**3. Methods for colloidal nanoparticles synthesis**

Remarkable morphological, structural, magnetic, electronic and physicochemical characteristics of colloidal NPs render them extraordinary for their uses in various fields such as physical, electrochemical, optical, environmental, biomedical fields etc. These peculiar properties of colloidal NPs depend on their source and route of synthesis process. Unremitting research in the field of nanotechnology have invented a range of ways to fabricate NPs. On the whole, these fabrication methods are segregated into three major groups, notably physical methods, chemical methods, and bio-assisted (also called biological and biogenic) methods in which NPs' synthesis is performed either by top-down approach or bottom-up approach. The top-down approach induces gradual trimming of bulk counterparts which invariably leads to the mass production of NPs. On the contrary, bottom-up approach deals with the consolidation of atoms and molecules to yield NPs with series of dimensions [54].

### **3.1 Physical methods**

Physical methods principally rely on top-down approach where high energy emissions, mechanical pressure, thermal or electrical powers are employed for melting, mitigation, abrasion of bulk materials to beget NPs. These techniques are devoid of solvent contamination, produce monodispersed and reproducible NPs making suitable for few specialized applications. However, generation of waste byproducts along synthesis is one of the flaws of physical methods [55]. Some of the most commonly used physical methods to generate NPs are high energy ball milling, laser ablation, electrospraying, inert gas condensation, physical vapor, deposition, flame spray pyrolysis etc. These methods are pictorially depicted in **Figure 1**.

#### **Figure 1.**

*Schematic representation of physical methods for synthesis of nanoparticles, (a) high energy ball milling, (b) laser ablation, (c) Electrospraying, (d) inert gas condensation, (e) physical vapor deposition,(f) flame spray pyrolysis.*

**7**

niques [63].

technique [66].

**3.2 Chemical methods**

*Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles*

in large scale production make this method not so popular [59].

are some of the shortcomings of electrospraying technique [61].

High energy ball milling is a high pressure and thermal, sturdy and energy effective synthesizing manner in which immensely movable balls pass on their kinetic energy to the bulk materials. The crushing process disrupts the chemical bonds of the materials and rifts it into tiny particles to raise NPs with diverse conformation and dimensionalities [56]. High contamination prominently due to wear and tear crushing by balls, polydispersity in terms of irregular dimensions of synthesized NPs, aggregation and long milling time [57] are few of the disadvantages associated

Laser ablation is another physical method that either employs continuous laser or pulsed laser to strike on the material opted to break down into NPs. It is a flexible mode which involves series of melting, evaporation and ionization of material onto collector surface. The continuous bombardment of laser beam results into ablation of targeted material to micro and nanostructure materials [58]. Even though, NPs with high purity can be obtained through this method, its high cost, long operational time for production high input of power for extirpation of matter, difficulty

The electrospraying mechanism is analogous to the electrospinning technique used to form fibers. In electrospraying, a blend of desired polymer solution and the solvent are filled in the syringe, subjected to high voltage electric field to split the solution into small charged nano-sized particles that are received by counter electrode. This technique provides flexibility over the size of NPs by varying the reaction conditions such as concentration of solution, electric field, conductivity, flow rate of liquid etc. [60]. Excess addition of cross linkages and low yield of NPs

Inert gas condensation is a very fundamental process that requires ultrahigh vacuum (UHV) conditions, inert gases like Helium (He) or Xenon (Xe) and a substrate cooled with liquid nitrogen. The target materials are first evaporated, then transferred along with inert gases and finally condensed on cooled substrate [62]. The agglomeration of condensed NPs, high cost associated with UHV conditions, difficulties related to maintaining clean vacuum situations, reproducibility and durability of working parameters etc. are some of the downsides of the tech-

Physical vapor deposition is an ecologically compatible route that incorporates

Flame spray pyrolysis is the recent and single step combustion process substantially operates to formulate compound and functional NPs. In this process, low volatile precursors are injected into highly sustainable flame with extreme temperature gradient where liquid precursor undergoes spray-to-particle or gas-to-particle pathway to form monodispersed NPs [55, 66]. The requirement of high stability and dispersibility of metal precursors and solvents, low volatility, relevant melting temperature limits the choice of materials and use of this

Chemical methods are certainly more favorable to synthesize colloidal NPs owing to their unaltered approach towards external stimuli. High yield and

three successive vital steps such as pyrolysis of solid materials to convert into vapors, transmission of vaporized materials followed by nucleation and growth process. This integrated group of processes have been widely designed and used to fabricate NPs in addition to deposit thin films of nanometers to micrometers [64]. Despite the fact that the technique delivers marked advantages, the instability of precursor gas at ambient temperature as well as reaction temperature and high cost

resulting from greatly controlled vacuum in chamber limits its use [65].

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

with high energy ball milling method.

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

*Colloids - Types, Preparation and Applications*

**3. Methods for colloidal nanoparticles synthesis**

molecules to yield NPs with series of dimensions [54].

