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Recent years the synthesis of gold nanoparticles has been the focus of intense interest because of their emerging applications in a number of areas such as bioimaging, biosensors, biolabels, biomedicines, and etc. Researches are now focusing on nanotechnology-based approaches to meet environmental challenges. The grave concern for human health due to scarcity of clean water has stimulated research for obtaining pure water free from contaminants such as pesticides and pathogenic organisms. Appropriate hygienic treatment eliminates pathogens from water; nevertheless a few may be present occasionally. The extent of pesticide contamination in water is a matter of great concern because of their potential health hazards and entry into the food chain of humans and animals. In view of extensive variations in the chemical structures of pesticides, it is hardly possible to find a single method suitable for reducing pesticide concentration of potable water to the permissible limit. Conventional practices such as adsorption on activated carbon or different biological materials, ultra filtration, reverse osmosis or electrochemical treatment suffer from number of limitations. These include cost effectiveness, low adsorption capacity or inadequate affinity toward target toxicant. Research activities are now concentrating on the development of nanotechnology-based methodologies to overcome these problems. The degradation of a wide variety of aromatic and aliphatic halogenated organic compounds by metal nanoparticles has recently been reported. This paper describes the microbial synthesis of gold nanoparticles using a native Rhizopus oryzae strain, and application of the generated nanogold-bioconjugate (NGBC) in a single-step removal of some model organophosphorus pesticides from water along with the some microorganisms.
1 Experiment and Result Analysis
Synthesis of Gold Nanoparticles by R. oryzae Mycelia:
Incubation of HAuCl4 solution with R. oryzae mycelia induces gradual color change of the biomass from light yellow to colorless and finally to purple within 24 h, indicating the formation of gold nanoparticles on the mycelial surface. The purple mycelia (gold particle immobilized mycelia) is collected by centrifugation (10 000 rpm for 10min), dried by lyophilization and dispersed in alcohol. The UV-vis spectra of the dispersed solution exhibited absorption maximum at about 540 nm (Figure 1A) due to the surface plasmon resonance (SPR) band of the gold nanoparticles With increasing initial concentration of the gold ions, the surface coverage of the gold nanoparticles on the mycelia increases with concomitant increment of the SPR band at 540 nm that reached saturation at 500 mg/L HAuCl4 concentration. TEM micrographs demonstrate the formation of gold nanoparticles on the surface of R. oryzae mycelia (Figure 1B). High-resolution image shows decoration of gold nanoparticles (10 nm average diameter) on the mycelial surface (Figure 1C). The micrograph also demonstrates that as-synthesized gold nanoparticles are well-dispersed with no conspicuous agglomeration and stable even up to 6 months; since the absorption band does not change over this period. This indicated that the mycelial surface acts both as reducing as well as capping agent. Figure 1D depicts the selected area electron diffraction (SAED) pattern obtained from the gold nanoparticles (Figure 1C). The Scherrer ring patterns characteristic of the facecentered cubic (fcc) gold is clearly observed, indicating that the structures seen in the TEMimages are nanocrystalline in nature.
(A) UV-vis spectra of the dispersed solution of goldembedded R. oryzae mycelia. (B) TEM micrograph of R. oryzae mycelia after reaction with aqueous HAuCl4 solution. (C) Highresolution images demonstrate formation of gold nanoparticles (10 nm average diameter) on the surface of mycelia. (D) SAED pattern of gold nanoparticles.
Adsorption of Pesticides by NGBC:
Removal of pesticides from water bodies using a single method is very difficult because of wide variations in their chemical structures. The adsorption behavior of different organophosphorus pesticides on NGBC material is tested in order to facilitate the eco-friendly removal of pesticides from aqueous solution. The adsorption of organophosphorus pesticides on NGBC increases significantly to 85-99% from 5-25% corresponding values of pristine mycelia (Figure 2A). Surface coverage of the mycelia with gold nanoparticles increases with increase in gold chloride concentration resulting in increase pesticide adsorption which attains a maximum value when the surface coverage reaches saturation level. However, adsorption of γ-BHC, an organochlorine pesticide, on both pristine and NGBC material remains almost the same (Figure 2A)
The rate of adsorption of all organophosphorous pesticides used in this experiment involving NGBC indicates (Figure 2B) that the adsorption process is very fast reaching equilibrium within 10 min. The micrographs of the control NGBC (Figure 3A) depict decorated gold nanoparticles throughout the surface.
However, post-adsorbed species are conspicuously different from that of the control NGBC. The micrographs demonstrate (Figure 3B-E) the formation of conglomerated island-type domains of pesticide molecules (as confirmed by EDXA) on the NGBC material along with the disappearance of gold nanoparticles. The surface roughness (rms) values of the NGBC material increase significantly to 60-75nm upon
(A) Adsorption of pesticides on the pristine and NGBC material. (B)Adsorption kinetics of organophosphorous pesticides on NGBCmaterial.Data represent an average of five independent experiments; (SD shown by error bar.
pesticides adsorption, corresponding to that (15.5 ( 2.5 nm) of the control (unadsorbed) NBGC. Following adsorption of pesticides, the gold nanoparticles disappeares (Figure 3A),
AFMimages (scan area 1.5 μm 1.5 μm) of (A) the pristine NGBC and the (B) malathione-, (C) parathione-, (D) chlorpyrifos-, and (E) dimethoate-adsorbedNGBCmaterial. (F)EDXAspectrumofNGBC material after adsorption of organophosphorous pesticide for 20 min.
and a few domain-like structures (as indicated by circles in the micrographs) are formed on the surface soon after initiation of the adsorption process (Figure 4). With increase in time of adsorption, more domain-like structures appear on the surface; the grain size increases laterally and eventually coalesced. The size of gold nanoparticles is 10 nm, whereas the domain size of the pesticides is much higher. The reproducibility of the images is confirmed by recording the images from five independent experiments.
