5.1 Ag nanoparticles (Ag NPs):
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 field such as antifungal, antibacterial, antioxidants as well as in agricultural and environmental remediation. These nanoparticles hold a specific place among other metals used in the biomedical field and have been widely explored via the greener routes (Jadoun and Dilfi 2021; Uthaman et al. 2021). Gevorgyan et al.(Gevorgyan et al. 2021) reported the use of Ag NPs as an excellent inhibitor of Gram-positive S. aureus and Gram-negative S. typhimurium. The synthesis, properties and applications of Ag NPs produced by microbes are discussed in this section.
5.1.1 Synthesis and properties
Synthesis of Ag NPs using the bacteria, Bacillus Licheniformis, as a reducing and capping agent was performed by adding the aqueous solution of silver nitrate (AgNO3) to the biomass of bacterial extract; the size of nanoparticles being 50 nm, (Figure 8a).(Kalimuthu et al. 2008) The culture supernatants of Staphylococcus aureus were also used for Ag NPs synthesis (Nanda and Saravanan 2009). Fungi have been used to procure Ag NPs where the reduction of AgNO3 was performed by the enormous amount of enzymes secreted by fungi and was further characterized and deployed in antimicrobial, antiviral, and wound dressing activities (Khan et al. 2018) (Figure 8b). The same nanoparticles were synthesized by using 5 mL of Botryococcus braunii algal extract when mixed with 1mM AgNO3 and stirred. After saturation of reaction, the mixture was centrifuged for 20 minutes and the pellets were separated with supernatant. The obtained nanoparticles were washed several times and dried at 55°C for 5 hours (Figure 9c).
Enterobacteriaceae sp. bacteria were found useful for the quick synthesis of Ag NPs as Ashraf et al. (Ashraf et al. 2020) described their preparation using Enterobacter cloacae (SMP1) bacteria’s cell-free supernatant protein. The bacterial strain was inoculated in liquid Luria Bertani (LB) broth for incubation in a rotatory shaker at 120 rpm at 37°C overnight followed by the centrifugation of the same at 600 g for 15 minutes. The supernatant was collected by filtering it with 0.22 µm pore size filter paper and stored at 4°C and the pellet was discarded. For the synthesis of nanoparticles, 1.5 mM AgNO3 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 confirmation of synthesis, UV-Visible spectroscopy was used which indicated the reduction of silver ions (Ag+) to zero-valent silver (Ag0) affirmed 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).
Santos et al. (Santos et al. 2021) adopted an extracellular biosynthetic route for Ag NPs assembly using 10 g of Entomopathogenic Fungi Biomass after the addition of deionized water (100 mL). The solution was incubated for three days at 25°C on a rotatory shaker at 100 rpm. Subsequently, the biomass was filtered and stored at 4°C for the synthesis of Ag nanoparticles. Afterward, 1 mL and 10 mM solution of an aqueous solution of AgNO3 was added with 9 mL of fungal extract biomass and the solution was magnetically stirred, away from the sunlight, for 72 hours at 25°C. The mean diameter of obtained nanoparticles was found between 40.14 and 289.13 nm using DLS. Recently, the spherical nanoparticles of Ag were obtained by using Trichoderma harzianum (Guilger-Casagrande et al. 2021) while 10-30 nm of spherical shaped nanoparticles with little agglomeration could be obtained by Bjerkandera sp. R1 white-rot fungus (Osorio-Echavarría et al. 2021).
Kashyap et al. (Kashyap et al. 2021) adopted an intracellular green synthetic route using the microalgae Scenedesmus sp. for Ag/AgCl nanohybrids synthesis. The 0.5 and 1 mM of AgNO3 precursor with extract of Scenedesmus sp. as a reducing agent was used for the synthesis; spherical particles with 10–20 nm and 10–50 nm in size were obtained, respectively. The change from transparent to deep brown color of the mixture of AgNO3 and bacterial strain solution indicated the formation of hexagonal shaped Ag nanoparticles using Bacillus anthracis PAFB2, showed 0.428 with − 15.5 mV Zeta potential value for polydispersity (PDI) index which indicated their good colloidal nature and long-term stability of nanoparticles; the size of nanoparticles being ~ 84 nm by AFM analysis (Banerjee et al. 2021). Some spherical shapes, ranging between 13–27 nm, were synthesized using Paenarthrobacter nicotinovorans MAHUQ-43 bacterial strain (Huq and Akter 2021).
