MATERIALS AND PRODUCT ENGINEERING

Chinese Journal of Chemical Engineering, 22(4) 474—479 (2014)

DOI: 10.1016/S1004-9541(14)60063-X

Synthesis and Antimicrobial Activity of Boron-doped Titania

Nano-materials*

WANG Yuzheng (王昱征)1,2,3,4, XUE Xiangxin (薛向欣)1,2,3,4,** and YANG He (杨合)1,2,3,4

1
Institute of Metallurgical Resources and Environmental Engineering, Northeastern University, Shenyang 110819, China
2
Liaoning Key Laboratory of Metallurgical Resources Recycling Science, Shenyang 110819, China
3
Liaoning Engineering and Technology Research Center of Boron Resources Comprehensive Utilization, Shenyang
110819, China
4
Liaoning Provincial Universities Key Laboratory of Boron Resources Ecological Utilization Technology and Boron
Materials, Shenyang 110819, China

Abstract Antibacterial activity of boron-doped TiO2 (B/TiO2) nano-materials under visible light irradiation and in
the dark was investigated. A simple sol-gel method was used to synthesize TiO2 nano-materials. X-ray diffraction
pattern of B/TiO2 nano-materials represents the diffraction peaks relating to the crystal planes of TiO2 (anatase and
rutile). X-ray photoelectron spectroscopy result shows that part of boron ions incorporates into TiO2 lattice to form
a possible chemical environment like Ti O B and the rest exist in the form of B2O3. The study on antibacterial
effect of B/TiO2 nano-materials on fungal Candida albicans (ATCC10231), Gram-negative Escherichia coli
(ATCC25922) and Gram-positive Staphylococcus aureus (ATCC6538) shows that the antibacterial action is more
significant on Candida albicans than on Escherichia coli and Staphylococcus aureus. Under visible light irradiation,
the antibacterial activity is superior to that in the dark.
Keywords boron doping, titania, antimicrobial activity

1 INTRODUCTION more attentions in the application studies of functional

Contamination by microorganisms is of great
concern in a variety of areas, such as medical devices,
healthcare products, water purification systems, hos-
pitals, dental office equipment, food packaging, food
storage, and household sanitation [1-3]. It is essential
to have appropriate antibacterial method. Photocataly-
sis is a promising technology based on the interaction
between light and solid semiconductor particles and is
able to produce highly oxidative species that destroy
bacteria and a large variety of chemical contaminants.
Among the photoactive semiconductors are TiO2, ZnO,
Fe2O3, WO3 and CdSe. TiO2 is most widely used in
different media as photocatalyst because of its high
stability, low cost and wide availability [4-6]. Matsu-
naga et al. [7] reported for the first time the microbio-
cidal effect of TiO2 photocatalytic reactions. Since
then, the research on photocatalytic killing of TiO2 has
been intensively conducted on a wide spectrum of
organisms including viruses, bacteria, fungi, algae and
cancer cells. From these experiments it is unclear as to
the primary mechanism for toxicity, with three possible
candidates: (i) oxidation of phospholipid membranes,
(ii) oxidation of Coenzyme A or (iii) direct cleavage of
DNA. The latter two mechanisms are reliant upon par-
ticulate TiO2 entering the cell structure [8].
However, the wide band gap of TiO2 greatly lim-
its its applications. Photocatalysts exhibiting reactivity
under visible light (λ>400 nm) could be obtained by
non-metal doping such as nitrogen [9], sulfur [10], car-
bon [11], fluorine [12] and boron [13, 14]. Among these
anion doped TiO2, boron doping TiO2 has attracted
materials due to its prompting creation of electron ac-
ceptor level. Zaleska et al. [15] synthesized boron modi-
fied TiO2 using boric acid and boric acid triethyl ester
by the sol-gel method and by grinding anatase powder
with boron dopant. They found that boron doping
could result in absorption of the visible light and these
B-TiO2 samples had higher activity for photo-oxidation of
phenol under the visible light irradiation than pure TiO2.
Boric acid is a well-known antibacterial and
anti-fungal agent, inhibiting a variety of bacteria and
fungi. However, boric acid is low melting and boiling,
so it cannot be directly applied in high temperature
products. This greatly limits its applications in antim-
icrobial material. No work has been reported on bo-
ron-doped TiO2 in antimicrobial field.
In this study, boron-doped TiO2 nano-materials are
prepared by a sol-gel method. Their antibacterial effect
is examined against Candida albicans, Escherichia coli
and Staphylococcus aureus. These microorganisms are
selected because of their relatively diverse make up in
terms of biological structure, in particular the cell walls
of the organisms, with high resistance to conventional
antibacterial techniques. Our aim is to better under-
stand the interaction of boron with anatase and poten-
tial effects in the antimicrobial ability.
2 EXPERIMENTAL
2.1 Preparation of boron-doped TiO2 nano-materials
The boron doped TiO2 was synthesized by sol-gel
method. 10 ml tetrabutyl titanate was dissolved into

Received 2012-10-19, accepted 2013-02-27.

