Facile fabrication of ZrO2 hollow porous microspheres with yeast as bio-templates

Article abstract of DOI:10.1016/j.materresbull.2011.03.024

ZrO₂ hollow porous microspheres have been fabricated successfully using living yeast cells as bio-templates through a facile ageing process at lower crystallizing temperature. XRD, SEM, FT-IR and N₂ adsorption-desorption were used to characterize ZrO₂ hollow microspheres. The results showed that the inorganic-organic hybrid hollow microspheres were fabricated at 100 °C and ZrO₂ hollow microspheres with tetragonal phase and porous structure were obtained at 300 °C. The crystallization temperature of ZrO₂ decreased obviously due to the inducting of bio-molecules. The as-prepared hollow microspheres are approximately ellipsoid. The shells of these hollow microspheres are porous, which are composed of some nanoparticles with size of ～20 nm. The formation mechanism of ZrO₂ hollow microspheres was proposed and discussed tentatively.

Full text of DOI:10.1016/j.materresbull.2011.03.024

Materials Research Bulletin 46 (2011) 1315–1319
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Materials Research Bulletin
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Short communication
Facile fabrication of ZrO2 hollow porous microspheres with yeast as
bio-templates
Xiaojuan Fan a, Xiuqin Song a, , Xiaohui Yang b,c, Lixue Hou a
*
a College of Chemistry and Materials Science, Hebei Normal University, Shijiazhuang 050016, China
b Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001, China
c Shijiazhuang University, Shijiazhuang 050801, China
A R T I C L E I N F O
A B S T R A C T
Article history:
ZrO2 hollow porous microspheres have been fabricated successfully using living yeast cells as bio-
templates through a facile ageing process at lower crystallizing temperature. XRD, SEM, FT-IR and N2
adsorption–desorption were used to characterize ZrO2 hollow microspheres. The results showed that the
inorganic–organic hybrid hollow microspheres were fabricated at 100 8C and ZrO2 hollow microspheres
with tetragonal phase and porous structure were obtained at 300 8C. The crystallization temperature of
ZrO2 decreased obviously due to the inducting of bio-molecules. The as-prepared hollow microspheres
are approximately ellipsoid. The shells of these hollow microspheres are porous, which are composed of
some nanoparticles with size of ꢀ20 nm. The formation mechanism of ZrO2 hollow microspheres was
proposed and discussed tentatively.
Received 12 October 2010
Received in revised form 26 February 2011
Accepted 28 March 2011
Available online 12 April 2011
Keywords:
A. Oxides
A. Microporous materials
A. Nanostructures
ß 2011 Elsevier Ltd. All rights reserved.
1. Introduction
al. [16] also found that lysozyme could catalyze the hydrolysis/
condensation of the precursor potassium hexafluorozirconate and
ZrO2 is one of the most intensively studied materials owing to its
technologically important applications in catalysts, catalytic sup-
ports, chromatography, and so on [1,2]. The size and morphology of
zirconia are closely related to their properties and influence their
potential applications [3–5]. Especially, the micro-/nanostructure
ZrO2 not only has some advantages of conventional size materials
but also retains the high surface areas and high activities of
nanoparticles [6]. Simultaneously, the double structure of nanome-
ter and micrometer probably produces some new properties and
characteristics [7]. Furthermore, ZrO2 with hollow micro-/nano-
structure exhibits even more advantages over their solid counter-
parts due to lower densities and peculiar cavum structure.
Several methods have been developed for the preparation of
zirconia with different sizes and morphologies, including nanopar-
ticles [8,9], wonderful morphologies [10,11], mesoporous [12,13]
and hollow spheres [1,14]. Especially, all these methods require
higher processing temperature to complete the crystallization of
zirconia. Recently, we find that the methods of biological molecules
inducing the nucleation and growth would help ameliorate this
feature. Bansal et al. [15] had shown that the fungus Fusarium
oxysporum secretes proteins which were capable of hydrolyzing
aqueous ZrF62À ions to form zirconia at room temperature. Jiang et
induced the formation of zirconia particles at room temperature.
In this paper, we reported a simple and mild method for the
synthesis of ZrO2 hollow porous microspheres using yeast as a bio-
template. This method exhibits a number of interesting features:
(i) Living yeast cell is the key factor for the preparation of ZrO2
hollow microspheres. (ii) Hollow structure could be obtained no
need to remove templates. (iii) ZrO2 crystallization temperature is
lower than the temperature required for the general template
methods. (iv) The resulting wall of hollow microspheres is porous
due to the stack of nanoparticles. A formation mechanism is
proposed which can explain the porosity and lower crystallization
temperature of the products. This bio-template method provides
an economical, green, and convenient strategy comparing with the
traditional template-directed method, which may open up a new
pathway to synthesize porous hollow inorganic spheres by means
of a facile bio-assisted method.
2. Experimental procedures
2.1. Preparation of ZrO2 hollow microspheres
An aqueous solution of ZrOCl2Á8H2O was prepared by mixing
the metal salt with distilled water. Acetylacetone solution was
added to the solution to control the hydrolysis rate of ZrOCl2Á8H2O
[17]. Then ammonia hydroxide was added drop by drop to adjust
the pH of solution between 4.5 and 5.0. The reaction system was
* Corresponding author.
E-mail address: xiuqinsong@gmail.com (X. Song).
0025-5408/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.materresbull.2011.03.024

