Nanomaterials for Sustainability: A Review on Green Synthesis of Nanoparticles Using Microorganisms

Nanotechnology has permeated all areas of sciences as one of the most propitious technology with the deployment of nanoparticles in environmental remediation and biomedical elds; their synthesis under greener conditions has been bourgeoned using microorganisms, plants, etc. to decrease the use of toxic chemicals. Synthesis of nanoparticles by exploiting microorganisms has opened up a new prospect at the interface of nanotechnology, chemistry, and biology enabling access via a biocompatible, safe, sustainable, eco-friendly, and reliable route; microorganisms offer crystal growth, stabilization, and prevention of aggregation thus performing a dual role of reducing and capping agent because of the presence of biomolecules such as enzymes, peptides, poly (amino acids), polyhydroxyalkanoate (PHA), and polysaccharides. Herein, the microorganisms-based synthesis of various nanoparticles comprising gold, silver, platinum, palladium, copper, titanium dioxide, zinc oxide, iron oxide, and selenium along with their appliances in waste treatment, biomedicine namely cancer treatment, antibacterial, antimicrobial, antifungal, and antioxidants, are deliberated.


Introduction
"Being green and clean is not just an aspiration but an action" is a small line delivered by "Christine Pelosi" (Jiwani et al. 2018) but has a bigger meaning which inspires everyone to be active in this domain (Varma 2016; Chen et al. 2020). Green and sustainable chemistry is a general strategy for the deployment of chemicals, reagents, solvents, and processes that helps in the reduction and use of hazardous chemicals to ensure the safety of the environment ( Ganesh et al. 2021). Greener biosynthesis pathways for nanomaterials has garnered immense interest as nanotechnology has spread its arms in all areas of sciences as one of the most propitious technology with the deployment of nanoparticles in environmental remediation and biomedical elds (Hu and Xianyu 2021). Nanotechnology combined with green technology provides a viable alternative to physical and chemical routes by utilizing safe, renewable, and non-toxic chemicals and eco-friendly means (Silva et al. 2020; Ajayi et al. 2021). This quest continues incessantly for the modi cation of shape and size for enhancing the properties of nanoparticles (Zare et al. 2020). Although, the synthesis of the nanoparticles has been accomplished by alternative activation methods such as sonochemical or microwave (MW)-assisted protocols (

Fungi
The synthesis of nanoparticles by fungi has more advantages as compared to other microorganisms (Dhillon et al. 2012). As compared to bacteria and plants, the fungal mycelial mesh can withstand agitation, ow pressure, and some other conditions in chambers and bioreactors, being easy to grow, handle, and fabricate. Fungi have outstanding metal bioaccumulation capacity with high binding capacity, their tolerance, and some other conditions such as intracellular uptake which is facile to handle under research conditions (Yadav et al. 2015). Nowadays, many fungi are in demand for nanoparticle synthesis such as Volvariella volvaceae ( Kaplan et al. (Kaplan et al. 2021) synthesized Ag NPs with the extract of Boletus edulis and Coriolus versicolor. They dried and pulverized these with a laboratory blender and 5 g of that powdered mushroom in 50 mL of distilled water was heated for 90 minutes at 60°C followed by ltration and centrifugation (10 minutes at 6000 rpm) of mushroom extract and stored at 4°C. For the synthesis of nanoparticles, 25 mL of mushroom extract were allowed to react in the microwave (MW) for 2 minutes at 475 W with AgNO 3 solution (25 mL of 10 mM) while pH was adjusted to 12 for Coriolus versicolor and 10 for Boletus edulis using NaOH. After that, the mixture was kept for centrifugation (20000 g for 10 min), the nanoparticles designated BE-Ag NPs and CV-Ag NPs were precipitated which were washed several times and dried for further use (Figure 7) (Kaplan et al. 2021). Recently synthesized several nanoparticles by adopting green routes via fungi are summarized in Table 2.

