5. Synthesis and characterization of lead metal nanoparticles from heavy metal hyperaccumulator plants
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1 CHAPTER - V 5. Synthesis and characterization of lead metal nanoparticles from heavy metal hyperaccumulator plants 5.1 Introduction Microscopic particles whose size is measured in nanometers (10-9 m) are called nanoparticles. These particles have very special properties which are used for the production and applications in technological devices. In recent years, nanotechnology is emerging as a revolutionizing field of research with multiple disciplines of science like physics, chemistry, materials science and biology. The synthesis of nanomaterials by the use of green chemicals has vital importance in medicinal and technological aspects. Lead metal nanoparticles have a wide range of applications in the field of environmental pollution control, drug delivery system, material chemistry and so on (Longoria et al., 2014). In conventional, various chemical and physical methods are utilized for the synthesis of nanomaterials, but due to the fact of usage of a large amount of hazardous chemicals, it becomes mandate to find an alternative eco-friendly methodology. In search of green chemistry approach, the usage of plants and microbes were of the prime target, which were considered safe, simple, nontoxic, biocompatible and environment friendly. Accumulation of metal ions in plants is largely used in phytoremediation, which has been regarded as a low-cost, eco-friendly and efficient approach for the cleanup of the polluted site. Synthesis of metal nanoparticles assisted by biological agents provides advantages over chemical and physical methods due to the fact that it is costeffective, environmentally friendly and easily scalable for large-scale synthesis and obviates the need for high pressure, energy, temperature, and toxic chemicals. However, research on this field has been focused mainly on gold and silver and many applications of these metallic nanostructures have been sufficiently explored. In the case of heavy metals such as lead, traditional chemical and physical procedures are employed to synthesize nanoparticles. Nevertheless, available procedures to obtain lead (Pb) nanoparticles are not simple. It has been generally 99
2 understood that lead particles are readily oxidized even at room temperature and under high vacuum and usually various techniques are needed to prevent oxidation, such as embedding the Pb particles in amorphous silicon oxide matrices or employing inorganic templates (Stella et al., 1994). The interest of lead nanoparticles is currently increasing due to their lubricant properties and superconducting fluctuation diamagnetism phenomena that lead nanoparticles present (Zhao et al., 2004; Guin et al., 2010). Lead is a kind of conventional lubricant, so the application of Pb nanoparticles in lubricating additives is potentially interesting. It was reported recently that Pb nanostructures present excellent antiwear performance in a broad concentration range where it is issued as lubricating oil additive (Zhao et al., 2004). In the presence of an external magnetic field, superconducting metals in the nanometer regime exhibit superconducting fluctuation diamagnetism when the particle diameter is smaller than the superconducting coherence length of the material. In the case of lead the useful reported size is 87 nm. Thus Pb nanoparticles are presently used as a model to study the quantum size effect of the superconducting properties, as well as fluctuation diamagnetism. For this reason, there is an increasing interest to synthesize Pb nanoparticles with size under 87 nm (Zhao et al., 2004; Karna et al., 2011). Plants and plant extracts have been successfully used to produce metallic nanostructures. The traditional methods for disposal of hyperaccumulator plants, such as incineration (Stuchi and Jakob, 1997; Bennet and Shaw, 2000; Reijnders, 2005), landfill (Zhao et al., 2000; Sarret et al. 2001) and ashing (Sas-Nowosielska et al. 2004), cannot reuse the metals in them and may lead to the secondary pollution. Because of the high concentrations of heavy metals in these plants, B. juncea and other metal hyperaccumulators cannot be used as composting materials (high levels of toxic metals in leachate pose a serious threat to soils and underground water). These plants do not necessarily remain in the environment as much as there are not good disposal options for them currently. However, the metals in these plants are also a kind of resource, and the reuse of the metals in hyperaccumulators is feasible and significant. 100
