(3)
CR and CS stand for the concentration of (R)-CHBE and (S)-CHBE.
2.3. Preparation of yeast cell lyophilized powder
The solid medium comprised of wort 10 g/L, yeast powder 3 g/L,
peptone 5 g/L, glucose 10 g/L, agar 20 g/L. The liquid medium con-
sisted of glucose 30 g/L, yeast powder 3 g/L, ammonium sulfate 5 g/L,
MgSO4 0.3 g/L, K2HPO4•3H2O 1 g/L, KH2PO4 1 g/L. S. cerevisiae
CGMCC No. 3361 was inoculated into a solid medium and cultured at
30 ℃ for 5 days to obtain a yeast colony. A loop of yeast colony was
transferred into the liquid medium. After cultivation in liquid medium
at 35 °C for 15 h, a seed liquid was obtained. The seed liquid was
Fig. 1. Batch (a) and continuous (b) reduction reaction in an alternating
magnetic field (Fe3O4-Arg-Cells, 1 Alternating current power supply, 2 Reactor,
3 Helmholtz coil, 4 Constant flow pump, 5 Inflow tank, 6 Effluent tank).
10

Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
inoculated into the liquid medium at a seed volume of 10 % for fer-
mentation. The fermentation broth was obtained after cultivation at
35 °C, 150 r/min for 24 h. Then, the fermentation broth was centrifuged
and the precipitate harvested. The lyophilized powder of S. cerevisiae
3361 was obtained after the precipitate was frozen at −80 °C for 8 h
and lyophilized in a freeze dryer for 12 h.
conditions were slightly different. The cell concentration corresponding
to the carrier Fe3O4-Glu was 0.075 g/mL, while Fe3O4-Lys and Fe3O4-
Asp were 0.15 g/mL. The immobilization time was 3 h for Fe3O4-Lys
and Fe3O4-Glu.
2.5.3. Effect of Fe3O4-AA amount on the activity recovery of immobilized
cells
2.4. Preparation of Fe3O4, Fe3O4-AA, and Fe3O4-AA-Cells
0.01 g, 0.015 g, 0.02 g, 0.025 g, and 0.03 g Fe3O4-AA were added
into five conical flasks containing a yeast cell solution (20 mL, pH 7).
The biomass was 0.075 g/mL for Fe3O4-Glu, while the biomass was
0.15 g/mL for Fe3O4-Arg, Fe3O4-Lys, and Fe3O4-Asp, respectively. The
immobilization process was carried out at 30 ℃, 150 r/min. The reac-
tion time was 3 h for Fe3O4-Lys and Fe3O4-Glu, while the reaction time
was 4 h for Fe3O4-Arg and Fe3O4-Asp.
Magnetic Fe3O4 nanoparticles were prepared. 0.03 M FeCl3•6H2O
(40 mL) and 0.02 M FeSO4•7H2O (40 mL) solutions were mixed with a
mechanical stirrer in a 250 mL flask containing 150 mL of deionized
water. This solution was chemically precipitated at 40 ℃ by adding
NH3•H2O dropwise until the pH reached 10. The above processes
needed vigorous stirring with nitrogen protection. The suspension was
heated to 80 ℃ for 1 h with continuous stirring. A black precipitate was
formed under the above conditions and was cooled at 20–30 ℃. The
black precipitate was collected by magnetic decantation and washed
with deionized water repeatedly until the washings were neutral. The
obtained black precipitates were pre-frozen in a -80 ℃ refrigerator for
8 h and then lyophilized in a freeze dryer for 12 h to obtain magnetic
Fe3O4 nanoparticles.
2.5.4. Effect of initial free cell concentration on the activity recovery of
immobilized cells
0.03 g Fe3O4-AA was added to five conical flasks containing
0.050 g/mL, 0.075 g/mL, 0.100 g/mL, 0.125 g/mL, and 0.150 g/mL of a
yeast cell solution (20 mL, pH 7), respectively. The immobilization
process was carried out at 30 ℃, 150 r/min shaker. The immobilization
time was 4 h for Fe3O4-Arg or Fe3O4-Asp, while the immobilization time
was 3 h for Fe3O4-Glu and Fe3O4-Lys.
Fe3O4-AA including Fe3O4-Arg, Fe3O4-Lys, Fe3O4-Glu, and Fe3O4-
Asp was prepared. 0.1 g magnetic Fe3O4 nanoparticles were added in
100 mL of the corresponding amino acid solution (0.1 g/100 mL) under
ultrasonication for 30 min. After the sonication treatment, the obtained
black precipitates were collected with magnetic decantation and wa-
shed with deionized water three times. Fe3O4-AA was obtained after
being frozen for 8 h in a −80 ℃ refrigerator and lyophilized in a freeze
dryer for 12 h.
