Modified magnetite nanoparticle as biocatalytic support for magnetically stabilized fluidized bed reactors

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Original Article
Modified magnetite nanoparticle as biocatalytic
support for magnetically stabilized fluidized bed
reactors
Ellen F. Bôa Morte a, Darlan S. Marum a, Elisa B. Saitovitch b,
Mariella Alzamora c, Sergio Neves Monteiro a,d,*,
Ruben J. Sanchez Rodriguez a
a Science and Technology Center e State University of the North Fluminense Darcy Ribeiro -UENF-, Campos dos
Goytacazes, Brazil
b Brazilian Center for Physical Research -CBPF- Dr. Xavier Sigaud Street, 150, Urca, Rio de Janeiro, Brazil
c Duque de Caxias Campus e Federal University of Rio de Janeiro -UFRJ-, Duque de Caxias, Brazil
d Department of Materials Science e Military Institute of Engineering -IME-, Rio de Janeiro, Praça General Tiburcio,
80, Rio de Janeiro, 22290-270, Brazil
a r t i c l e i n f o
Article history:
Received 7 April 2021
Accepted 30 June 2021
Available online 12 July 2021
Keywords:
Modified nanomagnetite
Magnetic biocatalyst supports
Relative catalytic activity
Magnetically stabilized fluidized bed
reactors
https://doi.org/10.1016/j.jmrt.2021.06.105
2238-7854/© 2021 The Author(s). Published
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a b s t r a c t
Magnetically stabilized fluidized bed reactor (MSFBR) is a sustainable and cost-effective
biotechnological process, which justifies the increasing search for biocatalytic super-
paramagnetic supports to enzyme immobilization. This work, investigates the effect of
modified nanomagnetite, Fe3O4 (nM), for biocatalytic support on properties associated with
catalytic behavior and potential use in MSFBR. The nM was either synthesized and then
modified with 3-aminopropyltriethoxysilano (nM-APTES) or stabilizedwith oleic acid (nM-OA)
and later modified with chitosan (nM-OA-Cs). A novel correlation study was carried out about
the support dimension effect, enzymatic surface loading and retained activity. Both nM-based
supports were morphologically, structurally and magnetically characterized by X-ray
diffraction, thermogravimetry, M€ossbauer and Fourier-transform Infrared spectroscopies, as
well as vibration sample magnetometry, scanning (SEM) and transmission (TEM) electron
microscopies. The nM-APTES and nM-OA samples disclosed crystal sizes of 8.07 and 8.69 nm,
respectively. From TEM imagens, the average particles sizes were 10.9 nm for nM-APTES and
12.6 nm for nM-OA. The nM-OA-Cs displayed an average particles size of 918.6 mm. These
magnetic supports displayed high saturated magnetization, 79.6 emu/g for nM-APTES and
72.7 emu/g for nM-OA. The amano lipase AK enzyme was immobilized on supports activated
with glutaraldehyde. The enzyme loading density of nM-APTES-GA-Lip (122 mg/g) with 61%
immobilization yield was higher than that of nM-OA-Cs-GA-Lip (46 mg/g) with 23% immo-
bilization yield. The catalytic activity of nM-APTES-GA-Lip (85%) was higher than that of nM-
OA-Cs-GA-Lip (46%), which reflected the correlation between enzyme loading efficiency on
the magnetic nanosupports and their relative activity, essential to potential use in MSFBR.
© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
* Corresponding author.
E-mail address: snevesmonteiro@gmail.com (S.N. Monteiro).
by Elsevier B.V. This is
).
an open access article under the CC BY-NC-ND license (http://
http://creativecommons.org/licenses/by-nc-nd/4.0/
mailto:snevesmonteiro@gmail.com
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1. Introduction
The catalytic process of obtaining biofuels is nowadays based
on biotechnologically more efficient and sustainable proced-
ures in relation to conventional refining processes, which
generate wastes that impact the environment [1]. The cata-
lytic route, in its common way, is associated with the use of
free enzymes, but has the shortcoming of a high cost owing to
the difficult in recovering the expensive enzyme after each
processing batch [2]. In order to overcome these limitations an
alternative has been proposed [2,3], which considers the
immobilization of enzymes on supports that have magnetic
properties, like F3O4, magnetite nanoparticle (nM). These
supports could be used in magnetically stabilized fluidized
bed reactor (MSFBR) offering additional advantages over con-
ventional fluidized bed reactors such as countercurrent
operation with higher flow rates [4]. These advantages facili-
tate the contact with the substrate, enhance the heat transfer
and reduce reaction times [5,6]. Moreover, they eliminate the
problem of dragging catalyst particles, since the reactor is
stabilized by the external magnetic field [7,8]. In addition, the
high operational flexibility of MSFBR allows the reuse of co-
valent immobilized enzymes, which makes the biotechno-
logical process more economically attractive [6].
