Synthesis of brushite particles in reverse microemulsions of the biosurfactant surfactin.
ABSTRACT In this study the "green chemistry" use of the biosurfactant surfactin for the synthesis of calcium phosphate using the reverse microemulsion technique was demonstrated. Calcium phosphates are bioactive materials that are a major constituent of human teeth and bone tissue. A reverse microemulsion technique with surfactin was used to produce nanocrystalline brushite particles. Structural diversity (analyzed by SEM and TEM) resulted from different water to surfactin ratios (W/S; 250, 500, 1000 and 40,000). The particle sizes were found to be in the 16-200 nm range. Morphological variety was observed in the as-synthesized microemulsions, which consisted of nanospheres (~16 nm in diameter) and needle-like (8-14 nm in diameter and 80-100 nm in length) noncalcinated particles. However, the calcinated products included nanospheres (50-200 nm in diameter), oval (~300 nm in diameter) and nanorod (200-400 nm in length) particles. FTIR and XRD analysis confirmed the formation of brushite nanoparticles in the as-synthesized products, while calcium pyrophosphate was produced after calcination. These results indicate that the reverse microemulsion technique using surfactin is a green process suitable for the synthesis of nanoparticles.
- SourceAvailable from: Mihir Janakrai Joshi[Show abstract] [Hide abstract]
ABSTRACT: Hydroxyapatite (Ca10(PO4)6(OH)2; HAP) is a major mineral component of the calcified tissues, and it has various applications in medicine and dentistry. In the present investigation, cobalt-doped hydroxyapatite (Co-HAP) nanoparticles were synthesized by surfactant-mediated approach and characterized by different techniques. The EDAX was carried out to estimate the amount of doping in Co-HAP. The transmission electron microscopy result suggested the transformation of morphology from needle shaped to spherical type on increasing the doping concentration. The powder XRD study indicated the formation of a new phase of brushite for higher concentration of cobalt. The average particle size and strain were calculated using Williamson–Hall analysis. The average particle size was found to be 30–60 nm. The FTIR study confirmed the presence of various functional groups in the samples. The antimicrobial activity was evaluated against four organisms Pseudomonas aeruginosa and Shigella flexneri as Gram negative as well as Micrococcus luteus and Staphylococcus aureus as Gram positive. The hemolytic test result suggested that all samples were non-hemolytic. The photoluminescence study was carried out to identify its possible applicability as a fluorescent probe.Journal of Nanoparticle Research 04/2013; 15:1644. · 2.18 Impact Factor
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ABSTRACT: Nano-sized crystals of alpha calcium sulfate hemihydrate (α-HH) with considerable morphology-dependent properties find promising applications in the clinical fields as a cementitious material. Towards this end, ultrasound-assisted rhamnolipid and surfactin biosurfactant-template route is explored to control the morphology and aspect ratio of nano-CaSO4 by adjusting the mass ratio of rhamnolipid/H2O, surfactin/H2O and rhamnolipid/surfactin. The change in the molar ratio of [SO4(2-)]:[Ca(2+)] results in modification in variable morphology and size of nano-CaSO4 including long, short rods and nanoplates. With increase in the rhamnolipid/H2O ratio from 1.3 to 4.5, the crystal length decreases from 3μm to 600nm with the corresponding aspect ratio reduced sharply from 10 to 3. Similarly, the crystal morphology gradually changes from submicrometer-sized long rod to hexagonal plate, and then plate-like appearance with increase in surfactin concentration. The preferential adsorption of rhamnolipid on the side facets and surfactin on the top facets contributes to the morphology control. The process using 50% amplitude with a power input of 45.5W was found to be the most ideal as observed from the high yields and lower average l/w aspect ratio, leading to more than 94% energy savings as compared to that utilized by the conventional process. As a morphology and crystal habit modifier, effects of Mg(2+) and K(+) ions on α-HH growth were investigated to find an optimal composition of solution for α-HH preparation. Mg(2+) ions apparently show an accelerating effect on the α-HH growth; however, the nucleation of α-HH is probably retarded by K(+) ions. Thus, the present work is a simple, versatile, highly efficient approach to controlling the morphology of α-HH and thereby, offers more opportunities for α-HH multiple applications.Ultrasonics Sonochemistry 12/2013; · 3.52 Impact Factor
