Received 30 October 2015; accepted 26 February 2016; published 29 February 2016
Materials in the nano dimensions (1 - 100 nm) have remarkable difference in the properties compared to the same material in the bulk. These differences lie in the physical and structural properties of atoms, molecules and bulk materials of the element due to difference in physiochemical properties and surface to volume ratio  . With advancement in nanotechnology, a large number of nanomaterials are appearing with unique properties, opening spectrum of applications and research opportunities  .
About 5000 years ago, many Greeks, Romans, Persians and Egyptians used silver in one form or other to store food products  . Use of silver ware during ancient period by various dynasties was common across the globe utensils for drinking and eating and storing various drinkable and eatable items probably due to the knowledge of antimicrobial action  . There are records regarding therapeutic application of silver in literature as earlier as 300 BC. In the Hindu religion, till date silver utensils are preferred for the “panchamrit” preparation using curd, Ocimum sanctum and other ingredients. The therapeutic potentials of various metals are mentioned in ancient Indian Aurvedic medicine book medicinal literature named “Charak Samhita”  . Until the discovery of antibiotics by Alexzander Flemming, silver was commonly used as antimicrobial agent.
In the recent past, silver nano particles (AgNps) have received enormous attention of the researchers due to their extraordinary defense against wide range of microorganisms and also due to the appearance of drug resistance against commonly used antibiotics  . The exceptional characteristics of AgNPs have made them applicable in various fields like biomedical  , drug delivery  , water treatment  , agricultural etc.  . AgNps are applied in inks, adhesives, electronic devises, pastes etc. due to high conductivity  . AgNps have been synthesized by physio-chemical techniques such as chemical reduction  , gamma ray radiation  , micro emulsion  , electrochemical method  , laser ablation  , autoclave  , microwave  and photochemical reduction  . These methods have effective yield, but they are associated with the limitations like use of toxic chemicals and high operational cost and energy needs. Considering the drawbacks of physio-chemical methods, cost-effective and energy efficient new alternative for AgNP synthesis using microorganisms  , plant extracts  and natural polymers  as reducing and capping agents are emerging very fast. The association of nanotechnology and green chemistry will unfold the range of biologically and cytologically compatible metallic nanoparticles   .
Over the past decade, few reviews focusing on green synthesis of AgNPs were published  -  . Most of these reviews focused on several plant and microbial sources for synthesis, several characterization techniques for analysis, certain tabular data representing source, shape and size and information regarding various applications. The present review, unlike the earlier ones, summarizes the synthesis procedure, parameters, characterizations, applications and predicted antibacterial mechanism in a systematic manner, focusing on various green routes for AgNPs synthesis.
2. Green Synthesis
The primary requirement of green synthesis of AgNPs is silver metal ion solution and a reducing biological agent. In most of the cases reducing agents or other constituents present in the cells acts as stabilizing and capping agents, so there is no need of adding capping and stabilizing agents from outside.
2.1. Metal Ion Solution
The Ag+ ions are primary requirement for the synthesis of AgNPs which can be obtained from various water soluble salts of silver. However, the aqueous AgNO3 solution with Ag+ ion concentration range between 0.1 - 10 mm (most commonly 1 mm) has been used by the majority of researchers.
2.2. Biological Reducing Agents
The reducing agents are widely distributed in the biological systems. The AgNPs have been synthesized using different organisms belonging to four kingdom out of five kingdom of living organisms i.e. Monera (prokaryotic organisms without true nucleus) Protista (unicellular organisms with true nucleus), fungi (eukaryotic, saprophyte/parasite), plantae (eukaryotic, autotrophs) and animalia (eukaryotic, heterotrophs). Data are not available regarding use of animal materials for the synthesis of AgNP’ till date to the best of our knowledge. Due to this limitation, green synthesis of AgNPs has been discussed under headings microorganisms, plants, and bio-poly- mers.