* Supported by the National Natural Science Foundation of China (51090384).
** To whom correspondence should be addressed. E-mail: xuexx@mail.neu.edu.cn
 Chin. J. Chem. Eng., Vol. 22, No. 4, April 2014
35 ml anhydrous ethanol (solution A), and solution B
consisted of 35 ml anhydrous ethanol, 4 ml acetic acid,
10 ml water, and 0.61 g boric acid. Then solution A
was added drop-wise to solution B under magnetic
stirring. The resultant mixture was stirred at room
temperature for 3 h until the transparent sol was ob-
tained. The sol was then aged for 1 day and the gel
was obtained. The gel was dried at 80 °C for 24 h, and
the dried powder was calcinated at various temperatures
(600, 700, 800 and 900 °C) at a heating rate of 5 °C
per minute and held at these temperatures for 1 h, then
cooled down naturally. The resulting materials were
labeled as B/TiO2-x, where x denotes the calcination
temperature (°C).
2.2 Characterization
X-ray diffractometry (XRD; Shimadzu) equipped
with a copper target ( λKα = 0.1541874 nm) was used
1
to identify the formation phase of the powder samples.
The morphology and crystallite size of the powder
were taken with TECNAI G2 F20 transmission electron
microscope (TEM). The binding energy was identified
by X-ray photoelectron spectroscopy (XPS) with Mg
Kα radiation (ESCALAB250). The BET surface area
was determined by N2 adsorption on a Quantachrome
NOVA 1200e apparatus at 77 K.
2.3 Determination of antimicrobial effect
Candida albicans (ATCC 10231), Escherichia
coli (ATCC 25922) and Staphylococcus aureus (ATCC
6538) were chosen as the bacteria in the antibacterial
examinations. Antibacterial performance was evalu-
ated by inhibition ring method. All glassware and ma-
terials were autoclaved at 120 °C for 15 min to ensure
the sterility for testing. Microorganisms were inocu-
lated and grew aerobically in 25 ml liquid nutrient
broth at 37 °C on a rotary shaker (120 r·min−1) for 18
h. They were adjusted to a concentration of 104-105
CFU·ml−1 in the antibacterial assay.
The experiments were carried out under two irra-
diation conditions: visible light and dark. The visible
light source was 15 common fluorescent tubes, mounted
on the inside of an artificial climate box (with total
power of 15×18 W). The experimental procedures
were as follows. Inhibition ring method was carried
out by pouring agar into Petri dishes to form 4 mm
thick layers and the Petri dishes were left for 10 min
to dry in the air. Dense inoculum of the tested micro-
organisms was added to Petri dishes, and then the
compacted powder (0.70 g of samples, 14 mm in di-
ameter) was arranged on the agar surface and incubated
at 37 °C for 24 h in the artificial climate box under the
visible light or in the dark. A vernier caliper was
adopted to measure the diameter of the width of inhi-
bition zone (mm).
475
3 RESULTS AND DISCUSSION
3.1 XRD analysis
Figure 1 shows the XRD patterns of boron-doped
TiO2 nano-materials with different calcination tem-
peratures. As the calcination temperature increases
from 600 to 800 °C, a diffraction peak corresponding
to the (101) plane (peak 25.3°) of anatase phase ap-
pears. The anatase is predominant in crystalline phases
of all calcinated samples. A small amount of rutile
(peak 27.4°) appears in B/TiO2 at 700 °C. It demon-
strates that the phase transformation from anatase to
rutile occurs at 700 °C, which is consistent with that
reported in literature [13]. At calcination temperature
of 900 °C, anatase phase nearly disappears, rutile
phase Ti0.924O2 is predominant and a small amount of
rutile TiB0.024O2 is present. The ratio between anatase
and rutile extracted from XRD spectra, which is often
used to quantify the anatase-to-rutile transformation,
is calculated with the empirical relationship:
R(T ) = 1/[1 + 0.8 ( I A / I R )] (1)
where R(T) is the percentage content of rutile at tem-
perature T, IA is the intensity for the main anatase re-
flection, and IR is the intensity for the main rutile re-
flection. Table 1 presents basic characteristic parame-
ters of the antimicrobial materials. It is well known
that the crystalline and the crystal phase are crucial
factors in the antimicrobial activity of TiO2, where the
crystalline anatase phase is considered as the most
active form of TiO2 [16], while rutile and amorphous
TiO2 are believed to be relatively inactive.
Figure 1 also shows that the intensity and width
of the diffraction peaks of anatase become higher and
narrower as the calcination temperature increases from
600 to 900 °C, suggesting that the crystalline of anatase
is greatly improved. According to the line width analy-
sis of anatase (101) based on the Scherrer equation,
the average grain sizes of the samples are estimated to
be about 18 nm, 48 nm and 73 nm for B/TiO2-600,
B/TiO2-700, B/TiO2-800, respectively. Apparently, the
calcination temperature has a significant effect on the
grain size of boron-doped titania particles.
Figure 1 XRD patterns of B/TiO2 calcinated at different
temperatures
■ anatase TiO2; ▲ rutile TiO2; ● rutile TiB0.024O2; ★ rutile Ti0.924O2
476 Chin. J. Chem. Eng., Vol. 22, No. 4, April 2014

