Biomimetic Synthesis of Nanoparticles: Science, Technology & Applicability

4.2.1. Use of organisms to synthesize nanoparticles

Biomimetics refers to applying biological principles for materials formation. One of the primary processes in biomimetics involves bioreduction.

Initially bacteria were used to synthesize nanoparticles and this was later succeeded with the use of fungi, actinomycetes and more recently plants.

4.2.2. Use of bacteria to synthesize nanoparticles

The use of microbial cells for the synthesis of nanosized materials has emerged as a novel approach for the synthesis of metal nanoparticles. Although the efforts directed towards the biosynthesis of nanomaterials are recent, the interactions between microorganisms and metals have been well documented and the ability of microorganisms to extract and/or accumulate metals is employed in commercial biotechnological processes such as bioleaching and bioremediation (Gericke & Pinches, 2006). Bacteria are known to produce inorganic materials either intra cellularly or extra cellularly. Microorganisms are considered as a potential biofactory for the synthesis of nanoparticles like gold, silver and cadmium sulphide. Some well known examples of bacteria synthesizing inorganic materials include magnetotactic bacteria (synthesizing magnetic nanoparticles) and S layer bacteria which produce gypsum and calcium carbonate layers (Shankar et al., 2004).

Some microorganisms can survive and grow even at high metal ion concentration due to their resistance to the metal. The mechanisms involve: efflux systems, alteration of solubility and toxicity via reduction or oxidation, biosorption, bioaccumulation, extra cellular complexation or precipitation of metals and lack of specific metal transport systems (Husseiny et al., 2007). For e.g. Pseudomonas stutzeri AG 259 isolated from silver mines has been shown to produce silver nanoparticles (Mohanpuria et al., 2007).

Many microorganisms are known to produce nanostructured mineral crystals and metallic nanoparticles with properties similar to chemically synthesized materials, while exercising strict control over size, shape and composition of the particles. Examples include the formation of magnetic nanoparticles by magnetotactic bacteria, the production of silver nanoparticles within the periplasmic space of Pseudomonas stutzeri and the formation of palladium nanoparticles using sulphate reducing bacteria in the presence of an exogenous electron donor (Gericke & Pinches, 2006).

Though it is widely believed that the enzymes of the organisms play a major role in the bioreduction process, some studies have indicated it otherwise. Studies indicate that some microorganisms could reduce silver ions where the processes of bioreduction were probably non enzymatic. For e.g. dried cells of Bacillus megaterium D01, Lactobacillus sp. A09 were shown to reduce silver ions by the interaction of the silver ions with the groups on the microbial cell wall (Fu et al., 1999, 2000). Silver nanoparticles in the size range of 10- 15 nm were produced by treating dried cells of Corynebacterium sp. SH09 with diammine silver complex. The ionized carboxyl group of amino acid residues and the amide of peptide chains were the main groups trapping (Ag(NH3)²⁺) onto the cell wall and some reducing groups such as aldehyde and ketone were involved in subsequent bioreduction. But it was found that the reaction progressed slowly and could be accelerated in the presence of OH- (Fu et al., 2006).

In the case of bacteria, most metal ions are toxic and therefore the reduction of ions or the formation of water insoluble complexes is a defense mechanism developed by the bacteria to overcome such toxicity (Sastry et al., 2003).

4.2.3. Use of actinomycetes to synthesize nanoparticles

Actinomycetes are microorganisms that share important characteristics of fungi and prokaryotes such as bacteria. Even though they are classified as prokaryotes, they were originally designated as ray fungi. Focus on actinomycetes has primarily centred on their exceptional ability to produce secondary metabolites such as antibiotics.