Green syntheses of AgNPs have been performed using plant extracts, microbial cell biomass or cell free growth medium and biopolymers. The plants used for AgNps synthesis range from algae to angiosperms; however, limited reports are available for lower plants and the most suitable choice are the angiosperm plants. Parts like leaf, bark, root, and stem have been used for the AgNP synthesis. The medicinally important plants like Boerhaavia diffusa  , Tinospora cordifolia  , Aloe vera  , Terminalia chebula  Catharanthus roseus  , Ocimum tenuiflorum  , Azadirachta indica  , Emblica officinalis  , Cocos nucifera  , common spices Piper nigrum  ), Cinnamon zeylanicum  . Some exotic weeds like Parthenium hysterophorus  growing in uncontrolled manner due to lack of natural enemies and causing health problems have also been used for AgNP’s synthesis. The other group includes alkaloids (Papaver somniferum) and essential oils (Mentha piperita) producing plants. All the plant extracts played dual role of potential reducing and stabilizing agents with an exception in few cases where external chemical agents like sodium-do-decyl sulphate were used for stabilization the AgNPs  ). Metabolites, proteins  and chlorophyll  present in the plant extracts were found to be acting as capping agents for synthesized AgNPs.
The preferred solvent for extracting reducing agents from the plant is water in most of the cases however, there are few reports regarding the use of organic solvents like methanol  -  , ethanol   and ethyl acetate  . Some researchers pre-treated the plants materials in saline  or acetone  atmospheres before extraction. On the whole, even though the extracting solvents differed, the nanoparticle suspensions have made in aqueous medium only. Synthesis using plant extracts generate nanoparticles of well-defined shape, structure and morphology in compared to those obtained through the utilization of bark, tissue and whole plant  .
The AgNPs synthesis by microbes is strenuous compared to the use of plant extracts and biopolymers as reducing and capping agents mainly due to the difficulty in growth, culture maintenance, and inoculums size standardization. Several fungal and bacterial species have been successfully used in the synthesis. The AgNPs synthesis mainly followed one of the two distinct routes, one utilizing extracellular materials secreted in the growth medium whereas the other utilizing microbial cell biomass directly. The microbes synthesize AgNP intracellularly as well as extracellularly. The Intracellular synthesis of AgNPs was observed by few researchers  .
AgNPs synthesis supports better control on size and shape of AgNPs, due to easy down streaming and larger adaptability to nano systems. However, extracellular AgNP synthesis is been widely reported   . One of the commonly used fungal genera for synthesizing AgNPs is Fusarium   -  . No special capping agent was used in the work of many researchers for stabilizing synthesized AgNPs, except Perni et al.  and Shahverdi et al.  who used L-cystine and piperitone as stabilizing agents, respectively. Among the wide varieties off bio-polymers used for AgNP synthesis, almost all played the dual role of reducing and stabilizing agents with an exception of using starch as a capping agent  .
3. Separation of AgNPs
Centrifugation technique is mostly used by researchers to obtain the pellet or powder form of synthesized silver nanoparticles. The AgNPs suspensions were also oven dried to obtain the product in powder form  .
Some common characterizations of AgNPs include UV-Vis Spectra, SEM, TEM, FTIR, XRD and EDAX or EDX/EDS. DLS study is mostly used for AgNPs synthesized from bio-polymers rather than plant extracts and microorganisms. Zeta potential values indicate the stability of synthesized AgNPs. Thermo-Gravimetric Analysis (TGA) is used to find the effect of AgNO3 and L-cystine on the organic composition of AgNPs  to find out the amount of organic material in synthesized AgNPs  and predict the thermal stability of AgNPs  . Inductive Coupled Plasma (ICP) analysis was performed to analyze the concentration and conversion of AgNPs  .
4. Monitoring of AgNPs
The appearance of yellow to slight brownish-yellow color in the colorless solution has been taken as indicative of AgNPs synthesis by almost all the researchers. The SPR peak of the synthesized AgNPs was witnessed in the range of 400 - 450 nm, the significant range for AgNPs  . The UV-Vis spectral analyses have been used to analyze the dependency of pH, metal ion concentration, extract content on the formation of AgNPs and reveal the size-stability of synthesized AgNPs by exhibiting red shift in the SPR peak with increase in size of nanoparticles and blue shift for decrease in size. The SEM morphological analysis in most of the studies revealed spherical AgNPs, whereas few authors reported irregular  , triangular  , hexagonal  , isotropic  , polyhedral  , flake  , flower  , pentagonal  , anisotropic  and rod like structures  . A pictorial representation of SEM/TEM images of AgNPs with different shapes is shown in Figure 1. Using XRD studies of almost all the researchers reported the formation of face centered cubic (FCC) crystalline structured AgNPs.
