crystals
Article
Preparation of Hydroxyapatite-Titanium Dioxide Composite
from Eggshell by Hydrothermal Method: Characterization and
Antibacterial Activity
Atiek Rostika Noviyanti 1, * , Efa Nur Asyiah 1 , Muhamad Diki Permana 1,2 , Dina Dwiyanti 1 , Suryana 3
and Diana Rakhmawaty Eddy 1
Citation: Noviyanti, A.R.; Asyiah,
E.N.; Permana, M.D.; Dwiyanti, D.;
Suryana; Eddy, D.R. Preparation of
Hydroxyapatite-Titanium Dioxide
Composite from Eggshell by
Hydrothermal Method:
Characterization and Antibacterial
Activity. Crystals 2022, 12, 1599.
https://doi.org/10.3390/
cryst12111599
Academic Editor: Leonid Kustov
Received: 29 September 2022
Accepted: 7 November 2022
Published: 10 November 2022
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Copyright: © 2022 by the authors.
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This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
Crystals 2022, 12, 1599. https://doi.org/10.3390/cryst12111599
1 Department of Chemistry, Faculty of Mathematics and Sciences, Universitas Padjadjaran,
Jl. Raya Bandung-Sumedang km. 21, Jatinangor, Sumedang 45363, Indonesia
2 Center for Crystal Science and Technology, University of Yamanashi, Yamanashi 400-8511, Japan
3 Department of Biology, Faculty of Mathematics and Sciences, Universitas Padjadjaran,
Jl. Raya Bandung-Sumedang km. 21 Jatinangor, Sumedang 45363, Indonesia
* Correspondence: atiek.noviyanti@unpad.ac.id; Tel.: +62-81221646415
Abstract: Hydroxyapatite (HA) has been widely used in biomedical applications. HA is prepared
from natural sources of eggshell. The obtained HA is composited with TiO2 using the hydrothermal
method at a temperature of 230 ◦ C. The structure and morphology of HA-TiO2 composites are
characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and a
scanning electron microscope (SEM). Meanwhile, its antibacterial activity was tested on Staphylococcus
aureus and Escherichia coli bacteria. The formation of the HA-TiO2 composite is evidenced by typical
peaks on the XRD pattern for HA and TiO2 . The FTIR spectrum shows that no bond formed between
TiO2 and HA which indicates the formation of composites. The smallest crystallite size and the
highest specific surface area were obtained from the composite with the composition of HA-TiO2
30:70. In addition, the composition of the composite also shows the smallest particle size distribution.
Therefore, the presence of TiO2 plays a significant role in determining the HA properties formed.
Furthermore, the HA-TiO2 composite showed good antibacterial activity using disk diffusion and
optical density (OD) methods. These results indicate that the synergistic combination of HA from
eggshell with TiO2 has favorable properties for antibacterial activity.
Keywords: natural source; eggshells; hydroxyapatite; composites; titanium dioxide; antibacterial
properties
1. Introduction
Hydroxyapatite (HA) is one of the inorganic compounds with the chemical formula
Ca10 (PO4 )6 (OH)2 [1,2]. HA has been widely used in biomedical applications, especially in
bone and dental implants [3,4]. This is because HA is a component of bone minerals and a
constituent of teeth in humans that has good bioactivity and biocompatibility [5,6]. HA
is usually synthesized from chemicals in the form of H2 SO4 and Ca(OH)2 , but in general,
the process is quite complicated and the product lacks biocompatible properties [7]. The
biocompatibility properties of the material for the purposes of biomedical application are
conditions that must be met. Some researchers have modified the sources of synthesis
materials used, as sourcing materials from nature is more often cheaper, easier to obtain,
and most importantly demonstrates biocompatible properties [8]. HA from natural sources
also contains ions such as cationic Na+ , Zn2+ , Mg2+ , K+ , Si2+ , Ba2+ , and anionic F− , Cl− ,
SiO4 4− , CO3 2− [9,10]. Some sources of HA from natural materials are cow bones, blood
clam shells, and fish bones [11]. Another study revealed the synthesis of HA from chicken
eggshells [12]. Eggshells are the easiest natural source to obtain, are relatively inexpensive,
and are easy to prepare [13,14]. Currently, many researchers are conducting research on
https://www.mdpi.com/journal/crystals
Crystals 2022, 12, 1599 2 of 15

