**2. Preparation of nanosilver**

#### **2.1 Conventional nanoparticle synthetic strategies**

Established technologies for AgNP synthesis and other metal preparations can be categorised distinctly into two approaches, namely: "top to bottom", which is normally employed by physicists and "bottom to up", a construction favourite of chemists [12, 13]. Both approaches converge at the nanodimension but vary drastically in the synthetic technology. "Top to bottom" approaches apply various physical methods such as grinding, milling, sputtering, evaporation-condensation and thermal/laser ablation to break down bulk solid materials to their nanoparticulate form. "Bottom to up" approaches entail various chemical and biological methods to synthesise nanoparticles by the self-assembly of atoms such as Ag<sup>+</sup> into nuclei that further develop into nano-sized particles [9].

Important physical "top to bottom" methods for nanoparticle preparation include evaporation-condensation and laser ablation techniques [14]. Evaporationcondensation applies a tube furnace at atmospheric temperature wherein primary material (metal Ag) contained in a boat; is centred in the furnace and vaporised into a carrier gas [9]. Several inadequacies have been identified with this technique, for example, the furnace occupies a large space, requires high energy input whilst raising the environmental temperature around the source material and requires long durations to achieve thermal stability. Additionally, a major drawback to this type of synthesis is the resulting imperfections in the surface structure of the derived nanoparticles which can ultimately alter their physical properties [9, 15]. In laser ablation, irradiation is used to remove material from a bulk metal in solution. The efficacy of this technique and characteristics of nascent particles is largely dependent on a number of parameters including the wavelength of the laser, duration of laser pulses, laser fluence, ablation duration and the effective liquid medium with or without surfactants [16, 17]. An important advantage of laser ablation for AgNP preparation is the absence of chemicals in solution which could potentially contaminate the nanoparticle preparation [18].

Regarding "bottom to up" approaches, wet chemical reduction is the most frequently practiced method for nanoparticle preparation [15] although, several other methods have been reported [19–22]. As the name suggests, wet chemical reduction

**255**

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

involves the reduction of a metal salt precursor in aqueous or organic solution. Various organic and inorganic compounds successfully utilised as reducing agents in the synthesis of AgNPs include: ascorbate; borohydride; citrate; elemental hydrogen; formaldehyde; N-N-dimethyl formamide (DMF); Tollen's reagent; and polyethylene glycol blocks [15, 23, 24]. In addition to reducing agents, protective stabilising agents are also included in the reaction solution to prevent agglomeration of nascent nanoparticles [25, 26]. With stability achieved, this method can be useful to produce high nanoparticle yields with low preparation costs [27]. However, the efficacy of this method is challenged by the potential contamination of nascent nanoparticles by precursor chemicals, the use of toxic solvents and the generation

Evidently, the aforementioned physical and chemical methods have certain limitations that restrict their use in the preparation of nanoparticles for biological applications [29]. In this regard, concerted efforts have been extended to develop nanoparticle synthetic strategies that are environmentally sound. Essentially, this would entail the use of benign, biotechnological tools and has given rise to the concept of green technology. This technology can best be described as the use of biological routes such as plants and microorganisms or their byproducts in the synthesis of nanoparticles [29–31]. These bio-inspired methods **(Figure 1)** are not only environmentally welcoming but are cost effective and can be easily up-scaled

As previously eluded, biological approaches for AgNP synthesis employ the use of living organisms or their extracts as capping/reducing agents in a synthetic reaction. To date, a variety of biological entities have been explored for their Ag<sup>+</sup> reducing abilities and include viruses, bacteria, plants, algae, fungi, yeast and mammalian cells [11, 13, 34–36]. Biological synthesis can be divided into two strategies, specifically: bioreduction and biosorption. Bioreduction occurs when metal ions undergo chemical reduction into biologically stable complexes. Many organisms have displayed dissimilatory metal reduction involving the coupling of reduction with oxidation of an enzyme. The resulting stable, inert nanoparticles can then be safely extracted from the reaction mixture. Alternatively, biosorption involves the attachment of metal ions onto an organism itself, such as on the cell wall. Various bacteria, fungi and plant species express peptides or possess modified cell wall structures that are capable of binding metal ions, thereby forming stable complexes

In this review, the use of plant and bacterial biological material for AgNP synthesis will be discussed. For a review on the use of alternative biological entities as AgNP factories, studies by the following authors are recommended [11, 36, 37].

Plants have shown the capacity to hyper-accumulate metals as a means to protect themselves from insects and herbivores. This observation has paved way for the technology known as phytoextraction, wherein plants are employed to extract minerals from various groundwater and soil sediments. Major applications of phytoextraction include the mining of precious metals from unfeasible ground sites (phytomining), stabilisation or recovery of non-naturally occurring contaminants (phytoremediation) and the addition of essential metals to growing crops. Interestingly, studies have unveiled that metals accumulated by

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

of hazardous by-products [13, 28].

for large productions [32].

in the form of nanoparticles [36].

