The document summarizes a novel technique called "bio-milling" that uses fungal biomass to break down chemically synthesized BiMnO3 nanoplates into smaller particles less than 10 nm in size while maintaining crystallinity and phase purity. Specifically: 1) Chemically synthesized BiMnO3 formed nanoplates 150-200 nm in size. 2) Exposure to the fungus Humicola sp. gradually broke the plates down over 120 hours to quasi-spherical particles mostly between 4-8 nm in size. 3) Transmission electron microscopy images show the breakdown process over time and X-ray diffraction confirms the crystalline structure and phase are preserved despite the size reduction.

1. PAPER www.rsc.org/materials | Journal of Materials Chemistry
Bio-milling technique for the size reduction of chemically synthesized
BiMnO3 nanoplates
Baishakhi Mazumder,a Imran Uddin,ac Shadab Khan,c Venkat Ravi,a Kaliaperumal Selvraj,b
Pankaj Poddar*a and Absar Ahmad*c
Received 24th April 2007, Accepted 19th July 2007
First published as an Advance Article on the web 3rd August 2007
DOI: 10.1039/b706154d
Wet-chemical techniques for the synthesis of complex oxide materials have advanced significantly;
however, achieving finely dispersed nanoparticles with sizes less than 10 nm still remains
challenging, especially for the perovskite family of compounds. On the other hand, a fungus-
mediated synthesis technique has recently shown potential to synthesize perovskites such as
BaTiO3 with sizes as small as 5 nm. Here we report, for the first time, the use of fungal biomass, at
room temperature, to break down chemically synthesized BiMnO3 nanoplates (size y150–200 nm)
into very small particles (,10 nm) while maintaining their crystalline structure and the phase
purity. This novel technique that we have named as ‘‘bio-milling’’ holds immense potential for
synergically utilizing both chemical and biological synthesis techniques to synthesize complex
oxide nanoparticles with particle sizes less than 10 nm with the proper crystalline phase.
Introduction advantages over these methods (especially in the case of oxide
nanoparticle synthesis) as the biological synthesis methods
There has been phenomenal success in the development of wet- avoid the use of harsh chemicals and the syntheses take place
chemical synthesis techniques of various nanomaterials such as in ambient conditions without the need of further treatment.
semiconductors, dilute magnetic semiconductors, core–shell In this technique, the as-synthesized particles are extremely
structured nanomaterials, ferroelectric ceramics and ferromag- stable due to the inherent coating at the surface by proteins,
netic nanomaterials with excellent control over the size and which enables them to be suspended in the aqueous medium.
shape.1–3 Among these materials, there is special interest in In this ‘‘bottom-up synthesis’’ approach, the metal salts are fed
synthesizing multifunctional nanomaterials, where there is to the biomass which in turn secretes enzymes to form the
coupling between various physical properties. For example, nanoparticles. So far, the microbial techniques have been used
from the application point of view, it is quite rewarding to synthesize a range of metal7 and binary oxides8–10 (TiO2, SiO2,
to synthesize materials such as magnetic semiconductors, ZrO2, Fe3O4). Recently, we reported the biosynthesis of BaTiO3,
nanocomposites of noble metals–ferromagnetic materials, with average particle sizes less than 5 nm in the stable tetragonal
metal–dielectric nanocomposites, piezoelectric–magnetostric- ferroelectric phase at room temperature.11 Moreover, it was the
tive nanocomposites, etc. The multiferroic oxides fall into the first reported synthesis of a ternary oxide (with perovskite
same category of materials with tunable physical properties.4 structure) using the microbial method. However, further
However, despite the advancement in the chemical techniques research needs to be done to fine-tune the synthesis parameters
to synthesize several complex-oxide nanomaterials, the syn- to meet the challenges related to the scale-up of synthesis, better
thesis of the perovskite family of compounds (particularly control over the particle size and shape and the synthesis of
with particle sizes less than 10 nm) remains a great challenge. various other complex ternary oxide phases. Moreover, the
It should be noted that several of these compounds exhibit synthesis mechanism and the involvement of various biomole-
interesting electrical and magnetic properties at small sizes. cules need to be understood completely over time.
