Biological Synthesis of Size-Controlled Cadmium Sulfide Nanoparticles Using ImmobilizedRhodobacter sphaeroides
© to the authors 2009
Received: 30 December 2008
Accepted: 24 March 2009
Published: 18 April 2009
Skip to main content
© to the authors 2009
Received: 30 December 2008
Accepted: 24 March 2009
Published: 18 April 2009
Size-controlled cadmium sulfide nanoparticles were successfully synthesized by immobilizedRhodobacter sphaeroides in the study. The dynamic process that Cd2+was transported from solution into cell by livingR. sphaeroides was characterized by transmission electron microscopy (TEM). Culture time, as an important physiological parameter forR. sphaeroides growth, could significantly control the size of cadmium sulfide nanoparticles. TEM demonstrated that the average sizes of spherical cadmium sulfide nanoparticles were 2.3 ± 0.15, 6.8 ± 0.22, and 36.8 ± 0.25 nm at culture times of 36, 42, and 48 h, respectively. Also, the UV–vis and photoluminescence spectral analysis of cadmium sulfide nanoparticles were performed.
Biosynthesis of nanomaterials as a novel nanoparticle synthesizing technology attracts increasing attention. It is well known that many organisms can provide inorganic materials either intra- or extracellularly [1, 2]. For example, unicellular organisms such as magnetotactic bacteria produce magnetite nanoparticles , and diatoms synthesize siliceous materials . Even live plants such as Alfalfa are able to produce gold clusters surrounded by a shell of organic ligands . Bansal et al.  have synthesized 4–5 nm barium titanate (BT) nanoparticles using a fungus-mediated approach. As far as the biosynthesis of cadmium sulfide (CdS) nanoparticles is concerned, a number of biosynthesis methods have been reported. For example, CdS nanoparticles can be synthesized intracellularly by the yeasts Schizosaccharomyces pombe. However, intracellular synthesis of CdS nanoparticles makes the job of downstream processing difficult and beats the purpose of developing a simple and economical process. The extracellular enzyme secreted by the fungus Fusarium oxysporum can mediate extracellular synthesis of CdS nanoparticles . But live organisms have the endogenous ability to exquisitely regulate synthesis of inorganic materials. For example, shape control of inorganic materials in biological systems was achieved either by formation of membrane vesicles  or through functional molecules such as aluminophosphates and polypeptides that bonded specifically to mineral surfaces . On the other hand, the size, shape, and yield of biosynthesized nanoparticles significantly depend on physiological parameters, and remarkably are affected by growth conditions (including pH, temperature, culture time, and metal ions concentration) of live organisms. For example, gold nanowires with a network structure can be synthesized with the change of HAuCl4 concentration by Rhodopseudomonas capsulate, and triangular gold nanoplates can be produced with adjusting the pH of initial solution by Rhodopseudomonas capsulate. The exploitation of size- and shape-controlled biosynthesis of CdS nanoparticles using live photosynthetic bacteria is so far unexplored and underexploited. In this study, prokaryote photosynthetic bacteria Rhodobacter sphaeroides, recognized as one of the ecologically and environmentally important microorganisms, commonly existing in the natural environment, were investigated for producing CdS at room temperature with a single step process. Especially CdS nanoparticles were formed intracellularly and then were transported into extracellular solution. In addition, immobilized R. sphaeroides can be separated from cadmium sulfide nanoparticles easily.
Rhodobacter sphaeroides were obtained from College of Life Science and Technology, Shanxi University, Taiyuan, China. R. sphaeroides were cultured in the medium containing (in 1 L) 2.0 g malic sodium, 0.15 g MgSO4 · 7H2O, 1.2 g yeast extract, and 1.5 g (NH4)2SO4 at pH 7 and 30 °C . The bacteria were cultured for 72 h and separated from broth by centrifugation (5000 rpm) at 4 °C for 10 min. The collected bacteria were washed five times with distilled water to obtain about 1 g wet weight of bacteria.
The concentrated pure-culture R. sphaeroides were then mixed with polyvinylalcohol (PVA) (10 g PVA/100 mL distilled water). The initial concentration of cells was 30 mg/L. The gel beads with wrapped microbial cells were formed in a solution of 10% H3BO3, and the average diameter was about 3 mm. The beads were “annealed” in the H3BO3 solution for 18 h. After activation in growth medium, the immobilized beads were washed twice with distilled water and were prepared for use .
Synthesis was conducted in a 1000 mL sterile serum bottle containing 20 g immobilizedR. sphaeroides and 500 culture medium of 1.0 mM CdCl2. The resulting solution was incubated at 30 °C under the dark and aerobic (DO = 5 mg L−1) conditions for 36 h. After the bio-transformation reaction was completed, the precipitate was washed several times with distilled water. The final precipitate was dried at 50 °C for 3 h in a vacuum kiln. The products were obtained in about 85% yield based on Cd.