**2.3 AgNP synthesis from plants**

**2.2 Biological nanoparticle synthetic strategies**

#### *Green Synthesis of Metal Nanoparticles for Antimicrobial Activity DOI: http://dx.doi.org/10.5772/intechopen.94348*

*Novel Nanomaterials*

could potentially result in fatalities [5]. Accordingly, concerted efforts have been extended by global pharmaceuticals to formulate new or improved antibiotics. However, despite high research cost-intensive investment in the last decade or so only two new classes of antibiotics have been introduced into the market [6, 7]. The imperative need for the uncovering of novel antimicrobial scaffolds has led to the

The antimicrobial activities of AgNPs are well established and currently researchers are striving to develop greener synthetic strategies for their production [1, 9]. The use of nanotechnology for the synthesis of AgNPs from environmentally compatible biomaterials is evolving into an important branch of science and technology [10]. To this end, a variety of biological extracts have been explored for the bottom-up synthesis of AgNPs [11]. However, there is an ongoing search to identify novel capping structures to produce AgNPs with increased bio-efficacies. In this context, this chapter points to highlight the use of plants as an alternative green technology for nanoparticle synthesis and their biomedical applications as potential

Established technologies for AgNP synthesis and other metal preparations can be categorised distinctly into two approaches, namely: "top to bottom", which is normally employed by physicists and "bottom to up", a construction favourite of chemists [12, 13]. Both approaches converge at the nanodimension but vary drastically in the synthetic technology. "Top to bottom" approaches apply various physical methods such as grinding, milling, sputtering, evaporation-condensation and thermal/laser ablation to break down bulk solid materials to their nanoparticulate form. "Bottom to up" approaches entail various chemical and biological methods to

Important physical "top to bottom" methods for nanoparticle preparation include evaporation-condensation and laser ablation techniques [14]. Evaporationcondensation applies a tube furnace at atmospheric temperature wherein primary material (metal Ag) contained in a boat; is centred in the furnace and vaporised into a carrier gas [9]. Several inadequacies have been identified with this technique, for example, the furnace occupies a large space, requires high energy input whilst raising the environmental temperature around the source material and requires long durations to achieve thermal stability. Additionally, a major drawback to this type of synthesis is the resulting imperfections in the surface structure of the derived nanoparticles which can ultimately alter their physical properties [9, 15]. In laser ablation, irradiation is used to remove material from a bulk metal in solution. The efficacy of this technique and characteristics of nascent particles is largely dependent on a number of parameters including the wavelength of the laser, duration of laser pulses, laser fluence, ablation duration and the effective liquid medium with or without surfactants [16, 17]. An important advantage of laser ablation for AgNP preparation is the absence of chemicals in solution which could potentially

Regarding "bottom to up" approaches, wet chemical reduction is the most frequently practiced method for nanoparticle preparation [15] although, several other methods have been reported [19–22]. As the name suggests, wet chemical reduction

into nuclei that

resurgence of silver, however, in its nano-particulate form [8].

biofactories for antibacterial, antifungal and anti-cancer agents.

synthesise nanoparticles by the self-assembly of atoms such as Ag<sup>+</sup>

**2.1 Conventional nanoparticle synthetic strategies**

further develop into nano-sized particles [9].

contaminate the nanoparticle preparation [18].

**2. Preparation of nanosilver**

**254**

involves the reduction of a metal salt precursor in aqueous or organic solution. Various organic and inorganic compounds successfully utilised as reducing agents in the synthesis of AgNPs include: ascorbate; borohydride; citrate; elemental hydrogen; formaldehyde; N-N-dimethyl formamide (DMF); Tollen's reagent; and polyethylene glycol blocks [15, 23, 24]. In addition to reducing agents, protective stabilising agents are also included in the reaction solution to prevent agglomeration of nascent nanoparticles [25, 26]. With stability achieved, this method can be useful to produce high nanoparticle yields with low preparation costs [27]. However, the efficacy of this method is challenged by the potential contamination of nascent nanoparticles by precursor chemicals, the use of toxic solvents and the generation of hazardous by-products [13, 28].

Evidently, the aforementioned physical and chemical methods have certain limitations that restrict their use in the preparation of nanoparticles for biological applications [29]. In this regard, concerted efforts have been extended to develop nanoparticle synthetic strategies that are environmentally sound. Essentially, this would entail the use of benign, biotechnological tools and has given rise to the concept of green technology. This technology can best be described as the use of biological routes such as plants and microorganisms or their byproducts in the synthesis of nanoparticles [29–31]. These bio-inspired methods **(Figure 1)** are not only environmentally welcoming but are cost effective and can be easily up-scaled for large productions [32].