**3.1 Physical methods**

Remarkable morphological, structural, magnetic, electronic and physicochemical characteristics of colloidal NPs render them extraordinary for their uses in various fields such as physical, electrochemical, optical, environmental, biomedical fields etc. These peculiar properties of colloidal NPs depend on their source and route of synthesis process. Unremitting research in the field of nanotechnology have invented a range of ways to fabricate NPs. On the whole, these fabrication methods are segregated into three major groups, notably physical methods, chemical methods, and bio-assisted (also called biological and biogenic) methods in which NPs' synthesis is performed either by top-down approach or bottom-up approach. The top-down approach induces gradual trimming of bulk counterparts which invariably leads to the mass production of NPs. On the contrary, bottom-up approach deals with the consolidation of atoms and

Physical methods principally rely on top-down approach where high energy emissions, mechanical pressure, thermal or electrical powers are employed for melting, mitigation, abrasion of bulk materials to beget NPs. These techniques are devoid of solvent contamination, produce monodispersed and reproducible NPs making suitable for few specialized applications. However, generation of waste byproducts along synthesis is one of the flaws of physical methods [55]. Some of the most commonly used physical methods to generate NPs are high energy ball milling, laser ablation, electrospraying, inert gas condensation, physical vapor, deposition, flame spray pyrolysis etc. These methods are pictorially depicted in **Figure 1**.

**6**

**Figure 1.**

*spray pyrolysis.*

*Schematic representation of physical methods for synthesis of nanoparticles, (a) high energy ball milling, (b) laser ablation, (c) Electrospraying, (d) inert gas condensation, (e) physical vapor deposition,(f) flame* 

High energy ball milling is a high pressure and thermal, sturdy and energy effective synthesizing manner in which immensely movable balls pass on their kinetic energy to the bulk materials. The crushing process disrupts the chemical bonds of the materials and rifts it into tiny particles to raise NPs with diverse conformation and dimensionalities [56]. High contamination prominently due to wear and tear crushing by balls, polydispersity in terms of irregular dimensions of synthesized NPs, aggregation and long milling time [57] are few of the disadvantages associated with high energy ball milling method.

Laser ablation is another physical method that either employs continuous laser or pulsed laser to strike on the material opted to break down into NPs. It is a flexible mode which involves series of melting, evaporation and ionization of material onto collector surface. The continuous bombardment of laser beam results into ablation of targeted material to micro and nanostructure materials [58]. Even though, NPs with high purity can be obtained through this method, its high cost, long operational time for production high input of power for extirpation of matter, difficulty in large scale production make this method not so popular [59].

The electrospraying mechanism is analogous to the electrospinning technique used to form fibers. In electrospraying, a blend of desired polymer solution and the solvent are filled in the syringe, subjected to high voltage electric field to split the solution into small charged nano-sized particles that are received by counter electrode. This technique provides flexibility over the size of NPs by varying the reaction conditions such as concentration of solution, electric field, conductivity, flow rate of liquid etc. [60]. Excess addition of cross linkages and low yield of NPs are some of the shortcomings of electrospraying technique [61].

Inert gas condensation is a very fundamental process that requires ultrahigh vacuum (UHV) conditions, inert gases like Helium (He) or Xenon (Xe) and a substrate cooled with liquid nitrogen. The target materials are first evaporated, then transferred along with inert gases and finally condensed on cooled substrate [62]. The agglomeration of condensed NPs, high cost associated with UHV conditions, difficulties related to maintaining clean vacuum situations, reproducibility and durability of working parameters etc. are some of the downsides of the techniques [63].

Physical vapor deposition is an ecologically compatible route that incorporates three successive vital steps such as pyrolysis of solid materials to convert into vapors, transmission of vaporized materials followed by nucleation and growth process. This integrated group of processes have been widely designed and used to fabricate NPs in addition to deposit thin films of nanometers to micrometers [64]. Despite the fact that the technique delivers marked advantages, the instability of precursor gas at ambient temperature as well as reaction temperature and high cost resulting from greatly controlled vacuum in chamber limits its use [65].

Flame spray pyrolysis is the recent and single step combustion process substantially operates to formulate compound and functional NPs. In this process, low volatile precursors are injected into highly sustainable flame with extreme temperature gradient where liquid precursor undergoes spray-to-particle or gas-to-particle pathway to form monodispersed NPs [55, 66]. The requirement of high stability and dispersibility of metal precursors and solvents, low volatility, relevant melting temperature limits the choice of materials and use of this technique [66].

#### **3.2 Chemical methods**

Chemical methods are certainly more favorable to synthesize colloidal NPs owing to their unaltered approach towards external stimuli. High yield and

reproducibility make them highly recommended. There are number of chemical methods, most of them are based on bottom up approach [67]. The chemical methods of colloidal NPs synthesis are diagrammatically represented in **Figure 2**. Sol–gel, plasma enhanced, chemical vapor deposition, polyol synthesis and hydrothermal synthesis are some of the primarily used chemical methods for synthesizing monodispersed NPs.

Colloidal solution of solid particles in liquid i.e. sol and liquid containing polymer i.e. gel are the two constituents of sol–gel method. The basic steps of the process are explicitly hydrolysis whereby the chemical bonds of precursors are deteriorated by water to form gel continued by condensation for genesis of sols in the liquid. In the end, the leftover liquid is drained to finalize the morphology of NMs [55, 68]. Owing to few flaws such as low abrasion resistance, poor bonding, exalted permeability and difficult control over porosity of technique, it becomes difficult to realize its industrial scale up [69].