2.5.5. Selection of optimal Fe3O4-AA-Cells for asymmetric reduction of
COBE
Four kinds of Fe3O4-AA-Cells were prepared under the optimization
conditions. The optimal Fe3O4-AA-Cells were selected by comparison of
reduction conversion.
Fe3O4-AA-Cells were prepared. The yeast cell lyophilized powder
was dispersed in 20 mL of 0.1 mol/L phosphate-buffered saline solution.
Fe3O4-AA was added to the above solution. The mixture was shaken at
30 ℃, 150 r/min. After 3–4 h, Fe3O4-AA-cells were collected by an ex-
ternal magnetic field and washed with deionized water until the upper
layer had no ultraviolet absorption at a wavelength of 260 nm. Fe3O4-
AA-cells were frozen at −80 °C for 8 h and lyophilized in a freeze dryer
for 12 h.
2.6. Characterization of Fe3O4, optimal Fe3O4-AA, and Fe3O4-AA-Cells
The FT-IR spectra of Fe3O4, optimal Fe3O4-AA, and Fe3O4-AA-Cells
were obtained using an FT-IR spectrophotometer (iS50, Nicolet). The
surface features of Fe3O4, optimal Fe3O4-AA, and Fe3O4-AA-Cells were
evaluated by a field emission scanning electron microscope (SEM)
(Zeiss Gemini 500). The content of elements in the samples were de-
termined by energy dispersive X-Ray spectroscopy (EDX) (XFlash 6/60,
Bruker). Fe3O4, optimal Fe3O4-AA, and Fe3O4-AA-Cells were analyzed
by X-ray powder diffraction (XRD). XRD measurements were conducted
on a Rigaku D/MAX-3B X-ray diffractometer employing Cu Kα radia-
tion (λ = 0.1542 nm).
2.5. Optimization of immobilized condition and selection of optimal Fe3O4-
AA-Cells for asymmetric reduction of COBE
To find an excellent Fe3O4-AA-Cells for asymmetric reduction of
COBE, the effects of immobilized conditions on activity recovery of
immobilized cells were studied during the preparation of four kinds of
Fe3O4-AA-Cells. Fe3O4-AA-Cells were collected by an external magnetic
field. The remaining liquid in the flask was analyzed to determine the
activity recovery of immobilized cells. Furthermore, Fe3O4-Arg-Cells,
Fe3O4-Glu-Cells, Fe3O4-Lys-Cells, and Fe3O4-Asp-Cells were prepared
under optimal conditions and used in the asymmetric reduction of
COBE to select the optimal Fe3O4-AA-Cells.
2.7. Optimization of the batch reduction conditions in a shaker
The effects of conditions on reduction were investigated. At the end
of the reaction, the conversion and enantiomeric excess of CHBE were
determined by the analytical methods used in Section 2.2.
2.7.1. Effect of reaction time on reduction
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline (pH 9) in five 50 mL small conical flasks.
9.408 mol/L of COBE ethanol solution (1 mL) was added in the flask.
The concentration of COBE in reaction mixture was 0.8553 mol/L. The
flask was shaken at 35 ℃, 150 r/min for 16 h, 24 h, 32 h, 40 h, and 48 h,
respectively.
2.5.1. Effect of time on the activity recovery of immobilized cells
0.03 g Fe3O4-Arg was added in five conical flasks containing a
0.15 g/mL yeast cell solution (20 mL, pH 7) at 30 ℃, 150 r/min shaker
for 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The immobilization processes
of other carriers of Fe3O4-AA were analogous except for the con-
centration of the yeast cells. The cell concentrations corresponding to
the carrier Fe3O4-Glu, Fe3O4-Lys and Fe3O4-Asp were 0.075 g/mL,
0.15 g/mL, and 0.15 g/mL, respectively.
2.7.2. Effect of substrate concentration on reduction
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline (pH 9) in five 50 mL small conical flasks.
1 mL of COBE ethanol solution (1.344 mol/L, 4.032 mol/L, 6.72 mol/L,
9.408 mol/L, and 12.096 mol/L) was added into 10 mL of 0.1 mol/L
phosphate-buffered saline (pH 9). The concentration of COBE in reac-
tion mixture were 0.1222 mol/L, 0.3665 mol/L, 0.6109 mol/L,
0.8553 mol/L, and 1.0996 mol/L. The reduction was carried out at 35
℃, 150 r/min for 24 h.