Bacteria
Bacteria is one of the best candidates for reducing metal ions to nanoparticles with its unique abilities because of its high growth rate and ease of handling. Bacteria is easy to genetically mold and manipulate for metal ion's biomineralization as compared to other microbes (Liu et al. 2011;Vaseghi et al. 2018). Due to high exposure to continual toxic and harsh environmental conditions, generally, its surroundings have a high concentration of heavy metal ions. They have developed some natural defense mechanisms such as extracellular precipitation, intracellular sequestration, change in metal ion concentration, and e ux pumps, which enables them to survive these harsh and stressful conditions and these mechanisms have been fruitfully utilized by bacteria for nanoparticle's synthesis.
When some bacteria such as Sulfolobus acidocaldarius, T. thiooxidans, and Thiobacillus ferrooxidans, were grown on elemental sulfur as an energy source, they could reduce ferric ions to the ferrous state. T. thiooxidans was able to complete the reduction at low pH medium aerobically but it was unable to oxidize the ferrous ions again, hence the ferrous ions were stable to autooxidation. The bio reduction by T. ferrooxidans from ferric ions was not aerobic because the presence of oxygen boosts the bacterial reoxidation of ferrous ions (Brock and Gustafson 1976 Table 3. Yeast extract plays the role of reducing and capping agent which encompasses carbohydrates, vitamins, and amino acids whereas metal ions serve as an electron acceptor. The organic capping agents provide stability to the synthesized monodispersed nanoparticles and as a result, these can be preserved without precipitation for more than a year (Boroumand Moghaddam et al. 2015;Skalickova et al. 2017). The synthesis of Ag NPs using yeast has been described by Shu and coworkers with the formation of yeast micelles in yeast extract by self-assembly of biomolecules followed by reduction of Ag + via in situ method which provided stabilization to the nanoparticles. A nity to the bacterial membrane is enhanced by the coating of the surface of Ag NPs wherein the permeability of the cell wall also increased. When peptidoglycan interacted with Ag NPs, the change in con guration of peptidoglycan occurred which resulted from the damage of bacteria by the apoptosis process ( Figure 8) (Shu et al. 2020). Yeast-assisted synthesis of nanoparticles and their applications are provided in Table 4. In old human civilizations, people used silver and silver salts, but in recent years the fabrication of Ag NPs has been in demand due to their outstanding applications in the biomedical eld such as antifungal, antibacterial, antioxidants as overnight followed by the centrifugation of the same at 600 g for 15 minutes. The supernatant was collected by ltering it with 0.22 µm pore size lter paper and stored at 4°C and the pellet was discarded. For the synthesis of nanoparticles, 1.5 mM AgNO 3 solution was added to 1% of cell-free culture supernatant, and the analysis of the samples was done for 6 days. In different time intervals after 24 hours, the harvesting of aliquots of 200 µL sample was performed. For con rmation of synthesis, UV-Visible spectroscopy was used which indicated the reduction of silver ions (Ag + ) to zero-valent silver (Ag 0 ) a rmed by the typical peak of Ag found at 450 nm, (Figure 10i). The TEM micrographs revealed spherical shape and 10-20 nm size of nanoparticles while X-ray diffraction pattern and elemental mapping suggested crystalline nature and showed the presence of four elements (Figure 10ii).

Applications
Ag NPs synthesized using Bacillus subtilis were studied against ve strains of multidrug-resistant microbes such as Candida albicans, Klebsiella. Pneumoniae, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli.

Au nanoparticles (Au NPs)
Gold nanoparticles (Au NPs) are the topic of interest and received much attention due to their simple synthesis and extensive applications. Initially, Beveridge and Murray used Bacillus subtilis for the synthesis of octahedral 5-25 nmsized Au nanoparticles (Beveridge and Murray 1980). Au NPs have been used as therapeutics, disease diagnostic materials, biocatalysts, and nanomedicine. Biocompatibility is the major concern for use in nanomedicine, hence, adopting green synthesis via microbes is an alternative to achieve this objective (Aminabad et al. 2019; Nejati et al. 2021); greener synthesis and the applications are discussed in this section.