In plasma enhanced chemical vapor deposition, also titled as plasma assisted chemical vapor deposition, plasma triggers the chemical reactions for formation of thin films and formulation of NPs as well. It is a well-known process conducted at lower temperature. The system is assembled by vacuum process unit, power supply, heater and precursor. The wide range of NPs can be formed via this method, for instance gallium nitride and so forth [70]. The expensive instrumentation, instability in damp conditions, presence of poisonous gases in plasma stream and lengthy process are some of the shortfalls of the method [71].

Polyol synthesis method fabricates colloidal NPs by using poly ethylene glycol as a medium to conduct the reaction. It also performs as solvent, reducing agent and integrating agent simultaneously with addition of protecting or capping agents externally [72]. The process is used to synthesize range of NPs of metals (platinum, palladium, silver, cobalt, etc.), metal oxide NPs (Zinc oxide, Cobalt oxide etc.) and magnetic, hybrid NPs as well [55]. However, the confined propensity of polyol to reduce precursors and slender stabilization of nonpolar metal surfaces by polar polyol are two major inadequacies with which the process has to dealt with and which diminishes the efficacy of the process [73].

#### **Figure 2.**

*Schematic representation of chemical methods for synthesis of nanoparticles, (a) sol–gel method, (b) polyol synthesis, (c) plasma enhanced chemical vapor deposition, (d) hydrothermal synthesis.*

**9**

*Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles*

*3.3.1 Advantage of nanoparticles synthesized via biological route*

Hydrothermal synthesis method explores various temperatures and pressure environments to change the behavior of water in the vicinity. During synthesis, NPs are synthesized from colloidal system that comprises of two or more states of compound from solid, liquid or gas and added together with controlled conditions of pressure and temperature. This method is carried out either by batch hydrothermal process or continuous hydrothermal process to create NPs of metal oxide, lithium iron phosphate etc. The batch hydrothermal executes reaction optimal ratios of phases while other allows faster mode of reaction. One of the incredible advantages of the method is its capability to produce large quantities of NPs at a time with preferable properties [74, 75]. The reaction requires water in supercritical state, higher pressure and temperature which in turn limits the onsite examinations to get

Despite the fact that chemical and physical methods of colloidal NPs synthesis are awfully proficient, these methods anyway own copious shortcomings just like use of acutely life-threatening chemicals, non-polar organic solvents, diversified synthetic capping, reducing agents, etc. therefore, hamper their engagement in biomedical purpose. On top of this, synthesis via physicochemical routes fetches contamination on the exterior of NPs post synthesis that has brought up solemn disquietude regarding the unfavorable upshots of the chemically synthesized NPs on the environment and living cells [76, 77]. These limitations has forced researchers to look for novel, environment friendly alternatives to synthesize colloidal NPs [78, 79]. Green synthesis or biosynthesis is the most feasible substitute that makes the use of microorganisms and parts of plants instead of toxic and pernicious chemicals. Bacteria, fungi, algae and yeast are frequently used as bio-reactors that can hire a batch of anionic functional groups proteins, enzymes, reducing sugars, etc. to reduce metals salts to corresponding colloidal NPs [80, 81]. The different methods of biological synthesis of colloidal NPs are diagrammatically represented

The routinely used NPs synthesis routes such as chemical and physical methods are not only energy and capital exhaustive but also employ the toxic chemicals and non-polar solvents for synthesis and synthetic additives or capping agents during the later process. These methods therefore rule out the application of such products in clinical and biomedical fields thereby creating a need for a safe, reliable, biocompatible and benign method for the production of NPs [82]. Worthy of the exceptional environment friendly nature, it has been reported that the NPs synthesis rate

For biomedical applications, it's obligatory that NPs must have depleted metal cytotoxicity and enhanced biocompatibility. Unlike physico-chemically synthesized NPs, green synthesized NPs are free from deleterious byproduct contamination that most often remain bound to the NPs surface and restraint their role in biomedical applications [86]. The decisive leverages that supports the biological routes for colloidal NPs synthesis are wide availability of key biological components, biocompatible reducing agents, capable of large scale synthesis with moderate temperature and pressure, dual working of enzymes or phytochemicals as reducing as well as stabilizing agents [87]. There are multiple superiorities concerned with bio-associated methods, uniquely expeditious and eco-friendly fabrication practices, less expensive and bio-tolerant nature of NPs. It does not demand for separate capping

via biogenic methods are comparable to that of chemical methods [83–85].

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

clarify with NPs synthesis [74].

**3.3 Biological methods**

in **Figure 3**.

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

Hydrothermal synthesis method explores various temperatures and pressure environments to change the behavior of water in the vicinity. During synthesis, NPs are synthesized from colloidal system that comprises of two or more states of compound from solid, liquid or gas and added together with controlled conditions of pressure and temperature. This method is carried out either by batch hydrothermal process or continuous hydrothermal process to create NPs of metal oxide, lithium iron phosphate etc. The batch hydrothermal executes reaction optimal ratios of phases while other allows faster mode of reaction. One of the incredible advantages of the method is its capability to produce large quantities of NPs at a time with preferable properties [74, 75]. The reaction requires water in supercritical state, higher pressure and temperature which in turn limits the onsite examinations to get clarify with NPs synthesis [74].