3 Heavy metals in soil and eco system pose a serious threat to the ecosystem and human health and therefore the implementation of appropriate remedial measures is required (Vaxevanidou et al., 2008). Hyperaccumulators are plants that can accumulate exceptionally high quantities of heavy metals. In consideration of high metals concentrations in hyperaccumulator plants, the plants cannot be usually used as composting, agricultural forage and feed. The metals in plants must be removed, otherwise, they will return to and retain in soils. It is natural to come up with such imaginations using the Pb-hyperaccumulator plants as the material for synthesis of Pb metal nanoparticles. Sesbania grandiflora has been reported to be highly efficient to remove and accumulate heavy metals like mercury (Hg) and lead (Pb) in both leaf and root tissues. However, this is the first study examining the cellular level of the accumulation of lead in S. grandiflora. While the capacity of some plants to remove heavy metals is widely known, only a limited number of studies (Chen et al., 2009; Jiang et al., 2010) have explored their potential uses reducing agents for the production of heavy metal nanoparticles. The results presented here serve as the basis to further explore the development of an optimal protocol using S. grandiflora as a reducing agent to biosynthesize heavy metal nanoparticles. 5.2 Materials and methods Plant culture and growth conditions Sesbania grandiflora seeds were collected from Kovai Marketing, Salem, Tamil nadu, India and the seeds were germinated on trays containing a mixture of coir waste and sand in a 3:1 ratio. After germination, seedlings (10 day-old) were used for hydroponics study. The seedlings were grown in plastic containers with full-strength Hoagland s nutrient solution (Hoagland and Arnon, 1950) for 7 days grown in the green house at room temperature (37 C) and natural sunlight (13h/11h, day/night). The solutions were continuously aerated Heavy metal treatment and nanoparticle synthesis After 7 days in hydroponics culture, lead treatment was initiated. The seedlings were treated with Pb(NO 3 ) 2 (1000 mg L -1 ). Seedlings without lead 101
4 treatment served as a control. The treatment groups along with the control were arranged in a randomized design and each contained five or six seedlings. After ten days of growth, seedlings were harvested and thoroughly washed with tap water followed by distilled water and washed three times with 10 mm chelating EDTA for desorption of surface bound lead. Approximately 5 g fresh weight of each sample was dried for XRD, FTIR and SEM with EDX analysis Characterization of lead nanoparticles After nanoparticle synthesis, approximately 5 g fresh weight of each sample was dried for FTIR, XRD, and SEM with EDX analysis. The lead nanoparticles were characterized by the following three methods: Dry samples before and after bioreduction, were analyzed by XRD measurements. The synthesized lead nanoparticles solution was drop coated on to glass substrate and carried out on Shimadzu-MODEL XRD 6000 operated at a voltage of 40 Kv and a current of 30 Ma with Cu, K radiation in a θ-2θ configuration. Further, fourier transform infrared (FT-IR) spectra of the samples were measured using Perkin-Elmer makesmodel spectrum RXI in the transmittable mode at the range of cm - 1 in KBr pellets to know the possible functional groups involved in the formation of nanoparticles. For the morphological analysis of the prepared nanostructured samples, scanning electron microscopy (JEOL JSM-5400 model) of the synthesized lead nanoparticles samples was performed. The average particle size of the prepared Pb nanoparticles was determined using the Sigma ScanPro Software. 5.3 Results Fourier Transform Infra Red Spectroscopy The FTIR spectroscopy measurements were carried out to identify the possible biomolecules present in the leaf extracts that bound specifically on the lead surface. The IR spectrum of lead nanoparticles obtained from this method showed the absorption peaks at 3278, 3186, 2921, 2857, 1655, 1527, 1426, 1363, 1243, 1102, 1067, 540 cm -1 for control leaf extracts; 3298, 3191, 2921, 2853, 1649, 1529, 1423, 1385, 1361, 1103, 1069, 893, 665, 618, 536 cm -1 for leaf synthesized lead nanoparticles; 3282, 2924, 2091, 1660, 1513, 1433, 1360, 1243, 1057, 605 cm -1 for 102
5 control root extracts; 3665, 2920, 2746, 1729, 1682, 1513, 1432, 1356, 1238, 1101, 1064, 805, 668, 618 cm -1 for root synthesized lead nanoparticles represents N- H stretch (1 per N-H bond) amines group, 3186 represents N-H stretch amides group, 2921 represents C-H stretch Alkenes group, 1655 represents C=O stretch amides group, 1527 represents N-H bend (1 ) amides group, 1426 denotes the O-H bend stretching carboxylic acids group, 1363 represents -NO 2 (aliphatic) nitro groups, 1243 represents C-O-C stretch dialkyl ethers group, 1102 represents C-C stretch ketones group, 1067 represents the C-N Stretch (alkyl) amines group, 540 cm -1 represents C-Cl stretch alkyl halides group represents N-H stretch (1 per N-H bond) amines group, 3191 represents N-H stretch amides group, 2921 represents C-H stretch Alkenes group, 1649 represents C=O stretch amides group, 1527 represents N-H bend (1 ) amides group, 1423 denotes the O-H bend stretching carboxylic acids group, 1361 represents -NO 2 (aliphatic) nitro groups, 1103 represents C-C stretch ketones group, 1069 represents the C-N Stretch (alkyl) amines group, 536 cm -1 represents C-Cl stretch alkyl halides group (Fig. 25). Therefore, this confirms that the IR band at 1385 cm 1 can be assigned to a C-O bond vibration associated with Pb moieties in a monodentate coordination mode. For roots, spectra show absorptions at 1377 cm 1 and 1322 cm 1 for material unexposed and exposed to lead solution. The intensity of the band for root exposed to Pb salts is increased and shifted; this could be related to metallic nitrates content in roots. These results together suggested that the synthesized lead nanoparticles were capped by the phytoconstituents, viz., flavonoids, alkaloids and tannins that were present in the aqueous extracts. The functional groups of many compounds proved to have potential to act as reducing agents in the synthesis of lead nanoparticles. Infrared spectroscopy (IR) results indicate that flavonoids, alkaloids and tannins are the major components that may be acting as the reducing agents for lead ions; these findings strongly suggest the potential use of this S. grandiflora to further explore the bioassisted synthesis of heavy metal nanostructures. 103