Covalent immobilization provides strong binding between
the functional groups of the enzyme and the surface of the
support, which minimizes enzyme leakage from the support
andmake the enzyme to stand several operational conditions,
such as temperature, pH and agitation [9,10]. Typically, in
covalent immobilization, the activation of supports with a
bifunctional agent, like glutaraldehyde (GA) is necessary, as a
spacer arm. It has the function of limiting non-specific in-
teractions, as well as providing additional degrees of freedom
for terminal functional groups [11,12]. Thus, these spacer
arms can reduce conformational changes in the ideal rear-
rangement of the enzyme's three-dimensional structure,
avoiding enzymatic inactivation [13,14].
Magnetic nanoparticles do not effectively interact with the
enzyme, requiring surface modification with addition of spe-
cific functional groups [15,16]. Various modifiers have been
used such as with 3-aminopropyltriethoxysilane (APTES)
[17e20] or coating with functional polymers such as chitosan
[21e24], allowing the formation of covalent bonds with spacer
arms and enzymes.
An important biocatalyst characteristic, the catalytic ac-
tivity, decreases in relation to free enzymes as a consequence
of covalent immobilization due to factors such support size, as
well as the topography and interface between the enzyme and
the support [25,26]. A particular parameters is the enzymatic
surface loading yield and distribution of the immobilized en-
zymes on the support surface, which is directly related to the
enzyme-support as well as enzymeeenzyme interactions and
conformation of the enzymes. From this perspective, the
surface/volume ratio of supports have a relevant influence on
the conformational enzyme mobility, the enzymeeenzyme
side interactions and diffusion barriers of the substrates
[11,27,28].
Different levels of enzymatic surface loading yield must be
associated with an impact on the conformational degrees of
freedom, which may or may not provide secondary in-
teractions with the surface of the support or between immo-
bilized enzymes. This includes shaping of clusters that might
form multilayers in regions of the surface, thus limiting the
access of the substrate to the active site. Consequently, the
catalytic potential of the enzyme is reduced [12,24,29].
The literature relates different immobilization yields and
enzyme charge densitywith apparently controversial retained
activity results[20e22,30]. The correlation between immobi-
lization yield and retained activity does not consider the sur-
face/volume ratio of the support, and consequently limits the
assessment of factors that may be affecting the activity of the
immobilized enzyme in relation to the free one [15,20,23,30].
In this work, two magnetic biocatalysts to use in MSFBR
from nanomagnetite synthetized and modified with 3-
aminopropyltriethoxsilano (nM-APTES) and chitosan (nM-
OA-Cs) were developed. The main objective was to obtain
supports with high saturation magnetization and perform an
original comparative analyses between different surface/vol-
ume supports and correlation to enzymatic loading yield per
support area and retained catalytic activity.
2. Experimental
2.1. Materials
The basic precursormaterials used in the investigations were:
ferrous sulfate heptahydrate (FeSO4.7H2O), iron chloride
hexahydrate (FeCl3.5H2O), ammoniumhydroxide (NaOH) (30%
solution), acetic acid, hydrochloric acid, sodium hydroxide,
glutaraldehyde (GA) (50% solution) supplied by Vetec Quı́mica
Fina, Brazil; oleic acid (OA) and Arabic gum powder supplied
by Labsynth Products for Laboratory, Brazil; 3-
aminopropyltriethoxysilane (APTES), chitosan (Cs) (PM 161 g/
mol), polyethylene glycol (PEG-1500) and amano lipase AK
(Pseudomonas Fluorescents) supplied by SigmaeAldrich, Brazil.
2.2. Magnetic biocatalysts formulation and
characterization
2.2.1. Magnetite nanoparticles (nM). Synthesis and
modification
In a reactor, under an inert atmosphere (N2) at 50 �C, 2.34 g
FeSO4.7H2O and 1.20 g FeCl3.5H2O (1:2 M ratio Fe
þ3:Feþ2) and
200 mL of distilled water were added for 5 min at 600 rpm.
NaOH (8 mol/L) was added until pH 10 was reached and kept
stirring for 5min. At 80 �C, pHwas corrected to 8with aqueous
solution of hydrochloric acid (4% v/v)to synthesize the nano-
particle of Fe3O4 (nM) and then, in sequence, two separated
modifications were made:
1 In situ modification with APTES:
Themodification as carried out by slow dripping 88.5 mL
of the APTES under 500 rpm constant agitation for 1 h at
80 �C.