## **2.2 Biological nanoparticle synthetic strategies**

As previously eluded, biological approaches for AgNP synthesis employ the use of living organisms or their extracts as capping/reducing agents in a synthetic reaction. To date, a variety of biological entities have been explored for their Ag<sup>+</sup> reducing abilities and include viruses, bacteria, plants, algae, fungi, yeast and mammalian cells [11, 13, 34–36]. Biological synthesis can be divided into two strategies, specifically: bioreduction and biosorption. Bioreduction occurs when metal ions undergo chemical reduction into biologically stable complexes. Many organisms have displayed dissimilatory metal reduction involving the coupling of reduction with oxidation of an enzyme. The resulting stable, inert nanoparticles can then be safely extracted from the reaction mixture. Alternatively, biosorption involves the attachment of metal ions onto an organism itself, such as on the cell wall. Various bacteria, fungi and plant species express peptides or possess modified cell wall structures that are capable of binding metal ions, thereby forming stable complexes in the form of nanoparticles [36].

In this review, the use of plant and bacterial biological material for AgNP synthesis will be discussed. For a review on the use of alternative biological entities as AgNP factories, studies by the following authors are recommended [11, 36, 37].

## **2.3 AgNP synthesis from plants**

Plants have shown the capacity to hyper-accumulate metals as a means to protect themselves from insects and herbivores. This observation has paved way for the technology known as phytoextraction, wherein plants are employed to extract minerals from various groundwater and soil sediments. Major applications of phytoextraction include the mining of precious metals from unfeasible ground sites (phytomining), stabilisation or recovery of non-naturally occurring contaminants (phytoremediation) and the addition of essential metals to growing crops. Interestingly, studies have unveiled that metals accumulated by

#### **Figure 1.**

*Different approaches for AgNP synthesis. Adapted from [9, 33].*

the plant are usually deposited in the form of nanoparticles. This has stimulated interest for the use of plants as factories for nanoparticle synthesis [35]. Whole plants have been explored for the synthesis of nanoparticles when grown on the appropriate metal enriched substrates. Species such as *Brassica juncae* (mustard greens) and *Medicago sativa* (alfalfa) have demonstrated the ability to accumulate AgNPs. For example, 50 nm sized AgNPs, at a high yield (13.6% of total plant weight) were reported for *M. sativa* when grown on silver nitrate (AgNO3) [38]. Additionally, icosahedral gold nanoparticles of 4 nm size were observed in *M. sativa* and semi-spherical copper nanoparticles of 2 nm size were observed in *Iris pseudacorus* when the plants were grown on gold and copper salt enriched substrates, respectively [39, 40].

Although whole plants can potentially serve as factories for nanoparticle synthesis, several disadvantages have been identified with this technology especially when up-scaling for industrial applications. For example, physical attributes of nanoparticles such as size and shape vary upon the localisation of the particles in the plant due to the differences in metal ion content in different plant tissues and the possibility of nanoparticle movement and penetration [39]. This heterogeneity of important bioactivity-determinants such as size and shape [41, 42] limit the use of these nanoparticles and especially in applications where mono-dispersed nanoparticle preparations are required. Furthermore, recovery of nanoparticles from living

**257**

**Figure 2.**

*(tryptophan, tyrosine). Adapted from [35].*

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

fruit and fruit peel have demonstrated the ability to reduce Ag+

plants entails laborious extraction, isolation and purification procedures and may

The use of plant broths/extracts in nanoparticle synthesis was introduced by Shankar *et al*., (2003). In their study, compounds responsible for the reduction of metal ions were extracted and used as reducing agents in a synthetic reaction mixture, resulting in the extracellular production of nanoparticles [43]. This strategy tentatively offers several advantages compared to the use of whole plants. For example, nanoparticle formation occurs considerably faster as opposed to whole plants which require diffusion of metal ions throughout the plant body. Additionally, the use of extracts would be more economical due to the ease of purification [35].

This *in vitro* approach has been actively developed and applied to a variety of plant flora for the synthesis of AgNPs [28]. Various organ extracts: stem, root, leaf, bark,

ecules **(Figure 2)** such as proteins, amino acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, saponins, terpenoids and vitamins present in the extracts act as

Terpenoids are a class of diverse organic polymers manufactured in plants from five-carbon isoprene units and display strong antioxidant activities. In a previous study by Shankar *et al*., involving gold nanoparticle synthesis from geranium leaf extracts, it was suggested that these polymers were actively involved in the reduction of gold ions into stable nanoparticles [44]. Later Singh

*Major plant metabolites involved in the synthesis of metal nanoparticles: (A)-terpenoids (eugenol); (B & C)-flavonoids (luteolin, quercetin); (D)-a reducing hexose with the open chain form; (E & F)-amino acids* 

. Particularly, biomol-

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

potentially result in low yields [35].

both reducing and stabilising agents [9].

#### *Green Synthesis of Metal Nanoparticles for Antimicrobial Activity DOI: http://dx.doi.org/10.5772/intechopen.94348*

*Novel Nanomaterials*

**256**

**Figure 1.**

substrates, respectively [39, 40].

