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

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 centrifuged to collect the NPs pellet [134].

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 NPs [139, 140].