Figure 1.Various shapes of AgNPs synthesized (from various sources).
However, cubic and hexagonal structures were also reported in some cases. EDS or EDAX, for analyzing elemental composition in the nanomaterials, exhibited a characteristic optical absorption band peak around 3 KeV with silver weight percentage ranging from 45% to 80%. The reported stability of synthesized AgNPs has varied from 1 day to 1 year depending upon reducing agents and other operating conditions.
5. Mechanism of AgNPs Synthesis
The synthesis of AgNP by biological entities is due to the presence of large number of organic chemical like carbohydrate, fat, proteins, enzymes& coenzymes, phenols flavanoids, terpenoids, alkaloids, gum, etc capable of donating electron for the reduction of Ag+ ions to Ag0. The active ingredient responsible for reduction of Ag+ ions varies depending upon organism/extract used. For nano-transformation of AgNPs, electrons are supposed to be derived from dehydrogenation of acids (ascorbic acid) and alcohols (catechol) in hydrophytes, keto to enol conversions (cyperaquinone, dietchequinone, remirin) in mesophytes or both mechanisms in xerophytes plants  . The microbial cellular and extracellular oxidoreductase enzymes can perform similar reduction processes. A schematic diagram showing the silver ion reduction, agglomeration and stabilization to form a particle of nano size is shown in Figure 2.
6. Factors Affecting AgNPs Synthesis
The major physical and chemical parameters that affect the synthesis of AgNP are reaction temperature, metal ion concentration, extract contents, pH of the reaction mixture, duration of reaction and agitation. Parameters like metal ion concentration, extract composition and reaction period largely affect the size, shape and morphology of the AgNPs  . Most of the authors have reported suitability of basic medium for AgNPs synthesis due to better stability of the synthesized nanoparticles in basic medium     . Some other advantages reported under basic pH are rapid growth rate    good yield and mono dispersity  and enhanced reduction process. Small and uniform sized nanoparticles were synthesized by increasing pH of the reaction mixture    -  . The nearly spherical AgNPs were converted to spherical AgNP by altering pH  , However, very high pH (pH > 11) was associated with the drawback of formation of agglomerated and unstable AgNPs  .
The Reaction conditions like time of stirring and reaction temperature are important parameters. Temperatures up to 100˚C were used by many researchers for AgNP synthesis using bio-polymers and plant extracts, whereas the use of mesophilic microorganism restricted the reaction temperature to 40˚C. At higher temperatures the mesophilic microorganism dies due to the inactivation of their vital enzymes. The temperature increase (30˚C - 90˚C) resulted in increased rate of AgNPs synthesis  and also promoted the synthesis of smaller size AgNPs  . On the whole, most of workers have synthesized AgNPs at room temperature (25˚C to 37˚C) range. A plot representing the size range of AgNPs synthesized in the room temperature range is elucidated in Figure 3.
Figure 2.Synthesis mechanism of AgNPs.
Figure 3. Size range of AgNPs synthesized at room temperature range (from various sources).
It has been found that the size range of AgNPs synthesized from algae, bryophytes, pteridophytes, gymnosperms and bio-polymer sources lie below 50 nm and that of AgNPs synthesized using from angiosperms, algae and bacterial sources ranged between 100 nm and more. The reaction mixture synthesizing AgNP using microorganisms and bio-polymers were continuously agitated to protect agglomeration compared to plant extracts without any suitable reason by the authors. Reaction mixture agitation achieved by applying external mechanical force might accelerate the formation of nanoparticles. Aging of the synthesized AgNP solution changed spherical nanoparticles into flower like structure  (Table 1).
7. Applications of AgNPs
The recent research results have shown that the AgNPs, due to their special characteristics, have immense potential for applications as anti-microbial, anti-parasitic and anti-fouling agents; as agents for site-specific medication, water purification systems, etc. The essential features of some of these applications are discussed in the following sections.