2 In situ stabilization with OA and further modification with
Cs:
The stabilization was conduct by slow dripping 4 mL of
OA under 500 rpm constant agitation for 1 h at 80 �C.
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The stabilized nanoparticles (nM-OA) were dispersed by
magnetic stirring for 30 min in a 2% (w/w) chitosan so-
lution previously solubilized in aqueous solution acetic
acid (5% v/v) for 1 h in a 40 �C bath and filtered. This
solution was dripped using an injection nozzle with the
aid of a peristaltic pump over a NaOH solution (3 mol/L).
The diameter and spherical morphology of the beads
were controlled by the drip height and speed. The
formed microspheres (nM-OA-Cs) remained in NaOH
for 12 h and were then washed until they reached pH 7.
The experimental parameters for co-precipitation and
surface stabilization and/or functionalized methods were
adapted from [5,31e36].
2.2.2. Characterization of nM
X-ray powder diffraction experiments were performed at
room temperature (RT) in the nM-APTES and nM-OA samples
using Bruker AXS D8 diffractometer with the Lynx Eye detec-
tor and Cu-Ka source radiation. The diffraction pattern was
collected in a BraggeBrentano configuration in the 10ºe90� 2q
range, with incremental steps of 0.02. From X-ray reflections,
WilliamsoneHall (WeH) graphs were obtained (bcosq vs. sinq;
where b is the integratedwidth of the peaks, and q is the Bragg
position of the reflections). From the slope, the crystal
microdeformations was calculated by using the following
equation:
MD¼S
4
(1)
where S is WeH straight angle and, the crystallite size (CS)
was calculated from the ordinate to the origin (Or) by using the
relationship
CS¼ l
Or
(2)
where l is the wavelength of the incident X-ray radiation,
lCuKa ¼ 1,5418 �A) [37].
Magnetic measurements were carried out using a vibrating
sample magnetometer (VSM) model Versa Lab Quantum
Design, and the 57Fe M€ossbauer spectroscopy experiments
were performed at RT in transmission geometry with the
Co�57 in Rh-matrix source moving in a sinusoidal mode. Iso-
mer shifts (IS) are reported relative to a-Fe at RT.
The analyses of size and dispersion of the nM particles
were carried out by assessing the images obtained by trans-
mission electron microscopy (TEM) JEOL, 1400Plus, (120 kV).
The nM samples weremixedwith isopropyl alcohol and taken
to an ultrasonic vat for dispersion. The dispersed solution was
dripped into 2 mm grids and placed in a desiccator for 24 h at
RT.
To generate the histogram particle size distribution, 150
particles were counted and measured in 5 different regions
using the ImageJ software.
Structural characterization of the coating and/or modifi-
cations was performed by Fourier transformed infrared
spectroscopy (FTIR). The FTIR spectra were obtained in an
IRPrestige-21 Shimadzu equipment and the samples prepared
in KBr compress disk. The spectra were recorded in the range
of 4000e400 cm�1 with resolution of ±0.1 cm.
Magneticmicrosphere (nM-AO-Cs)morphological analyses
were performed by scanning electronmicroscopy (SEM), ZEISS
Evo 40, high resolution operating at 10 kV. The microspheres
were deposited in a sample holder with graphite adhesive
tape and coated with approximately 20 nm of gold by sput-
tering using a Blazer's apparatus.
Thermogravimetric analyses (TGA) were carried out on a
model Q-5000 TA Instruments, with thermobalance sensi-
tivity of 0.1 mg, and platinum thermocouples sensitivity of
0.001 �C. Platinum pans were used, as well as flow of carrier
gas (nitrogen) of 25mL/min andheating rate of 10 �C/min up to
800 �C.
2.3. Covalent enzyme immobilization and activity
determination
2.3.1. Activation with glutaraldehyde (GA) space-arm
The nM-OA-Cs support was added in the proportion of 1:500
(w/v), to a GA 8% (v/v) in 0.1 M sodium phosphate buffer so-
lution at pH 7, under 150 rpm stirring for 5 h at 40 �C. The
previously mentioned system was later washed with the
buffer solution [16]. The nM-APTES support was activated by
adding a GA 2.7% (v/v) solution with 1:200 (w/v) ratio to 0.1 M
sodium phosphate buffer solution at pH 7 and then rest for 1 h
at RT. The supports were washed with alcohol, water and
buffer solution after activation, in this order [38].