The CdS nanoparticles synthesized by immobilized R. sphaeroides were used for powder X-ray diffraction (XRD) analysis. The spectra were recorded on a Rigaku Dmax-γA automatic instrument. The diffracted intensities were recorded from 10° to 70° 2θ angles. The sample was prepared by drop coating onto a carbon-coated copper grid for transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and selected area electron diffraction (SAED). TEM was performed on a Hitachi H-600 instrument operated at an accelerating voltage of 120 kV while HRTEM and SAED were performed on a Hitachi H-2010 instrument operated at a lattice image resolution of 0.14 nm. The cells were analyzed by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDXS), using a 100CX scanning transmission electron microscope and a Kevex 8000 EDX system. The cell samples were prepared as previously described . Ultraviolet and visible (UV–vis) absorption spectrum was collected at room temperature on Shimadzu UV-2101PC using BaSO4 powder as a standard. The photoluminescence emission and excitation spectra were recorded with a Hitachi F-850 fluorescence spectrometer.
Nanocrystal formation was initiated by adding CdCl2 (1 mM) to a cell sample (about 1 g wet weight) suspended in growing medium. The solutions were incubated on an orbital shaker at 30 °C and agitated at 150 rpm. Samples were taken at predefined time intervals (0, 12, 24, 36, 42, and 48 h). The sample was centrifuged at 4000×g for 20 min. The biomass pellet (P1) was collected and the medium without cells was centrifuged at 15000×g at 4 °C for 60 min. The supernatant (S1) was collected, and the pellet (P2) with the CdS-containing particles was washed with deionized water three times. Each experiment was repeated three times. The contents of cadmium in different forms of P1, S1, and P2 were determined using Shimadzu AA-6300 atomic absorption spectrophotometer in an air-acetylene flame at 228.8 nm wavelength .
Cysteine desulfhydrase activity of the cell was measured using a colorimetric assay adapted from Chu et al. . Samples of R. sphaeroides were centrifuged at 4000×g for 20 min. The pellet was resuspended in phosphate buffer (10 mM, 1 ml, pH 7.5). The reaction was started by the addition of Tris (0.1 M buffered to pH 7.6) and cysteine hydrochloride (100 mM, pH 8.6), then the mixture was incubated at 37 °C for 1 h. Sulfide formation was determined by adding N,N-dimethyl-p-phenylenediamine sulfate (20 mM, in 7.2 M HCl) and FeCl3 (30 mM, in 7.2 M HCl) to the reaction tubes. Absorbance was measured at 650 nm and the concentration of sulfide was determined according to a standard sodium sulfide calibration curve. Total protein was measured by the method of Chen et al. .
Previous studies indicated that cysteine desulfhydrase was an important factor in the biosynthesis of metal sulfide nanoparticles . Also, we had confirmed that R. sphaeroides could secrete cysteine desulfhydrase (C–S-lyase) being responsible for producing S2−. The result shows that the activity of cysteine desulfhydrase in R. sphaeroides depends on culture time, and the activities at 36, 42, and 48 h are 32.6, 45.1, and 50.8 U g−1, respectively. Namely, the activity of C–S-lyase at 36 h is lower than the ones at 42 h and 48 h. Hence, the reaction rate between cadmium ions and S2− is very slow at 36 h, resulting in the formation of CdS nanoparticles with small diameter. With the increasing culture time, the enzyme activities and reaction rate correspondingly increase, contributing to the formation of thermodynamic-favored spherical particles. Thus, the size-controlled biosynthesis of CdS nanoparticles using immobilized R. sphaeroides could be obtained by simply changing the culture time.
The present study demonstrated that size-controlled CdS nanoparticles had been synthesized by living immobilizedR. sphaeroides. Also, the result showed thatR. sphaeroides could transport Cd2+into cell from solution and then produced CdS. Finally, the CdS was carried to the extracellular solution and formed nanoparticles. The size of CdS nanoparticles biosynthesized by living immobilizedR. sphaeroides could vary with the culture time. The way of the size-controlled biosynthesis of CdS nanoparticles by simply changing culture time provides a fully green approach for the biosynthesis modulation of nanomaterials. Moreover, the UV–vis absorption spectra and photoluminescence spectra showed that CdS nanoparticles exhibited unique optical properties.
We acknowledge the service rendered by the Sophisticated Analytical Instrumentation Facility, Institute of Coal Chemistry, CAS, Taiyuan, China, in analyzing the samples by TEM. Financial supports from the Shanxi Provincial Key Technology R&D Program of Shanxi (No. 20080311027-1), and National Key Technologies R&D Program of China (No. 2001BA540C) are gratefully acknowledged.