Ag NPs synthesized using Bacillus subtilis were studied against five strains of multidrug-resistant microbes such as Candida albicans, Klebsiella. Pneumoniae, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli. The rate of MICs (minimum inhibitory concentrations) versus the clinical isolates revealed outstanding antimicrobial efficiency and revealed 100, 180, 200, 230, 300 µgmL−1 for Candida albicans, Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumonia, respectively. Ag NPs were indicated to be toxic for gram-positive and gram-negative bacteria. These nanoparticles showed high antifungal activity and could be used to treat multidrug-resistant microorganisms (Alsamhary 2020). Ag NPs synthesized from green algae showed excellent catalytic activity in the reduction of 2-nitroaniline. The reduction of 2-nitroaniline (100 mg, 0.724 mmol) into o-phenylenediamine was performed in presence of Ag NPs (0.10 mg, 10% w/w of 2-nitroaniline) and sodium borohydride (60 mg). The reaction mixture was adjusted to ∼6 pH using glacial acetic acid for the removal of sodium borohydride. The product was further cyclized for 10-12 hours at 80°C with substituted aldehydes (0.724 mmol) to produce 2-aryl benzimidazoles (Figure 11a) (Arya et al. 2019). On the other hand, Ag NPs synthesized from a new bacterial strain showed outstanding antibacterial effect against standard and multi-drug resistant strains according to the Clinical and Laboratory Standards Institute (CLSI) which were analyzed using a good diffusion method. The nanoparticles inhibited the growth of the strains including Salmonella paratyphi, Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 10536), Shigella dysenteriae (PTCC 1188), Proteus vulgaris (PTCC 1182), and Klebsiella pneumonia (ATCC 10031) around the wells, Figure 11 (b) (Nazari and Jookar Kashi 2021).
Ag NPs synthesized using Anabaena variabilis revealed antioxidant activity and antimicrobial activity (Ahamad et al. 2021). The IC50 value found in the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity was 13.22 ± 1.25 µg mL−1 while in the case of Ag nanoparticles synthesized by Ecklonia cava it was found 198 µg mL−1. These nanoparticles were used in anticancer activity (Venkatesan et al. 2016). In the case of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Ag NPs synthesized using Acutodesmus dimorphus, revealed an IC50 value of 14.41 µg mL−1 while using Anabaena variabilis, it was 2.67 ± 0.5 µg mL−1 (Chokshi et al. 2016). The antimicrobial activity against Bacillus amyloliquefaciens, Streptococcus pyogenes, Staphylococcus aureus, and Salmonella was analyzed by disc diffusion method and after incubation of 24 hours, the difference in the zone of inhibition was observed between control samples and green synthesized nanoparticles treated with microorganisms (Ghiuta et al. 2021).
Lactobacillus bulgaricus mediated Ag NPs showed antibacterial activity against Salmonella typhi, Staphylococcus epidermidis, and Staphylococcus aureus, which revealed 17-mm mean values of zone inhibition for the Salmonella typhi and Staphylococcus epidermidis while 15-mm for Staphylococcus aureus. The antibiotics effects were studied according to Birmingham Children’s Hospital guidelines (2014); antibiotic activities were varying against selected bacterial strains resulted in sensitivity to antibacterial activity in comparison of antioxidant activity (Naseer et al. 2021). Recently, The Ag NPs fabricated using Cedecea sp. showed immense potential in antibiofilm activity. These Ag NPs unveiled strong MIC and MBC (minimum bactericidal concentration) values against E. coli (12.5 µg/µl and 12.5 µg/mL) and P. aeruginosa 6.25 µg/µl and 12.5 µg/mL), respectively and were extremely stable for more than one year with strong antibacterial activity against biofilms of P. aeruginosa and E. coli. (Singh et al. 2021). Ag NPsacquired via extracellular synthesis using Gloeophyllum striatum were antibacterial. The cytotoxic and hemolytic activity was checked towards mammalian cells which revealed that after 24 hours, more than 30 µM triggered 50% hemolysis of RBC, and no toxicity was found at 0.5–10 µM concentrations, IC50 value at 24 hours being 28.76 µM. For the ecotoxicity study, the aquatic crustaceans Artemia franciscana and Daphnia magna were selected. In the saline ecosystem, Artemia franciscana showed higher tolerance than Daphnia magna towards Ag nanoparticles. The EC50 values for Daphnia magna and Artemia franciscana were found to be 0.275 and 61.97 µM, respectively (Zawadzka et al. 2021).