7.1. Anti-Microbial Activity
The AgNPs have been found to exhibit promising anti-micribial activity. Researchers have used several novel techniques to confirm and quantify the anti-micribial activity of AgNPs.
7.1.1. Disc/Well Diffusion Methods
The disc diffusion method, a most commonly used technique to access the antimicrobial activity of a liquid, has been employed by many researchers to confirm antimicrobial action of the AgNPs solution. In this method, uniform sized disc of adsorbent material are dipped in the increasing concentration of AgNP and placed over surface of the targeted microbe inoculated on the nutrient medium plates. An inhibition zone formation around the disc reflects antimicrobial action of the nanomaterials       and well diffusion 
Table 1. Summary of the work related AgNPs synthesis using green route.
Note: DLS―Dynamic light scattering, EDAX/EDS Energy Dispersive X-ray Analysis/Energy Dispersive Spectroscopy; FTIR―Fourier transform infrared spectroscopy, HRTEM―High Resolution Transmission Electron Microscopy; SEM―Scanning Electron Microscopy, TGA―Thermogra- vimetric analysis, UV-Vis―Ultra violet-visible spectroscopy; XRD―X Ray Diffraction, DEC―decahedral, sph―spherical, Tri―Triangular, R― Rod, Hex―Hexagonal, PD―Polydispersed, MD―monodispersed, WD―Well Dispersed, Cryst―Crystalline.
      . In the Well diffusion method instead of using discs, small disc shaped pits are created on the agar plate for filling the test solution. In both the techniques, the microbe inoculated plates are incubated under standard condition for the formation of clear inhibition zone. The inhibition zone diameter around the disc or well, directly relates the effects of AgNPs on the chosen microbe.
7.1.2. Minimum Inhibitory Concentration (MIC)/Minimum Bactericidal Concentration (MBC)
The MIC is defined as the minimum concentration of the analyte which inhibit 100% visible growth of the targeted microbe after 24 hours. The MIC is determined by monitoring growth of bacteria in culture tubes inoculated with the same amount of bacterial culture but increasing concentration of AgNPs in the growth medium. The minimum concentration of AgNP which checks growth of bacteria is called the minimum inhibitory concentration. For the determination of MBC, fixed AgNP concentration greater than MIC value is added to the nutrient mediums containing increasing bacterial inoculum and bacterial growth is monitored, using UV-Vis spectroscopy or plate analyzer, for change in the optical density of the samples    . The broth dilution test is also used to conduct MIC and MBC analysis, in which the results after experimentation are compared with a standard data   .
7.1.3. Analysis of SEM and TEM Micrographs
The SEM and TEM analyses have been used to monitor changes in the morphology of the bacterial cell before and after treatment with “AgNPs”; The visible alterations in the cell shape and perforations in the cell wall have been reported and used as indicator of the antimicrobial action of AgNPs by several workers    .
7.2. Antibacterial Action
The AgNPs have potent antibacterial action against gram positive bacteria, Lactobacillus fermentum  , Streptomyces sp.  . Bacillus cereus  Brevibacterium casei  , S. aureus  B. licheniromis  , and gram negative bacteria, E. coli  Entrobacteria  and Ureibacillus thermo sphaerius  . The antibacterial action of AgNPs on gram positive and gram negative bacterial strains is not the same but competes one over the other. There are contradictory reports regarding antibacterial action against gram positive and gram negative bacteria. According to some researchers the gram negative bacteria are reported to be more sensitive to AgNPs compared to gram positive bacteria     whereas reverse results were observed by other researchers     . The reported differential sensitivity of both the bacterial species could be attributed to the difference in structural characteristics of the bacterial species   as well as shape and size of AgNP, bacterial inoculum size, exposure time and nutrient medium used during analysis of antibacterial action  .