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ABSTRACT: This is for the first time that an attempt was made to enhance the performance of cross-flow ultrafiltration (CFUF) for the recovery and purification of lipopeptides by size-conditioning their micelles with Ca2+ ions and operating the system in diafiltration mode. In this study, polyether sulfone (PES) membranes with MWCOs 10, 30 and 50 kDa were evaluated for recovery and purification of lipopeptides produced by Bacillus sp. CFUF studies performed under batch mode showed poor recovery (68±2%) of F2 fraction comprising Fengycin isoforms, whereas F1 fraction comprising a mixture of surfactin and iturin isoforms showed a moderate recovery of 83.5±2.4% in 50 kDa MWCO membrane. To improve the recovery, lipopeptides were conditioned with Ca2+ ions. The CFUF purification of this size-conditioned lipopeptide solution under diafiltration mode was carried out in 50 kDa PES membrane. Continuous irrigation of Ca2+ to lipopeptide solution under diafiltration mode of CFUF resulted in the increased recovery of lipopeptides, F1 (surfactin+iturin) fraction about 90.5±2.1% and F2 about 95.3±2.0%. Further, the colored impurities with MW range of 2334–3506 Da were effectively removed from the solution. The results of CFUF purification of Ca2+conditioned lipopeptide solution under diafiltration mode indicate that this process can be readily scaled up to the commercial level, workable even with higher MWCO membranes.Journal of Membrane Science 03/2014; 454:436–443. · 4.09 Impact Factor
Int. J. Mol. Sci. 2011, 12, 3821-3830; doi:10.3390/ijms12063821
International Journal of
Synthesis of Brushite Particles in Reverse Microemulsions of the
Jyoti Prakash Maity 1, Tz-Jiun Lin 1, Henry Pai-Heng Cheng 2, Chien-Yen Chen 1,*,
A. Satyanarayana Reddy 1, Shashi B. Atla 1, Young-Fo Chang 1, Hau-Ren Chen 3,* and
Chien-Cheng Chen 4
1 Department of Earth and Environmental Sciences National Chung Cheng University,
168 University Road, Min-Hsiung, Chia-Yi 62102, Taiwan; E-Mails: email@example.com (J.P.M.);
firstname.lastname@example.org (T.-J.L.); email@example.com (A.S.R.);
firstname.lastname@example.org (S.B.A.); email@example.com (Y.-F.C.)
2 Taipei American School, 800 Chung Shan North Road, Section 6, Taipei 11152, Taiwan;
3 Department of Life Science, National Chung Cheng University, 168 University Road, Minhsiung,
Chia-Yi 62102, Taiwan
4 Department of Biotechnology, National Kaohsiung Normal University, No.62, Shenjhong Road,
Yanchao Township, Kaohsiung County 82444, Taiwan; E-Mail: firstname.lastname@example.org
* Authors to whom correspondence should be addressed; E-Mails: email@example.com (C.-Y.C.);
firstname.lastname@example.org (H.-R.C.); Tel.: +886-5-2720411 (ext. 66220); Fax: +886-5-2720807.
Received: 16 April 2011; in revised form: 10 May 2011 / Accepted: 2 June 2011 /
Published: 9 June 2011
Abstract: In this study the ―green chemistry‖ use of the biosurfactant surfactin for the
synthesis of calcium phosphate using the reverse microemulsion technique was
demonstrated. Calcium phosphates are bioactive materials that are a major constituent of
human teeth and bone tissue. A reverse microemulsion technique with surfactin was used
to produce nanocrystalline brushite particles. Structural diversity (analyzed by SEM and
TEM) resulted from different water to surfactin ratios (W/S; 250, 500, 1000 and 40,000).
The particle sizes were found to be in the 16–200 nm range. Morphological variety was
observed in the as-synthesized microemulsions, which consisted of nanospheres (~16 nm
in diameter) and needle-like (8–14 nm in diameter and 80–100 nm in length) noncalcinated
particles. However, the calcinated products included nanospheres (50–200 nm in diameter),
oval (~300 nm in diameter) and nanorod (200–400 nm in length) particles. FTIR and XRD
Int. J. Mol. Sci. 2011, 12
analysis confirmed the formation of brushite nanoparticles in the as-synthesized products,
while calcium pyrophosphate was produced after calcination. These results indicate that the
reverse microemulsion technique using surfactin is a green process suitable for the
synthesis of nanoparticles.
Keywords: biosurfactant; reverse microemulsion; brushite; surfactin; nanoparticle
Nanotechnology has had a great impact on several aspects of the food, drug, and electronic device
industries. In recent years, it has been studied extensively in the field of biological mineralization. The
majority of bone and teeth tissues naturally consist of calcium phosphate [1,2]. Through many
biological and medical studies, it has been found that artificially-made calcium phosphate has
characteristics such as biocompatibility and bioactivity, which arise from alteration of its size,
morphology, stoichiometry, or the composition of calcium phosphate [3,4]. The general formula
Ca10−x(HPO4)x(PO4)6−x(OH)2−x, with 0 ≤ x < 2, is non-stoichiometric apatite, while calcium phosphates
have a Ca/P molar ratio starting from 0.5 [Ca(H2PO4)2] and ending with 2.0 [Ca4(PO4)2O]. Compounds
such as brushite and hydroxyapatite have been the focus of many recent studies [5,6]. Calcium
phosphate nanoparticles of differing shapes and sizes can be used for different purposes [1,2,5,6].