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X. Fan et al. / Materials Research Bulletin 46 (2011) 1315–1319
samples were calcined respectively at 300 8C, 500 8C for 2 h to
remove organics. Thus, the ZrO2 hollow microspheres were
obtained.
2.2. Characterization
Scanning electron microscopy (SEM) measurement was per-
formed with a Hitachi S-4800 microscope. An X-ray diffraction
(XRD) study was carried out using a Bruker D8 ADVANCE X-ray
˚
diffractometer with Cu Ka radiation
(l = 1.5418 A). Infrared
spectra (IR) measurement was carried out on a SHIMADU FIRE-
8900 Fourier transform infrared spectrophotometer. The porous
structure of the samples was analyzed by N2 adsorption–
desorption in a NOVA4000e nitrogen adsorption apparatus.
3. Results and discussion
3.1. The morphology and structure of ZrO2 hollow microspheres
3.1.1. The morphology of ZrO2 hollow microspheres
Fig. 1. SEM images of original yeast cell.
Fig. 1 shows that the original morphology of yeast is
approximately spherical or ellipsoid with the diameter ranging
continuously stirring for 30 min at 30 8C and still was the solution
state. Then yeast-water suspension (1 g yeast/40 ml H2O) was
dropped into the above mixture solution under stirring. After the
ultimate solution aged at room temperature for 3 h, the
precipitates were filtered, washed with distilled water and then
ethanol for several times and dried at 100 8C. Finally, the dried
from 4.0
mm to 4.5 mm.
As shown in Fig. 2(a), the hollow microspheres were obtained
after the samples were dried at 100 8C. These hollow microspheres
should be a kind of organic/inorganic hybrid species and the
organics in the inner of living cell are attached to the internal wall
of the microspheres. The hollow spheres are ellipsoid with almost
Fig. 2. SEM images of (a) hybrid microspheres, (b) ZrO2 hollow microspheres calcined at 300 8C, (c) ZrO2 hollow microspheres calcined at 500 8C and (d) the surface of the
hollow microspheres.