It has been observed that a novel alkalothermophilic actinomycete, Thermomonospora sp. synthesized gold nanoparticles extracellularly when exposed to gold ions under alkaline conditions (Sastry et al., 2003). In an effort to elucidate the mechanism or the processes favouring the formation of nanoparticles with desired features, Ahmad et al. (2003), studied the formation of monodisperse gold nanoparticles by Thermomonospora sp. and concluded that extreme biological conditions such as alkaline and slightly elevated temperature conditions were favourable for the formation of monodisperse particles. Based on this hypothesis, alkalotolerant actinomycete Rhodococcus sp. has been used for the intracellular synthesis of monodisperse gold nanoparticles by Ahmad et al. (2003). In this study it was observed that the concentration of nanoparticles were more on the cytoplasmic membrane. This could have been due to the reduction of metal ions by the enzymes present in the cell wall and on the cytoplasmic membrane but not in the cytosol. The metal ions were also found to be non toxic to the cells which continued to multiply even after the formation of the nanoparticles.

4.2.4. Use of fungi to synthesize nanoparticles

Fungi have been widely used for the biosynthesis of nanoparticles and the mechanistic aspects governing the nanoparticle formation have also been documented for a few of them. In addition to monodispersity, nanoparticles with well defined dimensions can be obtained using fungi. Compared to bacteria, fungi could be used as a source for the production of large amount of nanoparticles. This is due to the fact that fungi secrete more amounts of proteins which directly translate to higher productivity of nanoparticle formation (Mohanpuria et al., 2007).

Yeast, belonging to the class ascomycetes of fungi has shown to have good potential for the synthesis of nanoparticles. Gold nanoparticles have been synthesized intracellularly using the fungi V.luteoalbum. Here, the rate of particle formation and therefore the size of the nanoparticles could to an extent be manipulated by controlling parameters such as pH, temperature, gold concentration and exposure time. A biological process with the ability to strictly control the shape of the particles would be a considerable advantage (Gericke & Pinches, 2006).

Extracellular secretion of the microorganisms offers the advantage of obtaining large quantities in a relatively pure state, free from other cellular proteins associated with the organism with relatively simpler downstream processing. Mycelia free spent medium of the fungus, Cladosporium cladosporioides was used to synthesise silver nanoparticles extracellularly. It was hypothesized that proteins, polysaccharides and organic acids released by the fungus were able to differentiate different crystal shapes and were able to direct their growth into extended spherical crystals (Balaji et al., 2009).

Fusarium oxysporum has been reported to synthesize silver nanoparticles extracellularly. Studies indicate that a nitrate reductase was responsible for the reduction of silver ions and the corresponding formation of silver nanoparticles. However Fusarium moniliformae did not produce nanoparticles either intracellularly or extracellularly even though they had intracellular and extracellular reductases in the same fashion as Fusarium oxysporum. This indicates that probably the reductases in F.moniliformae were necessary for the reduction of Fe (III) to Fe (II) and not for Ag (I) to Ag (0) (Duran et al., 2005).

Instead of fungi culture, isolated proteins from them have also been used successfully in nanoparticles production. Nanocrystalline zirconia was produced at room temperature by cationic proteins while were similar to silicatein secreted by F. oxysporum (Mohanpuria et al., 2007).

The use of specific enzymes secreted by fungi in the synthesis of nanoparticles appears promising. Understanding the nature of the biogenic nanoparticle would be equally important. This would lead to the possibility of genetically engineering microorganisms to over express specific reducing molecules and capping agents and thereby control the size and shape of the biogenic nanoparticles (Balaji et al., 2009).

Microbiological methods generate nanoparticles at a much slower rate than that observed when plant extracts are used. This is one of the major drawbacks of biological synthesis of nanoparticles using microorganisms and must be corrected if it must compete with other methods.

4.2.5. Use of plants to synthesize nanoparticles

The advantage of using plants for the synthesis of nanoparticles is that they are easily available, safe to handle and possess a broad variability of metabolites that may aid in reduction.