Sonochemical techniques are used in industrial applications, but these processes are expensive and
require complex systems. On the other hand, the microemulsion process is a simple and user-friendly
technique that can be used to produce various types of calcium phosphate [7–9]. The main components
of the microemulsion process are a mixture of oil, water and surfactant. Additionally, a cosurfactant is
used to create a low interfacial tension, which aids in the production of nanosized particles .
According to a previous report by Higgins , this technique is feasible for industrial applications to
produce nanoparticles. As a result, the reverse microemulsion process should be suitable for
synthesizing nanoscale brushite calcium phosphates with different morphological structures that
contain a water/oil interface in the presence of surfactin.
Surfactin is a type of lipopepetide produced by Bacillus subtilis that contains seven amino acids
bonded to the carboxyl and hydroxyl groups of a 13–15-carbon acid. It is considered one of the
most powerful biosurfactants, as it is able to reduce the surface tension from 72 to 27 mN m−1 at a
concentration as low as 0.005% . As a biosurfactant, it has the advantages of better biodegradability
and environmental-friendly properties.
In this study, brushite nanoparticles of calcium phosphate were prepared through the reverse
microemulsion process. Laboratory-made biological surfactin was used at different water/surfactin
molar ratios to create different brushite (calcium phosphate) nano-crystal formations. The resulting
crystals were then identified and characterized by assessing their structures.
Int. J. Mol. Sci. 2011, 12
2. Results and Discussion
2.1. Structural Characterization of Products
The XRD spectra shown in Figure 1 indicate that the maximum peak intensities are at 2θ values of
around 11.6°, 20.9°, 23.6°, 29.3°, 35.5°, and 48°, attributed to the Miller indices of (020), (121), (040),
(112), (231), and (080) of brushite (Figure 1a,b). The spectra confirmed that the products obtained
were of unique crystallinity and were mainly composed of the brushite form of calcium phosphate
(Figure 1a). The intensity of the diffraction peaks indicated that the particles were fairly well-crystallized.
The crystallinity of the particles increased as the W/S ratio of the as-synthesized products increased. In
comparison to the spectra of micro-emulsified calcium phosphate, the spectra of the as-synthesized
powders with different W/S ratios indicated that brushite was formed in the as-synthesized sets of
experiments. However, calcium pyrophosphate (peak at 29.6°) was found in the calcinated products
Figure 1. XRD patterns of nanoparticles obtained by the microemulsion process
with different water to surfactin ratios (W/S; 250, 500, 1000 and 40,000): (a) without
calcination, (b) with calcination at 800 °C.
Int. J. Mol. Sci. 2011, 12
Figure 1. Cont.
Brushite formation was also confirmed by FTIR spectral analysis (Figure 2). Figure 2a–d show the
FTIR spectra of products obtained at different water to surfactin ratios (W/S), in as-synthesized and
calcinated forms. The spectra exhibit easily distinguishable bands attributed to PO43− for the
non-calcinated form of micro-emulsified calcium phosphate powders (Figure 2a,b). Bands at around
525 cm−1 and 575 cm−1 were attributed to the ν4 bending vibrations of the P-O-P mode. Bands at
873 cm−1 and 1225 cm−1 assigned to the HPO2−4 group of brushite were observed in all samples. These
results were in agreement with the previous reports of Vallet-Regi and Gonzalez-Calbet  and
Lu et al. . A band at 985 cm−1 originated from the P-O(H) ν1 symmetric stretching vibration of
PO34−. In addition, the peak at around 1060 cm−1 was attributed to the ν3 vibration of the PO34− group.
A peak close to 1134 cm−1 was assigned to the ν′6 and ν″6 of HPO24− ions in brushite. A broad band
between 3400–3500 cm−1 was observed, due to the stretching (νs) mode of H-bonded OH− or water. A
sharp peak in the calcinated product (800 °C) due to P2O74− appeared at near 725 cm−1 indicating the
presence of calcium pyrophosphate (Ca2P2O7) (Figure 2c,d). It was clear that the latter was formed by
the loss of one H2O molecule from two brushite HPO24− groups under high-temperature conditions. The
FTIR spectra thus show that the nano-particles obtained in the as-synthesized samples were brushite,
whereas calcium pyrophosphate was obtained after calcination.