eggshells as an alternative treatment to replace bone damage in humans, since the eggshell
itself contains a fairly high element of CaCO3 which is useful as a biomaterial [15]. In
addition, the eggshell produced in a year weighs 138,956 tons, and the CaCO3 content
of eggshells can reach 94% [16]. Thus, the chicken eggshell has a high potential for the
synthesis of high-quality hydroxyapatite.
HA has the advantage that it can absorb bacteria by adsorbing molecules on its
surface [17,18]. However, HA cannot decompose a molecule [19,20]. In addition, HA has
properties that are resistant to ultraviolet radiation and X-rays. Thus, it is resistant to
interference, especially to radiation [21,22]. To overcome the shortcomings, HA needs to
be modified by adding other materials. Many studies have observed an increase in the
effectiveness of HA due to the formation of composites with polymers [23], metals [24],
or metal oxides [25]. The metal oxide that is commonly used is titanium dioxide (TiO2 ),
which is non-toxic, environmentally friendly, and has high mechanical stability, high
photocatalytic activity, and antibacterial activity [26,27]. TiO2 has the ability to deactivate
Gram-positive and Gram-negative bacteria [28].
Based on the results of previous studies, the increase in the performance of HA com-
posites is strongly influenced by the composition and modification of the structure as well
as the synthesis method. Therefore, many researchers have prepared HA-TiO2 compos-
ites by various methods such as sol-gel, solid state, and hydrothermal methods [29–31].
Ortiz et al. [32] synthesized TiO2 /HA composite by the sol-gel method followed by carbon
dioxide supercritical drying. In this study, the precursors used were Ti(OBu)4 and 2–5% mol
HA. In addition, other studies showed that the addition of 15 and 30% TiO2 to HA showed
changes in the behavior of the hydroxyapatite matrix such as the crystallinity, morphology,
and bioactivity [33,34]. In another study, HA-TiO2 composite with the addition of TiO2 by
25% can significantly increase the mechanical properties of HA [35,36].
In addition, several researchers have succeeded in synthesizing HA-TiO2 composites
to inactivate bacteria through photocatalytic degradation mechanisms [37]. By modifying
HA with TiO2 , HA can adsorb molecules which will then be broken down by TiO2 . HA-
TiO2 composites with a ratio of 50–50 showed a bacterial decomposition of about 50%
after 2 h of UV exposure [38,39]. In other research, the HA-TiO2 composite with 60% TiO2
showed good antibacterial activity [40]. Thus, the synthesis of HA and the formation of its
composite with TiO2 with the right composition can be used as an antibacterial material
and is the focus of this study.
However, although several studies have succeeded in synthesizing various HA/TiO2
composites [41], the degradation efficiency still needs to be improved because the research
does not fully discuss the effect of compositional variations of the two materials. In addition,
no reports have shown the synthesis of HA/TiO2 composites from natural sources, espe-
cially from chicken egg shells. Therefore, in this study, we conducted the HA synthesis via a
one-step hydrothermal method using the variant source of CaO from chicken eggshells [14].
The synthesized HA was then composited with various molar variations of TiO2 by the
hydrothermal method and observed for its antibacterial activity against Gram-positive and
Gram-negative bacteria. The use of the hydrothermal method has the advantage that the
synthesis results have high purity and crystallinity. In addition, the yield is more than 90%
with a homogeneous particle size distribution [42]. The effect of TiO2 addition on structure,
bond type, and morphology was observed using X-ray diffraction (XRD), Fourier-transform
infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The use of different
sources and synthesis methods is very likely to affect the structure, bonding, morphology,
particle size, and effectiveness of the composite as an antibacterial agent [23,43].

2. Materials and Methods

2.1. Synthesis of HA-TiO2 Composites by Hydrothermal Method
HA was synthesized by methods from our previous study [14,44]. HA was synthesized
from CaO obtained from eggshells. Chicken eggshells were washed with water to remove
contaminants and crushed using a planetary ball mill. After that, it was calcined at 1000 ◦ C
 2. Materials and Methods
2.1. Synthesis of HA-TiO2 Composites by Hydrothermal Method
HA was synthesized by methods from our previous study [14,44]. HA was synthe-
sized from CaO obtained from eggshells. Chicken eggshells were washed with water to
Crystals 2022, 12, 1599 remove contaminants and crushed using a planetary ball mill. After that, it was3calcined of 15

at 1000 °C for 5 h with an increase in temperature of 15 °C/min to produce CaO. The CaO
obtained is then used as a precursor for the synthesis of HA by mixing it with diammo-
for 5hydrogen
h with an phosphate
increase in temperature of4,15 ◦ C/min to produce CaO. The CaO obtained is
nium ((NH4)2HPO 99.0%, Merck, Kenilworth, NJ, USA) with a Ca:P
then used as a precursor for the synthesis
mole ratio of 1.67. The pH of the solution with acetic of HA by mixing
acid it(CH
with diammonium hydrogen
3COOH, 99.5%, Merck, Ken-
phosphate ((NH 4 )2 HPO 4 , 99.0%, Merck, Kenilworth,
ilworth, NJ, USA) was adjusted to pH 9. Then, the mixture was put NJ, USA) with a Ca:P
intomole ratio of at
an autoclave
1.67. The pH of the solution with acetic acid (CH3 COOH, 99.5%, Merck, Kenilworth, NJ,
120 °C for 48 h. After that, the formed HA was dried to obtain HA crystals.◦
USA) was adjusted to pH 9. Then, the mixture was put into an autoclave at 120 C for 48 h.
Composites were prepared by mixing HA and TiO2 (Anatase, 99.0%, Merck, Kenil-
After that, the formed HA was dried to obtain HA crystals.
worth, Composites
NJ, USA) with were various
preparedweight variations,
by mixing HA and namely HA:TiO
TiO2 (Anatase, 2 with ratios of 30:70
99.0%, Merck, Kenil-
(H3T7), 40:60 (H4T6), 50:50 (H5T5), 60:40 (H6T4), and
worth, NJ, USA) with various weight variations, namely HA:TiO2 with 70:30 (H7T3). Theratios
mixture was dis-
of 30:70
persed
(H3T7), 40:60 (H4T6), 50:50 (H5T5), 60:40 (H6T4), and 70:30 (H7T3). The mixture wasput
and stirred with 60 mL of deionized water using a magnetic stirrer, then dis-into
a 100 mLand
persed autoclave,
stirred withand60then
mL ofheated at 230
deionized °C using
water for 48a hmagnetic
to obtain a HA-TiO
stirrer, 2 composite
then put into a
100 mL autoclave, andsynthesis
then heated ◦ C for 48 h to obtain a HA-TiO composite [14,43].
at 230 for
[14,43]. The prepared process HA-TiO2 crystals is shown 2 in Figure 1.
The prepared synthesis process for HA-TiO2 crystals is shown in Figure 1.

Figure 1. Synthesis preparation for HA-TiO2 composites by hydrothermal method.