5.2 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 nm-sized 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.
5.2.1 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 HAuCl4·3H2O (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 (Senthilkumar et al. 2019).
Clarance et al. (Clarance et al. 2020) fabricated Au NPs using extract of Fusarium solani. They cultured the fungi in YEPD (yeast extract peptone dextrose) broth, and the culture was incubated at 28°C in the shaker at 120 rpm and kept for 9 days for further incubation. After that, it was filtered with cheesecloth and washed multiple times. Sterile Milli Q water (100 mL) was added to that biomass and kept undisturbed for 2 days. Further, it was filtered through Whatman No-1 filter paper and maintained the pH at 8.5 with 0.1 N NaOH. HAuCl4 (99 mL of 1 mM) solution was added to the above fungal extract (1.0 mL) and kept for incubation under dark for 48 hours. Formation of pink-ruby red color indicated the formation of nanoparticles of Au and the peak between 510 and 560 nm was observed for plasmon band while the peaks at 1413 cm−1 in FTIR attributed to the amine II bands of protein. The nanoparticles were flower-like and needles shaped with 40-45 nm size.
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 out and were separated by centrifugation, washed, and dried to achieve powder of nanoparticles. The 50-100 nm irregular monodispersed nanoparticles were suggested by TEM analysis (Shunmugam et al. 2021). Sargassum wightii, a marine alga was used for the extracellular synthesis of 8 to 12 nm-sized monodisperse Au NPs and a peak at 527 nm in the UV-visible spectrum suggested the plasmon absorbance of Au nanoparticles (Singaravelu et al. 2007). The same method was adopted by Mukherjee et al.(Mukherjee et al. 2002) for the fabrication of nanoparticles using fungus Fusarium oxysporum by exposing aqueous AuCl4− ions to the fungus extract. Three different fungi, Fusarium oxysporum, Fusarium sp., and Aureobasidium pullulans were used for the synthesis of Au NPs via mixing the fungal strain cells with AuCl4− ions solution (Zhang et al. 2011).
Salouti et al. (Zonooz et al. 2012) used Streptomyces sp. ERI-3 for the synthesis of Au NPs. The culture was incubated at 200 rpm for 48 hours at 28°C and HAuCl4 solution (50 mL of 1 mM) was added to the supernatant (10 mL) and kept on an orbital shaker at the same aforementioned conditions. TEM studies suggested spherical and cylindrical-shaped nanoparticles ranging in between 80-200 nm. The synthesis was optimized with different reaction conditions and the best optimum conditions were found to be, pH 6, incubation time 12-hours, temperature: 30°C, and HAuCl4 concentration 3 mM. Cladosporium cladosporioides (marine endophytic fungus) were used for 60 nm average-sized Au NPs synthesis and showed noteworthy antioxidant activity compared to ascorbic acid (M et al. 2017). Au NPs have been synthesized using various bacteria such as Bacillus subtilis, Shewanella algae, Pseudomonas aeroginosa, Escherichia coli, Lactobacillus sp., Thermomonospora sp., and Rhodococcus sp (Ahmad et al. 2003a, b; Mandal et al. 2006; He et al. 2007; Moghaddam 2010).