Table 1 Basic characteristic parameters of the antimicrobial materials

Catalysts Anatase phase percentage/% Rutile phase percentage/% BET surface/m2·g−1 XPS-determined boron content/%
B/TiO2-600 100 0 49.215 15.08
B/TiO2-700 99 1 14.994 13.96
B/TiO2-800 33 67 3.895 16.49
B/TiO2-900 2 98 2.604 13.21

Figure 2 TEM images of B/TiO2-600 (a) and SADP of B/TiO2-600 (b)

(a) B/TiO2-600 (b) B 1s regions

Figure 3 XPS spectra of B/TiO2-600

3.2 TEM analysis 3.3 XPS analysis

Figure 2 shows the TEM images of the B-doped
TiO2 sample calcinated at 600 °C. The morphology,
crystallite size and crystallographic planes of the par-
ticles are observed. The images show that the sample
consists of large number of small particles with the
size around 20 nm, which is in agreement with the
XRD results calculated by the Scherrer equation. A
corresponding selected area electron diffraction pat-
tern (SADP) shows the Debye rings, exhibiting a
polycrystalline nature of the particle. The Debye rings
with d-values corresponding to 0.3512 nm (marked as
101), 0.2293 nm (marked as 004), 0.1841 nm (marked
as 200), 0.1626 nm (marked as 211), 0.1442 nm
(marked as 204), and 0.1297 nm (marked as 215) are
assigned to the B/TiO2 anatase phase.
Figure 3 (a) shows the XPS spectra of
B/TiO2-600. It contains only Ti, O, B, and C elements.
Element C can be ascribed to the residual carbon from
the precursor. Fig. 3 (b) shows the XPS B 1s spectrum
of B/TiO2-600, which appears at around 191-193 eV.
Based on previous study reported [13], the standard
binding energy of B 1s in B2O3 or H3BO3 equals 193.0
eV (B O bond) and that in TiB2 equals 187.5 eV
(B Ti bond). The observed B 1s peak consists of two
peaks. The first peak (191.55 eV) is related to
Ti O B bonds and the second peak (193.28 eV) is
related to B O B bonds. XPS analysis confirms that
boron ions partially incorporate into TiO2 lattice after
heat treatment to form a possible chemical environ-
ment like Ti O B and the rest exist in the form of
 Chin. J. Chem. Eng., Vol. 22, No. 4, April 2014 477

Figure 4 Antimicrobial experiments of B/TiO2 and pure TiO2 nano-materials under visible light irradiation among different
strains. (a) Candida albicans; (b) Staphylococcus aureus; (c) Escherichia coli; (d) B/TiO2 nano-materials on Candida albicans
in the dark