Synthesis and properties
Synthesis of Au NPs using extract of Gelidiella acerosa marine algae (10 mL) was completed by mixing the algal extract with the aqueous solution of HAuCl 4 ·3H 2 O (gold chloride; 90 mL of 1 mM) and keeping the mixture under the static condition at 37°C. The Au NPs were washed and centrifuged for 15 minutes at 10,000 rpm to separate the nanoparticles which were dried at 50°C and kept at 4°C for further characterization and applications; crystalline nanoparticles had a size between 5.81 nm to 117.59 with spherical and hexagonal shapes. These Au NPs were found outstanding against inhibition of α-glucosidase and α-amylase enzyme with 2.8 ± 0.02, 4.1 ± 0.01 and 2.1 ± 0.01, 3.7 ± 0.01 µg/mL, respectively, Figure 12  Marine microbe (Vibrio alginolyticus) was used for the synthesis of Au NPs adopting the extracellular synthesis route. The culture was inoculated and incubated for 24 hours at 40°C on an orbital shaker at 120 rpm. After that, the mixture was centrifuged for 15 minutes at 8000 rpm and the supernatant was collected followed by the addition of aqueous HAuCl (chloroauric acid) (1mM) and again incubated under the same conditions. The nanoparticles were precipitated

Applications
The organic solvent-free nature and high photocatalytic dechlorination have been accomplished by the Au NPs synthesized using Saccharomyces cerevisiae yeast. These 9.99 ± 1.63 nm-sized mono-dispersed Au NPs revealed the conversion of quinclorac to 8-quinoline-carboxylic acid by dechlorination using sodium borohydride which followed pseudo-rst-order kinetics.

Pt nanoparticles (Pt NPs)
Platinum is a rare metal, used in cancer treatments, fuel cells, or catalytic converters. Its enormously low abundance makes it the topic of immense interest due to its unique structural, catalytic, and optical properties with huge potential in biomedical applications and catalysis (

Synthesis and properties
The cultivation of the control yeast and hydrogenase-displaying yeasts was performed anaerobically in AHLU + SDC medium (synthetic dextrose medium). The centrifugation of the cells was accomplished in 5 minutes at 3000×g. and and optimized temperature (38°C) and pH (7.5). For hydrogenase reactions, these conditions were found optimum as after 2 hours 30% reduction of PtCl 2 was observed, after 4 hours 70% and after 8 hours 90% reduction was noticed although for H 2 PtCl 6, these results didn't match, after 8 hours also, 96% platinum salt was remaining. It was concluded that during the redox mechanism, H 2 PtCl 6 functioned as an electron acceptor and indicated that the enzymes and metal/metal ions transferred electrons directly. Some hydrophobic active channels existed in between the molecular surfaces and the active site which were used as the passage by metal ions having 0.45-0.60 nm diameter. It was assumed that these channels were not small for PtCl 2 but were indeed very small for H 2 PtCl 6 ( Figure 15b).
The culture of Neurospora crassa extract was kept for 5 to 10 days at 28°C to attain macroconidia and harvested at 4°C in glycerol for further use, 100 mL of potato dextrose broth was inoculated with 100 µl of macroconidia for obtaining its biomass. Subsequently, the steps of incubation and ltration were performed at optimized temperature and time for starting the reduction process. The absorption spectra of both the control and Pt NPs were performed in which a peak at 530 nm was obtained for Pt NPs with quasi-spherical shape and 4-35 size by HRTEM analysis. From dark-eld images, the size distribution of nanoparticles showed a total of 234 nanoparticles in which 60% of particles were between 40 to 50 nm in size and more than 70 nm size was found only for 2% nanoparticles (Figure 14c

Pd nanoparticles (Pd NPs)
Palladium is a very precious metal and is endowed with signi cant thermal and chemical stability, optical and electronic properties, with ease of biofunctionalization for enhancing their medical applications (Fahmy et al. 2020).