The traditional techniques for the synthesis of perovskites Herein, for the first time, we have developed a novel ‘‘top-
such as sol–gel, hydrothermal, co-precipitation, etc. have been down’’ biosynthesis approach, while learning from nature,
extensively used, but often the as-synthesized particles need to where the degradation of rocks12 (such as granite, sandstone,
be calcined at high temperatures to get the proper crystalline bricks, etc.), in the form of fine particles is carried out over a
phase, which leads to the grain-growth and agglomeration.5,6 long period of time by microorganisms such as bacteria, fungi,
For the past few years, the biological methods such as yeast and algae.13 In some cases, these microorganisms are
microbial (fungi, yeast and bacteria), plant extract and found to actually bore paths into various materials.14,15
biomimetic synthesis routes have been gaining popularity However, in nature, due to the lack of nutrients, this process
over the traditional wet-chemical methods due to various is quite slow and uncontrolled. After learning from this ‘‘top-
down’’ approach used by nature, we have accelerated this
a
Materials Chemistry Division, National Chemical Laboratory, Pune, process in the laboratory environment by selectively using
411008, India. E-mail: p.poddar@ncl.res.in certain fungal species in the presence of nutrient media. For
b
Catalysis Division, National Chemical Laboratory, Pune, 411008, India
c
Biochemical Science Division, National Chemical Laboratory, Pune, the first time, we have named this process as ‘‘bio-milling’’,
411008, India. E-mail: a.ahmad@ncl.res.in which is equivalent to the ‘‘ball-milling’’ process commonly
3910 | J. Mater. Chem., 2007, 17, 3910–3914 This journal is ß The Royal Society of Chemistry 2007

2. used by materials scientists and engineers to break down fast solid-state detector, on a drop-coated sample prepared
large particles into smaller sizes. In this effort, we have on a glass substrate. The sample was scanned using the
chosen BiMnO3 as a model system, which is known to have X9celerator with a total number of active channels of 121.
multiferroic properties.16 ˚
Iron-filtered Cu Ka radiation (l = 1.5406 A) was used. The
XRD patterns were recorded in the 2h range of 20–80u with a
Materials and methods step size of 0.02u and a time of 5 seconds per step.
Synthesis of BiMnO3 nanoplates using the co-precipitation Fourier transform infrared spectroscopy (FTIR)
technique
FTIR spectroscopy measurement on the as-prepared and the
We have synthesized BiMnO3 nanoplates using the co- ‘‘bio-milled’’ BiMnO3 nanoparticles was carried out using a
precipitation method in which a simple hydroxide gel to oxide Perkin-Elmer Spectrum One instrument. The spectrometer
crystal conversion route was followed at 80–100 uC under operated in the diffuse reflectance mode at a resolution of
refluxing conditions. For this purpose, freshly prepared 2 cm21. To obtain a good signal to noise ratio, 128 scans of the
bismuth and manganese hydroxide gels were allowed to film were taken in the range 450–4000 cm21.
crystallize and react under refluxing and stirring conditions
for 4–6 hours. The as-obtained powder was calcined at 100 uC
for 12 hours to produce a pinkish material.11 Results and discussion
In Fig. 1 (A and B), we show TEM micrographs of the
‘‘Bio-milling’’ of chemically synthesized particles chemically synthesized BiMnO3 nanoparticles at different
For this purpose, we isolated an alkalotolerant and thermo- length scales. For this purpose, the as-synthesized particles
philic fungus, Humicola sp. (HAA-SHC-2), from self-heating were suspended in amyl acetate and were drop-cast on the
compost. We maintained this fungus on MGYP (malt extract, TEM grid. We noted that the dispersion of the particles in the
glucose, yeast extract, and peptone) agar slants. The stock solvent was quite poor due to the large size of the particles as
cultures were maintained by subculturing at monthly intervals. well as the absence of any capping agent. The TEM images
After growing the fungus at pH 9 and 50 uC for 4 days, the show that these particles are quite flat and almost square in
slants were preserved at 15 uC. After 4 days of incubation, we shape. The agglomeration of the particles is due to the absence
made fresh slants (at pH 9 and 50 uC) out of an actively of any capping agent at the particle surfaces. In Fig. 1(C), we
growing stock culture. Later on, we used these subcultures as show the selected area diffraction pattern which exhibits a
the starting material for further experiments. diffused ring pattern, while Fig. 1(D) shows the particle size
In order to break down the chemically synthesized BiMnO3 distribution histogram which show that the edge length of
nanoplates (edge lengths 150–200 nm) to small sizes, the these particles is quite long (in the range 150–250 nm). As
fungus was grown in 250 mL Erlenmeyer flasks containing mentioned above, these chemically synthesized particles
50 mL of the MGYP medium at pH 9 with shaking for were now fed to the alkalotolerant and thermophilic fungus,
96 hours. The fungul mycelia (20 g) separated from the culture
broth by centrifugation was resuspended in 100 mL of an
aqueous suspension of the BiMnO3 particles in 250 mL
Erlenmeyer flasks at pH 9 and kept on the shaker at 50 uC
(200 rpm) and maintained in the dark. The reduction in the
size of the BiMnO3 particles in the solution was monitored by
periodic sampling (over 120 hours) of aliquots of the aqueous
component for further characterization.