A number of plants are being currently investigated for their role in the synthesis of nanoparticles. Gold nanoparticles with a size range of 2- 20 nm have been synthesized using the live alfa alfa plants (Torresday et al., 2002). Nanoparticles of silver, nickel, cobalt, zinc and copper have also been synthesized inside the live plants of Brassica juncea (Indian mustard), Medicago sativa (Alfa alfa) and Heliantus annus (Sunflower). Certain plants are known to accumulate higher concentrations of metals compared to others and such plants are termed as hyperaccumulators. Of the plants investigated, Brassica juncea had better metal accumulating ability and later assimilating it as nanoparticles (Bali et al., 2006).

Recently much work has been done with regard to plant assisted reduction of metal nanoparticles and the respective role of phytochemicals. The main phytochemicals responsible have been identified as terpenoids, flavones, ketones, aldehydes, amides and carboxylic acids in the light of IR spectroscopic studies. The main water soluble phytochemicals are flavones, organic acids and quinones which are responsible for immediate reduction. The phytochemicals present in Bryophyllum sp. (Xerophytes), Cyprus sp. (Mesophytes) and Hydrilla sp. (Hydrophytes) were studied for their role in the synthesis of silver nanoparticles. The Xerophytes were found to contain emodin, an anthraquinone which could undergo redial tautomerization leading to the formation of silver nanoparticles. The Mesophyte studied contained three types of benzoquinones, namely, cyperoquinone, dietchequinone and remirin. It was suggested that gentle warming followed by subsequent incubation resulted in the activation of quinones leading to particle size reduction. Catechol and protocatechaldehyde were reported in the hydrophyte studied along with other phytochemicals. It was reported that catechol under alkaline conditions gets transformed into protocatechaldehyde and finally into protocatecheuic acid. Both these processes liberated hydrogen and it was suggested that it played a role in the synthesis of the nanoparticles. The size of the nanoparticles synthesized using xerophytes, mesophytes and hydrophytes were in the range of 2- 5nm (Jha et al., 2009).

Recently gold nanoparticles have been synthesized using the extracts of Magnolia kobus and Diopyros kaki leaf extracts. The effect of temperature on nanoparticle formation was investigated and it was reported that polydisperse particles with a size range of 5- 300nm was obtained at lower temperature while a higher temperature supported the formation of smaller and spherical particles (Song et al., 2009).

While fungi and bacteria require a comparatively longer incubation time for the reduction of metal ions, water soluble phytochemicals do it in a much lesser time. Therefore compared to bacteria and fungi, plants are better candidates for the synthesis of nanoparticles. Taking use of plant tissue culture techniques and downstream processing procedures, it is possible to synthesize metallic as well as oxide nanoparticles on an industrial scale once issues like the metabolic status of the plant etc. are properly addressed.

4.2.6. Work on the biomimetic synthesis of nanoparticles in India

There has been considerable significant research in India in the field of biomimetic synthesis of nanoparticles. More research has been found to be concentrated in the area of biomimetic synthesis using plants.

It has been observed that a novel alkalothermophilic actinomycete, Thermomonospora sp. synthesized gold nanoparticles extracellularly when exposed to gold ions under alkaline conditions (Sastry et al., 2003). The use of algae for the biosynthesis of nanoparticles is a largely unexplored area. There is very little literature supporting its use in nanoparticle formation. Recently stable gold nanoparticles have been synthesized using the marine alga, Sargassum wightii. Nanoparticles with a size range between 8nm to 12nm were obtained using the seaweed. An important potential benefit of the method of synthesis was that the nanoparticles were quite stable in solution (Singaravelu et al., 2007).

Yeast, belonging to the class ascomycetes of fungi has shown to have good potential for the synthesis of nanoparticles. Schizosaccharomyces pombe cells were found to synthesize semiconductor CdS nanocrystals and the productivity was maximum during the mid log phase of growth. Addition of Cd in the initial exponential phase of yeast growth affected the metabolism of the organism (Kowshik et al., 2002). Baker’s yeast (Saccharomyces cerevisiae) has been reported to be a potential candidate for the transformation of Sb₂O₃ nanoparticles and the tolerance of the organism towards Sb₂O₃ has also been assessed. Particles with a size range of 2- 10 nm were obtained.