2.5.2. Effect of temperature on the activity recovery of immobilized cells
0.03 g Fe3O4-Arg was added in five conical flasks containing a
0.15 g/mL yeast cell solution (20 mL, pH 7) at 20 ℃, 25 ℃, 30 ℃, 35 ℃,
and 40 ℃, 150 r/min shaker for 4 h respectively. The immobilization
processes of other carriers were analogous except the immobilization
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Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
2.7.3. Effect of temperature on reduction
apart) along the vertical axis and used to generate the external mag-
netic fields. Each coil had a 120 mm inner diameter, 160 mm outer
diameter, 20 mm thickness, and contained 600 wraps of AWG#21
copper wire [30]. The magnetic field intensity was controlled by ad-
justing the current, which was passed through the solenoid using a AC
power supplier. A constant flow pump was used for the flow of the
substrate solution through the reactor. The schematic diagram of the
device is shown in Fig. 1b.
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline (pH 9) in five 50 mL small conical flasks.
9.408 mol/L of COBE ethanol solution (1 mL) was added into the mix-
ture. The concentration of COBE in reaction mixture was 0.8553 mol/L.
The reduction was carried out at different temperatures (20 ℃, 25 ℃,
30 ℃, 35 ℃, 40 ℃), 150 r/min for 24 h.
2.7.4. Effect of pH of buffer on the reduction
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline (pH 5, pH 6, pH 7, pH 8, and pH 9) in five
50 mL small conical flasks. 9.408 mol/L of COBE ethanol solution
(1 mL) was added into the mixture. The concentration of COBE in re-
action mixture was 0.8553 mol/L. The reduction was carried out at 35
℃, 150 r/min for 24 h.
2.10.1. Effect of substrate flow rate on reduction in MFBRS
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline solution (pH 9) in the reaction tank.
1.5882 mol/L of COBE ethanol solution (10 mL) was added to the
mixture and a constant flow pump pumped the COBE ethanol solution
in tank 5 into the reaction tank. The substrate flow rate was 25 μL/min,
30 μL/min, 40 μL/min, 50 μL/min, 100 μL/min, and 200 μL/min. The
reaction mixture was pumped into the effluent tank at the same rate as
the substrate flow rate. The reduction in the reactor was performed at
35 °C, and magnetic field intensity of 12 Gs for 6 h.
2.8. Reuse of Fe3O4-Arg-Cells in the shaker reduction
The reduction was catalyzed by Fe3O4-Arg-Cells under optimum
conditions. 2.334 g of Fe3O4-Arg-Cells was dispersed in 10 mL of
0.1 mol/L phosphate-buffered saline (pH 9) in a 50 mL small conical
flask. 9.408 mol/L of COBE ethanol solution (1 mL) was added into the
mixture. The concentration of COBE in reaction mixture was
0.8553 mol/L. After a 24 h reduction at 35 ℃ in a 150 r/min shaker, the
Fe3O4-Arg-Cells were recovered using an external magnetic field and
washed 10 times with ethanol and deionized water to ensure that no
substrate or product residue left on Fe3O4-Arg-Cells. Fe3O4-Arg-Cells
was reused in the reduction and recycled under the above-mentioned
reduction conditions.
2.10.2. Effect of substrate concentration on reduction in MFBRS
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline solution (pH 9) in the reaction tank. 10 mL of
COBE ethanol solution (0.6109 mol/L, 0.8553 mol/L, 1.0996 mol/L,
1.3439 mol/L, 1.5882 mol/L and 1.8325 mol/L) was added to the tank
5 respectively. A constant flow pump with a substrate flow rate of
25 μL/min respectively pumped the solutions in tank 5 into the reaction
tank. The reaction mixture was pumped into the effluent tank at the
same rate as the substrate flow rate. The reduction was performed at
35 °C, a magnetic field intensity of 12 Gs for 6 h.
2.9. Optimization of batch reduction conditions in an alternating magnetic
field
3. Results and discussion
2.9.1. Effect of magnetic field strength on the activity of Fe3O4-Arg-Cells
An alternating magnetic field was generated using a lab-made de-
vice. A schematic diagram of the device for generating an electro-
magnetic alternating field is illustrated in Fig. 1a. The strength and
frequency of the magnetic field could be adjusted based on actual de-
mand. The reactions were conducted in an alternating magnetic field
with a specific frequency of 500 Hz for 8 h. 2.334 g of Fe3O4-Arg-Cells
were dispersed in 10 mL of 0.1 mol/L phosphate-buffered saline (pH 9).
9.408 mol/L of COBE ethanol solution (1 mL) was added to the mixture.