2.2. Composite Characterization

Figure 1. Synthesis preparation for HA-TiO2 composites by hydrothermal method.
Composite structures (H3T7, H4T6, H5T5, H6T4 and H7T3) were determined with
X-ray diffraction
2.2. Composite (XRD, PANalytical PW3040/X0 X’Pert PRO, Malvern, UK). Measurements
Characterization
were made in the range of 10–90◦ (2θ) with a step size of 0.01◦ and rate of 10◦ /min using
CuComposite
Kα radiation structures (H3T7,
(λ = 0.15406 H4T6,
nm) at roomH5T5, H6T4 and
temperature. The H7T3) were was
XRD pattern determined
analyzedwith
X-ray diffraction (XRD, PANalytical PW3040/X0 X’Pert PRO, Malvern, UK). Measure-
using HighScore Plus software (PANalytical 3.0.5) [45]. The crystallite size of the composite
ments were
crystal made in by
is calculated thethe
range of 10–90° (2θ)
Debye-Scherrer with as
equation a step size of 0.01°
in Equation and rate of 10°/min
(1) [46].
using Cu Kα radiation (λ = 0.15406 nm) at room temperature. The XRD pattern was ana-
lyzed using HighScore Plus software D =(PANalytical
(Kλ)/(Bcosθ) 3.0.5) [45]. The crystallite size(1)
of the
composite crystal is calculated by the Debye-Scherrer equation as in Equation (1) [46].
where D is the crystal size (nm), K is the crystal form factor (0.9), λ is the wavelength of
X-rays (0.15406 nm), B is the value of D Full Width at Half Maximum (FWHM) (rad), and (1)
= (Kλ)/(Bcosθ)
θ is angle of diffraction (rad). The crystallite size of HA was calculated from peaks at
where D is◦ (211),
2θ = 31.8 the crystal
while size
TiO2 (nm), K is the crystal
was calculated form
from peaks at factor ◦ (101).
(0.9),
2θ = 25.3 λ isThe
thecrystallinity
wavelength of
of synthesizing HA/TiO was calculated from the XRD data using
X-rays (0.15406 nm), B is2the value of Full Width at Half Maximum (FWHM) Equation (2) below [47]:
(rad), and θ
is angle of diffraction (rad). The crystallite size of HA was calculated from peaks at 2θ =
Crystallinity (%) = [(I300 − V112/300 )/I300 ] × 100% (2)
31.8°(211), while TiO2 was calculated from peaks at 2θ = 25.3°(101). The crystallinity of
synthesizing
where I300 isHA/TiO 2 was
the intensity ofcalculated frompeak
the diffraction the at
XRD data using
the (300), Equation
V112/300 (2) below
is the intensity [47]:
of the
(112) and (300). In this study, the calculated SSA is crystalline SSA with the definition as
Crystallinity (%) = [(I300 − V112/300)/I300] × 100% (2)
surface area (SA) of crystals per mass of crystals [48]. SSA (m2 /g) was calculated using
Sauter’s formula (Equation (3)), where D is the crystallite size (m) and ρ is the density of
crystals (g/m3 ).
SSA = 6/(D × ρ) (3)
Crystals 2022, 12, 1599 4 of 15

Ascertainment of the functional groups contained in HA-TiO2 is by Fourier-transform

infrared spectroscopy (FTIR, PerkinElmer Spectrum 100, Waltham, MA, USA) using the KBr
disc technique, at a frequency interval of 400–4000 cm−1 with a step size of 1 cm−1 . Each of
the collected spectrums is an average of 16 FTIR scans. A few micrograms of the HA/TiO2
composites were mixed with KBr, with a ratio KBr and composites 1:100, then pressed for
structural analysis. To determine its morphology, composite samples were characterized
using a scanning electron microscope (SEM, JEOL JSM-6360LA, Tokyo, Japan) with a
voltage of 20 kV at an ×1000 magnification. Several milligrams of the composite were fixed
in a sample holder and coated using gold. Then, the morphology and size were examined
and analyzed using ImageJ 1.52a software [49]. A total of approximately 300 particles were
analyzed for the area of each sample. The pixel area in the image is converted to the particle
area. After that, the length/diameter of each particle was calculated. In addition, the mean
and standard deviation values are also calculated.

2.3. Determination of Antibacterial Activity

2.3.1. Disc Diffusion Method
HA-TiO2 composites are suspended to test their antibacterial properties on E. coli
(Gram-negative bacteria) and S. aureus (Gram-positive bacteria). The method used is the
disc diffusion method. All tools used for antibacterial testing are sterilized first by washing
followed by heating using an autoclave at 121 ◦ C for 120 min. After sterilization, the
provision of test bacteria is then carried out, the test bacteria that have been inoculated are
taken 1 isolate using sterile nichrome wire, then suspended into a test tube containing 5 mL
of liquid media. Subsequently, the suspension of the test bacteria was incubated for 24 h
at a temperature of 37 ◦ C. Turbidity of the bacterial suspension was standardized to the
equivalent of 1 McFarland. The media for nutrient agar (NA) is to be put in a petri dish and
allowed to harden. The suspension of the test bacteria is taken and applied to the agar NA
medium. Disc paper is placed regularly on the substrate so that it is then incubated for 24 h
at a temperature of 37 ◦ C. After that, observations are made and measured inhibition zones
are formed using calipers. The inhibitory power is determined by reducing the diameter of
the formed inhibition zone by the diameter of the disc paper (ø = 6 mm).

2.3.2. Optical Density Method

Antibacterial activity test using the optical density (OD) method measured the decrease
in absorbance value at a wavelength of 600 nm. The composite sample, HA from eggshell,
pure TiO2 , and positive control (4.5% amoxicillin), were prepared in distilled water. Then
3 mL of S. aureus and E. coli cultures (∼106 cfu/mL) were added to each. The mixed
suspension was kept in an incubator with continuous shaking for different time periods.
The results of incubation were measured for the absorption value (OD) at a wavelength of
600 nm using a UV-visible spectrophotometer [50]. The graph was plotted of OD at 600 nm
versus time to predict the growth of bacteria after interacting with materials.