X. Fan et al. / Materials Research Bulletin 46 (2011) 1315–1319
1317
Fig. 3. XRD patterns of ZrO2 hollow microspheres calcinated at different
temperature (a) 300 8C and (b) 500 8C.
the same diameters as original yeast size. Obviously, hydrolysis of
zirconium chloride occurred on the surface of the yeast cells and
the yeast cells played a role of templates in the process of preparing
ZrO2 hollow microspheres. The morphology of ZrO2 hollow
microspheres change little but the size changed slightly when
the dried particles were calcined at different temperature, as
shown in Fig. 2(b) and (c). The size of ZrO2 hollow microspheres is
Fig. 4. Nitrogen adsorption–desorption isotherms and the corresponding pore size
distribution (inset) for the ZrO2 hollow microspheres.
pore structure shows benefits not only to the delivery of
macromolecules, but also to the shape selectivity of micro-
molecules.
about 2.0–3.0
mm. The shrinkage of the particles is attributed to
the removing of organic molecules during heat treatment. The
cracked spheres and apparent cavities show the hollow nature of
the products. The thickness of ZrO2 hollow microspheres shell is
estimated to be about 100 nm. In addition, it was found that the
thickness of the shells of ZrO2 hollow microspheres can be tuned
by the concentration of zirconium chloride rather than reaction
time at 30 8C. Careful observation shows that the surfaces of these
microspheres are constructed by nanoparticles as shown in
Fig. 2(d).
3.1.4. The infrared spectrogram analysis of hollow microspheres
Fig. 5 shows the FT-IR spectrum of ZrO2 hybrid microsphere
and original yeast templates. On the curve of the original yeast
templates, the bands at 1652.9 cmÀ1 could be attributed to the
C55O stretching vibration of the amide I band of proteins. The bands
at 3419.6 could be ascribed to the O–H, C–H, N–H stretching
vibration of the groups which possess hydrogen. On the basis of FT-
IR spectrum of original yeast, shows that active components of
yeast contain these groups. Compared Fig. 5(b) with (a), we found
that the shoulder peak at 1652.9 cmÀ1 in Fig. 5(b) corresponding to
the C55O stretching vibration splits to 1593.1 cmÀ1and
1529.4 cmÀ1 in Fig. 5(a) that belong to Zr–O–C deformation
vibration. The spectrum of original yeast exhibited clear shifts from
3.1.2. The structure of ZrO2 hollow microspheres
The XRD patterns of ZrO2 hollow microspheres synthesized via
the yeast bio-template route are shown in Fig. 3.
As shown here, the crystallization of zirconia has been achieved
almost perfect at 300 8C (Fig. 3(a)). The crystallization is
intensified with the increase of calcinations temperature as shown
in Fig. 3(b). Lower crystallization temperature may be confirmed
that the yeast cells not only are used templates but also the active
bio-molecules of yeast cells take part in the nucleation, growth and
crystallization of ZrO2. All the diffraction peaks can be indexed to
the tetragonal phase structure of ZrO2 (JCPDS 79-1769), and no
peaks of other materials or phases are observed, which indicates
the high purity of the products. The crystallite sizes, as estimated
by Scherrer formula, corresponding to (0 1 1) crystal plane are
6.9 nm (in Fig. 3(a)) and 11.4 nm (in Fig. 3(b)).
3419.6 cmÀ1
,
2927.7 cmÀ1
,
1456.2 cmÀ1 and 1130.2 cmÀ1in
2925.8 cmÀ1 1425.3 cmÀ1 and
Fig. 5(b) to 3415.7 cmÀ1
,
,
1132.1 cmÀ1 in Fig. 5(a) due to the interaction between reactive
functional groups such as carbonyls, hydroxyls, acid amides
andZr2+ by electrostatic force, hydrogen bond and covalent bond.
3.1.3. The pore structure of ZrO2 hollow microspheres
The ZrO2 hollow microspheres are further characterized by
nitrogen adsorption and desorption isotherms at 77 K, as shown in
Fig. 4.
Fig. 4 shows a type III-like isotherm with H3-hysteresis,
indicating the presence of slit-like type porosity in the micro-
spheres. The Barrett–Joyner–Halenda (BJH) method was used to
calculate the pore size distribution (as shown in Fig. 4 inset). The
result indicates that the specific surface area is 70.322 m2/g and
the pore volume is 0.077 cm3/g. However, for the solid micro-
spheres, the specific surface area is 57.708 m2/g and the pore
volume is 0.013 cm3/g. The hollow microspheres have a bimodal
pore structure: the smaller pores with a diameter of 18 nm and the
bigger pores of approximately 35 nm in diameter. This bimodal
Fig. 5. FT-IR spectrum of (a) ZrO2 hollow spheres without the calcined and (b)
original yeast templates.

