**6. Effect of different parameters on synthesis of metal nanoparticles**

#### **6.1 pH**

The pH is one of the most important biogenic synthesis reaction parameters that influence the particle size and morphology of NPs [141, 142]. The NPs can be tailored to the desired size by altering the pH of the reaction mixture which causes changes in the charge over secondary metabolites which has significant effect on their ability to adsorb the metal ions [86]. In case of microbial synthesis of NPs, the culture conditions play a significant role. The small-sized and monodispersed metal oxide NPs are formed in alkaline conditions rather than acidic conditions. This is

**15**

bagasse.

**6.2 Temperature**

*Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles*

because more functional groups are available at higher pH that increase the binding ability and stability during nucleation and growth stages favoring the formation of less aggregated NPs [112]. Singh and Srivastava [143] observed a gradual blue shift (towards lower wavelength) in absorption maxima as the pH was increased from 3 to 7 indicating decrease in sizes of the NPs. Also, a red shift (towards higher wavelength) was observed when the pH was increased further from 7 to 11 The further increase in the pH was found to increase the NPs size. The reaction pH also has significant effect on particle morphology in terms of shape of the synthesized NPs [144]. Gericke and Pinches et al. [145] synthesized gold NPs from fungal cultures and observed that at pH 3, uniform sized spherical NPs of 10 nm were synthesized. When the pH was increased to 5, fewer smaller spherical particles were obtained, the morphology of most of the NPs changed to larger well defined triangular, hexagonal, spherical and rod like structures were also obtained. At higher reaction mixture pH 7 and 9, similar undefined structures were observed. Abeywardena et al. [146] employed sucrose solution based extraction of calcium to precipitate calcium carbonate nanostructures to study the effect of pH to yield nanostructures with different morphologies and sizes. The precipitation reaction was carried out at pH values of 7.5, 10.5 and 12.5 using CO2 bubbling for carbonation as it promoted formation of smaller particles. Different morphologies such as catkin-like structure, spherical particles and rod-like formations; and tiny particles aggregated into large spheres were observed at pH 12.5, 10.5 and 7.5 respectively. Thus concluding that the alkaline pH is suitable for the formation of stable and less agglomerated nanoparticles. Aguilar et al. [147] studied the effect of different pH to yield stable silver nanoparticles using sugar cane bagasse extract. It was observed that acidic conditions (pH 3.5) were not favorable for the production of nanoparticles as the reaction yields mixture of submicron-sized silver (Ag) and silver chloride (AgCl) particles. At neutral pH, though the size of resultant nanoparticles dropped down in the range 8-30 nm, the mixture of Ag and AgCl particles still existed. In alkaline conditions i.e. pH 12, pure silver nanoparticles were obtained exhibiting excellent bactericidal and bacteriostatic properties against Gram positive and Gram negative bacteria. After the thorough inspection of the X-ray diffraction patterns and X-ray energy dispersive spectra (EDS) of the biosynthesized silver nanoparticles, it was evident that the Cl and S in the bagasse induces formation of side products such as AgCl and Ag@AgCl nanoparticles. While, at alkaline pH, the formation of such side products is avoided due to the interaction of Na ions of NaOH with Cl ions cane

Temperature is an equally important reaction parameters that significantly influence the biogenic synthesis of NPs [86]. For instance, the rate of synthesis increases at an elevated temperature as compared to that at room temperature eventually leading to higher product yield and crystalline NPs [148]. At elevated temperatures, the rate of reduction of metal ions increases and homogeneous nucleation of metal nuclei occurs facilitating the synthesis of NPs [149, 150]. Noteworthy, the required temperatures in physical methods of synthesis are greater than 350°C, while chemical reaction take place at temperatures as high as 350°C. On contrary, biogenic synthesis occur at considerably low temperatures in the range of 37°C-100°C [151]. In case of NPs synthesis using microorganisms, it is recommended that the microorganisms must be grown at the maximum possible temperature for optimal growth as the enzyme responsible for NPs synthesis shows enhanced catalytic activity at high temperatures and thus is more active [152]. Jameel et al. [153] highlighted the fact that reaction temperature affects the size,

*DOI: http://dx.doi.org/10.5772/intechopen.94853*

#### *Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.94853*

*Colloids - Types, Preparation and Applications*

fuged to collect the NPs pellet [134].

such as temperature, stirring speed etc. After a defined time span, the mixture is centrifuged at high speed, filtered using muslin cloth or syringe filters and stored in chilled conditions until future use. The filtrate is then diluted according to optimized conditions and used as a source of reducing and capping agents for the synthesis of NPs [131]. The plant extract thus prepared is mixed with defined ratio of metal salts at optimum conditions for defined time period resulting in NPs [132]. Not only the reaction conditions, but also the nature of extract and its concentra-