2.3.2. Enzyme immobilization
Amano lipase in amounts of 200 mg per gram of dry activated
support was added in pH 7 buffer solution, PEG-1500 was
added at a value of 5mg/g of each support, stirringwas carried
out for 4 h, at 40 �C followed by 16 h in static conditions at 4 �C
and washed in buffer solution [39].
2.3.3. Protein (Lipase AK) determination
The Bradford method [40] was used to determine the con-
centration of proteins in the commercial product Amano
Lipase AK from Pseudomonas Fluorescentes. A solution of lipase
using 5 mg/mL of the commercial product in buffer solution
was analyzed. Using this same method, concentrations were
determined after the immobilization process carried out on
the solutions used for the immobilization on supports [41e43].
The percentages of immobilization yield (IY), and enzymatic
surface loading (ESL) were calculated by:
IY ð%Þ¼ Amount of immobilized enzyme
Amount of enzyme added in immobilization process
� 100
(3)
ESL ðmg = gÞ¼Amount of immobilized enzyme
Amount of support
(4)
2.3.4. Hydrolytic activity determination
Hydrolytic activity was determined using the olive oil hydro-
lysis process as indicated in reference [44]. A 1:1 olive oil
emulsion in Arabic gum solution (8%w/w) was prepared. 5mL
of the emulsion, 2 mL of buffer solution and 5 mL of the
enzyme solution were added to an Erlenmeyer flask, which
was subjected to stirring (150 rpm) at 40 �C for 5min. This step
was performed in triplicates.After this period, the reaction
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Fig. 1 e X-ray diffractograms of nM-APTES and nM-OA
samples. XRD (a) and WilliamsoneHall graphs obtained
from the X-ray reflections (b) of nM-APTES and nM-OA
samples.
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was stopped by the addition of 10 mL of an ethanol and
acetone solution (1:1 v/v). The final solution was titrated with
a 0.1 mol/L NaOH solution, and the volumes were registered
calculating both the hydrolytic activities (Ai), and the retained
catalytic activities (RA) [45].
Ai ðU =mgÞ ¼ ðVa � VbÞ � M � 10
6
t � m (5)
RA ð%Þ¼Ai of immobilized enzyme
Ai of free enzyme
� 100 (6)
3. Results and discussion
3.1. Characterization of magnetic core of supports
3.1.1. Identification of Fe3O4 phases
Fig. 1a shows the X-ray diffraction (XRD) patterns of nM-
APTES and nM-OA samples. All diffraction peaks can be
indexed as a cubic inverse spinel structure of Fe3O4 (JCPDS
no.19-0629).
The WilliamsoneHall (WeH) graphs are shown in Fig. 1b,
and provide qualitative and semiquantitative information
about the existence of microdeformations in the crystal. If the
peak broadening was solely due to the crystallite size effect,
there would be a horizontal line; but the observed slope in-
dicates the existence of microdeformations (MD). From the
slope, the crystal microdeformations and crystallite size (CS)
were calculated using Eqs. (1) and (2) [37].
The linear fit to the WeH graphs corresponding to the nM-
APTES and nM-OA samples showed a slope associated with
the microdeformations (MD) in the crystal lattice of the crys-
tals. These deformations may be associated with surface
disorder due to the small CS. The MD calculated values were
0,0025 and 0,0018 for the nM-APTES and nM-OA samples,
respectively. While the CS calculated value was 8,07 nm for
nM-APTES sample and 8,69 nm for the nM-OA. The values of
the calculated parameters are similar in both cases of the two
samples because the Fe3O4 magnetic nanoparticle was ob-
tained by the same synthesis method in both cases.
Magnetite and maghemite (g-Fe2O3) are iron oxides with
the same cubic spinel structure. In this case, it is difficult to
distinguish one phase from another by XRD; such an ambi-
guity could be, however, eliminated by using M€ossbauer
spectroscopy. The observed diffractograms allow to rule out
the presence of other iron oxides and impurities in concen-
trations that could be detected by this technique.
In the spinel structure, Fe ions occupy two inequivalent
sites assigned as A and B. These positions have tetrahedral
and octahedral symmetry, respectively. In magnetite the Fe2þ
ions places at site B and Fe3þ ions at both sites A and B [46].
The RT 57Fe M€ossbauer spectra of the samples are shown in
Fig. 2. Both compounds display broadened magnetic spectra
and no superparamagnetic subespectrum (singlet or doublet)
were observed. The magnetically broadened M€ossbauer
spectra were properly fitted with two crystalline sextets and a
hyperfine field distribution. The hyperfine field distribution
reflects the presence of small particles while the crystalline
subspectra could be attributed to larger particles of magnetite
[47]. This result clearly indicates a moderate distribution in
the particle sizes.