The concentration of COBE in reaction mixture was 0.8553 mol/L. The
influence of magnetic field strength on reduction was investigated
when the magnetic field strengths were 4 Gs, 8 Gs, 12 Gs, 16 Gs, and
20 Gs.
3.1. Optimization of the preparation conditions for Fe3O4-AA-Cells
3.1.1. Effect of time on the activity recovery of immobilized cells
The cells are not initially completely bound to the carrier. With the
prolongation of time, the active groups on the surface of the carrier
were sufficiently connected with the cells to reach a saturation level.
Bare Fe3O4 could only immobilize 11.43 % of the cells in 2 h, while the
activity recovery of immobilized cells with Fe3O4-Arg reached 47.24 %.
Although increasing the reaction time within a certain range would
improve the activity recovery of immobilized cells, only 23.68 % of the
cells were immobilized on bare Fe3O4 while 77.3 % of the cells were
immobilized on Fe3O4-Arg within 4 h. It was evident that the mod-
ification of Fe3O4 with amino acids greatly enhanced the activity re-
covery of immobilized cells and cell capture efficiency. Fig. 2 showed
that the activity recovery of immobilized cells reached the maximum
value at 4 h with Fe3O4, Fe3O4-Asp, and Fe3O4-Arg as carriers for the
cell immobilization. The optimal time was 4 h for the preparation of
Fe3O4-Asp-Cells and Fe3O4-Arg-Cells. Meanwhile, the optimal time was
3 h for Fe3O4-Lys-Cells and Fe3O4-Glu-Cells formation.
2.9.2. Effect of reaction time on reduction in an alternating magnetic field
2.334 g of Fe3O4-Arg-Cells were dispersed in 10 mL of 0.1 mol/L
phosphate-buffered saline (pH 9) in five 30 mL reaction flasks in the
apparatus shown in Fig. 2a. 9.408 mol/L COBE ethanol solution (1 mL)
was added into the mixture. The concentration of COBE in reaction
mixture was 0.8553 mol/L. The reaction was carried out in the alter-
nating magnetic field at 35 ℃ for 2 h, 4 h, 6 h, 8 h, and 10 h, respec-
tively. The reactions were conducted in an alternating magnetic field
with a specific frequency of 500 Hz and magnetic field strength of
12 Gs.
3.1.2. Effect of temperature on the activity recovery of immobilized cells
A low activity recovery of immobilized cells was obtained at a low
temperature (< 30 ℃) because the unit carriers were not saturated. The
immobilization reaction proceeded at a faster rate with the increase of
temperature. However, the activity recovery of immobilized cells de-
creased when the temperature reached above 30 ℃. Fig. 2 showed that
the optimal immobilization temperature was 30 ℃. When the tem-
perature was 30 ℃, all four types of Fe3O4-AA could immobilize more
than 43 % of the cells. Particularly, the activity recovery of immobilized
cells of Fe3O4-Arg was 77.3 %. It had a significant advantage over bare
Fe3O4 that had an activity recovery of immobilized cells of 23.68 %.
The isoelectric points of Fe3O4-Arg increased compared with bare Fe3O4
[29]. The enhanced isoelectric points of Fe3O4-Arg could ensure a
2.10. Optimization of continuous reduction conditions in a magnetic
fluidized bed reactor system (MFBRS)
The MFBRS consisted of a jacketed glass tube reactor (Din = 1.5 cm,
H = 20 cm) and three copper wire coils, which were connected to a
power supply (0–10 A, 0.1 A sensitivity) and produced an axially uni-
form magnetic field to prevent the escape of the magnetic particles from
the bed. A set of three Helmholtz coils were evenly spaced (16 mm
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ProcessBiochemistry99(2020)9–20
Fig. 2. Effect of immobilized condition on the activity recovery of immobilized cells.
stronger electrostatic force between nanoparticles and cells to increase
activity recovery of immobilized cells [29]. Amino acids interacted with
nanoparticle surface through the carboxyl groups and side chains ex-
posed to the exterior. Fe3O4-Arg with side-chain functional groups also
provide an active group for interaction with multiple biological mole-
cules and ligands [31,32]. The hydrogen bonding interactions between
the carboxyl oxygen of the cell membrane and the amino/imino of
amino acids on the surface of Fe3O4-Arg nanoparticles, along with hy-
drophobic attractions of the carbon chains of amino acids with methyl
side chains in the cell membrane, might also contribute to the activity
recovery of immobilized cells by Fe3O4-Arg [33].
the maximum activity recovery of immobilized cells was obtained with
Fe3O4-Arg as a carrier. A higher activity recovery of immobilized cells
illustrated that more biomass of cells can be immobilized per unit mass
carrier. Four types of Fe3O4-AA-Cells were prepared under their optimal
preparation conditions and used in asymmetric reduction of COBE.
