Mechanistic aspects of biogenic synthesis of CdS nanoparticles using Bacillus licheniformis

The bacterial strain of B. licheniformis MTCC 9555 was obtained from the Department of Microbiology, College of Life Sciences, Gwalior, India. Cadmium chloride (CdCl₂) and sodium sulfide (Na₂S) was purchased from Qualigens Fine Chemicals, Mumbai, India. Nutrient broth media was purchased from Hi-media, Mumbai, India. All chemicals were used as received.

B. licheniformis MTCC 9555 was grown in Erlenmeyer flasks containing essential sterilized nutrient broth media to obtain the biomass for nanoparticles synthesis. The culture was incubated at 37 ± 1 °C temperature with continuous stirring (200 rpm) for 36 h. After completion of incubation period, the grown biomass was extracted by centrifugation at 8000 rpm for 15 mins. The collected bacterial biomass was washed with deionized water 4 times. The extracted biomass was used for synthesis of CdS nanoparticles.

Bacterial biomass (0.5 g) of B. licheniformis MTCC 9555 was added into an Erlenmeyer flask containing 45 ml of deionized water and stirring at 200 rpm for 30 mins. Then 0.1 mM of cadmium chloride (CdCl₂) was added into it. Subsequently, sodium sulfide (Na₂S) solution (0.01 mM) was added into it drop wise. Then the above solution was left for stirring (200 rpm) at 37 ± 1 °C for 48 h and pH was maintained at 8 ± 1. A control sample was also run without bacterial biomass to compare the results. After incubation, the solution was centrifuged to separate the bacterial biomass from the nanoparticles suspension and used further for characterization.

The formation of CdS nanoparticles using bacterial biomass of B. licheniformis MTCC 9555 was determined with the help of a UV-Vis spectrophotometer (UV-1601pc Shimadzu). UV-Vis spectroscopy reveals a strong absorption peak at 368 nm corresponding to the formation of CdS nanoparticles (figure 1). Further, the band gap of synthesized nanoparticles was calculated by using the equation

$$\begin{eqnarray*}&&{{E}_{g\;\,nano}}=hc/\lambda ,\end{eqnarray*}$$

where h is Planck's constant, c is speed of light and λ is cutoff wavelength (475 nm).

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The band gap of synthesized nanoparticles was found to be 2.61 eV, which is larger than the band gap of bulk CdS (2.42 eV) [20]. This increase in the band gap arises due to shifting of absorption edge towards lower wavelengths (blue shift) as a consequence of quantum size effect. Further, the size of semiconductor CdS nanoparticles is estimated by the Brus equation

$$\begin{eqnarray*}&&{{E}_{g\;\,CdS}}={{E}_{g\;\,bulkCdS}}+\frac{{{h}^{2}}}{2d_{p}^{2}}\left( \frac{1}{{{m}_{e}}}+\frac{1}{{{m}_{h}}} \right)-\frac{3.6{{e}^{2}}}{4\pi \epsilon {{d}_{p}}},\end{eqnarray*}$$

where d_p is the diameter of the CdS nanoparticles, E_gCdS is the energy band gap of CdS nanoparticles (2.61 eV), E_gbulkCdS is the energy band gap of bulk CdS (2.42 eV), e = 1.60 × 10⁻¹⁹ C, ₀ = 8.85 × 10⁻¹² C² Nm⁻²,  = 5.7 ₀, m_e  = 1.73 × 10⁻³¹ kg, m_h  = 7.29 × 10⁻³¹ kg. According to the Brus equation, the size of the CdS nanoparticles having a band gap of 2.61 eV was estimated to be 5.1 ± 0.5 nm, thus signifying the fact that band gap increases with decrease in particle size.