*Different approaches for AgNP synthesis. Adapted from [9, 33].*

the plant are usually deposited in the form of nanoparticles. This has stimulated interest for the use of plants as factories for nanoparticle synthesis [35]. Whole plants have been explored for the synthesis of nanoparticles when grown on the appropriate metal enriched substrates. Species such as *Brassica juncae* (mustard greens) and *Medicago sativa* (alfalfa) have demonstrated the ability to accumulate AgNPs. For example, 50 nm sized AgNPs, at a high yield (13.6% of total plant weight) were reported for *M. sativa* when grown on silver nitrate (AgNO3) [38]. Additionally, icosahedral gold nanoparticles of 4 nm size were observed in *M. sativa* and semi-spherical copper nanoparticles of 2 nm size were observed in *Iris pseudacorus* when the plants were grown on gold and copper salt enriched

Although whole plants can potentially serve as factories for nanoparticle synthesis, several disadvantages have been identified with this technology especially when up-scaling for industrial applications. For example, physical attributes of nanoparticles such as size and shape vary upon the localisation of the particles in the plant due to the differences in metal ion content in different plant tissues and the possibility of nanoparticle movement and penetration [39]. This heterogeneity of important bioactivity-determinants such as size and shape [41, 42] limit the use of these nanoparticles and especially in applications where mono-dispersed nanoparticle preparations are required. Furthermore, recovery of nanoparticles from living

plants entails laborious extraction, isolation and purification procedures and may potentially result in low yields [35].

The use of plant broths/extracts in nanoparticle synthesis was introduced by Shankar *et al*., (2003). In their study, compounds responsible for the reduction of metal ions were extracted and used as reducing agents in a synthetic reaction mixture, resulting in the extracellular production of nanoparticles [43]. This strategy tentatively offers several advantages compared to the use of whole plants. For example, nanoparticle formation occurs considerably faster as opposed to whole plants which require diffusion of metal ions throughout the plant body. Additionally, the use of extracts would be more economical due to the ease of purification [35].

This *in vitro* approach has been actively developed and applied to a variety of plant flora for the synthesis of AgNPs [28]. Various organ extracts: stem, root, leaf, bark, fruit and fruit peel have demonstrated the ability to reduce Ag+ . Particularly, biomolecules **(Figure 2)** such as proteins, amino acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, saponins, terpenoids and vitamins present in the extracts act as both reducing and stabilising agents [9].

Terpenoids are a class of diverse organic polymers manufactured in plants from five-carbon isoprene units and display strong antioxidant activities. In a previous study by Shankar *et al*., involving gold nanoparticle synthesis from geranium leaf extracts, it was suggested that these polymers were actively involved in the reduction of gold ions into stable nanoparticles [44]. Later Singh

#### **Figure 2.**

*Major plant metabolites involved in the synthesis of metal nanoparticles: (A)-terpenoids (eugenol); (B & C)-flavonoids (luteolin, quercetin); (D)-a reducing hexose with the open chain form; (E & F)-amino acids (tryptophan, tyrosine). Adapted from [35].*

*et al*. reported that eugenol, the main terpenoid found in *Szyygium aromaticum* (clove), played an important role in reducing AgNO3 and HAuCL4. The Fourier transform infrared (FTIR) spectroscopy analysis of their study suggests that the dissociation of the proton from the OH group in eugenol leads to the formation of intermediate resonance structures which can undergo further oxidation. This latter reaction may be coupled to the reduction of Ag<sup>+</sup> and subsequent formation of stable AgNPs [45].

Flavonoids are made up of a large group of polyphenolic compounds containing various classes such as anthocyanins, isoflavonoids, flavonols, chalcones, flavones and flavanones. There are several functional groups present on flavonoid compounds that can participate in nanoparticle formation. It has been hypothesised that the tautomerization of flavonoids from the enol to keto form releases a reactive hydrogen atom that can participate in the reduction of metal ions. For example, studies involving AgNP synthesis from *Ocimum sanctum* extracts indicate that synthesis is likely to be the result of tautomerization of the flavonoids luteolin and rosmarinic acid [46]. Additionally, some flavonoids can chelate metal ions with their carbonyl groups or π-electron. Quercetin is an example of a flavonoid with strong chelating activity [35]. These mechanisms may explain the prevalence of flavonoid groups adsorbed on to the surface of AgNPs derived in previous studies [47, 48]. Further indication of flavonoid involvement in nanoparticle synthesis is provided by a study using *Lawsonia inermis,* in which the flavonoid apiin was extracted and successfully employed in the synthesis of gold and Ag nanoparticles [49].