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-first-order kinetics. (Figure 13a) (Shi et al. 2017). Qu et al. (Qu et al. 2017b) synthesized Au NPs from Trichoderma sp. for their potential application in azo dyes decolorization in contaminated water; these dyes absorb and reflect the sunlight which affects the aquatic organism’s growth as well as the photosynthesis process. The above nanoparticles could decolorize the Acid Brilliant Scarlet GR up to 94.7% in 120 minutes (Figure 13b) at various concentrations of dye; at a dye concentration of 25 mg/L, degradation was more than 90% in 40 minutes while at 50 mg/L, 90% of decolorization was observed in 100 minutes (Qu et al. 2017b).
Psychrotolerant Antarctic mediated synthesis of Au NPs showed antimicrobial activity against sulfate-reducing bacteria (Desulfovibrio desulfuricans) which was assessed by the optical density of bacteria culture. The nanoparticles reduced the Desulfovibrio desulfuricans numbers to 12% (106 to 103cells mL−1) along with the reduction of sulphate reducing activity to 7% (0.0246 nanomoles mL−1 day−1 to 0.0016 nanomole mL−1 day−1); MIC was calculated to be 200 µg mL−1 concentrations. The nanoparticles revealed the antimicrobial activity against Bacillus, Pseudomonas aeruginosa, Escherchia coli, Klebsiella pneumonia, and Staphylococcus aureus. The Au NPs deteriorate the cells of microbes by connecting with the surface, creating an aperture in the cell wall which induces seeping of cell contents resulting in death. They could inhibit the transcription by inhibiting the DNA (Shunmugam et al. 2021).
The Au NPs showed immense potential in anticancer activity which was assessed against colon cancer cell line by a dose-dependent inhibition activity. These nanoparticles were synthesized as a chemotherapeutic alternative to escape from drugs that are toxic and exhibit numerous side effects in the body; IC50 value was found to be 15 µg/mL by comparing it with standard (Figure 14a). The morphological analysis revealed a cytotoxicity effect on the HCA-7 (human colon cancer) cell line when treated with Au NPs. There was a clear difference seen in treated and untreated cell organelles and unveiled noteworthy cell damage by Au NPs with undistinguished cell debris which indicated a significant contribution of these biogenic Au NPs for human colon cancer cells (Figure 14b) (Shunmugam et al. 2021). Ecklonia cava, a marine alga mediated spherical (20-50 nm size) Au NPs showed antimicrobial activity against some pathogenic organisms and revealed the diameter of the zone of inhibition (20 µL) for Candida albicans ATCC 10231, Aspergillus niger ATCC 1015, Bacillus subtilis ATCC 6633, Aspergillus fumigates ATCC 1022, Escherichia coli ATCC 10536, Aspergillus brasiliensis ATCC 16404, Staphylococcus aureus ATCC 6538 and, Pseudomonas aeruginosa ATCC 27853 about 23.3 ± 0.25, 24.6 ± 0.23, 19.7 ± 0.21, 21.5 ± 0.25, 31.8 ± 0.32, 19.3 ± 0.26, 16.6 ± 0.30 and 21.3 ± 0.28 mm, respectively. The highest antimicrobial activity was shown against Aspergillus niger ATCC 1015 and Escherichia coli ATCC 10536 (Venkatesan et al. 2014). Sargassum incisifolium (brown algae)-assisted secured Au NPs were studied for antimicrobial and anticancer activity (Mmola et al. 2016).
5.3 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 (Yamada et al. 2015; Siddiqi and Husen 2016; Pedone et al. 2017).