6 Fig. 25. FT-IR spectra recorded from dry leaves (A) and roots (B) of S. grandiflora seedlings grown for 10 days period in hydroponics medium supplemented with Pb(NO 3 ) 2 104
7 5.3.2 X-ray Diffraction analysis X-ray diffraction studies were performed to confirm the crystalline structure of the synthesized lead nanoparticles. The crystalline nature of PbNPs was confirmed by XRD analysis. A number of Bragg s reflection peaks at 2θ values of 21.61, 28.31, 46.05, 50.35, 58.53, and corresponding to the (200), (101), (202) and (220) planes for leaves, for root synthesized lead nanoparticles reflection peaks at 2θ values of 28.31, 46.05, 50.22, and corresponding to (101), (200), (202), (220) planes (Fig. 26). The synthesized nanoparticles were crystalline in nature. Furthermore, the diffraction peaks were few to be narrow, which showed the crystalline nature of lead metal nanoparticles. The presence of very short and broad peaks indicates that the synthesized lead metal nanoparticle was amorphous. The unindexed peaks of and was reported in the previous literature works related to the lead nanoparticles. Appearances of these peaks were due to the presence of phytochemical compounds in the leaf and root extracts SEM and EDX analysis SEM images were measured and topographical analysis was performed based on the surface study. The SEM studies provide the information on the morphology and size of the synthesized lead metal nanoparticles. According to the SEM micrograph, the morphology of the lead nanoparticles was observed as spherical in shape. The sizes of the lead nanoparticle were found to be in the ranges of nm for leaf and nm for root. Most of the nanoparticles aggregated and only a few of them were scattered, as observed under SEM image system (Fig. 27). The confirmation of metallic lead in the synthesized product was further confirmed by the EDAX analysis where a strong signal from the lead atoms in the nanoparticles and weaker signals from oxygen and other atoms were provenients from proteins/enzymes present in the leaf and root extracts. Metallic lead nanocrystals showed strong absorption spectra in the range 1-3 kev. The sharp signal peak of lead strongly confirmed the reduction of lead nitrate to lead nanoparticles. Strong signals from the Pb atoms in the nanoparticles were observed and signals from O, Si, C, Na, Mg and Cl atoms were also recorded. The EDX attachment present with the SEM is known to provide information on the biochemical analysis of the fields that are being investigated or the composition at specific locations (spot EDX). 105
8 Fig. 26. XRD spectra recorded from dry leaves (A) and roots (B) of S. grandiflora seedlings grown for 10 days period in hydroponics medium supplemented with Pb(NO 3 ) 2 106
9 Fig. 27. SEM with EDX spectra recorded from leaves (A) and roots (B) of S. grandiflora seedlings grown for 10 days period in hydroponics medium supplemented with Pb(NO 3 ) 2 107
10 5.4 Discussion S. grandiflora has been used in phytoremediation of pollutants because it can be easily grown and could produce high biomass in terrestrial environment. S. grandiflora and other metal hyperaccumulators cannot be used as composting materials (high levels of toxic metals in leachate pose a serious threat to soils and underground water). Thus, these plants remain in the natural food chain. The lead in S. grandiflora plants returns to the soil after the plants have died and decomposed, which poses a serious threat to the ecosystem and to human health. However, the metals in these plants are also a kind of resource, and the recycling of the metals in hyperaccumulators is feasible, significant and useful. The growth inhibition rate was varied according to the Pb concentration used. Growth reduction was positively correlated with Pb treatment and the maximum reduction of shoot growth noticed was 47%, while the root growth was decreased by 59% at 1000 mg L -1 Pb treatment (Table 7). Furthermore, several visual toxic symptoms such as chlorosis, withering and falling of leaves were also noticed in Pb treated seedlings while control plant did not show such toxicity symptoms. Accumulation of Pb content level was greater in roots than in shoots. The shoots accumulated mg g -1 dry weight, while roots accumulated mg g -1 dry weight. A 5.13 fold increase was noticed in roots compared to the shoots at 1000 mg L -1 Pb exposure. Plants were analyzed and all showed higher amounts of lead (weight %) in roots than in leaves. Also the concentration of glutathione (GSH), the activity of glutathione synthase (GS) and the expression levels of the SmGS gene are all higher in leaves than in roots, suggesting that GSH play an important role in protecting leaves from the detrimental effects of lead (Estrella-Gomez et al., 2009). In the present study, the S. grandiflora plant was exposed to a much higher concentration of lead in the medium; therefore it is not surprising that the roots also had high concentrations of this metal. It is well documented that S. grandiflora is able to accumulate high amounts of lead in its tissues, in fact it is considered to be a Pb-hyper-accumulator. However, in the present work it is revealed that lead accumulates in the form of nanostructured material. Accumulation 108