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X. Fan et al. / Materials Research Bulletin 46 (2011) 1315–1319
Fig. 6. SEM images of (a) ZrO2 core–shell microspheres and (b) ZrO2 solid microsphere.
3.2. The mechanism for the formation of ZrO2 hollow microspheres
reproduce prolifically at appropriate environment, such as pH 4.5–
5.0, T = 30 8C. The wall of the living cell of yeasts can prevent
inorganic salt or ions from entering the inner of the cells. (ii) The
cell wall of yeast is primarily made up of mannan and glucan [18].
Thus, there are many reactive functional groups on the cell wall,
such as hydroxyls, acid amides and carbonyls (as shown in
Fig. 5(b)) which are able to bind mental ions through coordination
or electrostatic attractions. Consequently, tens of thousands of the
cells provide nucleate sites for ZrO2 crystals and ZrO2 crystals will
grow in the surfaces of the cells when the concentration of
ZrOCl2Á8H2O is fixed. These groups not only have interaction with
crystal element but also guide nucleation, growth and crystalliza-
tion of ZrO2. It results in the crystallization temperature of ZrO2
inclining to drop obviously as shown in Fig. 3(a). (iii) The yeast cell
is dead when it is dried at 100 8C. Then, the hydration water in the
inner cell is escaped and the free water is evaporated together.
Other compositions (such as cell nucleus, mitochondria and so on)
adhere to inner wall of cytomembrane to form inorganic–organic
hybridization precursors of ZrO2 hollow microstructure. The
organics is volatilized and precursors of ZrO2 hollow microstruc-
ture are crystallized at different calcinations temperature. At the
end, ZrO2 hollow microspheres with the porous shell are formed.
The influences of experimental parameters on the morphology
of products, such as the reaction temperature, the reaction time,
the concentration of zirconium chloride and pH of the solution,
were investigated. It was found that the hydrolysis of zirconium
chloride was accelerated with increasing the reaction temperature.
However, when the temperature was higher than 30 8C, the
mixture of hollow and core–shell microspheres were obtained
because yeast cells were deactivated and the walls of the yeast cells
became loose and porous. At 80 8C, ZrO2 core–shell microspheres
and solid microspheres were synthesized following different
reaction time, as shown in Fig. 6.
When the initial temperature was 80 8C and the reaction times
were between 0.5 h and 3.0 h, ZrO2 core–shell microspheres were
synthesized (as shown in Fig. 6(a)). When the reaction time was
longer than 5.0 h, solid microspheres were synthesized (as shown
in Fig. 6(b)). The main reason for such results might be that yeast
cells are deactivated at 80 8C and the longer reaction time the more
serious inactivation. At the same time the walls of the yeast cell
become loose and porous. The inorganic salt or ions enter to the
inner of cell through these porous and combine with some
biomolecules. At this point, nucleation and growth of ZrO2 occur
not only in the surface of yeast cell, but also in internal. Core–shell
structure comes into being. With the reaction time extension, solid
microspheres are finally formed. The living yeast cell with a dense
wall is the necessary condition for the formation of hollow
microspheres.
4. Conclusions
In summary, a facile technique for synthesizing ZrO2 hollow
porous microspheres was developed using living yeast cells as bio-
templates. It was demonstrated that the dense wall of living yeast
cell is the necessary condition for the formation of hollow
microspheres. The structure of ZrO2 hollow microspheres could
be obtained only when pH 4.5–5.0, T = 30 8C of the solution, while
other conditions will lead to the formation of core–shell or solid
The mechanism model for the formation of ZrO2 hollow
microspheres is tentatively proposed as shown in Scheme 1.
The whole process can be described as: (i) the yeasts keep
characteristic of unicellular eukaryotic microorganism and would
Scheme 1. Schematic illustration of ZrO2 hollow microspheres formation.

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This work was supported by the Hebei Natural Foundation
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