Int. J. Mol. Sci. 2011, 12
Figure 2. FTIR spectra of nanoparticles obtained by the microemulsion process with
different water to surfactin ratios (W/S; 250, 500, 1000 and 40,000): without calcination
(a) 4000–400 cm−1, (b) 1300–400 cm−1; with calcination at 800 °C (c) 4000–400 cm−1,
(d) 1300–400 cm−1.
2.2. Morphological and Particle Size Characterization
Figure 3 shows that the morphology of the particles changed with differing W/S ratios. At a W/S
ratio of 250, the particles had an irregular, long needle-like structure (90–200 nm) (Figure 3a–c). A
microemulsion at a W/S ratio of 500 produced mixed-type hexagons with a large irregular structure
(Figure 3d–f). As the W/S ratio increased further, the inside pool of water was enlarged. The droplet
size increased to a much greater dimension, and even deformed the micelle shape. Figure 3j,l shows
that an irregular nano-sphere was formed at a W/S ratio of 40,000. However, the calcinated sample
induced a recrystallization process to form irregular shapes deviating from spherical shapes and a
nanosphere-like structure (Figure 3b) with a diameter of 50–200 nm was observed for the calcinated
particles (800 °C) at a W/S ratio of 250. Figure 3b,e,h, and k indicate an increase in particle size after
calcination. It was also found that there was an increase in width (irregular plate-like structure) as the
W/S ratio increased.
Int. J. Mol. Sci. 2011, 12
Figure 3. SEM images of nanoparticles obtained by the microemulsion process with
different water to surfactin ratios (W/S): (a) 250, (d) 500, (g) 1000 and (j) 40,000 without
calcination; (b) 250, (e) 500, (h) 1000 and (k) 40,000 with calcination at 800 °C. TEM
images of nanoparticles obtained by the microemulsion process with different water to
surfactin ratios (W/S): (c) 250, (f) 500, (i) 1000 and (l) 40,000 without calcination.
At a W/S ratio of 500, the particle width increased to ~300 nm. The particle shape changed
from irregular nano-spheres (200–400 nm) to long aggregated irregular round-edged rods (length
1000–1500 nm) as the W/S ratio varied from 1000 to 40,000. The results suggest that the size of the
Int. J. Mol. Sci. 2011, 12
nanoparticles is mainly controlled by the W/S ratio and reaction temperature. However, the literature
reports using P123 and F 127 do not mention any change in the morphology with different surfactant
concentration . The results showed that the as-synthesized products were brushite particles and the
calcinated (800 °C) products were calcium pyrophosphate particles.
2.3. Mechanism of Crystallization in the Reverse Microemulsion Process
Regarding the solubility of calcium phosphate in the crystalline phases of brushite, it was noticed
that at a normal pH it precipitates at room temperature. FTIR spectra and XRD analysis indicated that
the as-synthesized products obtained at a lower temperature were brushite particles . However, the
products obtained after calcination at the higher temperature of 800 °C were calcium pyrophosphate
particles . The formation of a solid crystalline phase of calcium phosphate depends on kinetic
rather than thermodynamic factors. Recent reports have indicated that the crystallization rate of
brushite is much higher than that of HAP at room temperature . The precipitation process of reverse
micelles is indicated by the following equation in equilibrium at room temperature:
(NH4)2HPO4 ↔ 2(NH4)+ + HPO42− (1)
Ca2+ + HPO42− ↔ CaHPO4 (2)
Ca(NO3)2·4H2O + (NH4)2HPO4 → CaHPO4 + (NH4)2NO3 + 2H2O (3)
An increase in the crystal growth of brushite is due to the utilization of phosphate ions, which
depends on the amount of ammonium ions present in the solution. With an increasing W/S ratio, the
number of ammonium ions is increased as a result of (NH4)2HPO4.
The presence of calcium pyrophosphate in a calcinated sample indicates the presence of HPO42−.
Calcium pyrophosphate is formed by the loss of one H2O molecule from two HPO42− groups
of brushite under high-temperature conditions. A possible equation of the equilibrium (at 800 °C)
according to the results is given below:
CaHPO4·2H2O → CaHPO4 + 2H2O ↑ (4)
The transition of monetite into pyrophosphate occurs at temperatures above 400 °C:
2CaHPO4 → Ca2P2O7 + H2O↑ (5)
The results of the present study are in agreement with previous reports that indicate that the mass
loss at 800 °C produces calcium pyrophosphate through a decomposition reaction .