Fig. 3 showed that conversion with Fe3O4-Arg-Cells as catalyst reached
the optimal value compared with the other three immobilized cells
(Fe3O4-Lys-Cells, Fe3O4-Glu-Cells, and Fe3O4-Asp-Cells). Therefore, the
Fe3O4-Arg-Cells was selected as the optimal catalyst and used for fur-
ther study.
3.2. Characterization of Fe3O4, Fe3O4-Arg, and Fe3O4-Arg-Cells
3.1.3. Effect of Fe3O4-AA amount on the activity recovery of immobilized
cells
The FT-IR spectra of Fe3O4, Fe3O4-Arg, and Fe3O4-Arg-Cells in the
range of 400−4000 cm−1 were recorded and presented in Fig. 4. The
characteristic absorption peak was observed at around 570−635, 1630,
and 3400 cm−1 in FT-IR spectra. The strong absorption bands at around
570−635 cm−1 reflect the vibration of FeeO [29,34,35], indicating
that the preparation of Fe3O4 was successful and Fe3O4 existed in the
structure of Fe3O4-Arg and Fe3O4-Arg-Cells. In aqueous medium, MNP
surface is modified by OH groups owing to the interaction between
unsaturated surface Fe atoms and hydroxylions or water molecules.
These OH groups absorb IR waves at approximately 1630 cm−1 (de-
forming) and 3400 cm−1 (stretching) [36,37]. In Fe3O4-Arg nano-
particles, C]O stretching vibrations are observed at 1633 cm-1 [32].
For Fe3O4-Arg and Fe3O4-Arg-Cells, OH stretching overlaps with NeH
stretching vibration at 3420 cm-1. Similar results were reported by
Antal et al. [36] and Ebrahiminezhad et al. [32].
Due to the difference in the molecular structure of different amino
acids, there is also a difference in immobilization ability between the
carriers (Fe3O4-AA) modified by different amino acids. Fig. 2 showed
that the activity recovery of immobilized cells increased with the in-
crease of carrier amounts for bare Fe3O4 and the four types of Fe3O4-
AA. In contrast with Fe3O4-Arg and Fe3O4-Asp, the activity recovery of
immobilized cells of Fe3O4-Lys and Fe3O4-Glu were lower within carrier
amounts from 0.01 g to 0.03 g. Fe3O4-Arg could provide more active
binding points for cell immobilization. Fig. 2 showed that the optimal
carrier amount was 0.03 g for bare Fe3O4 and various types of Fe3O4-
AA. The higher activity recovery of immobilized cells was observed for
Fe3O4-Arg.
3.1.4. Effect of initial biomass on the activity recovery of immobilized cells
The initial biomass was studied in the range of 1–3 g because the
cells cannot be evenly dispersed in 20 mL of PBS when the dry weight of
cells exceeds 3 g. Generally, the activity recovery of immobilized cells
increased with the increase of biomass until the carriers were saturated.
Fig. 2 showed that the optimal initial biomass was 1.5 g for the pre-
paration of Fe3O4-Glu-Cells, while the optimal initial biomass was 3 g
for the preparation of Fe3O4-Asp-Cells, Fe3O4-Arg-Cells, and Fe3O4-Lys-
Cells. This difference may be due to the saturation of the active sites on
the surface of Fe3O4-Glu. When the initial cell weight was 3 g, even the
Fe3O4-Lys with the lowest activity recovery of immobilized cells among
the four Fe3O4-AA carriers had a higher activity recovery of im-
mobilized cells (43.26 %) than that of bare Fe3O4 (23.68 %).
XRD patterns of Fe3O4, Fe3O4-Arg, and Fe3O4-Arg-Cells were shown
in Fig. 5. The six peaks (2θ of 30.1, 35.5, 43.1, 53.4, 57.0 and 62.6)
were observed in the spectra of Fe3O4-Arg. Fe3O4-Arg-Cells were con-
sistent with the six characteristic peaks observed in the pure Fe3O4
spectra (JCPD standards: Fe3O4, 89–3854, 2θ = 30.088, 35.439, 43.07,
53.432, 56.958, and 62.546). This indicates that the crystal form
transformation did not occur during the amino acid modification pro-
cesses and all three materials contained Fe3O4. The immobilization
process did not have a destructive effect on the crystal structure of
magnetite. The magnetic particles could preserve their magnetic
properties during the separation process, which was beneficial for
conducting bioseparation.
Fe3O4, Fe3O4-Arg, and Fe3O4-Arg-Cells were characterized using
SEM/EDX. The results were shown in Fig. 6. It can be observed in
Fig. 6(A) that the appearance of Fe3O4 was nearly spherical with a
diameter of 10−20 nm. Fe3O4 is uniform in size. The results were
consistent with Unal et al’s prior study [38]. After Fe3O4 dispersed into
3.1.5. Selection of optimal Fe3O4-AA-Cells for asymmetric reduction of
COBE
The optimal conditions for the preparation of the four types of
Fe3O4-AA-Cells were summarized in Table 1. The results showed that
13

Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
Table 1
Optimal condition for the preparation of Fe3O4-AA-Cells.