Synthesis and properties
Biogenic Pd NPs fabricated using green alga, Botryococcus braunii revealed the formation of 4.89 nm-sized and truncated triangular, spherical, cubical shaped nanoparticles which were synthesized by stirring the solution of algal extract (20 mL) and an aqueous solution of Pd (OAc) 2 (80 mL of 1 mM) for 3 hours at 60°C while maintaining pH in the range 6-7. A positive (with alga extract and Pd (OAc) 2 ) and negative control (without alga extract) were maintained for the comparison. The solution bearing Pd NPs was centrifuged for 30 minutes and washed for the removal of impurities. For better separation of nanoparticles, the process was repeated 3 times and nanoparticles were dried at 70°C in a hot air oven, the entire process of Pd NPs synthesis is depicted in Figure 17 (Anju et al. 2020).
Genetically modi ed Pichia pastoris fungus was found as the factory for producing Pd NPs. Parental and modi ed species were cultivated in YPD media (Yeast, Peptone, and Dextrose in 1,2,2% w/v) and inoculated with the covering of the ask by cheese cloth (two-folded) for oxygen transfer; 0.5% methanol was daily added to the asks for AOX1 promoter induction. To reach optical cell density up to 0.1, the cells were cultivated in a shaker incubator at 30°C at 250 rpm. Subsequently, PdCl 2 was added to the ask to attain 60 mM nal concentration and after the time intervals of 0-, 6-, 12-, 24-, 36-, 48-, 60-and 72-hour, 10 mL of aliquots were collected for recording the absorbance value at 600 nm.

Applications
Pd NPs are generally used as a homogenous and heterogeneous catalyst in many of the organic reactions such as For the synthesis of Cu NPs using Escherichia sp., the cultivation and incubation were accomplished in nutrient broth for 24 hours at 150 rpm followed by the addition of CuSO 4 (5 mM) and incubation again. The color change was noticed from bluish-green to dense green and it was phenotypic con rmation for the synthesis of Cu nanoparticles; in the UV region, it showed a peak at 325.89 a rming the synthesis of nanoparticles ( Figure 18) (Noman et al. 2020). Shewanella loihica PV-4 mediated Cu NPs were synthesized by extracellular bioreduction of Cu (II) and the size of nanoparticles was found to be10-16 nm by TEM analysis while a strong Cu signal was observed by EDX (Energydispersive X-ray spectroscopy) analysis to con rm the synthesis (Lv et al. 2018).

Applications
Penicillium chrysogenum assisted spherical CuO NPs unveiled antibio lm, antifungal, and antibacterial activity.  (Figure 19a). However, they showed no effect on bio lm formation by Pseudomonas aeruginosa (Figure 19b). These nanoparticles showed antibacterial activity against Gram-positive and Gram-negative bacteria but the more effective diameter of clear zone was found against Gram-positive than Gram-negative bacteria. The formed clear zone diameters were 11.66 ± 0.33, 11.93 ± 0.52, 13.6 ± 0.4, 16.26 ± 0.63 and 22 ± 0.57 mm for Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus, respectively at 5 mg/mL of CuO nanoparticles, the same process was repeated with the ZnO NPs synthesized using the same bacteria which revealed that CuO NPs exhibited better inhibitory effects against all bacteria in comparison of ZnO NPs probably due to their interaction with bacterial protein via the SH groups thus inactivating the growth of bacteria (Figure 19c) (Mohamed et al. 2021).
The textile wastewater has a high percentage of contaminants such as hardness, high turbidity, pH, TSS (total suspended solids), TDS (total dissolved solids), COD (chemical oxygen demand), sulphates, chlorides, and many other impurities which causes the death to aquatic organisms. The treatment with biogenic Cu NPs obtained using