The anti-bacterial action of AgNPs is quite complex and not well studied. Its mechanism is onlytentatively explained. The antimicrobial action of AgNPs can be categorized in two types: the inhibitory action and bactericidal action. In the former strategy bacterial cells are not killed but their division is prevented whereas in the later bacterial cells will die due to the action of AgNP  . The antibacterial action mechanism of AgNP is summarized in Figure 4. The graphical presentation shown in Figure 4 is the result of bacterial growth loaded with AgNPs synthesized from different green sources. Probable mechanism leading the differential behavior in the cases “a” to “e” is shown on the right hand part. The reason behind the bacterial cells resuming their growth after certain period of inhibitory action in cases “b”, “c”, “d” respectively was assumed to due to the unaffected cells, which in turn promote the growth (figure shown in inset). On the other hand a complete inhibition/bactericidal effect as in the case “e” is attributed to the complete death of cells. A shift from inhibitory action to nearly bactericidal action was observed with an increase in concentration of AgNPs loading   . The experimental support in the form of morphological changes and perforations in cell wall has been presented as shown in Figure 5. The mechanism behind the bactericidal action of AgNP was illustrated by release of Ag+ ions, which serves as reservoirs for anti-microbial action  . The Ag+ cations produced interacts with the negative charge on the cell wall and affects the membrane permeability. The nano-silver cations which have greater affinity towards sulphur and phosphorus containing compounds present in the outer membrane, respiratory enzymes, proteins and DNA, penetrate through the cell wall and plasma membrane by destabilizing them and cause protein denaturation by dissipating proton motive force, respiratory inhibition, intracellular ATP depletion
Figure 4. Mechanism of antibacterial action of AgNPs.
Figure 5. Morphological change and cell wall damage of bacterial cell.
and DNA damage. The above stated mechanism is in agreement with the reports of many authors       .
7.3. Anti-Fungal Action
The AgNPs exhibited antifungal action against various fungi   . Actual mechanism behind the antifungal activity is not fully. The disrupting the structure of the cell membrane by destructing the membrane integrity, thereby the inhibition of the budding process has been attributed to be responsible for the antifungal action of AgNPs against C. albanicans species  . The shape of the AgNPs has a significant effect on the anti-micr- obial activity 
7.4. Anti-Parasitic Action
The AgNPs have been found to be effective larvicidal agents against dengue vector Aedes aegypt  , and Culex quinquefasciatus  , filariasis vector C. quinquefasciatus  and malarial vector A. subpictus  , Aedes aegypti  , A. subpictu  and other parasites   . No attempt has been made to propose a proper mechanism for anti-parasitic action of AgNPs. Denaturation of sulfur containing proteins and phosphorus containing DNA by AgNPs, leading to denaturation of organelles and enzymes is believed to be responsible for the larvicidal activity  .
7.5. Anti-Fouling Action
The AgNPs synthesized from Rhizopus oryzae fungal species have been used for treating contaminated water and adsorption of pesticides  and that from Lactobacillus fermentum cells have been used as anti-bio fouling agent  . The AgNPs are being used to treat many environmental concerns like; air disinfection, water disinfection, ground water and biological water disinfection and surface disinfection  .
7.6. Other Applications
There have been several reports on the use of AgNPs in the field of medicine. The AgNPs have been used as therapeutic agents  , as glyconano sensors for disease diagnosis  and as nano carriers for drugs delivery  . Reports are also available on the use of AgNPs in radiation therapy  , in H2O2 sensor  , in ESR- Dosimetry  , as heavy metal ion sensors  and as catalyst for reduction of dyes such as methylene blue  .
Sufficient volume of published literature is available on the synthesis of AgNPs through green routes. Among plants, angiosperm species has been widely used in comparison with the other sources. Several characterizations methods and techniques have been used for AgNPs synthesis and confirmation. The AgNPs synthesized using biological reducing and capping agents have shown wide variation in shape and size. Among applications, the anti-microbial action of AgNPs has been widely studied. Various methods used to carry out antibacterial study and elucidate mechanism of anti-microbial have been developed. The results, however, are conflicting and there is a need for more work to resolve this issue. The potential of AgNPs for their use as drug carriers in cancer therapy, as biosensors for metabolites and pollutants, as catalyst etc. is quite high and requires intensive and integrated research activity for harnessing it.
One of the Authors (SNU) is grateful to the Department of Atomic Energy, GoI, Mumbai for the award of Raja Ramanna fellowship. The financial support to DDG in the form of Dr DS Kothari Postdoc fellowship from the UGC, New Delhi is gratefully acknowledged. Authors are also grateful to Head of the Department of Chemical Engineering and Technology, IIT (BHU) for providing necessary encouragement and facilities.
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