Carriers
Time(h)
Temperature(℃)
Amount of Fe3O4-AA (g)
Dry yeast(g)
Weight of Fe3O4-AA-Cells(g)
Activity recovery of immobilized cells(%)
Fe3O4-Arg
Fe3O4-Lys
Fe3O4-Glu
Fe3O4-Asp
4
3
3
4
30
30
30
30
0.03
0.03
0.03
0.03
3
2.334
76.8
3
1.3078
0.7002
1.7973
42.59
44.68
58.91
1.5
3
Fig. 3. Selection of optimal Fe3O4-AA-Cells for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE).
(Reduction time 24 h, 35 ℃, initial COBE concentration 0.8553 mol/L, pH 9).
Fig. 5. XRD patterns for Fe3O4, Fe3O4-Arg, Fe3O4-Arg-Cells.
Fig. 4. FT-IR spectra of Fe3O4, Fe3O4-Arg, Fe3O4-Arg-Cells.
3.3. Optimization of the reaction condition for asymmetric reduction of
COBE
water, the dispersion effect was not ideal and the size of Fe3O4 was
found to be larger. This was due to homoaggregation between the Fe3O4
nanoparticles because of the formation of a large number of hydrogen
bonds between the surface-rich hydroxyl groups. Fig. 6(B) was the
SEM/EDX image of the Fe3O4-Arg. From the SEM image of Fe3O4-Arg,
near-spherical appearance was observed. The overall size of Fe3O4-Arg
was slightly larger than that of Fe3O4. In the EDX spectrum of Fig. 6(B),
the presence of N element provided a basis for the successful connection
of arginine with Fe3O4 nanoparticles. It can be seen in Fig. 6(C) that
some rod-like substances were attached to the surface of the spherical
carrier (Fe3O4-Arg). According to the morphology and FT-IR spectra of
Fe3O4-Arg-Cells, the cells were successfully immobilized on the carrier
(Fe3O4-Arg).
3.3.1. Effect of reaction time on reduction
Fig. 7 illustrated that the reaction time affected the conversion and
enantiomeric excess of (R)-CHBE. COBE could be gradually converted
by Fe3O4-Arg-Cells with a reaction time extension. The conversion in-
creased with the increase of reaction time at < 24 h. Conversion
reached 100 % at 24 h. The enantiomeric excess of (R)-CHBE was
maintained above 99.9 %. The optimal reaction time was 24 h.
3.3.2. Effect of substrate concentration on reduction
Fig. 7 showed that conversion reached 100 % when the substrate
concentration was below 0.8553 mol/L. The conversion decreased with
14

Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
Fig. 6. SEM/EDX image of Fe3O4 (A), Fe3O4-Arg (B), Fe3O4-Arg-Cells (C).
the increase of substrate concentrations at above 0.8553 mol/L of
COBE. At low substrate concentrations, the active site of Fe3O4-Arg-
Cells was not fully saturated and its catalytic potential was not fully
exploited. With further increases in the substrate concentration, con-
version decreased slightly, possibly owing to the substrate inhibition of
the Fe3O4-Arg-Cells activity. The presence of the carrier may have made
it difficult for the substrate to contact the active site of the immobilized
cells, which led to a conversion decrease at high substrate concentra-
tions.
15

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ProcessBiochemistry99(2020)9–20
Fig. 7. Effect of reaction condition on batch reduction in the shaker.
Fig. 8. Reuse of immobilized cells for batch reduction in the shaker.
3.4. Reuse of Fe3O4-Arg-Cells in the batch reduction in a shaker
3.3.3. Effect of temperature on reduction
Fig. 7 indicated that conversion increased with the increase of
temperature at 20–35 ℃ and decreased at above 35 ℃. The optimal
reduction temperature was 35 ℃. Enzyme activity was lost or inhibited
at high temperatures due to protein denaturation. For the Fe3O4-Arg-
Cells catalyst, the temperature had little effect on the enantiomeric
excess of (R)-CHBE, which remained above 99.9 %.
Fe3O4-Arg-Cells can be recovered by an external magnetic field and
reused easily in reduction. Fig. 8 indicated that Fe3O4-Arg-Cells reused
11 times achieved 99.8 % conversion. The enantiomeric excess of (R)-
CHBE was maintained above 99 % in the reduction with reused Fe3O4-
Arg-Cells as catalysts. The reuse of Fe3O4-Arg-Cells was a powerful
method for the asymmetric reduction of COBE; it can enhance pro-
ductivity improvement.
