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 1b).

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 [12] and Lu et al. [13]. 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.

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.

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 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 [14]. The results showed that the as-synthesized products were brushite particles and the calcinated (800 °C) products were calcium pyrophosphate particles.

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 [8]. However, the products obtained after calcination at the higher temperature of 800 °C were calcium pyrophosphate particles [15]. 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 [8]. The precipitation process of reverse micelles is indicated by the following equation in equilibrium at room temperature:

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:

The transition of monetite into pyrophosphate occurs at temperatures above 400 °C:

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 [14].

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.

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.

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).

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.