Aspergillus flavus has been found to accumulate silver nanoparticles on the surface of its cell wall when challenged with silver nitrate solution. Monodisperse silver nanoparticles with a size range of 8.92+/- 1.61nm were obtained and it was also found that a protein from the fungi acted as a capping agent on the nanoparticles (Vigneshwaran et al., 2007).

Aspergillus fumigatus has been studied as a potential candidate for the extracellular biosynthesis of silver nanoparticles. The advantage of using this organism was that the synthesis process was quite rapid with the nanoparticles being formed within minutes of the silver ion coming in contact with the cell filtrate. Particles with a size range of 5- 25nm could be obtained using this organism (Bhainsa & D Souza, 2006).

In addition to the synthesis of silver nanoparticles, Fusarium oxysporum has also been used to synthesize zirconia nanoparticles. It has been reported that cationic proteins with a molecular weight of 24- 28 kDa (similar in nature to silicatein) were responsible for the synthesis of the nanoparticles (Bansal et al., 2004).

Recently, scientists in India have reported the green synthesis of silver nanoparticles using the leaves of the obnoxious weed, Parthenium hysterophorus. Particles in the size range of 30- 80nm were obtained after 10 min of reaction. The use of this noxious weed has an added advantage in that it can be used by nanotechnology processing industries (Parashar et al., 2009). Mentha piperita leaf extract has also been used recently for the synthesis of silver nanoparticles. Nanoparticles in the size range of 10-25 nm were obtained within 15 min of the reaction (Parashar et al., 2009). Table 1 denotes the use of various organisms for the synthesis of nanoparticles.

Azadirachta indica leaf extract has also been used for the synthesis of silver, gold and bimetallic (silver and gold) nanoparticles. Studies indicated that the reducing phytochemicals in the neem leaf consisted mainly of terpenoids. It was found that these reducing components also served as capping and stabilizing agents in addition to reduction as revealed from FT IR studies. The major advantage of using the neem leaves is that it is a commonly available medicinal plant and the antibacterial activity of the biosynthesized silver nanoparticle might have been enhanced as it was capped with the neem leaf extract. The major chemical constituents in the extract were identified as nimbin and quercetin (Shankar et al., 2004, Tripathy et al., 2009). Figure 2 and 3 show the TEM micrograph of the biosynthesized silver nanoparticles (unpublished data, Prathna T.C. et al., 2009).

4.2.7. Some of the mechanistic aspects of nanoparticle formation

Though there are many studies reporting the biosynthesis of various nanoparticles by bacteria, there is very little information available regarding the mechanistic aspects of nanoparticle production.

The mechanisms of gold bioaccumulation by cyanobacteria (Plectonema boryanum UTEX 485) from gold (III) chloride solutions have been studied and it is found that interaction of cyanobacteria with aqueous gold (III) chloride initially promoted the precipitation of nanoparticles of amorphous gold (I) sulfide at the cell walls and finally deposited metallic gold in the form of octahedral (III) platelets near cell surfaces and in solutions (Lengke et al., 2006).

Scientists in Iran have investigated the extracellular biosynthesis of silver nanoparticles by the cells of Klebsiella pneumoniae. They hypothesize that the reduction of the metallic ions in the solution by the cell free supernatant is most likely due to the presence of nitroreductase which is produced by some members of Enterobacteriaceae. It has been widely studied that nitrate reductase is necessary for some metallic reduction.

Recently cadmium sulfide nanoparticles have been biosynthesized using the photosynthetic bacteria, Rhodopseudomonas palustris. The work indicated that the cysteine desulfhydrase (C-S lyase) could control crystal growth, because cysteine rich proteins can produce S2- through the action of C-S lyase. The content of C-S lyase in R.palustris was suggested to be responsible for nanocrystal formation. C-S lyase is found to be an intracellular enzyme located in the cytoplasm and it was indicated that R.palustris synthesized CdS nanoparticles intracellularly, later discharging it (Bai et al., 2009).