3.3.4. Effect of buffer pH on reduction
The stable catalytic activity of Fe3O4-Arg-Cells was observed at pH
8–10 in Fig. 7. At pH 5–9, the conversion gradually increased with the
increase of pH until the maximum conversion reached 100 %. 100 %
conversion was achieved at pH 9 while conversion was only 53.71 % at
pH 7. The optimal reduction pH was pH 9. Arginine is a basic amino
acid. The dissociation state of arginine is affected by the pH value of the
buffer solution. Therefore, the catalytic activity of Fe3O4-Arg-Cells was
also affected by the pH of the buffer. The enantiomeric excess of (R)-
CHBE was not affected by the pH of the buffer and was maintained
above 99 %.
3.5. Optimization of batch reduction reaction conditions under alternating
magnetic field
3.5.1. Effect of magnetic field strength on the batch reduction in the
alternating magnetic field
Fig. 9 demonstrated that with the Fe3O4-Arg-Cells as catalysts,
conversion increased initially and then decreased with increasing
magnetic field intensity. The alternating magnetic field can accelerate
the movement of Fe3O4-Arg-Cells to improve the reaction rate of re-
duction. When the magnetic field strength was less than 12 Gs, Fe3O4-
16

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ProcessBiochemistry99(2020)9–20
Fig. 9. Effect of magnetic field strength on reduction.
(Magnetic field frequency 500 Hz, Reaction time 8 h).
Fig. 10. Effect of reaction time on reduction in alternating magnetic field.
(Magnetic field frequency 500 Hz, Magnetic field strength 12 Gs).
Arg-Cells were in an activated state and the conversion increased with
the increase of the magnetic field strength. However, when the mag-
netic field intensity exceeded 12 Gs, the conversion started to decrease.
Fe3O4-Arg-Cells generated an induced magnetic field under the action
of the external magnetic field. The interaction between these magnetic
fields affected the spatial conformation of the enzyme and brought
about changes in the activity of Fe3O4-Arg-Cells. The enantiomeric
excess of (R)-CHBE was above 99 % under different magnetic field
strengths. The optimal magnetic field frequency and magnetic field
intensity were determined to be 500 Hz and 12 Gs, respectively.
3.6. Continuous reduction reaction in a magnetic fluidized bed reactor
system (MFBRS)
MFBRS is considered an efficient system when coupled with mag-
netic immobilized cells or enzymes for biocatalysis and bio-
transformation process intensification [39,40].
3.6.1. Effect of substrate flow rate on reduction reaction in MFBRS
Fig. 11 indicated that the conversion gradually decreased with the
increase of substrate flow rate because of the insufficient retention time
in which the substrate molecule stayed in the reactor. When the sub-
strate flow rate increased from 25 μL/min to 200 μL/min, the conver-
sion decreased from 100 % to 38.93 % and the enantiomeric excess of
(R)-CHBE was above 99.0 %. Therefore, the optimal substrate flow rate
was 25 μL/min.
3.5.2. Effect of reaction time on the batch reduction in the alternating
magnetic field
Under the same condition, 0.8553 mol/L COBE can be converted
completely in 8 h in the alternating magnetic field, while in the shaker
for 24 h. Fe3O4-Arg-Cells were uniformly distributed in the alternating
magnetic field; they could sufficiently make contact with the COBE
solution and mass transfer was not required by shaking and stirring.
The reaction time was remarkably shortened and the conversion effi-
ciency was greatly improved. The specific results were shown in Fig. 10.
3.6.2. Effect of substrate concentration on reduction in MFBRS
Fig. 12 revealed that the conversion decreased with the increase of
substrate concentration. The enantiomeric excess of (R)-CHBE re-
mained above 99 %. The active site of the cell was not fully saturated
17

Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
Fig. 11. Effect of substrate flow rate on reduction in magnetic fluidized bed reactor.
(Magnetic field strength 12 Gs, Reaction time 6 h, Temperature 35 ℃, Substrate concentration 1.5882 mol/L).
Fig. 12. Effect of substrate concentration on reduction in magnetic fluidized bed reactor.
(Magnetic field strength 12 Gs, Reaction time 6 h, Temperature 35 ℃, Substrate flow rate 25 μL/min).
Table 2
Optimal reduction condition in different three systems.