3. Results and Discussions

3.1. XRD Characterization
The XRD pattern of the HA-TiO2 composite shows peaks corresponding to the In-
organic Crystal Structure Database (ICSD) 98-005-6311 for HA (hexagonal, P63/m) [51]
and ICSD 98-015-4602 for TiO2 anatase (tetragonal, I41/amd) [52], which is shown in
Figure 2. The conformity of the sample peaks by reference confirms the presence of merg-
ing or composting [53]. The diffraction peaks at 2θ = 25.9◦ (002), 31.8◦ (211), 46.7◦ (222), and
49.5◦ (213) are typical peaks from pure HA, while the peak of anatase TiO2 was evident at
2θ = 25.3◦ (101), 37.8◦ (004), and 48.0◦ (200). The highest peak of HA composite HA-TiO2
resulted from the synthesis of H3T7, H4T6, H5T5, H6T4, dan H7T3 is the same as the
highest peak of pure HA (31.8◦ ). The peak intensity of HA increased with increasing HA
concentration, as well as the peak of TiO2 which increased with increasing TiO2 content [17].
Based on the XRD pattern, all composites did not obtain other phase peaks. Thus, it can
 Figure 2. The conformity of the sample peaks by reference confirms the presence of
merging or composting [53]. The diffraction peaks at 2θ = 25.9°(002), 31.8°(211), 46.7°(222),
and 49.5°(213) are typical peaks from pure HA, while the peak of anatase TiO2 was evident
at 2θ = 25.3°(101), 37.8°(004), and 48.0°(200). The highest peak of HA composite HA-TiO2
resulted from the synthesis of H3T7, H4T6, H5T5, H6T4, dan H7T3 is the same as the
Crystals 2022, 12, 1599 highest peak of pure HA (31.8°). The peak intensity of HA increased with increasing 5HA of 15
concentration, as well as the peak of TiO2 which increased with increasing TiO2 content
[17]. Based on the XRD pattern, all composites did not obtain other phase peaks. Thus, it
can
be be concluded
concluded that
that thereisisno
there noimpurity
impurityininthe
the HA-TiO
HA-TiO22 composite. This
Thisindicates
indicatesthat
that
nonochemical
chemical reaction occurred that caused other products to form betweenthe
reaction occurred that caused other products to form between theHA
HAandand
TiO
TiO2 phases.
2 phases.

Figure 2. XRD pattern of (a) TiO2 ICSD 01-071-1166; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; (f) H7T3
Figure 2. XRD pattern of (a) TiO2 ICSD 01-071-1166; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; (f) H7T3
and; (g) HA ICSD 00-009-0432.
and; (g) HA ICSD 00-009-0432.
The percentage of crystal structure was calculated by the Rietveld refinement method
The
using percentage
HighScore of crystal
Plus softwarestructure was calculated
(PANalytical 3.0.5). Theby the Rietveld
goodness of fit refinement methodto
(GoF) is calculated
using HighScore Plus software (PANalytical 3.0.5). The goodness of fit (GoF)
confirm the accuracy of the Rietveld refinement. Figure S1 shows the Rietveld refinement of is calculated
toXRD
confirm theThe
pattern. accuracy of thethat
results show Rietveld refinement.
the percentage of theFigure S1 shows
phase formed the
in the XRDRietveld
pattern
refinement of XRD pattern. The results show that the percentage of the phase
corresponds to the amount of precursor added (Table 1). In addition, in this calculation, formed in
the XRD pattern corresponds to the amount of precursor added (Table 1).
an accurate GoF value is obtained with a value close to 1 [54]. Then, the crystallinity ofIn addition, in
this
thecalculation,
sample wasan accurate based
calculated GoF value
on theis peaks
obtained with
in the XRDa value close
pattern. Thisto crystallinity
1 [54]. Then,value
the
crystallinity of the sample was calculated based on the peaks in the XRD
is important because it determines its properties in further applications. It is known that pattern. This
crystallinity valuecomposites
HA-TiO2 50:50 is important because
have it determines
the highest its properties
crystallinity comparedin further applications.
to other composites
It(Table
is known that HA-TiO
1). However, H3T7,2 50:50
H4T6,composites
H5T5, and H6T4have composites
the highest had
crystallinity comparedthan
higher crystallinity to
other composites (Table 1). However, H3T7, H4T6, H5T5, and H6T4 composites
pure HA (56%) [44]. High crystallinity will cause the material to be more stable and have had
higher crystallinity
increased mechanicalthanstrength
pure HA (56%)
when [44]. High
applied crystallinity will cause the material to
[55,56].
be more stable and have increased mechanical strength when applied [55,56].
Table 1. Percentage of phase composition, Rietveld refinement parameters, and crystallinity of
the samples.

Crystal Phase (%) * Rietveld Refinement Parameters Crystallinity

Sample
HA TiO2 Rexp Rwp GoF (%)
H3T7 29.3 ± 0.0 70.7 ± 2.2 22.60 16.29 1.92 60
H4T6 40.1 ± 0.0 59.9 ± 2.6 22.59 16.97 1.77 61
H5T5 52.6 ± 0.0 47.4 ± 2.2 21.70 16.63 1.70 63
H6T4 61.2 ± 0.0 38.8 ± 2.8 23.33 17.64 1.75 58
H7T3 73.0 ± 0.0 27.0 ± 1.4 18.52 15.60 1.41 36
* Mean ± standard deviation.
Crystals 2022, 12, 1599 6 of 15

The crystallite size was calculated using the Debye-Scherrer equation. The crystallite
size of HA was calculated from peaks at 2θ = 31.8◦ (211), while TiO2 was calculated from
peaks at 2θ = 25.3◦ (101). In Table 2, it can be seen that the crystallite size of the TiO2
decreases with the increasing amount of TiO2 in the composite. As for the crystallite size
of HA, the highest crystallite size is in the H4T6 composite and tends to decrease in other
composites. However, the difference is not significant. The calculation results show a
relatively large measurement error with the difference between samples that are not too far
apart. The smallest crystallite size for HA is in the H3T7 composite [57]. The crystallinity
size will affect the specific surface area (SSA) properties. SSA is a very important material
property for adsorption, heterogeneous catalysis, and reactions at the surface [58–60]. The
H3T7 composite has the highest SSA value compared to other composites. This SSA value
is directly proportional to the size of the crystallite obtained. The SSA values for HA and
TiO2 crystals are shown in Table 2.

Table 2. Crystallite size and specific surface area (SSA) calculated using Scherrer method from all
peaks of HA and TiO2 .