Transmission electron microscopy (TEM) measurements
The size and shape analysis of the BiMnO3 nanoparticles was
done using a JEOL model 1200EX TEM operated at a voltage
of 120 kV. For this purpose, we prepared the samples by drop-
coating the particles suspended in aqueous medium on carbon
coated copper grids.
High-resolution transmission electron microscopy measurements
High-resolution TEM (HRTEM) was performed on a JEOL
JEM-2010 UHR instrument operated at a lattice image
resolution of 0.14 nm.
X-Ray diffraction pattern (XRD) Fig. 1 Transmission electron micrographs of the chemically synthe-
sized BiMnO3 (A and B; B shows a higher magnification image), (C)
Powder XRD patterns were recorded using a PHILIPS selected area electron diffraction curve and (D) particle size distribu-
X9PERT PRO instrument equipped with an X9celerator-,a tion histogram.
This journal is ß The Royal Society of Chemistry 2007 J. Mater. Chem., 2007, 17, 3910–3914 | 3911

3. Fig. 2 Photographs of the BiMnO3 particles suspended in the water:
(A) as-synthesized, (B) and (C) after 18 and 48 hours of reaction with
the fungal biomass respectively.
Humicola sp. To monitor the effect of the fungus on the
particles, we picked up the samples from the flask containing
the fungal biomass after 18, 48 and 120 hours. We observed
that the particles dispersed in the flask which initially formed a
cloudy and unstable suspension in the aqueous medium
(Fig. 2A) were slowly (over a period of 18–120 hours) taken
into the solution by the biomass, with no precipitate left in the
bottom of the reaction flask after almost 18 hours. In Fig. 2B
and C, we show images of the suspensions of the particles after
18 and 48 hours of reaction with the fungus where a clear
suspension of particles can be observed. It should be noted
Fig. 3 (A–D) Transmission electron micrographs of the chemically
that this particle suspension was quite stable over a period of
synthesized BiMnO3 particles reacted with the fungus for 18 hours. (E)
several months.
Particle size distribution histogram.
To further investigate the effect of the fungus on the surface
morphology of these particles, we also imaged the particles
taken from the reaction flask after 18, 48 and 120 hours. In
Fig. 3(A–D), we present various TEM micrographs of the
supernatant solution after 18 hours. As can be seen from these
images, the nanoplates start fragmenting and form spherical
particles of smaller sizes due to the reaction with the biomass.
The tendril like structure as seen in some of these images
(especially A and C), are possibly formed by the fungal
mycelia.
In Fig. 4(A,B), we show TEM images of the particles taken
from the flask after 48 hours of the reaction. The particle size
histogram presented in Fig. 4(C) shows that the particle size
decreases significantly from hundreds of nanometers to around
50 nm. Further, in Fig. 5, we show the TEM images of the
particles after nearly 120 hours of reaction. It should be noted
that the particles, which were initially much larger in size, have
broken down to particles with sizes between 4 and 8 nm
with a quasi-spherical geometry as shown in the histograms
(Fig. 5(B,D)) with the average particle size at around 6 nm,
which is quite remarkable.
As we mentioned earlier, in nature various kinds of Fig. 4 (A,B) Transmission electron micrographs of the BiMnO3
microorganisms, including fungus, are known to degrade particles reacted with the fungus for 48 hours. (C) Particle size
rocks to form smaller particles over a very long period of distribution histogram.
time; however, to the best of our knowledge, this is the first
time that this process has been applied in a research lab. In our laser deposition, etc.), which are quite expensive and the bio-
opinion, this process carries huge technical advantages over milling process provides a very simple and economical route to
traditional top-down methods (such as lithography, pulsed form smaller particles while maintaining proper crystallinity.
3912 | J. Mater. Chem., 2007, 17, 3910–3914 This journal is ß The Royal Society of Chemistry 2007

4. Fig. 7 Powder X-ray diffraction patterns of BiMnO3: as-synthesized
by chemical methods and reacted with the fungus for 18, 48 and
120 hours.
the fungal biomass. This is the same for the case of the pattern
Fig. 5 (A,C) Transmission electron micrographs of the BiMnO3
particles reacted with the fungus for 120 hours. (B,D) Particle size corresponding to the sample treated for 48 hours. However,
distribution histograms. the changes in the relative intensities of the 010 and 100
reflections are due to the possible isotropic size reduction
We believe that the scaling up of this synthesis process could of the crystallites during the course of fungal treatment.