The crystalline sextet with larger hyperfine magnetic field
(Bhf) and smaller IS (0.33 mm/s) corresponds to the Fe3þ ions
located at the tetrahedral A sites, while the second sextet,
with smaller Bhf, is interpreted as the average signal fromFe3þ
and Fe2þ ions at the octahedral B sites, seen effectively (due to
the electron hopping) as Fe2.5þ ions. The Fe3þA =Fe
3þ;2þ
B ratio of
nM-APTES and nM-OA samples obtained from the M€ossbauer
subespectra areas were found as 0.75 and 0.77 respectively, in
contrast to the theoretical ratio of 0.50. Such difference in
ratios could be associated with cationic vacancies [48]. Va-
cancies are also typical in nanostructures and probably the B
sites are more structural affected than sites A generating
structural changes around the Fe ions in B sites [49].
On the other hand, the hyperfine field distribution fitting
procedure gives a mean magnetic hyperfine field of approxi-
mately 29 T for both samples. As presented in Table 1, the
mean isomer shifts (IS) are 0.42 and 0.46 mm/s for nM-APTES
and nM-OA samples, respectively. The value of IS is an
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Fig. 2 e M€ossbauer spectra for nM-APTES and nM-OA samples.
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indicative of the presence of Fe2þ. These components can be
associated with themagnetic inter-particle interaction [49,50].
The obtained hyperfine parameters, shown in Table 1,
suggest that both nM-APTES and nM-OA samples correspond
to the magnetite structure. The magnetic fields for the syn-
thesized magnetite were smaller than those reported in the
literature, 49.1 T for site A and 46 T for site B [51], probably due
to their size and coating of the nanoparticles.
Table 1 e Obtained parameters from M€ossbauer spectrum.
Component IS (mm/s) QS (m
nM- APTES Sexted A 0.33 0
Sexted B 0.45 0
Dist 0.42
nM-OA Sexted A 0.33 0
Sexted B 0.48 0
Dist 0.46
3.1.2. Magnetization behavior
In Fig. 3, the nM particles show characteristic curves associ-
ated with superparamagnetic behavior [52], since no rema-
nence and coercivity could be observed in the magnetic loops
for both samples. Also, in this figure, saturated magnetization
(MS) of nanoparticles of nearly 79.6 emu/g for nM-APTES and
72.7 emu/g for nM-OA can be noted. These MS values are
higher than several reported results in the literature [19,53],
which are normally below 30 emu/g. The relative high
m/s) Bhf (kOe) G (mm/s) Area (%)
.01 45.9 0.65 31.7
.02 42.3 1.1 42.3
29.4 0.65 25.9
.01 46.4 0.65 34.4
.02 42.8 1.04 44.2
29.7 0.70 21.4
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Fig. 4 e Schematic representation of magnetite (a) functionaliza
different types of interactions between the carboxylate head an
Fig. 3 e Magnetization curves of nM-APTES and nM-OA
supports.
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saturation magnetization observed in Fig. 3 can be related to
the average size (13-10 nm) of nanoparticles synthesized
herein [54e56].
The superparamagnetic behavior presented by the Fe3O4
nanoparticles aswell as the high saturationmagnetization are
favorable properties for the intended application in fluidized
bed reactors assisted by external magnetic fields. These
properties offer the possibility to operate this type of reactor,
MSFBR, with relatively high flows and effective recovery of the
biocatalyst [57].
3.1.3. Structural characterization of nanomagnetite (nM)
samples
Van der Walls forces associated with dipoleedipole in-
teractions between the nanoparticles lead to the formation of
clusters which limit their applications, particularly as cata-
lytic supports [58e60]. Aiming to restrain the existence of
these clusters, the surface stabilization of nM was carried out
[10,61]. Fig. 4 shows two schematic representation of
tion with APTES and (b) stabilization with oleic acid with
d metal oxide surface. Adapted from [63e65].
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Fig. 5 e FTIR spectra of: (a) nM-APTES and nM-OA particles; (b) nM-APTES-GA, nM-APTES-GA-Lip and nM-OA-GA, nM-OA-
GA-Lip particles.
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superficial coating of hydroxylated magnetite nanoparticle
(nMeOH), in (a): the silanization reaction with APTES andin
(b): the 3 possible interaction the COOe group of oleic acid. In
addition, this coating provides greater resistance to the
nanoparticles in contact with air and stability to pH [10,62].
Fig. 6 e Typical TEM micrographs and size distribution histogra
as modified with APTES (c,d).