Batch reaction in shaker
Batch reaction in magnetic field
Continuous reaction in magnetic field
Optimal substrate concentration(mol/L)
Optimal reaction time(h)
Substrate volume(mL)
0.8553
24
0.8553
8
1.5882
6
1
1
10
conversion(%)
100
99.9
0.392
100
99.9
1.176
100
99.9
2.647
Enantiomeric excess of (R)-CHBE(%)
Productivity(mmol/h)
and its catalytic activity was not fully exploited at low substrate con-
centrations. The number of COBE molecules in contact with Fe3O4-Arg-
Cells increased with the substrate concentration increase indicating that
the conversion of COBE probably increased. During the continuous
transformation, the substrate concentration increase could maximize
the use of the catalyst within a certain range and probably reduced the
conversion because of high substrate concentration inhibition.
3.7. Comparison of optimization results of the three systems
Productivity was defined as conversion per unit time. Table 2 re-
vealed the detailed productivities of batch reduction in the shaker,
batch reduction in the alternating magnetic field, and continuous re-
duction in MFBRS. MFBRS is considered an efficient system for in-
tensification of biocatalysis and biotransformation processes using
magnetically immobilized cells (Fe3O4-AA-Cells) as a catalyst. The
18

Y. Lu, et al.
ProcessBiochemistry99(2020)9–20
productivity of continuous reaction in the magnetic field was much
higher than the other two systems; this provides insights for the effi-
cient production of (R)-CHBE.
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Magnetic carriers are of interest because of their small particle size,
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through external magnetic fields. In our study, an alternating magnetic
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(R)-CHBE conversion and enantiomeric excess (e.e.) was achieved.
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% conversion. The enantiomeric excess of (R)-CHBE was maintained
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ductivity reduction. MFBRS is an efficient system for the intensification
of biocatalysis and biotransformation processes. This study provides a
new basis for further research on the application of immobilization
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CRediT authorship contribution statement
Hongqian Dai: Writing - original draft. Hanbing Shi: Writing -
original draft. Lan Tang: Writing - review & editing. Xingyuan Sun:
[15] Y.C. He, Z.C. Tao, J.H. Di, L. Chen, C.L. Ma, Effective symmetric bioreduction of
ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate by re-
combinant E. coli CCZU-A13 in [Bmim]PF6-hydrolyzate media, Bioresource
Technol. 214 (2016) 411–418, https://doi.org/10.1016/j.biortech.2016.04.134.
[16] P. Wei, J.X. Gao, G.W. Zheng, H. Wu, W.Y. Lou, Engineering of a novel carbonyl
reductase with coenzyme regeneration in E. coli for efficient biosynthesis of en-
antiopure chiral alcohols, J. Biotechnol. 230 (2016) 54–62, https://doi.org/10.
1016/j.jbiotec.2016.05.004.
Writing
-
review
&
editing. Zhimin Ou: Conceptualization,
Methodology.
Declaration of Competing Interest
[17] Y.P. Zhang, H.L. Wang, L.F. Chen, K. Wu, D.Z. Wei, Efficient production of ethyl (R)-
4-chloro-3-hydroxybutanoate by a novel alcohol dehydrogenase from Lactobacillus
curieae S1L19, J. Mol. Catal. B-Enzym. 134 (2016) 51–60, https://doi.org/10.1016/
j.molcatb.2016.09.010.
The authors report no declarations of interest.
Acknowledgements
[18] G.G. Chong, J.H. Di, C.L. Ma, D.J. Wang, C. Wang, L.L. Wang, P.Q. Zhang, J. Zhu,
Y.C. He, Enhanced bioreduction synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate
by alkalic salt pretreatment, Bioresource Technol. 261 (2018) 196–205, https://doi.
org/10.1016/j.biortech.2018.04.015.
We thank the National Nature Science Foundation of China
(21978267) and the Natural Science Foundation of Zhejiang Province
(LY15B060005, LY15B060007) for the financial support.
[19] T. Gu, B.Q. Wang, Z.W. Zhang, Z.T. Wang, G.G. Chong, C.L. Ma, Y.J. Tang, Y.C. He,
Sequential pretreatment of bamboo shoot shell and biosynthesis of ethyl (R)-4-
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(2019) 112–118, https://doi.org/10.1016/j.procbio.2019.02.002.
Appendix A. Supplementary data
[20] X. Xu, Y. Cai, Z. Song, X. Qiu, J. Zhou, Y. Liu, T. Mu, D. Wu, Y. Guan, J. Xing,
Desulfurization of immobilized sulfur-oxidizing bacteria, Thialkalivibrio versutus, by
magnetic nanaoparticles under haloalkaliphilic conditions, Biotechnol. Lett. 37
(2015) 1631–1635, https://doi.org/10.1007/s10529-015-1845-x.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2020.07.027.
[21] J. Hou, F. Liu, N. Wu, J. Ju, B. Yu, Efficient biodegradation of chlorophenols in aqu-
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