Sugars contained in plant extracts are also capable of inducing nanoparticle formation. It is known that monosaccharides in the linear form containing an aldehyde (e.g. glucose), are capable reducing agents [35]. Monosaccharides harbouring a keto-group may act as antioxidants upon tautomeric transformation from a ketone to an aldehyde (e.g. fructose). In this regard, glucose is reportedly more efficient at metal ion reduction than fructose due to the kinetics of tautomerism from a ketone to an aldehyde which limits the reducing potential of fructose. Disaccharides and polysaccharides may also participate in the reduction of metal ions however, this is largely dependent on the ability of their monosaccharide components to take on an open chain configuration within an oligomer. Examples include lactose and maltose. In contrast, sucrose is unable to participate in metal ion reduction because the linkage of its glucose and fructose monomers restrict the formation of open chains. However, when sucrose was placed in tetrachloroauric and tetrachloroplatinic acids, nanoparticle formation proceeded [50]. This may be due to the acidic hydrolysis of sucrose yielding glucose and fructose. In general, it is suggested that nanoparticle formation by sugars occurs by the oxidation of an aldehyde group into a carbonyl group which subsequently leads to the reduction of metal ions and nanoparticle formation [44].

FTIR analysis of plant derived metal nanoparticles have revealed the presence of proteins on their surface, suggesting that proteins may also possess metal ion reducing ability. However, amino acids have displayed differences in their potential for metal ion reducing and binding efficiencies. For example, lysine, cysteine, arginine and methionine have been shown to bind Ag<sup>+</sup> . In a separate study, aspartate was used to reduce tetrachloroauric acid forming nanoparticles, whilst valine and lysine did not possess this ability. Amino acids capable of binding metal ions are thought to do so through their amino or carboxyl groups or through side chain groups: carboxyl groups of aspartic and glutamic acid, imidazole ring of histidine, thiol of cysteine, thioether of methionine, hydroxyl group of serine; threonine and tyrosine, carbonyl groups of asparagine and glutamine [35].

**259**

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

Linkage of amino acids in a peptide chain may also affect the ability of individual amino acids to bind and reduce metal ions. For example, the R-carbon of amines and carboxylic acids in a peptide bond are inaccessible for association with metal ions. However, the free side chains of individual amino acids can still participate in binding and reduction of metal ions although, this is largely dependent on the amino acid sequence. Tan *et al*. demonstrated that synthesised peptides derived from amino acids with strong binding abilities and high reducing activities displayed lower reduction than expected [51]. A previous study suggested that protein molecules capable of nanoparticle formation display a strong attraction of metal ions to the regions on the molecule responsible for reduction however, their chelating activity is limited [52]. It was also suggested that the amino acid sequence of a protein can influence the size, shape and yield of derived nanoparticles. For example, the synthetic peptide GASLWWSEKL was found to rapidly reduce metal ions forming a large number of small nanoparticles (˂10 nm), however, replacement of the N- and C- terminal residues forming the peptide SEKLWWGASL led to slower reduction and formation of larger nanospheres and nanotriangles (40 nm). These findings seemingly suggest that peptides and proteins present in plant extracts probably play a vital role in determining nanoparticle size and shape and potentially

There exists a vast array of literature pertaining to the use of bacteria as factories for nanoparticle synthesis [53, 54]. Bacteria have a marked advantage over other microbial systems such as fungi due to their abundance, rapid growth rate, cheap cultivation and the relative ease of their manipulation [55]. Their ubiquitous nature has led to their exposure and proliferation in many environmental extremes and ultimately depends on the natural defence mechanisms of these microorganisms to resist the effects posed by environmental stresses [56]. Bacteria have demonstrated these defence mechanisms in a few non-optimal growth conditions including

AgNP synthesis by bacteria can occur intracellularly or by the use of their extracts [53]. Several studies have reported intracellular synthesis by a variety of bacterial species and as similarly reported for the use of whole plants, this technology is associated with long duration periods for nanoparticle synthesis. For example, Pugazhenthiran *et al*. reported an incubation time of 7 days for AgNP synthesis from *Bacillus* sp. [57]. Kalimuthu *et al*. reported a reaction time of 24 hours for AgNP synthesis by *Bacillus licheniformis* [58]. Although this reaction time was more industrially significant, the authors reported an additional extraction to acquire the derived nanoparticles. Synthesis of AgNPs by the use of bacterial cell free supernatant (CFS) extracts was reported by Shahverdi *et al*., (2007).

Interestingly, nanoparticle synthesis occurred within five minutes of Ag+

*2.4.1 Bacterial metabolites involved in nanoparticle synthesis*

into contact with the CFS [59]. Thus, this method presents the greatest potential for industrial production of AgNPs from bacteria. Several other studies have reported on the production of AgNPs from bacterial CFS extracts but not at the previously stated formation rate [60, 61]. This seemingly suggests that bacterial extracts differ in their metal ion reducing abilities and may require an external energy source to

As previously stated, metal nanoparticle synthesis in bacteria may potentially occur through resistance mechanisms attained by these organisms to overcome the

coming

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

affect the overall yield of the nanoparticles [51].

environments contaminated with metal ions.

accelerate nanoparticle formation.

**2.4 AgNP synthesis from bacteria**

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity DOI: http://dx.doi.org/10.5772/intechopen.94348*

*Novel Nanomaterials*

of stable AgNPs [45].

and Ag nanoparticles [49].