The two alternative surface modifications of nM, with
APTES as well as chitosan from nM-OA (nM-OA-Cs) were
analyzed after GA activation in the FTIR spectra shown in
Fig. 5. The spectra present bands at 570 and 440 cm�1, which
are characteristic of the FeeO stretch. Between 600 and
ms of nanomagnetite stabilized with oleic acid (a,b) as well
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Fig. 7 e SEM micrographs of nM-OA-Cs support (a) and size
distribution histogram (b).
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400 cm�1 bands associated with stretches n1 and n2 of the
tetrahedral and octahedral sites in the crystalline structure of
the inverse spinel are also observed.
In particular, the nM-OA spectrum displays bands, which
are characteristic of the oleic acid structure present on the
surface at 2852 and 2918 cm�1, as well as bands belonging to
the vibration of both symmetric and asymmetric elongation of
methylene (-CH2
-), respectively [66e68]. Bands at 3420 and
1632 cm cm�1 were attributed to OeH vibrations, indicating
that the Fe3O4 nanoparticle surface was covered with hy-
droxyl groups. A sharp characteristic band at 1710 cm�1 was
not observed as a result of the absence of the C]O stretch
from oleic acid, replaced by the appearance of two bands at
1514 and 1416 cm�1 that correspond to the asymmetric nas
(COO�) and the symmetric ns (COO
�) [64,66,68]. Thus, oleic
acid existed as a carboxylate when chemically bound to the
Fe3O4 nanoparticles. These two bands might be utilized to
predict the types of binding interaction between the carbox-
ylate head and iron oxide surface [63,64,66] The types of the
interaction can be distinguished by the wave number sepa-
rations (D) of the asymmetric nas (COO
�) and symmetric ns
(COO�) bands: D ¼ 200�320 cm�1 correspond to monodentate
interaction; D ¼ 140e190 cm cm�1 correspond to bridging
bidentate interaction; and D < 110 cm�1 correspond to
chelating bidentate interaction as displayed in Fig. 4b. The
binding interaction between oleic acid and hematite, found by
calculating the D of 98 cm�1 was consistent with chelating
bidentate interaction between the carboxylate head and the
particle surface [63,64]. At 885 cm�1 it is possible to observe a
band related to double bonds, C]C, which is present in the
oleic acid chain [68,69].
The nM-APTES spectrum in Fig. 5a shows bands at 1527
and 1635 cm�1 referring to NH2 flexion of the free NH2 groups.
On the other hand, the band concerning NH axial deformation
of secondary amines cannot be identified because it overlaps
the OH-associated region, around 3480 cm�1. Furthermore,
the characteristic bands of silane modification were recorded
at 1042 and 1114 cm�1 due to the asymmetric stretching of the
SiOeH and SieOeSi groups, as well as due to FeeOeSi vibra-
tion at 580 cm�1, which overlaps the FeeO bands [70e73].
Fe3O4 modified oxides samples, nM-APTES and nM-OA-Cs,
have the appropriate functionality (-NH2) for the establish-
ment of a covalent link with the enzyme using glutaraldehyde
GA. In addition, GA acts as an enzyme extender arm in rela-
tion to the support surface, which causes less restriction in
the conformational mobility of the immobilized enzyme and
eventual loss of catalytic activity in relation to the free
enzyme.
Modification with GA and covalent immobilization of
amano lipase AK were characterized with the aid of FTIR
spectroscopy. In the magnified spectra region in Fig. 5b, the
bands between 1650 and 1600 cm�1 are related to the pri-
mary amine (-NH2) and attributed to the amino group pre-
sent in the structures of APTES and Cs. The establishment of
links between the modified oxides and the GA originates the
bands between 1750 and 1650 cm�1, characteristic of the
formation of a Schiff base, imines, (-C]N-) [74]. This would
be expected in a support activation process with GA.
After activating and immobilizing amano lipase AK on the
nM-APTES-support, respectively nM-APTES-GA and nM-
APTES-GA-Lip, the subsequent appearance of a band refer-
ring to the imine clusters (-C]N-) around 1680 cm�1 is
observed. Concerning the nM-OA-Cs-GA and nM-OA-Cs-GA-
Lipase supports, the band also appears in the spectrum at
1680 cm�1 (-C]N-) with subsequent increase in intensity.
3.1.4. Morphological and size distribution
Fig. 6 shows TEM images of the nM synthetized particles sta-
bilized with OA and APTES with visible limits between
neighboring particles, despite the discreet agglomeration
caused by grids deposition in the TEM analysis. From
M€ossbauer (3.1.1) analysis, the hyperfine field distribution
observed is characteristic of the small particles.