Microbial route of synthesis of NPs has garnered enormous interest of researchers in the field of nanobiotechnology. Microorganisms including bacteria, fungi, actinomycetes, yeasts, and viruses are considered as bio-factories, owing to their inherent potential to produce NPs via. Extracellular or intracellular route of synthesis [102]. In case of extracellular synthesis, the microorganisms after subsequent growth of 1-2 days in shaking condition and optimum growth conditions are centrifuged to remove the biomass. The filter-sterilized metal salt solution is then added to the supernatant and incubated. The mixture is then centrifuged to collect the NPs pellet. For intracellular synthesis, the biomass is collected by centrifuging the micro-organisms culture grown in optimum conditions. The biomass pellet is washed and mixed with filter-sterilized solution of metal salt. Color changes in the reaction mixture are observed as a preliminary confirmation of NPs synthesis and further confirmed by spectrophotometric observations and highly sophisticated techniques. Further, similar to that of extracellular synthesis, the mixture is centri-

In case of algae-mediated synthesis of metal NPs, the algal extract is prepared in sterile distilled water or an appropriate organic solvent by boiling it for specified duration. Further, the algal extract and the metal precursors are stirred at optimum conditions. Finally depending upon the mode of synthesis of NPs via algae, i.e. extracellular or an intracellular, the supernatant and biomass are used for the further process [135]. The bioactive agents such as polysaccharides, polyphones, proteins, and/or other reducing factors reduce the metal ions in case of extracellular synthesis of NPs [96, 98, 135, 136] while in case of intracellular synthesis, the algal metabolism via photosynthesis and respiration causes reduction of metal ions [135, 137, 138]. Eventually, the chromatic changes determine the synthesis of NPs as preliminary confirmation. In mycosynthesis i.e. fungi based synthesis, the metal precursors are used to treat fungus mycelium resulting in production of fungi metabolites and enzymes. These bioactive substances reduce toxic metal ions into non-toxic metal NPs [129]. The fungi are usually cultured on an agar plate and further transferred into a liquid medium. Depending upon the route of synthesis, either the biomass or the supernatant is mixed along with metal precursor to yield

**6. Effect of different parameters on synthesis of metal nanoparticles**

The pH is one of the most important biogenic synthesis reaction parameters that influence the particle size and morphology of NPs [141, 142]. The NPs can be tailored to the desired size by altering the pH of the reaction mixture which causes changes in the charge over secondary metabolites which has significant effect on their ability to adsorb the metal ions [86]. In case of microbial synthesis of NPs, the culture conditions play a significant role. The small-sized and monodispersed metal oxide NPs are formed in alkaline conditions rather than acidic conditions. This is

tion has a significant effect on the NPs synthesis and its quality [133].

**14**

NPs [139, 140].

**6.1 pH**

because more functional groups are available at higher pH that increase the binding ability and stability during nucleation and growth stages favoring the formation of less aggregated NPs [112]. Singh and Srivastava [143] observed a gradual blue shift (towards lower wavelength) in absorption maxima as the pH was increased from 3 to 7 indicating decrease in sizes of the NPs. Also, a red shift (towards higher wavelength) was observed when the pH was increased further from 7 to 11 The further increase in the pH was found to increase the NPs size. The reaction pH also has significant effect on particle morphology in terms of shape of the synthesized NPs [144]. Gericke and Pinches et al. [145] synthesized gold NPs from fungal cultures and observed that at pH 3, uniform sized spherical NPs of 10 nm were synthesized. When the pH was increased to 5, fewer smaller spherical particles were obtained, the morphology of most of the NPs changed to larger well defined triangular, hexagonal, spherical and rod like structures were also obtained. At higher reaction mixture pH 7 and 9, similar undefined structures were observed. Abeywardena et al. [146] employed sucrose solution based extraction of calcium to precipitate calcium carbonate nanostructures to study the effect of pH to yield nanostructures with different morphologies and sizes. The precipitation reaction was carried out at pH values of 7.5, 10.5 and 12.5 using CO2 bubbling for carbonation as it promoted formation of smaller particles. Different morphologies such as catkin-like structure, spherical particles and rod-like formations; and tiny particles aggregated into large spheres were observed at pH 12.5, 10.5 and 7.5 respectively. Thus concluding that the alkaline pH is suitable for the formation of stable and less agglomerated nanoparticles. Aguilar et al. [147] studied the effect of different pH to yield stable silver nanoparticles using sugar cane bagasse extract. It was observed that acidic conditions (pH 3.5) were not favorable for the production of nanoparticles as the reaction yields mixture of submicron-sized silver (Ag) and silver chloride (AgCl) particles. At neutral pH, though the size of resultant nanoparticles dropped down in the range 8-30 nm, the mixture of Ag and AgCl particles still existed. In alkaline conditions i.e. pH 12, pure silver nanoparticles were obtained exhibiting excellent bactericidal and bacteriostatic properties against Gram positive and Gram negative bacteria. After the thorough inspection of the X-ray diffraction patterns and X-ray energy dispersive spectra (EDS) of the biosynthesized silver nanoparticles, it was evident that the Cl and S in the bagasse induces formation of side products such as AgCl and Ag@AgCl nanoparticles. While, at alkaline pH, the formation of such side products is avoided due to the interaction of Na ions of NaOH with Cl ions cane bagasse.