Crystallite Size (nm) * SSA (m2 /g)

Sample
HA TiO2 HA TiO2
H3T7 32.5 ± 2.9 13.5 ± 2.2 58.4 113.7
H4T6 42.8 ± 3.8 16.5 ± 3.4 44.4 93.0
H5T5 35.7 ± 3.8 13.6 ± 2.3 53.1 112.8
H6T4 39.2 ± 3.6 16.6 ± 3.3 48.6 92.4
H7T3 35.3 ± 7.1 17.5 ± 3.5 54.0 88.4
* Mean ± standard deviation.

The HA crystal from the synthesized composite had a smaller lattice volume as the
amount of TiO2 was added (Table 3). This indicates that TiO2 causes shrinkage of the lattice
volume of HA. This phenomenon complies with another study we found demonstrating
that the presence of TiO2 can decrease the length of the lattice constant in the c direction [32].
The smallest HA lattice volume was obtained on the H3T7 composite with a volume of
528.211 Å3 . In addition, in the anatase TiO2 , the smallest lattice volume value is also shown
in the H3T7 composite, which means that the small amount of TiO2 gives the effect of
decreasing the lattice volume. The volume of the TiO2 anatase lattice in the H3T7 composite
is 135.834 Å3 . The crystal structure of HA and anatase TiO2 was shown in Figure 3.

Table 3. Crystal lattice parameters of the samples.

HA * Anatase TiO2 *
Sample
a = b (Å) c (Å) V (Å3 ) a = b (Å) c (Å) V (Å3 )
H3T7 9.420(3) 6.873(3) 528.211 3.786(1) 9.475(5) 135.834
H4T6 9.422(3) 6.875(2) 528.577 3.785(2) 9.482(6) 135.871
H5T5 9.422(2) 6.875(2) 528.578 3.786(2) 9.477(7) 135.835
H6T4 9.425(2) 6.876(2) 528.981 3.786(2) 9.477(10) 135.878
H7T3 9.428(2) 6.879(2) 529.525 3.788(2) 9.487(8) 136.112
* Values in parentheses represent estimated standard deviations in the last quoted place.

3.2. FTIR Analysis

FTIR analysis was carried out to analyze the functional groups in the HA-TiO2 com-
posite. The FTIR spectrum of HA, TiO2, and variations in the HA-TiO2 are shown in
Figure 4. The peak at wavenumber 3399–3571 cm−1 for all HA-TiO2 composites appears
with a pointed shape indicating the presence of a –OH group in HA [44]. Meanwhile, the
peak at wavenumber 567–1091 cm−1 is a response to the absorption for PO4 3− [61]. In the
wavenumber range of 500–700 cm−1 , peak widening is seen, which is observed as the con-
centration of HA decreases [17]. Meanwhile, the peak at the wavenumber 1420–1458 cm−1
 Crystals 2022, 12, 1599 7 of 15
Crystals 2022, 12, x FOR PEER REVIEW 7 of 15

is characteristic of the presence of a carbonate group (CO3 2− ). Carbonates identified on

H5T5 the FTIR spectrum
9.422(2) can be atmospherically
6.875(2) 528.578 derived3.786(2)
and adsorbed with HA [62,63]. The
9.477(7) 135.835
Figure 3. of
addition Crystal
TiO2 structure
to HA didofnot
HAsignificantly
and anatase show
TiO2. a shift in the FTIR spectrum. There is
H6T4 9.425(2) 6.876(2) 528.981 3.786(2) 9.477(10) 135.878
no bond between TiO2 and HA, but that only indicates the presence of HA and TiO2 that
H7T3 9.428(2) 6.879(2) 529.525 3.788(2)
the formation of9.487(8) 136.112
3.2. FTIR
do not bindAnalysis
to each other, which is a characteristic of composites of both.
* Values2in
HA-TiO parentheses
composite represent
FTIR estimated
spectrum data for standard deviations
ratio variations in the
can be seenlast quoted
in Table 4.place.
FTIR analysis was carried out to analyze the functional groups in the HA-TiO2 com-
posite. The FTIR spectrum of HA, TiO2, and variations in the HA-TiO2 are shown in Figure
4. The peak at wavenumber 3399–3571 cm–1 for all HA-TiO2 composites appears with a
pointed shape indicating the presence of a –OH group in HA [44]. Meanwhile, the peak
at wavenumber 567–1091 cm–1 is a response to the absorption for PO43– [61]. In the wave-
number range of 500–700 cm–1, peak widening is seen, which is observed as the concen-
tration of HA decreases [17]. Meanwhile, the peak at the wavenumber 1420–1458 cm–1 is
characteristic of the presence of a carbonate group (CO32–). Carbonates identified on the
FTIR spectrum can be atmospherically derived and adsorbed with HA [62,63]. The addi-
tion of TiO2 to HA did not significantly show a shift in the FTIR spectrum. There is no
bond between TiO2 and HA, but that only indicates the presence of HA and TiO2 that do
not bind to each other, which is a characteristic of the formation of composites of both
HA-TiO 2 composite FTIR spectrum data for ratio variations can be seen in Table 4.
Figure 3. Crystal structure of HA and anatase TiO .2
Figure 3. Crystal structure of HA and anatase TiO2.

3.2. FTIR Analysis

FTIR analysis was carried out to analyze the functional groups in the HA-TiO2 com-
posite. The FTIR spectrum of HA, TiO2, and variations in the HA-TiO2 are shown in Figure
4. The peak at wavenumber 3399–3571 cm–1 for all HA-TiO2 composites appears with a
pointed shape indicating the presence of a –OH group in HA [44]. Meanwhile, the peak
at wavenumber 567–1091 cm–1 is a response to the absorption for PO43– [61]. In the wave-
number range of 500–700 cm–1, peak widening is seen, which is observed as the concen-
tration of HA decreases [17]. Meanwhile, the peak at the wavenumber 1420–1458 cm–1 is
characteristic of the presence of a carbonate group (CO32–). Carbonates identified on the
FTIR spectrum can be atmospherically derived and adsorbed with HA [62,63]. The addi-
tion of TiO2 to HA did not significantly show a shift in the FTIR spectrum. There is no
bond between TiO2 and HA, but that only indicates the presence of HA and TiO2 that do
not bind to each other, which is a characteristic of the formation of composites of both
HA-TiO2 composite FTIR spectrum data for ratio variations can be seen in Table 4.