Synthesis and properties
Synthesis of TiO 2 NPs using Streptomyces sp. HC 1 was studied by Ağçeli and coworkers when they cultured the bacterial colony in sterile nutrient broth (50 mL) and incubated it for 24 hours in a shaker with 150 rpm at 37°C. This bacterial culture was used after the appearance of turbidity and added to TiO(OH) 2 solution (20 mL, 0.025 M), followed by incubation in a steam bath for 30 minutes at 60°C. After incubation, white clusters were noticed at the bottom of the ask which was collected by centrifugation, and the precipitate was washed with distilled water to maintain neutral pH (Ağçeli et al. 2020). Bacillus sp. bacteria was used for the synthesis of 50 nm-sized spherical shapes TiO 2 NPs. The synthesis of these bacteria-mediated nanoparticles was performed using the Taguchi method (which increases the reliability of production by optimizing the process parameters) and obtained nanoparticles revealed their highly dense spherical shape which was con rmed by TEM and SEM micrographs. The TGA studies suggested the weight loss up to 670°C in which the evaporation of water was responsible for rst weight loss at below 150°C temperature while in second weight loss, TiO 2 nanoparticles and organic compounds were decomposed in the range of 250 to 670°C (

Applications
TiO 2 NPs exhibit outstanding contributions in photoelectrochemical energy production, and their biocompatible and non-toxic properties, render them a suitable candidate for biomedical applications and pharmaceutical industries. The shape of nanoparticles was found spherical within the range of 10.23 ± 2.48 nm size. The change in color to golden brown with some precipitate indicated the formation of nanoparticles which was centrifuged for 10 minutes at 10,000 g to separate the precipitate; their formation was determined by UV Visible spectroscopy which suggested the absorption maxima at 360 nm (characteristic peak for ZnO) and quantum size effect was analyzed with a blue shift which is responsible for the diminution of size and wavelength due to widening of the bandgap. The 34.98 nm-sized and spherical-shaped nanoparticles were a rmed by TEM analysis while SAED analysis suggested crystals of 7 nm average size (Rafeeq et al. 2021).
Periconium sp. was used for ZnO NPs synthesis by applying the sol-gel process via dissolution of Zn (NO 3 ) 2 (20 g) in deionized water (100 mL) at 90°C with constant stirring. The addition of fungal extract (25 mL) was done at this stage and could form a sol by evaporation while the pH was maintained at 5. The sol was kept for more than 24 hours in a hot air oven at 45°C for evaporation of water resulting in the gel formation and even dispersal of Zn 2+ ions. After drying for 12 more hours at 125°C, the color of gel changed to brittle yellow and porous ZnO NPs ensued after calcination for 4 hours at 700°C under the aerobic condition in a mu e furnace. The process of synthesis and morphology (studied by TEM micrographs) of ZnO NPs are depicted in Figure 22

Applications
The antioxidant activity of ZnO NPs synthesized via Marinobacter sp.2C8 and Vibrio sp. bacteria was evaluated by DPPH scavenging radicals. When the ZnO NPs react with DPPH radicals, pale yellow color ensues from the deep purple which showed the existence of 1,1-diphenyl-2-picrylhydrazine as a result of receiving electrons. The two sets, ZnO-2C8 and ZnO VLA, of nanoparticles, showed different activity from 31.2 µg/mL to 2500 µg/mL concentration; at increasing concentration of ZnO concentration, the DPPH radical scavenging activity percent also increased, suggesting a dosedependent antioxidant activity. The maximum antioxidant activity was observed at 2500 µg/mL was 89% for ZnO-2C8 NPs and 86% for ZnO-VLA NPs; EC 50 values for both being at 600 µg/mL, Figure 24

Synthesis and properties
For the synthesis of Fe 2 O 3 NPs by three strains of fungus, i.e., Fusarium incarnatum, Phialemoniopsis ocularis, and Trichoderma asperellum, initially, fungal cell ltrate (10 mL) of fungal strains were mixed with the salt solution of FeCl 3 and FeCl 2 (2:1 mM). The mixture was allowed to agitate at room temperature for 5 minutes when the change in color of the reaction mixture, indicated the formation of nanoparticles with selected fungal strain. Later, the synthesized Fe 2 O 3 NPs were centrifuged for 20 minutes at 12,000 rpm and washed thoroughly with deionized water; the entire process was described by Mahanty et al. (Mahanty et al. 2019) in the owchart (Figure 25). Aqueous extract of Aegle marmelos (5g) was used for the synthesis of Fe 2 O 3 NPs with 100 mL distilled water and stirred with heating for 1 hour followed by the ltration of the extract using Whatman lter paper. 10 mL of this extract was mixed with 90 mL of ferric nitrate and stirred for 1 hour and then kept in a hot air oven while the temperature was maintained at 150°C. Later, the powder was calcinated for 5 hours at 400°C to generate Fe 2 O 3 NPs (Sriramulu et al. 2021).