11 occurs mainly in the cell wall of leaves and roots and closer analysis revealed that PbNPs accumulate differently in the cell wall structures. In general, cell walls of plant consist of 3 types of layers, middle lamella, primary wall and secondary wall; the middle lamella is rich in pectin and free of cellulose, the primary wall is composed of polysaccharides (cellulose, hemicellulose and pectin) and proteins while the principal components of secondary walls are cellulose, hemicellulose and lignin. Although primary and secondary cell wall boundaries are not clearly distinguished in the micrographs we present, it is clear that the concentration of PbNPs is not the same in the entire cell wall of roots and leaves cells; they appear to be arranged in bands. The intensity of the band for root exposed to Pb salts is increased and shifted; this could be related to metallic nitrates content in roots. Similar results were also reported by McCluskey et al. (1990). These results together suggested that the synthesized lead nanoparticles were capped by the phytoconstituents, viz., flavonoids, alkaloids and tannins that were present in the aqueous extracts. The functional groups of many compounds proved to have potential to act as reducing agents in the synthesis of lead nanoparticles. Either free amine groups or cystein residues in the proteins which ultimately stabilizes the lead nanoparticles (Gole et al., 2001). Biological components are known to interact with metal salts via these functional groups and mediate their reduction to nanoparticles. Infrared spectroscopy (IR) results indicate that flavonoids, alkaloids and tannins are the major components that may be acting as the reducing agents for lead ions; these findings strongly suggest the potential use of this S. grandiflora to further explore the bioassisted synthesis of heavy metal nanostructures. The synthesized nanoparticles were crystalline in nature. Furthermore, the diffraction peaks were few to be narrow, which showed the crystalline nature of lead metal nanoparticles. The presence of very short and broad peaks indicates that the synthesized lead metal nanoparticle was amorphous. The unindexed peaks of and were reported in the previous literature works related to the lead nanoparticles. Appearances of these peaks were due to the presence of 109
12 phytochemical compounds in the leaf and root extracts. Similar results were also reported earlier by Longoria et al. (2014). The SEM studies provide the information on the morphology and size of the synthesized silver nanoparticles. According to the SEM micrograph, the morphology of the lead nanoparticles was observed as spherical in shape. The sizes of the lead nanoparticle were found to be in the ranges of nm for leaf and nm for root. Most of the nanoparticles aggregated and only a few of them were scattered, as observed under SEM. The sharp signal peak of silver strongly confirmed the reduction of lead ion to lead metal nanoparticles. Strong signals from the Pb atoms in the nanoparticles were observed and signals from S, O and Cl atoms were also recorded. The signals were likely due to X-ray emission from carbohydrates/proteins/enzymes present in the cell wall of the biomass (Mishra et al., 2010). Xu and Kall (2002) demonstrated that the shape of metal nanoparticles considerably changed their optical and electronic properties due to the presence of various bioactive molecules. The EDX attachment present with the SEM is known to provide information on the biochemical analysis of the fields that are being investigated or the composition at specific locations (spot EDX). In the presence study, the metallic lead nanocrystals showed strong absorption spectra in the range 1-3 kev. Recently, Longoria et al. (2014) obtained quasi-spherical shape of lead metal nanoparticles at 1-3 kev by using Salvinia minima fern. 5.5 Summary The concentration of lead in S. grandiflora shoots and roots were greater than 10,000 mg kg -1 (dry weight). The plant has a high tolerance for lead in soil, and S. grandiflora can be classified as a lead hyperaccumulator and used to remediate lead contaminated sites. Lead metal nanoparticles were synthesized from S. grandiflora seedling and characterized by FTIR, XRD and SEM with EDS. FTIR results confirmed the presence of various phytochemicals viz., Phosphines, Sulfonates, Amides and Alky halides in lead treated of S. grandiflora. The presence of various phytochemical constituents in lead treated seedlings had reduced the lead ion into metallic lead nanoparticles compared to untreated control seedlings. The XRD pattern and EDS spectrum clearly showed that crystalline substances of the 110
13 lead metal nanoparticles were synthesized. The sizes of the lead nanoparticle were found to be in the ranges of nm for leaf and nm for root. These results indicated that S. grandiflora plant possess the great potential for lead accumulation and nanoparticle synthesis and also shows promising results for further development of a green process for nanoparticle synthesis using this fast growing S. grandiflora plant. 111
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