Figure
Figure 4.4.Infrared
Infraredspectra
spectra of (a)
of (a) TiOTiO 2; H3T7;
2 ; (b) (b) H3T7; (c) H4T6;
(c) H4T6; (d)(e)
(d) H5I; H5I; (e) (f)
H6T4; H6T4; (f)and
H7T3; H7T3; and (g) HA.
(g) HA.

Table 4. Types of vibrations in HA, TiO2 , and HA/TiO2 composites based on the peaks that appear
in each wavenumber.

Vibration Wavenumber (cm−1 )

Ref.
Type HA TiO2 H3T7 H4T6 H5T5 H6T4 H7T3
Ti–O–Ti - 800–450 800–450 800–450 800–450 800–450 800–450 [64]
PO4 3− 1095–472 - 1091–744 1089 567 1089–600 1089–570 1089–567 [61]
O–H (HA) 3573 - 3571–3399 3570–3419 3571–3400 3571–3435 3571–3413 [44]
OH free - 1635 1635 1635 1634 1636 1639 [64]
CO3 2− 1456–1403 - 1456–1403 1456–1404 1456–1404 1456–1407 1456–1410 [62,63]

Figure 4. Infrared spectra of (a) TiO2; (b) H3T7; (c) H4T6; (d) H5I; (e) H6T4; (f) H7T3; and (g) HA.
 Vibration Type Ref.
HA TiO2 H3T7 H4T6 H5T5 H6T4 H7T3
Ti–O–Ti - 800–450 800–450 800–450 800–450 800–450 800–450 [64]
PO43– 1095–472 - 1091–744 1089 567 1089–600 1089–570 1089–567 [61]
O–H (HA) 3573 - 3571–3399 3570–3419 3571–3400 3571–3435 3571–3413 [44]
Crystals 2022, 12, 1599 8 of 15
OH free - 1635 1635 1635 1634 1636 1639 [64]
CO32– 1456–1403 - 1456–1403 1456–1404 1456–1404 1456–1407 1456–1410 [62,63]

3.3.
3.3.SEM
SEMAnalysis
Analysis
Figure
Figure55shows
showsthe themorphology
morphologyof ofpure
pureHA
HAfromfromeggshells
eggshellsandandHA-TiO
HA-TiO22 composite.
composite.
The surface structure of HA samples from chicken eggshells has a surface
The surface structure of HA samples from chicken eggshells has a surface shape shape in the in
form
the
of agglomeration of fine sub-particles. After adding TiO , relatively less agglomeration
form of agglomeration of fine sub-particles. After adding TiO2, relatively less agglomera-
2
occurs. All composite
tion occurs. morphologies
All composite have spherical
morphologies and porous
have spherical particleparticle
and porous shapes.shapes.
The entire
The
composite sample looks inhomogeneous to each other. In addition, in
entire composite sample looks inhomogeneous to each other. In addition, in each compo- each composite
sample, therethere
site sample, is a very
is a large
very particle size that
large particle sizeisthat
agglomerated and fused
is agglomerated and with
fusedeach
withother.
each
The addition of TiO 2 led to an increase in the density of the composite layer [53,57].
other. The addition of TiO2 led to an increase in the density of the composite layer [53,57].

Figure 5. SEM image of (a) HA from chicken eggshell; (b) H3T7; (c) H4T6; (d) H5T5; (e) H6T4; and
(f) H7T3.

From the particle size analysis using ImageJ 1.52a software (Figures S2 and S3) [65],
all composites have different particle sizes between 90–1000 nm (Figure 6). Data regarding
the value of the mean, mode, median, standard deviation, and polydispersity index (PI)
are shown in Table 5. The amount of addition of TiO2 to the composite causes the average
particle size to be smaller. Mode values of each composite variation H3T7, H4T6, H5T5,
H6T4, and H7T3 were 90.8, 193.7, 194.1, 196.5, and 193.7 nm, respectively.
 (f) H7T3.
From the particle size analysis using ImageJ 1.52a software (Figures S2 and S3) [65],
all composites have different particle sizes between 90–1000 nm (Figure 6). Data regarding
the value of the mean, mode, median, standard deviation, and polydispersity index (PI)
are shown in Table 5. The amount of addition of TiO2 to the composite causes the average
Crystals 2022, 12, 1599 9 of 15
particle size to be smaller. Mode values of each composite variation H3T7, H4T6, H5T5,
H6T4, and H7T3 were 90.8, 193.7, 194.1, 196.5, and 193.7 nm, respectively.

Figure 6.6.Particle
Figure Particlesize
sizedistribution
distribution from
from SEM
SEM image
image using
using ImageJ
ImageJ 1.52a1.52a software
software of (a) of (a) H3T7;
H3T7; (b)
(b) H4T6;
H4T6;
(c) (c) (d)
H5T5; H5T5;
H6T4;(d)and
H6T4;(e) and (e) H7T3.
H7T3.

Table 5. Statistical data calculation from particle size distribution using ImageJ from SEM image.