be easily demonstrated by optimizing parameters such as the However, the XRD pattern of the final sample after 120 hours
type of microorganism, medium, pH and temperature as well of treatment matches that of the chemically synthesized initial
as by using large fermenters for the reaction. To further powder in terms of the 2h positions as well the peak intensities.
examine the use of this technique for other materials, recently Further, the low background and sharper peaks suggest that
we found that even other oxides such as Gd2O3 can be bio- the particles retain their crystallinity (BiMnO3 has a highly
milled to form smaller particles. distorted perovskite structure) even after the bio-milling
process.4,19 As we indicated earlier the change in the preferred
In Fig. 6, we show the HRTEM images of the BiMnO3
orientation with digestion time (seen as the change in the
nanoparticles after nearly 120 hours of reaction where the
˚ ˚ relative peak intensities) is not surprising in the present work
lattice planes exhibit spacings of y1.62 A and y1.81 A corres-
because the chemically synthesized particles show a plate-like
ponding to the lattice planes S112T and S200T respectively.
structure and after reacting the particles with the fungus, the
To further check the effect of the exposure of the fungus on
particle morphology changes from flat to sphere-like structures
the crystallinity of the chemically synthesized particles at
thereby exposing various other crystalline planes for the
various time-scales, we performed powder XRD on the
incident X-rays which results in the change in the line
samples after 18, 48 and 120 hours reaction time. The results
intensities. Additionally, it has been seen that the difference
are shown in Fig. 7. The XRD profile of the chemically
in the sample preparation for powder X-ray diffraction can
synthesized BiMnO3 nanoplates matches very well with that
significantly contribute towards the overall texture of the
reported in the literature.17,18 It is known that BiMnO3 has a
sample. It should be noted here that we did not attempt to
triclinic structure with reported unit cell parameters a = c =
calculate the particle size from Scherrer’s formula as in this
˚ ˚
3.923 A, b = 3.981 A, a = c = 91.4u and b = 91.0u. The XRD
case the calculation of the crystallite sizes from the line
pattern in Fig. 6 corresponding to the sample treated for
broadening of the XRD peaks will be prone to errors because
18 hours shows an elevated background due to the presence of
during the ‘‘bio-milling process’’ it is not certain that there is
100% fragmentation of the chemically synthesized particles.
There might be a few particles remaining in the samples picked
up for XRD which are still not fully fragmented, leading to
particular line widths. Additionally, due to the smoothing
process of the raw XRD data to get rid of the protein
background, using the peak heights for further analysis might
not be error-free.
In Fig. 8 are shown the FTIR spectra in different regions for
the chemically synthesized particles (curves 1) and the bio-
milled particles after 120 hours (curves 2). In Fig. 8A is shown
the presence of the absorption band around 538 nm due to the
stretching of the Bi–O bond.20 The bands around 630 nm,
Fig. 6 HRTEM micrographs of the BiMnO3 nanoparticles reacted 670 nm, and 725 nm in Fig. 8B show the presence of Mn–O
with the fungus for 120 hours. bond formation in BiMnO3.21,22 In Fig. 8C, curve 1 shows the
This journal is ß The Royal Society of Chemistry 2007 J. Mater. Chem., 2007, 17, 3910–3914 | 3913

5. We believe that this novel approach of using micro-
organisms in the laboratory environment to break up large
particles into small particles holds tremendous potential in
materials science.
Acknowledgements
The authors P.P. and A.A. would like to acknowledge and
thank the financial support from the Department of Science
and Technology (DST), India to set up a unit on Nanoscience.
One of the authors, P.P., would also like to acknowledge
Fig. 8 FTIR spectra for BiMnO3: (1) as-synthesized by the chemical
a separate grant from SERC, DST, India under the
method, (2) reacted with the fungus.
Nanomission. We acknowledge Mr Gholap, Centre for
Materials Characterization, NCL Pune for assistance with
TEM imaging and Dr P. V. Satyam (Institute of Physics
Bhubneshwar, India) for the HR-TEM imaging.
References
1 C. B. Murray, C. R. Kagan and M. G. Bawendi, Annu. Rev. Mater.
Sci., 2000, 30, 545–610.
2 M. Niederberger, N. Pinna, J. Polleux and M. Antonietti, Angew.
Chem., Int. Ed., 2004, 43, 2270–2273.
3 Y. Sahoo, P. Poddar, H. Srikanth, D. W. Lucey and P. N. Prasad,
J. Phys. Chem. B, 2005, 109, 15221.
4 N. A. Hill, J. Phys. Chem. B, 2000, 104, 6694.
5 X. Li and W. H. Shih, J. Am. Ceram. Soc., 1997, 80, 2844.
6 V. R. Calderone, A. Testino, M. T. Buscaglia, M. Bassoli,
Fig. 9 Preliminary gel electrophoresis (SDS PAGE, pH 8.8) for C. Bottino, M. Viviani, V. Buscaglia and P. Nanni, Chem.