#### **3.3 Biological methods**

*Colloids - Types, Preparation and Applications*

ing monodispersed NPs.

realize its industrial scale up [69].

process are some of the shortfalls of the method [71].

which diminishes the efficacy of the process [73].

reproducibility make them highly recommended. There are number of chemical methods, most of them are based on bottom up approach [67]. The chemical methods of colloidal NPs synthesis are diagrammatically represented in **Figure 2**. Sol–gel, plasma enhanced, chemical vapor deposition, polyol synthesis and hydrothermal synthesis are some of the primarily used chemical methods for synthesiz-

Colloidal solution of solid particles in liquid i.e. sol and liquid containing polymer i.e. gel are the two constituents of sol–gel method. The basic steps of the process are explicitly hydrolysis whereby the chemical bonds of precursors are deteriorated by water to form gel continued by condensation for genesis of sols in the liquid. In the end, the leftover liquid is drained to finalize the morphology of NMs [55, 68]. Owing to few flaws such as low abrasion resistance, poor bonding, exalted permeability and difficult control over porosity of technique, it becomes difficult to

In plasma enhanced chemical vapor deposition, also titled as plasma assisted chemical vapor deposition, plasma triggers the chemical reactions for formation of thin films and formulation of NPs as well. It is a well-known process conducted at lower temperature. The system is assembled by vacuum process unit, power supply, heater and precursor. The wide range of NPs can be formed via this method, for instance gallium nitride and so forth [70]. The expensive instrumentation, instability in damp conditions, presence of poisonous gases in plasma stream and lengthy

Polyol synthesis method fabricates colloidal NPs by using poly ethylene glycol as a medium to conduct the reaction. It also performs as solvent, reducing agent and integrating agent simultaneously with addition of protecting or capping agents externally [72]. The process is used to synthesize range of NPs of metals (platinum, palladium, silver, cobalt, etc.), metal oxide NPs (Zinc oxide, Cobalt oxide etc.) and magnetic, hybrid NPs as well [55]. However, the confined propensity of polyol to reduce precursors and slender stabilization of nonpolar metal surfaces by polar polyol are two major inadequacies with which the process has to dealt with and

*Schematic representation of chemical methods for synthesis of nanoparticles, (a) sol–gel method, (b) polyol* 

*synthesis, (c) plasma enhanced chemical vapor deposition, (d) hydrothermal synthesis.*

**8**

**Figure 2.**

Despite the fact that chemical and physical methods of colloidal NPs synthesis are awfully proficient, these methods anyway own copious shortcomings just like use of acutely life-threatening chemicals, non-polar organic solvents, diversified synthetic capping, reducing agents, etc. therefore, hamper their engagement in biomedical purpose. On top of this, synthesis via physicochemical routes fetches contamination on the exterior of NPs post synthesis that has brought up solemn disquietude regarding the unfavorable upshots of the chemically synthesized NPs on the environment and living cells [76, 77]. These limitations has forced researchers to look for novel, environment friendly alternatives to synthesize colloidal NPs [78, 79]. Green synthesis or biosynthesis is the most feasible substitute that makes the use of microorganisms and parts of plants instead of toxic and pernicious chemicals. Bacteria, fungi, algae and yeast are frequently used as bio-reactors that can hire a batch of anionic functional groups proteins, enzymes, reducing sugars, etc. to reduce metals salts to corresponding colloidal NPs [80, 81]. The different methods of biological synthesis of colloidal NPs are diagrammatically represented in **Figure 3**.

#### *3.3.1 Advantage of nanoparticles synthesized via biological route*

The routinely used NPs synthesis routes such as chemical and physical methods are not only energy and capital exhaustive but also employ the toxic chemicals and non-polar solvents for synthesis and synthetic additives or capping agents during the later process. These methods therefore rule out the application of such products in clinical and biomedical fields thereby creating a need for a safe, reliable, biocompatible and benign method for the production of NPs [82]. Worthy of the exceptional environment friendly nature, it has been reported that the NPs synthesis rate via biogenic methods are comparable to that of chemical methods [83–85].

For biomedical applications, it's obligatory that NPs must have depleted metal cytotoxicity and enhanced biocompatibility. Unlike physico-chemically synthesized NPs, green synthesized NPs are free from deleterious byproduct contamination that most often remain bound to the NPs surface and restraint their role in biomedical applications [86]. The decisive leverages that supports the biological routes for colloidal NPs synthesis are wide availability of key biological components, biocompatible reducing agents, capable of large scale synthesis with moderate temperature and pressure, dual working of enzymes or phytochemicals as reducing as well as stabilizing agents [87]. There are multiple superiorities concerned with bio-associated methods, uniquely expeditious and eco-friendly fabrication practices, less expensive and bio-tolerant nature of NPs. It does not demand for separate capping

**Figure 3.**

*Schematic representation of biological methods for synthesis of nanoparticles, (a) plant based synthesis, (b) microbial synthesis.*

agents considering the potential of the plant's and the microorganism's components to act so [5]. On top of that, when NPs came in proximity of biological fluids while synthesis, they gradually and electively imbibe biomolecules establishing corona on the superficies that bestow additional potency and make them more efficient over uncovered NPs [88]. Precisely, medicinal plants are supposed to furnish the NPs with strengthened adequacy by entitling them with ample metabolites having pharmacological values [5, 89, 90]. As biosynthesized NPs are highly equipped with functional groups over the course of reaction, it eliminates additional steps required for physicochemical processes which automatically shorten the time period [91]. All of these supremacies make biosynthesis or green synthesis worth applicable.