AFM images (scan area 1.5 μm 1.5 μm) of NGBC on adsorption of (A) malathione, (B) parathione, (C) chlorpyrifos, and (D) dimethoate at different incubation times.
The same growth pattern is observed in the case of all the pesticide molecules confirming good adsorption of organophosphorous pesticides on the NGBC.
Antibacterial Activity of NGBC:
In order to make potable water free from microbial pathogens, Researchers explored the antimicrobial activity of NGBC. The antimicrobial activity of the dispersed NGBC solution against P. aeruginosa, E. coli, B. subtilis, S. aureus, Salmonella sp., S. cerevesiae, and C. albicans is tested by the cup-plate method.
Antimicrobial activity of NGBC material against (A) E. coli, (B) P. aeruginosa, (C) B. subtilis, (D) S. aureus, (E) S. cerevisiae, and (F) C. albicans. Cup containing (I) dispersed solution of R. oryzae and (II) NGBC; concentration of bioconjugate in each plate was 50 μg/mL.
Observations showed a clear zone of inhibition around the cup (II) in the plate (Figure 5) containing an absorbed dispersed solution of NGBC, indicating the antimicrobial activity of NGBC against these organisms; the control experiment with a dispersed solution of pristine R. oryzae in the cup (I) exhibited no zone of inhibition. The viability of these pathogens following interaction with NGBC is also studied by incubating the organisms with dispersed solution of NGBC for 30 min. Upon completion of the incubation period, the microbial cell suspension is stained using a LIVE/DEAD kit following the manufacturer's instructions. Figure 6 shows the fluorescent microscopic images of microbial cells following NGBC treatment and after being stained with LIVE (green)/DEAD (red) stains following the manufacturer's instructions. Exposure of microbial cells to NGBC resulted in a significant decrease in cell viability compared to the control cells. Quantification of the viability of the cells is done by containing live (green) versus dead (red) stains. There was 90% reduction in cell viability with corresponding increase in the number of red dead cells. This observation exhibits the microbicidal activity of NGBC. The extent of microbial cell membrane disruption following interaction with NGBC was examined by SEM study. The SEM images of cells exposed to NGBC (Figure 7, middle panel) reveal
Viability of (A) E. coli, (B) P. aeruginosa, (C) B. subtilis, (D) S. aureus, (E) S. cerevisiae, and (F)C. albicans cells following treatment with NGBC for 30 min. Cells were stained with SYTO 9 and propidium iodide and imaged at 100 magnification using a fluorescence microscope.Green fluorescent cells are representative of live cells,while red fluorescent cells are representative of dead or compromised cells.
distinct morphological changes compared to that of the control one (Figure 7, left panel).
SEM micrographs of control (left panel) and NGBC-treated (middle panel) (A) E. coli, (B) P. aeruginosa, (C) B. subtilis,
(D) S. aureus, and (E) S. cerevisiae cells, indicating morphological changes of microbial cells. Higher magnified images (right panel) of the post-treated cells clearly depict severe membrane damage.
After incubation with NGBC, the integrity of most of the microbial cells is lost, indicating irreversible cell damage and ultimate cell death. High-resolution images (Figure 7, right panel) indicate that the smooth surface of the control cells changes to an irregular one upon treatment with NGBC.
Treatment of Simulated Contaminated Water with NGBC:
Upon successful removal of organophosphorous pesticides and inactivation of microorganisms in separate experiments, we can consider that the NGBC may be used to obtain potable water free from pathogens with pesticide concentrations below the safety level in a single operation. By preparing simulated contaminated water relevant to the environmental condition containing E. coli (103 cells/mL) and 10 μg/L malathion, 5 μg/L parathion, 12 μg/L chlorpyrifos, and 8 μg/L dimethoate. 5 mg of NGBC is added to 100 mL of this water and incubated with gentle shaking at room temperature (30 C) for different time intervals. At the end of the desired incubation period, NGBC was separated aseptically by filtration through glass wool, and the cell count of E. coli and pesticide concentration in the filtrate can then be determined by plating on MacConkey agar and GC analysis respectively.
Results noted that the concentration of the pesticides (Table 1) and E. coli density (Figure 8B-C) in the treated water fall significantly within 10 min compared with the control (Figure 8A).
The pesticides levels decreased (Table 1) below detectable limit (<1 μg/L)while E. coli could not be detected (Figure 8D) after 30 min of incubation. The control experiment with pristine R. oryzae mycelia (without embedded gold nanoparticles) failed to kill E. coli. The experiment was repeated five times and obtained similar results. The proficient bactericidal activity as well as organophosphorous pesticide adsorption capacity of NGBC suggests the use of this conjugated material for a one-step water purification process.
Micrographs of E. coli cell following treatment with NGBC material at different incubation periods: (A) 0 min,(B) 5 min, (C) 10 min, and (D) 30 min.
The synthesis of gold nanoparticles on the surface of R. oryzae by a one-pot green chemical approach is described. The NGBC strongly adsorbs different organophosphorous pesticides and, in addition, exhibits antimicrobial activity against different bacteria and yeasts. The interaction of NGBC with microbial cells causes rupture of the cell membrane, resulting in cell death. The removal of pesticides and E. coli from simulated contaminated water in a single step using NGBC suggests a significant advancement in the development of a nanotechnology-based green chemical approach for water purification.
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