Humicola sp. (HAA-SHC-2) showing four distinct protein bands. Mater., 2006, 18(16), 1627–1633.
7 M. Sastry, A. Ahmad, M. I. Khan and R. Kumar, Curr. Sci., 2003,
85, 2.
absence of amide bands in the chemically synthesized BiMnO3 8 V. Bansal, D. Rautaray, A. Bharde, K. Ahire, A. Sanyal, A. Ahmad
whereas in curve 2 two absorption bands centered around 1658 and M. Sastry, J. Mater. Chem., 2005, 15, 2583.
and 1535 cm21 are attributed to the amide I and II bands 9 V. Bansal, D. Rautaray, A. Ahmad and M. Sastry, J. Mater.
Chem., 2004, 14, 3303.
respectively due to the presence of proteins.23 10 A. Bharde, A. Wani, Y. Shouche, P. A. Joy, B. L. V. Prasad and
To identify the number of proteins secreted by the fungus M. Sastry, J. Am. Chem. Soc., 2005, 127, 9326.
and their molecular weights, the fungus biomass [20.0 g of wet 11 V. Bansal, P. Poddar, A. Ahmad and M. Sastry, J. Am. Chem.
mycelia] was resuspended in 100 mL of sterile distilled water Soc., 2006, 128, 11958–11963.
12 H. L. Ehrlich, Chem. Geol., 1996, 132, 5–9.
for a period of 3 days. The mycelia were then removed by 13 P. Hirsch, F. E. W. Eckhardt and R. J. Palmer, J. Microbiol.
centrifugation, and the aqueous supernatant thus obtained Methods, 1995, 23, 143.
was concentrated by ultra-filtration using a YM3 (molecular 14 V. Bansal, A. Sanyal, D. Rautaray, A. Ahmad and M. Sastry, Adv.
weight cutoff 3000) membrane and then dialyzed thoroughly Mater., 2005, 17, 889.
15 C. N. Mulligan and M. Kamali, J. Chem. Technol. Biotechnol.,
against distilled water using a 3000 cutoff dialysis bag. This 2003, 78, 497.
concentrated aqueous extract containing protein was analyzed 16 J. Y. Son, B. G. Kim, C. H. Kim and J. H. Cho, Appl. Phys. Lett.,
by PAGE (polyacrylamide gel electrophoresis) carried out at 2004, 84, 4971.
pH 8.8.24 In Fig. 9, the preliminary gel electrophoresis 17 V. Samuel, S. C. Navale, A. D. Jadhav, A. B. Gaikwad and
V. Ravi, Mater. Lett., DOI: 10.1016/j.matlet.2006.06.046.
measurement indicates that the fungus secretes four distinct 18 H. Chiba, Solid State Ionics, 1998, 108, 193–199.
proteins ranging in weight from 97 to 20 kDa. One or more 19 Z. H. Chi, C. J. Xiao, S. M. Feng, F. Y. Li and C. Q. Jina, Chem.
of these proteins might be the enzymes that reduce the size Mater., 2005, 17, 1765–1773.
of BiMnO3 and cap BiMnO3 nanoparticles formed by the 20 R. Irmawati, M. N. Noorfarizan Nasriah, Y. H. Taufiq-Yap and
S. B. Abdul Hamid, Catal. Today, 2004, 701, 93–95.
reduction process. It is possible that more than one protein 21 R. A. Nyquist and R. O. Kagel, Infrared spectra of Inorganic
might take part in the capping and stabilization of the BiMnO3 Compounds, Academic Press, New York and London, 1971,
nanoparticles. The exact mechanism leading to the reduction pp. 216–217.
of nanosize BiMnO3 is yet to be elucidated for this fungus. 22 F. A. Alsagheer, M. A. Hasan, L. Pasupulety and M. I. Zaki,
J. Mater. Sci. Lett., 1967, 121, 309.
We are currently separating and concentrating the different 23 D. Rautaray, A. Sanyal, S. D. Adyanthaya, A. Ahmad and
proteins released by the fungus Humicola sp. to test and M. Sastry, Langmuir, 2004, 20, 6827–6833.
identify the ones active in the above processes. 24 U. K. Laemmli, Nature, 1970, 227, 680.
3914 | J. Mater. Chem., 2007, 17, 3910–3914 This journal is ß The Royal Society of Chemistry 2007