*et al*. reported that eugenol, the main terpenoid found in *Szyygium aromaticum* (clove), played an important role in reducing AgNO3 and HAuCL4. The Fourier transform infrared (FTIR) spectroscopy analysis of their study suggests that the dissociation of the proton from the OH group in eugenol leads to the formation of intermediate resonance structures which can undergo further oxidation. This

Flavonoids are made up of a large group of polyphenolic compounds containing various classes such as anthocyanins, isoflavonoids, flavonols, chalcones, flavones and flavanones. There are several functional groups present on flavonoid compounds that can participate in nanoparticle formation. It has been hypothesised that the tautomerization of flavonoids from the enol to keto form releases a reactive hydrogen atom that can participate in the reduction of metal ions. For example, studies involving AgNP synthesis from *Ocimum sanctum* extracts indicate that synthesis is likely to be the result of tautomerization of the flavonoids luteolin and rosmarinic acid [46]. Additionally, some flavonoids can chelate metal ions with their carbonyl groups or π-electron. Quercetin is an example of a flavonoid with strong chelating activity [35]. These mechanisms may explain the prevalence of flavonoid groups adsorbed on to the surface of AgNPs derived in previous studies [47, 48]. Further indication of flavonoid involvement in nanoparticle synthesis is provided by a study using *Lawsonia inermis,* in which the flavonoid apiin was extracted and successfully employed in the synthesis of gold

Sugars contained in plant extracts are also capable of inducing nanoparticle formation. It is known that monosaccharides in the linear form containing an aldehyde (e.g. glucose), are capable reducing agents [35]. Monosaccharides harbouring a keto-group may act as antioxidants upon tautomeric transformation from a ketone to an aldehyde (e.g. fructose). In this regard, glucose is reportedly more efficient at metal ion reduction than fructose due to the kinetics of tautomerism from a ketone to an aldehyde which limits the reducing potential of fructose. Disaccharides and polysaccharides may also participate in the reduction of metal ions however, this is largely dependent on the ability of their monosaccharide components to take on an open chain configuration within an oligomer. Examples include lactose and maltose. In contrast, sucrose is unable to participate in metal ion reduction because the linkage of its glucose and fructose monomers restrict the formation of open chains. However, when sucrose was placed in tetrachloroauric and tetrachloroplatinic acids, nanoparticle formation proceeded [50]. This may be due to the acidic hydrolysis of sucrose yielding glucose and fructose. In general, it is suggested that nanoparticle formation by sugars occurs by the oxidation of an aldehyde group into a carbonyl group which subsequently leads to the reduction of metal ions and nanoparticle

FTIR analysis of plant derived metal nanoparticles have revealed the presence of proteins on their surface, suggesting that proteins may also possess metal ion reducing ability. However, amino acids have displayed differences in their potential for metal ion reducing and binding efficiencies. For example, lysine,

study, aspartate was used to reduce tetrachloroauric acid forming nanoparticles, whilst valine and lysine did not possess this ability. Amino acids capable of binding metal ions are thought to do so through their amino or carboxyl groups or through side chain groups: carboxyl groups of aspartic and glutamic acid, imidazole ring of histidine, thiol of cysteine, thioether of methionine, hydroxyl group of serine; threonine and tyrosine, carbonyl groups of asparagine and

cysteine, arginine and methionine have been shown to bind Ag<sup>+</sup>

and subsequent formation

. In a separate

latter reaction may be coupled to the reduction of Ag<sup>+</sup>

**258**

formation [44].

glutamine [35].

Linkage of amino acids in a peptide chain may also affect the ability of individual amino acids to bind and reduce metal ions. For example, the R-carbon of amines and carboxylic acids in a peptide bond are inaccessible for association with metal ions. However, the free side chains of individual amino acids can still participate in binding and reduction of metal ions although, this is largely dependent on the amino acid sequence. Tan *et al*. demonstrated that synthesised peptides derived from amino acids with strong binding abilities and high reducing activities displayed lower reduction than expected [51]. A previous study suggested that protein molecules capable of nanoparticle formation display a strong attraction of metal ions to the regions on the molecule responsible for reduction however, their chelating activity is limited [52]. It was also suggested that the amino acid sequence of a protein can influence the size, shape and yield of derived nanoparticles. For example, the synthetic peptide GASLWWSEKL was found to rapidly reduce metal ions forming a large number of small nanoparticles (˂10 nm), however, replacement of the N- and C- terminal residues forming the peptide SEKLWWGASL led to slower reduction and formation of larger nanospheres and nanotriangles (40 nm). These findings seemingly suggest that peptides and proteins present in plant extracts probably play a vital role in determining nanoparticle size and shape and potentially affect the overall yield of the nanoparticles [51].