Schizosaccharomyces pombe cells were found to synthesize semiconductor CdS nanocrystals and the productivity was maximum during the mid log phase of growth. Addition of Cd in the initial exponential phase of yeast growth affected the metabolism of the organism (Kowshik et al., 2002). A possible mechanism for this could be that when Cd is initially added, it causes stress to the organism triggering a series of biochemical reactions. Firstly, an enzyme phytochelatin synthase was activated to synthesize phytochelatins (PC) that chelated the cytoplasmic Cd to form a low molecular weight PC- Cd complex and ultimately transport them across the vacuolar membrane by an ATP binding cassette type vacuolar membrane protein (HMT- 1). In addition to Cd, sulfide could also be added to this complex in the membrane and this could result in the formation of high molecular weight PC- CdS complex that allows it to be ultimately sequestered into the vacuole (Mohanpuria et al., 2007).

Baker’s yeast (Saccharomyces cerevisiae) has been reported to be a potential candidate for the transformation of Sb₂O₃ nanoparticles and the tolerance of the organism towards Sb2O3 has also been assessed. Particles with a size range of 2- 10 nm were obtained. It has been hypothesized that membrane bound oxido reductases and quinones may have played a role in the biosynthesis. The oxidoreductases are pH sensitive and work in alternative manner. At a lower pH, oxidase gets activated while a higher pH value activates reductase. This along with a number of simple hydroxy/ methoxy derivatives of benzoquinones and toluquinones mainly found in lower fungi (and hypothesized to be present in yeast) may facilitate the redox reaction due to its tautomerization. The transformation appears to be negotiated at two levels, one at the cell membrane level immediately after the addition of Sbcl₃ solution which triggers tautomerization of quinones and low pH sensitive oxidases which thereby makes molecular oxygen available for transformation. Also when Sb³⁺ enters the cytoplasm, it might trigger the family of oxygenases harboured in the ER, meant for cellular level detoxification by a process of oxidation/ oxygenation (Jha et al., 2009).

The synthesis of gold and silver nanoparticles has also been reported using black tea leaf extracts. Black tea leaf extracts are known to contain more amounts of flavonones and polyphenols. It was found that the reduction of metal ions was accompanied by oxidation of polyols (Begum et al., 2009). Table 2 gives a summary of some aspects of nanoparticle formation.

The latex of Jatropha curcas, a plant whose seeds are used to extract biodiesel has also been used for the synthesis of silver nanoparticles. Some of the major components in the latex of Jatropha curcas were identified as curcain, curacycline A and curacycline B. The silver nanoparticles obtained using this source had two broad distributions- one having particles in the range of 20- 40 nm and the other having larger and uneven particles. Molecular modeling studies of the peptides in the latex revealed that the silver ions were first entrapped in the core structure of the cyclic structure of the protein and were then reduced and stabilized insitu by the amide group of the peptide. This resulted in particles with radius similar to the peptides. It was also found that the larger particles with uneven shapes were stabilized by the enzyme curcain (Bar et al., 2009).

Li et al (2007) synthesized silver nanoparticles using the Capsicum annum L. extract. Capsicum annum L. extract is known to contain a number of biomolecules such as proteins, enzymes, polysaccharides, amino acids and vitamins. These biomolecules could be used as bioreductants to react with metal ions and they could also be used as scaffolds to direct the formation of nanoparticles in solution. The mechanism responsible for the reduction was postulated as follows: the silver ions were trapped on the surface of proteins in the extract via electrostatic interactions. This stage was the recognition process. The silver ions were then reduced by the proteins leading to changes in their secondary structure and the formation of silver nuclei. The silver nuclei subsequently grew by the further reduction of silver ions and their accumulation of the nuclei.