Carboxylic clusters of OA interact with hydroxyls on the
surface of the nM, Fig. 6a and, as a consequence, a steric hin-
drance originates between the nanoparticles, which limits the
clustering of these nanoparticles [75].The silanized nM nano-
particles surface with APTES in Fig. 6b, show a similar effect
and their limits are defined. For each sample, using the ImageJ
software, the average sizes were 10.9 and 12.6 nm for nM-
APTES and nM-OA samples, respectively. In the distribution
graphic, the largest fraction of nanoparticles is ranging from 6
to 15 nm. The nanoparticles modified with APTES display 91%
of diameter under 21 nm while those stabilized with AO pre-
sent a fraction of 84% of diameters smaller than 21 nm. These
measurements have a good correlation with crystallite size
calculated by WeH without the amorphous contribution,
which would not be observed if the nanoparticles were
agglomerated.
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Fig. 8 e Thermogravimetric analysis of nM-APTES and nM-OA-Cs supports: (a) TG and (b) DTG curves.
Fig. 9 e Correlation of retained catalytic activity, immobilization yield and enzymatic surface loading reported by different
authors: Wang et al. [25]; Osuna et al. [22]; Samo et al. [17] as well as the present work, nM-APTES.
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Table 2 e Immobilized enzymes, immobilization yield and retained hydrolytic activity.
Biocatalyst formulations Enzymatic surface loading (mg/g) Immobilization yield (%) Retained hydrolytic activity (%)
nM-APTES-GA-Lipase 122 61 85
nM-OA-Cs-GA-Lipase 46 23 46
Table 3 e Support size, immobilization yield, enzymatic surface loading and retained hydrolytic activity.
Support [References]a Average size (nm) Immobilization
Yield (%)
Enzymatic Surface
Loading (mgenz/gsupp)
Retained Activity (%)
nM-APTES-GA 11 61 122 85
Fe3O4/APTES/GA [17] 6 52 52
a 77
nM/Cs/GA [22] 10 90 5a 65
Fe3O4@Cs/EDC [25] 30 81 16
a 75
a Calculated from reference data.
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Fig. 7 shows both SEM images and sizes distribution of nM-
OA-Cs microparticles. The external morphology of these mi-
croparticles, Fig. 7a, reveals a regular spherical shape and a
slightly rough surface with absence of cavities or micropo-
rosity. This is an appropriate characteristic for the support on
which the immobilization and accommodation of enzyme
molecules will only be located on the exposed surface, which
facilitates the interaction with substrates [9].
As observed in Fig. 7b, the coating of nM-OA particles,
aiming to activate and immobilize the enzyme, changes the
average size of the nM-OA-Cs support in comparison to OA-
coated nanomagnetite(nM-OA). They show a relatively nar-
row distribution between 800 and 1050 mm, with its largest
fraction (32%) with a diameter of 925e950 mm and average
sizes of 918.6 mm.
3.1.5. Thermal characterization of nM supports
The thermal behavior of the nM samples, shown in Fig. 8,
reveals that the hydrophilic trait of these surfaces is associ-
ated with loss of superficially adsorbed water by the formu-
lations nM-APTES (3.1%) and nM-OA-Cs (6.4%). When it
comes to the chitosan-modified oxide, water is adsorbed as a
consequence of the interaction with the hydroxyl (-OH) and
amine (-NH2) groups present in the chitosan structure. In
APTES-modified oxide, water is absorbed by interaction with
the amine groups (-NH2) of the silane structure. In nM-OA
gravimetric curve, two weight loss characteristics at 252
and 360 �C were displayed associated with oleic acid-coated
Fe3O4 nanoparticles which may be attributed to two kinds
of bonding between COO groups and Fe3O4 nanoparticles [76].
The maximum rate weight loss (Tmax) shown in Fig. 9 for
nM-OA-Cs structure occurs at 310 �C due to the thermal
degradation of the chitosan structure [77]. Regarding nM-
APTES sample, the maximum weight loss is registered at
281.5 and 365 �C associated with thermal fragmentation of the
amino silane “extensor arm” and at 728 �C by fragment
degradation and silicon oxide formation [78]. Both supports
could be stable up to 200 �C in biotechnologies process by
enzymatic catalysis, which for lipases generally has an
optimal temperature range of 30e60 �C [13].
3.2. Lipase AK immobilization and retained catalytic
activity
Table 2 discloses that the immobilization yield percentages
(%) on surface of the magnetic supports nM-APTES-GA and
nM-OA-Cs-GA, both with space-arms (GA), are markedly
different. Apparently, as a consequence of the different GA
concentration used in the support activation, difference in GA
availability in the support surface and enzymatic immobili-
zation are introduced.