#### **2.4 AgNP synthesis from bacteria**

There exists a vast array of literature pertaining to the use of bacteria as factories for nanoparticle synthesis [53, 54]. Bacteria have a marked advantage over other microbial systems such as fungi due to their abundance, rapid growth rate, cheap cultivation and the relative ease of their manipulation [55]. Their ubiquitous nature has led to their exposure and proliferation in many environmental extremes and ultimately depends on the natural defence mechanisms of these microorganisms to resist the effects posed by environmental stresses [56]. Bacteria have demonstrated these defence mechanisms in a few non-optimal growth conditions including environments contaminated with metal ions.

AgNP synthesis by bacteria can occur intracellularly or by the use of their extracts [53]. Several studies have reported intracellular synthesis by a variety of bacterial species and as similarly reported for the use of whole plants, this technology is associated with long duration periods for nanoparticle synthesis. For example, Pugazhenthiran *et al*. reported an incubation time of 7 days for AgNP synthesis from *Bacillus* sp. [57]. Kalimuthu *et al*. reported a reaction time of 24 hours for AgNP synthesis by *Bacillus licheniformis* [58]. Although this reaction time was more industrially significant, the authors reported an additional extraction to acquire the derived nanoparticles. Synthesis of AgNPs by the use of bacterial cell free supernatant (CFS) extracts was reported by Shahverdi *et al*., (2007). Interestingly, nanoparticle synthesis occurred within five minutes of Ag+ coming into contact with the CFS [59]. Thus, this method presents the greatest potential for industrial production of AgNPs from bacteria. Several other studies have reported on the production of AgNPs from bacterial CFS extracts but not at the previously stated formation rate [60, 61]. This seemingly suggests that bacterial extracts differ in their metal ion reducing abilities and may require an external energy source to accelerate nanoparticle formation.

#### *2.4.1 Bacterial metabolites involved in nanoparticle synthesis*

As previously stated, metal nanoparticle synthesis in bacteria may potentially occur through resistance mechanisms attained by these organisms to overcome the toxic effects of metals. These strategies include redox state changes, efflux systems, intracellular precipitation, metal accumulation and extracellular formation of complexes **(Figure 3)** [56]. In an early study, Slawson *et al*. observed that the Ag resistant strain *Pseudomonas stutzeri* AG259, was capable of accumulating AgNPs (35–46 nm) within its periplasmic space. The formation of these nanoparticles was thought to have occurred by a mechanism involving the NADH-dependent reductase enzyme which undergoes oxidation to form NAD<sup>+</sup> . The lost free electron may potentially reduce Ag<sup>+</sup> to AgNPs [62]. Later, He *et al*. reported that the NADH-dependent reductase enzyme may similarly participate in the extracellular formation of gold nanoparticles by the bacterium *Rhodopseudomonas capsulata* [63]. Other studies have reported nanoparticle formation without the use of biological enzymes. Non-enzymatic nanoparticle synthesis by a *Corynebacterium* sp. was reported by Sneha *et al*. [64]. Organic functional groups present at the cell wall were thought to induce metal ion reduction [64]. Sintubin *et al.* proposed a two-step mechanism for AgNP formation by several lactic acid bacteria, involving biosorption of Ag<sup>+</sup> on the cell wall which is coupled to the subsequent reduction of these ions to form the nanoparticles [65]. Parikh *et al*. identified a gene homologue in a Ag-resistant *Morganella* strain with a 99% nucleotide sequence similarity to a periplasmic Ag-binding protein-encoding gene [66]. Johnston *et al*. further reported the production of a small non-ribosomal peptide, delftibactin by *Delftia acidovorans* which they believed to be associated with a resistance mechanism. By producing inert gold nanoparticles bound to delftibactin, gold ions no longer caused toxicity to the cells [67].

#### **Figure 3.**

*Metabolites and mechanisms involved in AgNP synthesis in bacteria: (a)-uptake of Ag+ and activation of reduction machinery; (b)-electron shuttle system involving various cofactors and enzymes; (c & d)- intra or extracellular localisation of AgNPs; (e)-electrostatic interaction between Ag<sup>+</sup> and cell wall peptides/proteins & (f)-extracellular reduction by enzymes or other metabolites released in solution. Adapted from [53].*

**261**

resulting in rapid reduction of Ag<sup>+</sup>

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

**3. Mechanism of nanoparticle synthesis using plants and microbes**

There are three main phases in the synthesis of metal nanoparticles from plants and plant extracts. Initially, an activation phase takes place during which metal ions are reduced from mono or divalent oxidation states to zero-valent states, followed by nucleation of the reduced atoms. This step is immediately followed by a growth phase where small neighbouring nanoparticles coalesce into larger particles with greater thermodynamic stability while further biological reduction occurs. As growth proceeds nanoparticles aggregate to form various shapes such as: cubes, spheres, triangles, hexagons, pentagons, rods and wires [68]. Lastly, a termination phase follows in which nanoparticles acquire the most energetically favourable conformation, which ultimately determines the final shape of the particles **(Figure 4)** [69]. This step is largely influenced by the ability of the plant extract to stabilise the resulting nanoparticles. For example, the high surface energy of nanotriangles results in their decreased stability. Such nanoparticles would then acquire a more stable morphology such as a truncated triangle to minimise Gibbs free energy unless the stability is supported by the given extracts. It can be tentatively suggested that a similar mechanism occurs by the use of bacterial extracts since proteins and