#### *3.3.2 General mechanisms of biological synthesis*

#### *3.3.2.1 Algal synthesis*

Algae, either unicellular or multicellular, are autotrophic and aquatic photosynthetic organisms belonging to kingdom Protista. Depending upon their sizes that range from micrometer to macrometer, they are distinguished as microalgae or macroalgae and serve as extreme source of vitamins, minerals, and proteins. They have successfully drawn the utter attention by virtue of their competency to diminish the toxicity of metals accompanied by presence of bioactive components to stabilize the NPs; nonetheless the reports for algal synthesis of NPs are merely few, exclusively on iron oxide and zinc oxide [92]. Regardless the ongoing research on synthesis of NPs through different biological sources at greater extent, the detailed mechanism for the synthesis by algae is not revealed yet wholly. Studies so

**11**

*Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles*

far disclose that cell walls of seaweed are comprised of polysaccharides that carry hydrophilic surface groups like carboxyl, hydroxyl and sulfate groups [93]. Further, it holds abundant biomolecules, intrinsically proteins and enzymes which play the role of biocatalyst to convert metal ions into NPs, meanwhile, the other larger amphiphilic biomolecules act as capping agents to stabilize the NPs [94–97]. Some of the reported examples of algal synthesized NPs include AuNPs synthesized by brown seaweeds *Fucus vesiculosus* [98], and *Turbinaria ornate* [99]. A report by Khanehzaei et al. [100] explains the algal synthesis of copper and copper oxide NPs

Fungal synthesis is the quite pertinent among remaining bio-synthesis methods, even than bacteria, in the wake of their phenomenal properties adeptness of NPs' synthesis with various dimensions [101]. Fungi viz. yeasts or molds are eukaryotic organisms that bear mycelia which allocate them extended surface area for metal ions acquaintance. Fortuitously, cell surface of fungi possesses chain of biomolecules and reducing agents which offers them numerous additional privileges [102]. Moreover, the NPs' configuration due to fungi is rapid considering the fact that fungi biomass proliferate rapidly than bacteria, and contrary to bacteria, fungi have superior endurance and metal bioaccumulation. In supplement, it provokes monodispersed synthesis of NPs with quite defined structures. Typically, fungal manufacture of metal NPs is judiciously cheaper, eco-friendly, engage uncomplicated down-streaming operation and no need of external stabilizing agent as fungal biomass itself function as capping agent as well [103, 104]. Bhainsa and D′ Souza in 2006 reported the synthesis of AgNPs by the fungus *Aspergillus fumigates* [105]. Metal oxide NPs have also been synthesized through the fungus synthesis for example, silicon dioxide (SiO2), TiO2 and ferric oxide (Fe2O3) NPs by fungus

Amidst other microorganisms that fall into kingdom fungi, yeast is the most examined species given the fact that extracellular synthesis is simpler to regulate and to manipulate in laboratory scenario [104]. As an example, Bharde and his coworkers have reported the reduction of TiO2 to NPs by means of fugal extract of

There are copious number of bacteria that smoothly sustain with harsh environmental conditions. Moreover, they can multiply and grow at extreme speed, their maintenance is cost effective and are easy to manipulate for synthesis. For this sake, they are being employed for the biogenic synthesis of colloidal NPs. Furthermore, the bacterial growth parameters especially temperature, oxygen supply and incubation time can be monitored with ease as they might affect the sizes of NPs [107]. The synthesis of NPs from bacteria is either intracellular or extracellular, depending on the site of synthesis. The intracellular synthesis deals with carrying of metal ions inside the microbial cell while in case of extracellular synthesis, metal ions are entrapped by the surface of cell to reduce it into corresponding NPs in presence of enzymes and other biomolecules [81]. The mechanism for formation of NPs differs with respect to the bacteria. When metal ions that are almost poisonous to bacteria, come in proximity, bacteria secrete specific proteins, enzymes and other biochemicals as a safeguard provision. To rectify the detrimental effect, bacteria modify metal ions into NPs by assorting not only dissolution of metal ions but also their redox behavior and extracellular sorption. Bacteria with S-layer and

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

by extract of red seaweed *Kappaphycus alvarezii*.

*3.3.2.2 Fungal synthesis*

*Fusarium Oxysporum* [106].

*Saccharomyces cerevisiae* [106].