3. Experimental Section
The chemical precursors used for the synthesis of nanoparticles were: (i) Ca(NO3)2·4H2O
(calcium nitrate tetrahydrate), (ii) (NH4)2HPO4 (biammonium hydrogen phosphate), (iii) surfactin, and
(iv) n-hexane. All chemicals used were supplied at analytical grade (Sigma-Aldrich, USA), while the
surfactin was laboratory-madet.
Int. J. Mol. Sci. 2011, 12
3.2. Calcium Nitrate Tetrahydrate and Ammonium Phosphate Solutions
Two solutions were made for the microemulsion (Table 1). The first solution contained
Ca(NO3)2·4H2O (2.36 g) and water (100 mL). The second solution contained (NH4)2HPO4 (1.584 g)
and water (100 mL). Both solutions were stirred separately for 5 min.
Table 1. Recipe for reverse microemulsion with a water/surfactin ratio (W/S) from 250
3.3. Surfactin Production
Bacillus subtilis (BBK006) was used to produce surfactin. The microorganism was cultured in M9
medium (1 g/L NH4Cl, 3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 mmol/L MgSO4, and 0.1 mmol/L
CaCl2) at 37 °C for sufficient growth. Afterwards, the bacteria and supernatant were separated using
high-speed centrifugation at 13,000 rpm for 30 min (Himac CR-22G, R20A2Rotor, Hitachi, Japan).
The supernatant was acidified to pH <2 using hydrochloric acid. Surfactin was collected using
high-speed centrifugation at 13,000 rpm for 30 min. The precipitated surfactin was then dried to
surfactin powder using a freeze drier (Biotron 3180C).
3.4. Synthesis of Nanoparticles by the Reverse Microemulsion Process
The reverse microemulsion process was used to prepare calcium phosphate nanoparticles. Surfactin
was added to produce an emulsion layer that acted as a potential reaction space. The ratio of water
to surfactin (W/S) used varied from 250 to 40,000 (250, 500, 1000 and 40,000, Table 1). Calcium
nitrate tetrahydrate (2.25 mL, 0.2 M) and ammonium phosphate (0.12 M) were separately added to
10 mL n-hexane in two centrifuge tubes ―A‖ and ―B‖. Different amounts (g dry weight) of surfactin
(Table 1) were then added. These two solutions were mixed together by rigorous shaking. After 4 days
of reaction, a white product was formed and was collected by centrifugation, washed repeatedly with
methanol, and dried overnight at 50 °C. All synthesized powders were calcinated at 800 °C for 3 h.
The calcinated powders were labeled W/S250@800 °C, W/S500@800 °C, W/S1000@800 °C and
W/S40000@800 °C, and the synthesized powders were labeled W/S250@RT, W/S500@RT,
W/S1000@RT and W/S40000@RT.
Int. J. Mol. Sci. 2011, 12
3.5. Characterization of Particles
Morphological study by TEM and SEM
The powders were dispersed in ethanol before analysis. The morphology of the synthesized
powders was characterized by TEM (JEOL JEM 2010, Japan) using an accelerating voltage of 200 kV.
The powders were also analyzed by SEM (Hitachi S4800, Japan) with a coating (Pt) operated at
Assessment of functional groups by FTIR
The compositions of the synthesized powders were identified by FTIR (Jasco FT/IR-460 plus) with
the assistance of KBr. The functional groups of the samples were observed in the spectral region of
4000~1300 cm−1 and the fingerprint region of 1300~400 cm−1.
Assessment of phase composition and crystallinity using XRD
The phase composition and crystallinity of both the calcinated and non-calcinated powders were
analyzed by X-ray diffraction (LabX XRD6000) with CuKa radiation (λ = 0.15418 nm) at 40 kV and
30 mA. The angle was set to 10–70 degrees, four degrees per minute.
It has been shown that microemulsions created by surfactin successfully produced calcium
phosphate of unique shapes and sizes. The reverse microemulsion process resulted in the formation
and growth of brushite nanoparticles under non-calcinated conditions at room temperature. On the
other hand, calcium pyrophosphate particles were formed under calcinated (800 °C) conditions. The
crystalline structure of the calcium phosphate depended on the ratio of the calcium nitrate tetrahydrate
and ammonium phosphate water solution to surfactin. The different structures obtained included
hexagonal-shaped, thin-layered, needle-shaped, and small roundish-shaped crystals. All the particles
produced were nano-sized (16–200 nm), and the technique is inexpensive and eco-friendly. This novel
approach can potentially be used in a wide range of technological applications.
The authors are very grateful to the Science Council of Taiwan for sponsoring this research.
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