The concentration of GA (8%) used in nM-OA-Cs support
activation must have induced the bi-anchoring of GA mole-
cules onto the chitosan surface, which leads to a decrease in
the availability of active sites and, therefore, in the ability to
immobilize enzymes, indicated in Table 2. By the same
immobilization experimental conditions (times, temperature,
enzyme solution concentration, ph), a low immobilization
yield of the immobilization process and a low immobilized
enzyme on the surface of the nM-OA-Cs-GA support could be
noticed. On the other hand, due tomultipoint interactionwith
support, the enzyme loading immobilization must be affected
and, consequently, the retained hydrolytic activity of nM-OA-
Cs-GA-Lipase is lower than nM-APTES-GA-Lipase [79,80].
Some reports on enzyme immobilization yield and
retained catalytic activity, shown in Table 3 and Fig. 9 do not
apparently have clear correlation. Wang et al. [25] and Osuna
et al. [22] obtained both nM/Cs/GA and Fe3O4@Cs/EDC
respectively retained catalytic activity and higher enzyme
immobilization yield lower than nM-APTES-GA.
The relative catalytic activity of this novel modified nano-
metric Fe3O4 particlesMSFBR support nM-APTES-GA, retained a
high percentage (85%) of activity of free enzyme. Moreover, the
experimental data reveals an effective correlationwith enzyme
surface immobilization (Fig. 9), which could cause effects
associated with enzymatic surface loading as conformational
restrictions, enzymeeenzyme and surfaceeenzyme in-
teractions. Additionally, diffusion limitations due to
enzymeeenzyme interactions lead to multilayer or clusters in
the support surface, which generally occurs at high enzyme
concentrations [12,15,24,29].
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4. Conclusions
Magnetite, Fe3O4, nanoparticles (nM) as support for magneti-
cally stabilized fluidized bed reactor (MSFBR) were prepared by
co-precipitation and modified with APTES (nM-APTES). This
support was compared with incorporated chitosan micro-
sphere (nM-OA-Cs) previously stabilized by oleic acid (nM-
AO).
Modified nM presented different size distribution, nM-
APTES (~11 nm) and nM-OA-Cs (~920 mm) as well as high
saturated magnetization of ~70 emu/g for both nM-APTES
and nM-AO. This is suitable to be used as support in
MSFBR operating with relatively high flows. Particularly the
nM-APTES-GA-Lip presented a low mass transfer resistance
associated with high surface/volume relation. The amano
lipase (AK) enzyme, immobilized by covalent bonding
coupling with glutaraldehyde (GA) space-arms on both nM-
APTES-GA-Lip and nM-OA-Cs-GA-Lip supports with
different surface/volume relation, presented a drastic
reduction of retained activity to nM-OA-Cs-GA-Lipase (46%).
This could be justified by considering the original analysis
from enzyme density immobilization on the support surface
in contrast to traditional analyses from immobilization yield
(%). The magnetic saturation and retained catalytic activity
of nM-APTES-GA-Lipase revealed an effective potential
biocatalyst for magnetically stabilized fluidized bed
reactors.
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.
Acknowledgements
The funding from LDRX-UFF as well as the Brazilian agencies
CAPES (Coordenaç~ao de Aperfeiçoamento de Pessoal de Nı́vel
Superior), CNPq (Conselho Nacional de Desenvolvimento Cien-
tı́fico e Tecnol�ogico) (310108/2017-9) and FAPERJ (Fundaç~ao de
Amparo �a Pesquisa do Estado do Rio de Janeiro) (CNE 239703) is
gratefully acknowledged.
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	Modified magnetite nanoparticle as biocatalytic support for magnetically stabilized fluidized bed reactors
	1. Introduction
	2. Experimental
	2.1. Materials
	2.2. Magnetic biocatalysts formulation and characterization
	2.2.1. Magnetite nanoparticles (nM). Synthesis and modification
	2.2.2. Characterization of nM
	2.3. Covalent enzyme immobilization and activity determination
	2.3.1. Activation with glutaraldehyde (GA) space-arm
	2.3.2. Enzyme immobilization
	2.3.3. Protein (Lipase AK) determination
	2.3.4. Hydrolytic activity determination
	3. Results and discussion
	3.1. Characterization of magnetic core of supports
	3.1.1. Identification of Fe3O4 phases
	3.1.2. Magnetization behavior
	3.1.3. Structural characterizationof nanomagnetite (nM) samples
	3.1.4. Morphological and size distribution
	3.1.5. Thermal characterization of nM supports
	3.2. Lipase AK immobilization and retained catalytic activity
	4. Conclusions
	Declaration of Competing Interest
	Acknowledgements
	References