*3.3.2.3 Bacterial synthesis*

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

far disclose that cell walls of seaweed are comprised of polysaccharides that carry hydrophilic surface groups like carboxyl, hydroxyl and sulfate groups [93]. Further, it holds abundant biomolecules, intrinsically proteins and enzymes which play the role of biocatalyst to convert metal ions into NPs, meanwhile, the other larger amphiphilic biomolecules act as capping agents to stabilize the NPs [94–97]. Some of the reported examples of algal synthesized NPs include AuNPs synthesized by brown seaweeds *Fucus vesiculosus* [98], and *Turbinaria ornate* [99]. A report by Khanehzaei et al. [100] explains the algal synthesis of copper and copper oxide NPs by extract of red seaweed *Kappaphycus alvarezii*.

## *3.3.2.2 Fungal synthesis*

*Colloids - Types, Preparation and Applications*

agents considering the potential of the plant's and the microorganism's components to act so [5]. On top of that, when NPs came in proximity of biological fluids while synthesis, they gradually and electively imbibe biomolecules establishing corona on the superficies that bestow additional potency and make them more efficient over uncovered NPs [88]. Precisely, medicinal plants are supposed to furnish the NPs with strengthened adequacy by entitling them with ample metabolites having pharmacological values [5, 89, 90]. As biosynthesized NPs are highly equipped with functional groups over the course of reaction, it eliminates additional steps required for physicochemical processes which automatically shorten the time period [91]. All

*Schematic representation of biological methods for synthesis of nanoparticles, (a) plant based synthesis,* 

of these supremacies make biosynthesis or green synthesis worth applicable.

Algae, either unicellular or multicellular, are autotrophic and aquatic photosynthetic organisms belonging to kingdom Protista. Depending upon their sizes that range from micrometer to macrometer, they are distinguished as microalgae or macroalgae and serve as extreme source of vitamins, minerals, and proteins. They have successfully drawn the utter attention by virtue of their competency to diminish the toxicity of metals accompanied by presence of bioactive components to stabilize the NPs; nonetheless the reports for algal synthesis of NPs are merely few, exclusively on iron oxide and zinc oxide [92]. Regardless the ongoing research on synthesis of NPs through different biological sources at greater extent, the detailed mechanism for the synthesis by algae is not revealed yet wholly. Studies so

*3.3.2 General mechanisms of biological synthesis*

*3.3.2.1 Algal synthesis*

**Figure 3.**

*(b) microbial synthesis.*

**10**

Fungal synthesis is the quite pertinent among remaining bio-synthesis methods, even than bacteria, in the wake of their phenomenal properties adeptness of NPs' synthesis with various dimensions [101]. Fungi viz. yeasts or molds are eukaryotic organisms that bear mycelia which allocate them extended surface area for metal ions acquaintance. Fortuitously, cell surface of fungi possesses chain of biomolecules and reducing agents which offers them numerous additional privileges [102]. Moreover, the NPs' configuration due to fungi is rapid considering the fact that fungi biomass proliferate rapidly than bacteria, and contrary to bacteria, fungi have superior endurance and metal bioaccumulation. In supplement, it provokes monodispersed synthesis of NPs with quite defined structures. Typically, fungal manufacture of metal NPs is judiciously cheaper, eco-friendly, engage uncomplicated down-streaming operation and no need of external stabilizing agent as fungal biomass itself function as capping agent as well [103, 104]. Bhainsa and D′ Souza in 2006 reported the synthesis of AgNPs by the fungus *Aspergillus fumigates* [105]. Metal oxide NPs have also been synthesized through the fungus synthesis for example, silicon dioxide (SiO2), TiO2 and ferric oxide (Fe2O3) NPs by fungus *Fusarium Oxysporum* [106].

Amidst other microorganisms that fall into kingdom fungi, yeast is the most examined species given the fact that extracellular synthesis is simpler to regulate and to manipulate in laboratory scenario [104]. As an example, Bharde and his coworkers have reported the reduction of TiO2 to NPs by means of fugal extract of *Saccharomyces cerevisiae* [106].

#### *3.3.2.3 Bacterial synthesis*

There are copious number of bacteria that smoothly sustain with harsh environmental conditions. Moreover, they can multiply and grow at extreme speed, their maintenance is cost effective and are easy to manipulate for synthesis. For this sake, they are being employed for the biogenic synthesis of colloidal NPs. Furthermore, the bacterial growth parameters especially temperature, oxygen supply and incubation time can be monitored with ease as they might affect the sizes of NPs [107].

The synthesis of NPs from bacteria is either intracellular or extracellular, depending on the site of synthesis. The intracellular synthesis deals with carrying of metal ions inside the microbial cell while in case of extracellular synthesis, metal ions are entrapped by the surface of cell to reduce it into corresponding NPs in presence of enzymes and other biomolecules [81]. The mechanism for formation of NPs differs with respect to the bacteria. When metal ions that are almost poisonous to bacteria, come in proximity, bacteria secrete specific proteins, enzymes and other biochemicals as a safeguard provision. To rectify the detrimental effect, bacteria modify metal ions into NPs by assorting not only dissolution of metal ions but also their redox behavior and extracellular sorption. Bacteria with S-layer and

Magnetotactic bacteria are best suited to harvest metal NPs whose cell wall surfaces are shielded with protein rich components [108, 109].