Measurement Parameters
Sample
Mean (nm) Mode (nm) Median (nm) Standard deviation (nm) PI
H3T7 220.4 90.8 194.1 143.9 1.42
H4T6 344.2 193.7 293.7 223.1 1.42
H5T5 313.5 194.1 294.2 176.7 1.32
H6T4 292.2 196.5 290.1 167.7 1.33
H7T3 353.6 193.7 296.9 196.7 1.31

Furthermore, it is known that if the value of the polydispersity index (PI) is more than 1,
it indicates that the sample has a wide nanoparticle distribution and non-uniform nanoparti-
cle diameter size [66]. It is very difficult to make particles of uniform size (monodispersion).
The PI values for the H3T7, H4T6, H5T5, H6T4, and H7T3 composites were 1.42, 1.42, 1.32,
1.33, and 1.31, respectively. The H3T7 composite has the smallest average particle size. It is
 nanoparticle diameter size [66]. It is very difficult to make particles of uniform size (m
odispersion). The PI values for the H3T7, H4T6, H5T5, H6T4, and H7T3 composites w
1.42, 1.42, 1.32, 1.33, and 1.31, respectively. The H3T7 composite has the smallest aver
particle size. It is understood that the amount of TiO2 affects the sample size, whereas
Crystals 2022, 12, 1599 10 of 15
addition of TiO2 can cause a decrease in particle size. Size reduction can increase the re
rate for electrons and holes during surface photocatalytic processes. It also decreases p
toelectron and hole recombination thereby increasing reactivity [66].
understood that the amount of TiO affects the sample size, whereas the addition of TiO
2 2
can cause a decrease in particle size. Size reduction can increase the redox rate for electrons
3.4.holes
and Antibacterial Activity
during surface photocatalytic processes. It also decreases photoelectron and hole
recombination thereby increasing
The results obtained from reactivity [66].
this antibacterial test are inhibitory diameter which can
seen
3.4. in FigureActivity
Antibacterial 7. The inhibition zone showed bactericidal activity on the Gram-posi
bacteria (S. aureus) and Gram-negative bacteria (E. coli). The highest inhibition zon
The results obtained from this antibacterial test are inhibitory diameter which can be
found
seen in the7.H3T7
in Figure composite,
The inhibition zonewhere
showedGram-positive bacteria
bactericidal activity on theare more extensive t
Gram-positive
Gram-negative
bacteria (S. aureus)bacteria. These results
and Gram-negative correspond
bacteria to previous
(E. coli). The studies that
highest inhibition zonereported
is
the antibacterial
found in the H3T7 activity
composite,of where
the HA-TiO 2 composite
Gram-positive is more
bacteria pronounced
are more against Gr
extensive than
Gram-negative
positive bacteria bacteria.
due toThese
the results correspond
plasmolysis of thetocell
previous
wall orstudies that reported
the separation ofthat
the cytopla
the antibacterial activity of the HA-TiO2 composite is more pronounced against Gram-
from the cell wall [40]. Some studies have reported that Gram-negative bacteria are u
positive bacteria due to the plasmolysis of the cell wall or the separation of the cytoplasm
ally more resistant. This resistance is associated with the cell walls of Gram-negative b
from the cell wall [40]. Some studies have reported that Gram-negative bacteria are
teria that
usually moreareresistant.
more complex than those
This resistance of Gram-Positive
is associated with the cellbacteria. However, under cer
walls of Gram-negative
conditions,
bacteria Gram-negative
that are more complexbacteria canofbeGram-Positive
than those more resistant to chemical
bacteria. agents
However, than to Gr
under
positive
certain bacterialGram-negative
conditions, cells [67,68].bacteria
Another canreason
be moreisresistant
that the to external membrane
chemical agents than of Gr
to Gram-positive
negative bacteriabacterial cells
consists [67,68].ofAnother
mainly a strongreason
layeris of
that the external membrane
lipopolysaccharides, of
which are c
Gram-negative bacteria consists mainly of a strong layer of lipopolysaccharides,
sidered a barrier. Thus, Gram-positive bacteria have a high sensitivity due to differen which are
considered a barrier. Thus, Gram-positive bacteria have a high sensitivity due to differences
in the composition of their cell walls compared to Gram-negative bacteria [69,70].
in the composition of their cell walls compared to Gram-negative bacteria [69,70]. The
antibacterial
antibacterial test
test image
image can can be seen
be seen in Figure
in Figure S4. S4.

Figure 7. Inhibition zone test of HA-TiO2 composite against Escherichia coli and Staphylococcus
aureus bacteria.