B2O3. This is probably because the concentration of
doped boron is beyond its solubility limit in the TiO2
anatase structure. The boron ions expelled from the
anatase structure could form nanoclusters on the sur-
face of TiO2 nanoparticles and grow slowly [13]. The
B contents are given in Table 1. The influence of cal-
cination temperature on boron content can be almost
ignored. The average boron content in the surface
layer is 14.5%.
3.4 Study of antimicrobial effect of boron ions
doped nano-TiO2
We tested the antimicrobial efficiency of B/TiO2
by checking the ability of attached bacteria to form
colonies in agar. Bacteria attached to a surface are able
to duplicate, move and form colonies beyond the
biofilm. Bacteria from the biofilm formed on the TiO2
surface move and colonize the surrounding agar area
forming a halo. From growing halo assays it is possi-
ble to have an indication about the viability of the
bacteria attached to the surface [17].
The antimicrobial results are given in Fig. 4.
Formation of inhibition zone confirms that all the
B-doped TiO2 nano-materials have antimicrobial ac-
tivity, while pure TiO2 nano-materials do not have.
However, antimicrobial abilities of B-doped TiO2
nano-materials prepared at different calcination tem-
peratures are not the same. Calcinated at 600 and 700 °C,
B/TiO2 shows higher antimicrobial activity. B/TiO2-800
and B/TiO2-900 have moderate activity. Fig. 4 (d) shows
that antimicrobial activity of B/TiO2 nano-materials
on Candida albicans in the dark is weaker than that in
the visible light and the average inhibition zones
against Candida albicans are 8.28, 7.31, 6.59, and
4.30 mm for B/TiO2-600, B/TiO2-700, B/TiO2-800, and
B/TiO2-900, respectively.
478 Chin. J. Chem. Eng., Vol. 22, No. 4, April 2014
Figure 5 Antimicrobial experiment of B/TiO2 nano-materials
For different strains, antimicrobial abilities of
B/TiO2 nano-materials are different (see Fig. 5).
B/TiO2 nano-materials present about 16 mm average
inhibition zone against Candida albicans, 10 mm zone
against Escherichia coli and 7 mm zone against
Staphylococcus aureus. We can conclude that the an-
timicrobial action of the B/TiO2 is more significant on
fungal than on Gram-negative and Gram-positive
bacteria. In one of the most important publications
related to TiO2 [18, 19], the reduction efficiency of
TiO2 coated Plexiglass depends on the cell wall thick-
ness, with Gram-negative microorganisms killed eas-
ily and Gram-positive microorganisms more resistant.
C. albicans is highly resistant to photocatalytic degra-
dation due to its thick eukaryotic cell wall, while in
our study, the antibacterial effect of boron-doped TiO2
nano-materials on C. albicans is the best. This is a
promising result especially for simple preparation
route with incorporation of nano-materials.
In general, the bactericidal effect of TiO2 is at-
tributed to the decomposition of outer membranes of
bacteria by reactive oxygen species, primarily hy-
droxyl radicals, which leads to phospholipid peroxida-
tion and ultimately cell death. It is proposed that
nano-materials that can physically attach to a cell are
bactericidal when they contact with this cell. If the
membrane of a bacterium is compromised, the cell
may repair itself; if the scratch is severe, the cell
component may release and the cell will die eventu-
ally [20, 21].
The boron-doped TiO2 nano-particles under visi-
ble light have strong antimicrobial activity, probably
due to the following points. (1) XPS show that part of
B exists in the form of Ti O B with boron atoms
incorporated into the TiO2 lattice, so B p and O 2p
form with mixed valence band and the band gap nar-
rows, leading to visible light response [22]. (2) Due to
B3+ electron deficient character, Lewis acid on the
B/TiO2 surface enhances and OH− increases. OH− can
capture electron hole pair and transform into free
groups (·OH), causing the rupture of cell membrane
[23]. (3) B atoms incorporated into TiO2 lattice result
in a charge imbalance and lattice distortion, so that
TiO2 generates surface oxygen vacancy, which also
improves TiO2 antimicrobial activity [24].
4 CONCLUSIONS
In this study, highly effective boron-doped TiO2
antimicrobial materials were synthesized by a sol-gel
method. The anatase is predominant in crystal planes
of the TiO2 calcinated at 600 and 700 °C, and rutile
phases are predominant at calcination temperature of
900 °C. Part of boron ions incorporate into TiO2 lat-
tice, forming a possible chemical environment like
Ti O B and the rest exist in the form of B2O3. The
B/TiO2 nano-materials exhibit high antibacterial effi-
ciency. Their antibacterial action is more significant
on fungal than on Gram-negative and Gram-positive.
Antimicrobial activities of B/TiO2 materials on Can-
dida albicans in the dark are weaker than that in the
visible light.
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