Several controlling factors affect the synthesis and morphology of derived nanoparticles. Several researchers have associated these variations with the choice of adsorbate and catalyst used in the synthetic process [29, 70]. However, reaction parameters have also been shown to strongly affect the synthesis of nanoparticles

Studies have revealed that the pH of a reaction solution strongly influences the formation of the produced nanoparticles. Variances in reaction pH tend to induce variability in the shape and size of the produced nanoparticles. Lower acidic pH values tend to produce larger particles when compared to higher pH values. In a study employing *Avena sativa* (oat) biomass for the production of gold nanoparticles, larger particles (25–85 nm) where formed at pH 2 whilst smaller particles (5–20 nm) were formed at pH 3 and 4 [71]. The researchers suggested that at pH 2, fewer functional groups were available for particle nucleation resulting in aggregation of the particles. A similar finding was observed in the synthesis of gold nanoparticles from the bacterium *Rhodopseudomonas capsulate.* At an increased pH of 7, spherical particles in the range of 10–20 nm in size were observed. In contrast,

lowering the reaction pH to 4 resulted in the formation of nanoplates [63].

elevation (10–50°C) was correlated with enhanced reduction of Ag+

Temperature is an important factor in any synthesis. With respect to nanoparticle formulation with the use of biological entities, temperature elevation has demonstrated catalytic behaviour by increasing the reaction rate and efficiency of nanoparticle formation. For example, a study on the influence of reaction temperature in the synthesis of AgNPs from neem leaf extracts suggested that temperature

also noted that smaller sized AgNPs were produced at 50°C, similar to the finding of Kaviya *et al.* in the production of AgNPs from *Citrus sinensis* peel extracts using varying temperatures [73]. Similarly, this trend was observed in the production of AgNPs from the spent culture supernatants of *Escherichia coli* [61]. The authors tentatively suggested that the increased reaction rate might be because of temperature on a key enzyme participating in nanoparticle synthesis. However, the study importantly revealed that temperature elevation above 60°C contrastingly favoured

the production of larger sized particles. The reason for this observation was reported as follows: at high temperatures, kinetic energy of the molecules increase

(facilitating reduction and nucleation), to the

reduction as previously stated.

[72]. It was

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

metabolites may also participate in Ag+

from biological extracts.

*Novel Nanomaterials*

biosorption of Ag<sup>+</sup>

tron may potentially reduce Ag<sup>+</sup>

caused toxicity to the cells [67].

toxic effects of metals. These strategies include redox state changes, efflux systems, intracellular precipitation, metal accumulation and extracellular formation of complexes **(Figure 3)** [56]. In an early study, Slawson *et al*. observed that the Ag resistant strain *Pseudomonas stutzeri* AG259, was capable of accumulating AgNPs (35–46 nm) within its periplasmic space. The formation of these nanoparticles was thought to have occurred by a mechanism involving the NADH-dependent

NADH-dependent reductase enzyme may similarly participate in the extracellular formation of gold nanoparticles by the bacterium *Rhodopseudomonas capsulata* [63]. Other studies have reported nanoparticle formation without the use of biological enzymes. Non-enzymatic nanoparticle synthesis by a *Corynebacterium* sp. was reported by Sneha *et al*. [64]. Organic functional groups present at the cell wall were thought to induce metal ion reduction [64]. Sintubin *et al.* proposed a two-step mechanism for AgNP formation by several lactic acid bacteria, involving

these ions to form the nanoparticles [65]. Parikh *et al*. identified a gene homologue in a Ag-resistant *Morganella* strain with a 99% nucleotide sequence similarity to a periplasmic Ag-binding protein-encoding gene [66]. Johnston *et al*. further reported the production of a small non-ribosomal peptide, delftibactin by *Delftia acidovorans* which they believed to be associated with a resistance mechanism. By producing inert gold nanoparticles bound to delftibactin, gold ions no longer

. The lost free elec-

 *and activation of* 

 *and cell wall peptides/proteins &* 

to AgNPs [62]. Later, He *et al*. reported that the

on the cell wall which is coupled to the subsequent reduction of

reductase enzyme which undergoes oxidation to form NAD<sup>+</sup>

*Metabolites and mechanisms involved in AgNP synthesis in bacteria: (a)-uptake of Ag<sup>+</sup>*

*extracellular localisation of AgNPs; (e)-electrostatic interaction between Ag<sup>+</sup>*

*reduction machinery; (b)-electron shuttle system involving various cofactors and enzymes; (c & d)- intra or* 

*(f)-extracellular reduction by enzymes or other metabolites released in solution. Adapted from [53].*

**260**

**Figure 3.**