Antibacterial testing on TiO2 does not produce an inhibitory zone. In contrast to TiO2 ,
HA in the test produces an inhibitory zone, this is because HA can adsorb bacteria, in other
words, HA deactivates bacteria by an adsorption mechanism. Adhered bacteria are believed
to be killed due to the disruption of their cell membrane by the composites, which reach
across the microbial membrane [71]. However, the combination of compounds, namely the
HA-TiO2 composite, makes the inhibition zone vary. Since this antibacterial activity is a
surface reaction [72,73], this activity is determined by surface area and particle size. Samples
with higher surface area and small particle sizes showed greater antibacterial activity.
After that, the composite antibacterial activity test was carried out using the optical
density method. The positive control used was amoxicillin and the negative control was
S. aureus and E. coli without the addition of composites. Amoxicillin was chosen as a positive
control in the antibacterial activity test because amoxicillin is an antibiotic that is widely
 activity is a surface reaction [72,73], this activity is determined by surface area and particle
size. Samples with higher surface area and small particle sizes showed greater antibacte-
rial activity.
After that, the composite antibacterial activity test was carried out using the optical
Crystals 2022, 12, 1599 density method. The positive control used was amoxicillin and the negative control 11 wasof 15
S. aureus and E. coli without the addition of composites. Amoxicillin was chosen as a pos-
itive control in the antibacterial activity test because amoxicillin is an antibiotic that is
widely
used in usedthe in the treatment
treatment of various
of various infectious
infectious diseases diseases
causedcaused by pathogenic
by pathogenic bacteria
bacteria [74]. In
[74]. In this study, the difference between the OD method
this study, the difference between the OD method and the disc diffusion method and the disc diffusion method
is that
isin
that
theinODthe method,
OD method, lightlight is used
is used to initiate
to initiate thethe photocatalytic
photocatalytic process.
process. Figure
Figure 8 shows
8 shows the
the photocatalyticantibacterial
photocatalytic antibacterialactivity activitybased
basedon on the
the percentage
percentage of of inhibition
inhibitionofofE.E.colicoliand
and
S.S.aureus
aureusbacteria.
bacteria.All Allcomposites
compositesapplied applied to to each bacterium were
each bacterium wererecorded
recordedinintime timeintervals.
inter-
vals.
Based Based on the
on the testtest
results,results,
the the antibacterial
antibacterial activity
activity of the
of the composite
composite waswas higher
higher thanthanpure
pure HA, pure TiO , and the positive control of amoxicillin. The
HA, pure TiO2 , and the positive control of amoxicillin. The greatest inhibition was obtained
2 greatest inhibition was
obtained
from thefrom H3T7the H3T7 composite
composite for E. colifor E. coli In
bacteria. bacteria.
addition,In the
addition, the H3T7 composite
H3T7 composite also has the
also has the
smallest smallest
particle sizeparticle
and thesize and specific
largest the largest specific
surface area,surface
therebyarea, therebythe
increasing increasing
active site
the
and active
surfacesitereactivity.
and surface H3T7 reactivity.
composites H3T7 havecomposites have high
high antibacterial antibacterial
activity comparedactivity
to other
compared
composites, to other
this iscomposites,
supportedthis is supported
by XRD by XRD
data showing data showing
a smaller a smaller
crystalline crystal-
size and high
line size area,
surface and highand by surface
SEM data area,which
and by SEMsmaller
shows data which shows
particle sizes smaller
[75,76]. A particle sizes
large specific
[75,76].
surfaceAarea largewillspecific
lead to surface area will
an increase in thelead to ansite
active increase in the reactivity
and surface active site[72].
and However,
surface
for S. aureus
reactivity [72]. bacteria,
However,the forbest antibacterial
S. aureus bacteria,activity
the best was shown by activity
antibacterial the H7T3 wascomposite
shown
byfollowed
the H7T3 bycomposite
H3T7. This is in accordance
followed by H3T7.with Thistheis inantibacterial
accordancedata withusing the disk diffusion
the antibacterial data
method
using that the
the disk H3T7 method
diffusion and H7T3 thatcomposites
the H3T7 and provide
H7T3 the largest zone
composites of inhibition.
provide the largest The
growth
zone of bacteria The
of inhibition. decreasedgrowth drastically
of bacteria overdecreased
the period.drastically
This studyoverdemonstrates
the period.thatThis
both
materials’
study synergistic
demonstrates thateffect
bothin a short time
materials’ is well proven.
synergistic effect in a short time is well proven.

Figure 8. Optical density measurements of bacteria at a wavelength of 600 nm (OD 600) for (a) Es-
cherichia
Figure coli; and
8. Optical (b) Staphylococcus
density measurementsaureus.
of bacteria at a wavelength of 600 nm (OD 600) for (a) Esch-
erichia coli; and (b) Staphylococcus aureus.
4. Conclusions
HA-TiO2 composites from eggshells have been successfully synthesized using the
4. Conclusions
hydrothermal method. It can be concluded that the composition of the HA-TiO2 composite
HA-TiO2 composites from eggshells have been successfully synthesized using the
from eggshell affects the crystallinity, crystal size, specific surface area, and particle dis-
hydrothermal method. It can be concluded that the composition of the HA-TiO2 composite
tribution. The H3T7 composite had the smallest crystallite size and the highest specific
from eggshell affects the crystallinity, crystal size, specific surface area, and particle dis-
area, namely 32.5 ± 2.9 nm and 58.4 m2 /g for HA, and 13.5 ± 2.2 nm and 113.7 m2 /g
tribution. The H3T7 composite had the smallest crystallite size and the highest specific
for TiO2 , respectively. The FTIR spectrum shows that there is no chemical bond formed
between TiO2 and HA which indicates the formation of a composite. The morphology
of the HA-TiO2 composite has an irregular particle shape, and agglomerates, and is not
homogeneous for all compositions. The size distribution shows that the H3T7 composite
has the smallest particle size. This indicates that the addition of TiO2 to HA significantly
reduces the particle size.
Antibacterial activity using the disk diffusion method showed that the H3T7 composite
had the highest antibacterial activity against Staphylococcus aureus and Escherichia coli
bacteria compared to other composites. In addition, the optical density (OD) method also
showed that all composites had better antibacterial activity than pure HA and pure TiO2 .
These results indicate that the synergistic combination of HA from eggshell with TiO2 has
favorable properties for antibacterial activity.
Crystals 2022, 12, 1599 12 of 15

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/cryst12111599/s1, Figure S1: Rietveld refinement of XRD pattern
of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3; Figure S2: SEM image with ×10,000
magnification of (a) H3T7; (b) H4T6; (c) H5T5; (d) H6T4; and (e) H7T3; Figure S3: SEM Image
after processing in ImageJ 1.52a software for particle distribution analysis of (a) H3T7; (b) H4T6;
(c) H5T5; (d) H6T4; and (e) H7T3; Figure S4: Antibacterial test results against Escherichia coli (1) and
Staphylococcus aureus (2) bacteria using disc diffusion method of (a) HA; (b) TiO2 ; (c) H3T7; (d) H4T6;
(e) H5T5; (f) H6T4; and (g) H7T3.
Author Contributions: Conceptualization, A.R.N. and D.R.E.; methodology, E.N.A.; software, E.N.A.
and M.D.P.; validation, A.R.N. and D.R.E.; formal analysis, E.N.A.; D.D. and M.D.P.; investigation,
E.N.A.; D.D. and M.D.P.; resources, E.N.A.; data curation, E.N.A.; writing—original draft prepa-
ration, E.N.A.; writing—review and editing, E.N.A. and M.D.P.; visualization, E.N.A. and M.D.P.;
supervision, A.R.N. and D.R.E.; project administration, A.R.N. and S.; funding acquisition, A.R.N.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Riset Data Pustaka dan Daring (RDPD) 2021, Universitas
Padjadjaran, grant number 1959/UN6.3.1/PT.00/2021.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank to Kiki Maesaroh for preparing the materials. We also thank
to Annisa Luthfiah and Lintang Kumoro Sakti for discussing the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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