XRD (X'Pert PRO PANanalytical x-ray Diffractometer) of synthesized nanoparticles was done by making a thin film of CdS nanoparticles over a glass substrate calcined at 100 °C for the removal of organic substances. The 2θ values were measured over a wide range (3° ≦̸ 2θ ≦̸ 70°), strong peaks occurred at 2θ = 26.7°, 36.6°, 54.9° and 67.2° corresponding to (002), (102), (004) and (203) lattice planes, respectively (figure 2). These sets of lattice planes indicate the hexagonal structure of CdS nanoparticles, which is in good agreement with JCPDS File No.80-0006. Broadening of peaks indicates that the formed particles are in a narrow region. The intensity of the peaks shows that the particles are crystalline in nature.

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Drop coated film of CdS nanoparticles was prepared onto the carbon coated copper grid for the analysis of size and morphology of the biosynthesized CdS nanoparticles using TEM (Philips CM-10). The average size of nanoparticles was found to be 5.1 ± 0.5 nm having a spherical morphology (figure 3(a)). Further, the HRTEM shows that the particles have a d-spacing of 2.439 Å (figure 3(b)).

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Energy dispersive x-ray (EDX) spectroscopy was used to analyze the elements present in the sample. EDX (Sigma) analysis shows the peaks of the elemental signal of Cd and S from the emission of the energies of these elements which are being displayed in the spectrum plotted between x-ray counts and energy (keV) and the additional two peaks of Cu are obtained due to the copper grid used for the sampling (figure 4).

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The synthesized nanoparticles were analyzed by Fourier transform infrared (FTIR) spectroscopy (Perkin-Elmer) to determine the possible biomolecules involved in the biosynthesis of nanoparticles (figure 5). An absorbance band of peaks occurring from 3929.00 − 3412.08 cm⁻¹ corresponds to the –OH stretching with the presence of –NH (amide) group which shows the presence of protein/enzyme in the sample. Another peak was also obtained at 2121.70 cm⁻¹ which can be assigned to the stretching of C≡⃥C bond. An absorbance peak occurring at 1641.42 cm⁻¹ and 1560.62 cm⁻¹ corresponds to amide I and amide II adsorption of proteins molecule, respectively. Three peaks occurring at 1462.04 cm⁻¹, 1429.25 cm⁻¹, 1365.60 cm⁻¹ correspond to the bending of –CH group. A single peak at 1230.58 cm⁻¹ corresponds to the C-O stretch of ester. A single peak at 1072.42 cm⁻¹ corresponds to the C-O stretch of ether. Two peaks obtained at 711.73 cm⁻¹ and 619.15 cm⁻¹ can be assigned to the stretching of C-Cl bond. The absorbance band occurring at 572.86 cm⁻¹ and 505.35 cm⁻¹ can be assigned to the stretching of the C-Br bond. The absorbance band occurring from 499.56–374.19 cm⁻¹ can be assigned to the stretching of C-I bond. Earlier, it was reported that amine group helps in the binding of proteins with the CdS nanoparticles [21]. Thus, from this we can conclude that protein released by the bacterial biomass is mainly responsible for the stabilization of CdS nanoparticles.

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A plausible mechanism has been made to understand the synthesis of CdS nanoparticles (figure 6). At the initial phase of the reaction, CdCl₂ dissociates into Cd²⁺ and accumulates around the bacterial membrane because of the negative potential of the bacterial membrane [22]. Cd²⁺ being a heavy metal ion creates stress conditions for bacteria, thus leading bacterial biomass to initiate a defense mechanism [23, 24]. This leads bacteria to secrete certain enzymes/proteins in order to detoxify the metal ions that created the metal stress condition [25]. The secreted protein by bacterial biomass binds up with Cd²⁺. Subsequently, Na₂S being added dissociates into S²⁻ in the solution and it also binds with the protein. The CdS nuclei then grow following the process of Ostwald ripening [26] leading to the formation of CdS nanoparticles. Thus, protein secreted in this process becomes incorporated as it serves a capping layer for synthesis of CdS nanoparticles. Further, the detailed mechanism of biosynthesis of CdS nanoparticles is under study.

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