For *in vitro* synthesis of NPs using bacteria, initially convenient bacterial species is cultured for 1-2 days in shaking incubator or orbital shaker at optimal parameters incorporating temperature, pH, media concentration, shaking speed etc. The culture is then centrifuged to separate biomass. For intracellular synthesis biomass is collected, washed thoroughly with deionized water and dissolved in sterile water which in turn acts as a bacterial extract to reduce metal ions. Conversely, supernatant after centrifugation can also be used for extracellular synthesis of NPs [110].

#### *3.3.2.4 Plant-based synthesis*

As phytomining practices, plants with ability to hyper accumulate metals are planted on metal contaminated soils for uptake of metal ions. The metal ions disseminate into plant and travel to the convinced plant parts where primary and secondary metabolites such as terpenoids, flavonoids, phenolic acids, proteins, polysaccharides, organic acids etc. remold ions into metal NPs [5, 111, 112]. This approach is merely time consuming, tedious and retrieval of synthesized NPs is strenuous [111, 113]. The biogenic synthesis of NPs using plant extract or biomass is one of the most effective, rapid, absolute non-hazardous and ecofriendly methods. Nanoparticlesof noble metals, metal oxides, bimetallic alloys, etc. have been mainly synthesized *in vitro* by harnessing this method which is well reviewed by Iravani in 2011 [114].

For phytomediated biogenic synthesis, plant extracts are prepared from different parts of plants largely leaves, flowers, fruits, stem, roots, peels etc. [111, 113] and used as a source of reducing and capping agents. To accomplish this, the metal ion solution is subsequently added to extract where co-precipitation of metal ions with accessible functional groups is favored. The reaction parameters such as reaction time, temperature, pH, ration and concentration of metal salt influence the synthesis [115], therefore can be fine tunes. For example, ZnO NPs cane be formulated through leaf extract of *Corymbia citriodora* [116]*,* peel extract of *Nephelium lappaceum* [117], root extract of *Polygala tenuifolia* [118]*.*

#### **4. Selection of biological agents for the synthesis of nanoparticles**

Two main criteria for the selection of suitable plants for synthesis are selection of plant part on the basis of enzyme activities and biochemical pathways (for example: plants with heavy metal accumulations and detoxification properties); and setting the optimal conditions for enhanced cell growth and high enzyme activity [114].

According to Das and Brar [119], the plants are majorly preferred due to their exceptional reduction ability, yet, only the ethanobotanical conclusions are not the only basis for the selection of plants for the synthesis of NPs. These authors pointed out the fact that, the bio-reduction of the metallic cations can be a part of the plant's defensive reaction towards ionic stress. The chemical evolution of phytochemicals must thus be reconsidered to probe the possibility of exploiting different plant groups for biogenic synthesis. It was therefore suggested that, some representatives for each plant group must be picked and the protocol be standardized keeping in mind the process parameters and laboratory scale to commercial scale scaling up. It is important that the plant encompassing the desired properties must not fail at large scale level [119]. Some of the NPs synthesized from plants have the extreme potential in biomedical fields and should be considered for

**13**

*Optimization of Biogenic Synthesis of Colloidal Metal Nanoparticles*

have a broader perspective about the following parameters:

2.Phylogenetic studies to set a reference plant.

3.In vitro study including cellular damage studies.

6.Genetic aspects, which is still an unexplored area.

biogenic synthesis more scalable.

4.Precision in identifying the part of plant and mechanism

scaling up purposes [120–122]. Das and Brar [119], also mentioned that instead of focusing on the advantages and disadvantages of biogenic synthesis routes such as efficiency, dual functionality, propensity, broad application etc.; it is important to

1.Clinical relevance must be checked by studying the previous well documented

5.Geographical distribution studies to select a plant that does not have a very

7.This selection criteria is very feasible and applicable to all the plant groups and can bridge the gap exploitation of nature's ability and possibility to make

Some bacterial species such as *Pseudomonas stutzeri* and *P. aeruginosa* have the ability to recourse specific defense mechanisms in order to deal with stress conditions like toxicity of heavy metal ions to survive and grow at high metal ion concentrations [123, 124]. Algae are economical contenders for the bioremediation and bioconversion of precious toxic metals into non-toxic nano forms due to their ability to accumulate and reduce metal ions into NPs [125]. Algae are preferred as they are convenient to handle, pose lower toxic effects to the environment and synthesize NPs at lower temperatures with great efficiency. Different algae widely used for the synthesis of NPs are: *Lyngbya majuscule, Spirulina platensis, Rhizoclonium hieroglyphicum, Phaeophyceae, Cyanophyceae, Rhodophyceae,* and *Chlorella vulgaris* [126, 127]*.* Fungi act as ideal biocatalysts for NPs synthesis and are preferred over bacteria due to greater potential of biologically active substances production [128]. Furthermore, fungal biomass are suitable for use in bioreactors as they can resist flow pressure, agitation and harsh conditions in chambers such as bioreactors and can exude extracellular reductive proteins suitable for employment in further steps of synthesis [102]. *Fusarium oxysporum* is one such fungi used for manufacturing

**5. Preparation of extract and biomass for the synthesis of nanoparticles**

The potential of phytosynthesis, a "green" synthesis approach is not yet completely utilized in full throttle for the colloidal NPs synthesis. As plants harbor a wide range of metabolites, it is possible to utilize plant tissue culture methods and optimizing the downstream processing techniques for the industrial production of NPs [130]. The part of the plant is chosen on the basis of desired application and the widely used plant parts of the part for extract preparation are leaf, seed, stem, fruit, root and flower. Initially, the test plant samples are collected, washed, dried and weighed. These are then chopped down into smaller pieces and soaked into sterile distilled water. This mixture is eventually incubated at optimized conditions

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

scientific reports.

narrow distribution.