Applications
The

Se nanoparticles (Se NPs)
Se nanoparticles are attractive due to their low toxicity, good biocompatibility, excellent biological activities, and being essential for mammalian's life; as a trace element, they exhibit preventive properties for disease and exhibit good antitumor activity (Sun et al. 2014). It plays a key role in ghting against oxidative stress by participating in the antioxidant defense system of the liver. (Kondaparthi et al. 2019) On the other hand, selenium sul de is an antifungal medicine and bioactive chemical. However, its biosynthesis is always an issue of controversy when one discusses nanoparticle forms.

Synthesis and properties
Hashem et al. (Hashem et al. 2021) synthesized Se NPs using Bacillus megaterium bacteria wherein the bacteria were cultured and incubated at 37°C for 48 h with shaking aerobically followed by the removal of bacterial cells and macromolecules by ltration through 0.44 µm PVDF lter and centrifugation at 10,000 rpm. Later, selenious acid suspension (1 mM) was mixed with cell-free supernatant and stirred at 25°C. The synthesis of Se NPs by reduction was indicated by a color change from colorless to reddish color when they were centrifuged for more than 30 minutes at 12,000 rpm. DLS and TEM studies suggested the synthesis of 41.2 nm-sized monodispersed spheres. The fungus, Saccharomyces cerevisiae was used to fabricate the SeS nanoparticles via the addition of selenium salts in 1 mM concentration into the 24 hours culture of Saccharomyces cerevisiae and incubation for 4.5 hours at 35°C with 180 rpm shaking using a shaking incubator Next, the medium changed the color from yellow to brownish red for S 1 (synthesized using sodium selenosul de salt) in 18 hours while for S 2 (using selenous acid/sodium sul te) in 4.5 hours. The presence of nanoparticles was con rmed by optical microscopy and these results were supported by TEM and SEM analysis which revealed 6.0 size for S 1 and 153 nm size for S 2 nanoparticles. The characteristic peaks of SeS nanoparticles were con rmed by XRD spectra as well as mass spectroscopy. The process of SeS NPs fabrication has been given in Figure  Lactobacillus casei ATCC 393 assisted prepared Se NPs inhibited the colon cancer cell growth which was studied in BALB/c mice's CT26 syngeneic colorectal cancer model. The nanoparticles showed in vitro antiproliferative activity, induced apoptosis, and raised ROS levels in cancer cells (Spyridopoulou et al. 2021).

Advantages And Disadvantages Of The Nanoparticle's Synthesis Using Microorganisms
Several microorganisms have been used for the sustainable synthesis of nanoparticles such as metals, quantum dots, semiconductors, etc. having different sizes and shapes (Narayanan and Sakthivel 2010). In comparison to conventional synthesis, green synthesis is cheaper, eco-friendly, and non-toxic (Mahmoud 2020). Adopting the microbial route has numerous advantages such as the microbes having a high growth rate and being inexpensive to cultivate (Prasad et al. 2016). They are easy to handle and can be genetically manipulated or modi ed without much di culty (Bhattacharya and Gupta 2005). The process for the synthesis of nanoparticles using microbes is very simple, stable, and robust that leading to higher production rates (Nikolaidis 2020). In addition, the nanoparticles synthesized using microbes revealed high surface area, as well as these, were monodispersed (Singaravelu et al. 2007).
Although the microbial synthesis route showed several advantages yet some disadvantages needed to be noticed such as low repeatability and the process for getting the clear ltrates from colloidal broths involving the use of sophisticated equipment. However, genetic manipulation is in demand but for the fungal platform is still challenging (Grasso et al. 2020). Moreover, pure nanoparticles are hard to obtain that are lack biomolecules and capping agents.
Also, there is a need for thorough research for large-scale production and applications. (Prasad et al. 2016)

Conclusion And Future Perspective
In recent years, sustainable or green synthesis has received considerable attention due to its economic importance as it provides a clean, facile, effectual, non-toxic, and eco-friendly route for the synthesis of nanoparticles. Microbesassisted metal or metal oxide nanoparticles with extremely ordered structures demonstrated their potential for numerous applications in waste treatment, biological and therapeutic elds due to their biocompatibility, controlled morphology, and other useful endowed properties in nano form. Some unique features of microbial cells that promote their use in the synthesis of nanoparticles as reducing and capping agents comprise easy maintenance, fast growth, and safer use. The greener synthesis addresses the bottlenecks for the synthetic methods due to the coating of nanoparticles with the biomolecule or lipid layer which endows physiological solubility, processibility, and stability to the nanoparticles as it enables surface functionalization for applications in biomedical elds.
However, the nanoparticles synthesized adopting the green route face some challenges which need to be addressed namely the slow rates of synthesis and stability of nanoparticles. The problems can be circumvented by augmenting the methods of cultivation of microbes and techniques of extraction and optimization via numerous combinatorial approaches, for example, photobiological methods. The other challenge is the production rate which is quite low in the case of biosynthesis (1/3 in comparison of chemical synthesis) which needs to be surmounted for their applications in real-world systems for large-scale applications. In addition, the lack of monodispersity, variations from batch to batch, and time-consuming process also limit their use in the commercial world. All the underlying mechanisms for the synthesis of nanoparticles including biochemical, cellular, and molecular mechanisms should be researched in detail for enhancing the properties, rate of synthesis, and applications of these nanoparticles. Biological protocols should be kept in mind before the synthesis of such nanoparticles namely the type and inheritable properties of organisms, ideal conditions for enzyme activity and cell growth as well as the reaction conditions. Although researchers are focusing on the therapeutic effect of nanoparticles yet the other important aspect is their toxic side effects. In the absence of biodegradation, delayed elimination trailed by the intercellular reactive oxygen species generation, damage to DNA, apoptotic cell death, and long-term toxicity can be caused by nanoparticles. Till now, most of the microbes-assisted nanoparticles have been examined in in-vitro studies for biomedical use and clinical trials on large scale to assess their safety is an important aspect for their effects in in-vivo. Hence, some factors such as reduction or removal of toxicity, doses, and response to host immune system throughout treatment are some aspects that still need to be addressed before the commercialization of these nanoparticles. The "green chemistry" concept combined with the "white biotechnology" approach can lead to a major achievement in many sustainable industrial developments with the use of genetically modi ed organisms by understanding the mechanistic aspects and related metabolic pathway culminating in the enhancement of e ciency for the synthesis of nanoparticles with reduced toxicity (Iravani and Varma 2019). Hopefully, with further thorough investigations, the microbes-assisted nanoparticles will attain their immense potential in various sectors of nanotechnology using genetically engineered organisms. The authors declare no con ict of interest.  The number of articles published on green synthesis of nanoparticles using algae, fungi, bacteria, yeast, and other microorganisms over the last decades (Data from the ISI Web of Knowledge database).  TEM images. X-ray elemental mapping of (a2) Ag (red), (a3) nitrogen (blue), (a4) carbon (green), (a5) oxygen (yellow), and (a6) sulfur (orange) in which the area covered by Ag NPs is shown with the pink dotted line and the presence of characteristic elements are shown by intense color for respective elements detected (c) the crystalline nature of Ag NPs is shown by X-ray diffraction pattern and (d) the presence of silver is shown by energy dispersive spectroscopy (EDS) spectrum (Reproduced from Ref. (     The numerous therapeutic and other bene ts of Se NPs