NPs at industrial scale [129].

*Colloids - Types, Preparation and Applications*

*3.3.2.4 Plant-based synthesis*

Iravani in 2011 [114].

activity [114].

are shielded with protein rich components [108, 109].

Magnetotactic bacteria are best suited to harvest metal NPs whose cell wall surfaces

As phytomining practices, plants with ability to hyper accumulate metals are planted on metal contaminated soils for uptake of metal ions. The metal ions disseminate into plant and travel to the convinced plant parts where primary and secondary metabolites such as terpenoids, flavonoids, phenolic acids, proteins, polysaccharides, organic acids etc. remold ions into metal NPs [5, 111, 112]. This approach is merely time consuming, tedious and retrieval of synthesized NPs is strenuous [111, 113]. The biogenic synthesis of NPs using plant extract or biomass is one of the most effective, rapid, absolute non-hazardous and ecofriendly methods. Nanoparticlesof noble metals, metal oxides, bimetallic alloys, etc. have been mainly synthesized *in vitro* by harnessing this method which is well reviewed by

For phytomediated biogenic synthesis, plant extracts are prepared from different parts of plants largely leaves, flowers, fruits, stem, roots, peels etc. [111, 113] and used as a source of reducing and capping agents. To accomplish this, the metal ion solution is subsequently added to extract where co-precipitation of metal ions with accessible functional groups is favored. The reaction parameters such as reaction time, temperature, pH, ration and concentration of metal salt influence the synthesis [115], therefore can be fine tunes. For example, ZnO NPs cane be formulated through leaf extract of *Corymbia citriodora* [116]*,* peel extract of

*Nephelium lappaceum* [117], root extract of *Polygala tenuifolia* [118]*.*

**4. Selection of biological agents for the synthesis of nanoparticles**

Two main criteria for the selection of suitable plants for synthesis are selection of plant part on the basis of enzyme activities and biochemical pathways (for example: plants with heavy metal accumulations and detoxification properties); and setting the optimal conditions for enhanced cell growth and high enzyme

According to Das and Brar [119], the plants are majorly preferred due to their exceptional reduction ability, yet, only the ethanobotanical conclusions are not the only basis for the selection of plants for the synthesis of NPs. These authors pointed out the fact that, the bio-reduction of the metallic cations can be a part of the plant's defensive reaction towards ionic stress. The chemical evolution of phytochemicals must thus be reconsidered to probe the possibility of exploiting different plant groups for biogenic synthesis. It was therefore suggested that, some representatives for each plant group must be picked and the protocol be standardized keeping in mind the process parameters and laboratory scale to commercial scale scaling up. It is important that the plant encompassing the desired properties must not fail at large scale level [119]. Some of the NPs synthesized from plants have the extreme potential in biomedical fields and should be considered for

For *in vitro* synthesis of NPs using bacteria, initially convenient bacterial species is cultured for 1-2 days in shaking incubator or orbital shaker at optimal parameters incorporating temperature, pH, media concentration, shaking speed etc. The culture is then centrifuged to separate biomass. For intracellular synthesis biomass is collected, washed thoroughly with deionized water and dissolved in sterile water which in turn acts as a bacterial extract to reduce metal ions. Conversely, supernatant after centrifugation can also be used for extracellular synthesis of NPs [110].

**12**

scaling up purposes [120–122]. Das and Brar [119], also mentioned that instead of focusing on the advantages and disadvantages of biogenic synthesis routes such as efficiency, dual functionality, propensity, broad application etc.; it is important to have a broader perspective about the following parameters:


Some bacterial species such as *Pseudomonas stutzeri* and *P. aeruginosa* have the ability to recourse specific defense mechanisms in order to deal with stress conditions like toxicity of heavy metal ions to survive and grow at high metal ion concentrations [123, 124]. Algae are economical contenders for the bioremediation and bioconversion of precious toxic metals into non-toxic nano forms due to their ability to accumulate and reduce metal ions into NPs [125]. Algae are preferred as they are convenient to handle, pose lower toxic effects to the environment and synthesize NPs at lower temperatures with great efficiency. Different algae widely used for the synthesis of NPs are: *Lyngbya majuscule, Spirulina platensis, Rhizoclonium hieroglyphicum, Phaeophyceae, Cyanophyceae, Rhodophyceae,* and *Chlorella vulgaris* [126, 127]*.* Fungi act as ideal biocatalysts for NPs synthesis and are preferred over bacteria due to greater potential of biologically active substances production [128]. Furthermore, fungal biomass are suitable for use in bioreactors as they can resist flow pressure, agitation and harsh conditions in chambers such as bioreactors and can exude extracellular reductive proteins suitable for employment in further steps of synthesis [102]. *Fusarium oxysporum* is one such fungi used for manufacturing NPs at industrial scale [129].