5.3.1 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 incubated in 7.4 pH PBS comprising 100 µM PtCl42− salt. The mixture was allowed to react anaerobically at a rotatory shaker at 37°C for 72 hours and the black precipitate was separated from the solution and reduced Pt NPs were collected.(Ito et al. 2016) Green synthesis of Pt NPs has been reviewed by Puja et al. (Puja and Kumar 2019) wherein the syntheses are described by various biological species using H2PtCl6 as a precursor and the indication of nanoparticles synthesis by a color change and their potential application in biomedical fields (Figure 15a). Fusarium oxysporum was incubated with H2PtCl6 for extracellular synthesis of stable Pt NPs of 5-30 nm size (Syed and Ahmad 2012). The same fungi were used by Govender et al.(Govender et al. 2009) by isolating 10 mL of 120 nmol min−1 mL−1 purified hydrogenase and reacting with 10 mL of 1 mM solution of PtCl2 or H2PtCl6 under the hydrogen atmosphere and optimized temperature (38°C) and pH (7.5). For hydrogenase reactions, these conditions were found optimum as after 2 hours 30% reduction of PtCl2 was observed, after 4 hours 70% and after 8 hours 90% reduction was noticed although for H2PtCl6, these results didn’t match, after 8 hours also, 96% platinum salt was remaining. It was concluded that during the redox mechanism, H2PtCl6 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 PtCl2 but were indeed very small for H2PtCl6 (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 filtration 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-field 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) (Castro-Longoria et al. 2012) Acinetobacter calcoaceticus mediated synthesis of Pt NPs was performed by Gaidhani et al. (Gaidhani et al. 2014) where they described the reduction of H2PtCl6. The entrenchment of nanoparticles within the cells was seen by AFM analysis and average surface roughness was found to change when compared to control cells which indicated the formation of Pt NPs. The size of nanoparticles was 2-3.5 nm while the shape was found cuboidal by HRTEM analysis.
The nanoparticles fabricated using P. chrysogenum were assessed for anticancer activity on myoblast C2C12 cancer cells. To evaluate the mitochondrial activity, death of cell and survival in presence of biogenic Pt NPs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed as it is the most vigorous method for analysis of nanotoxicology which elucidated the response of cells to metal toxicity along with evidence for the death of the cell, metabolic activities, and survival. The Pt NPs revealed a significant decrease in cell viability and mitochondrial reduction (90.4%) at 100 µg concentration while the control cell unveiled no mitochondrial reduction with maximum cell viability; nanoparticles of size less than 20 nm showed no cytotoxic against cancer cells. With the pre-treatment by Pt NPs, cell viability was reduced in human squamous carcinoma and A431 at 24 hours; IC50 values were found to be 41.09%, (Subramaniyan et al. 2018). The Pt NPs were synthesized using various bacterial strains such as Pseudomonas kunmingensis, Psychrobacter faecalis, Vibrio fischeri, Jeotgalicoccus coquina, Sporosarcina psychrophile, Kocuria rosea, Pseudomonas putida, Rhodotorula mucilaginosawhich exhibited antioxidant activity against the DPPH radical scavenging assay. The purple color of DPPH radicals changed to pale yellow by interacting with nanoparticles as Pt NPs provide transferability of electrons/hydrogen atoms to neutralize DPPH. It was a dose-dependent activity as the inhibition percent increased with the increment of Pt concentration. The degree of antioxidant activity of 1000 µg/mL nanoparticles were ordered from lowest to highest was MN23 < ADR19< KT2440 < KC19 < B-11177 < CCV1 < FZC6 < ZC15 (Figure 16) (Eramabadi et al. 2020).
5.4 Pd nanoparticles (Pd NPs)
Palladium is a very precious metal and is endowed with significant thermal and chemical stability, optical and electronic properties, with ease of biofunctionalization for enhancing their medical applications (Fahmy et al. 2020).
5.4.1 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 modified Pichia pastoris fungus was found as the factory for producing Pd NPs. Parental and modified species were cultivated in YPD media (Yeast, Peptone, and Dextrose in 1,2,2% w/v) and inoculated with the covering of the flask by cheese cloth (two-folded) for oxygen transfer; 0.5% methanol was daily added to the flasks 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, PdCl2 was added to the flask to attain 60 mM final 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. The nanoparticles were collected by centrifugation at 5000×g for 10 min and washed thoroughly. The maximum absorption in recombinant yeast was found at 79.79%. The best equation of Pd NPs was found y = 177.78× Ln(t) − 233.23 and y = 6.87× Ln(t) + 49.91, for the Pd biosorption capacity (mg/g) and formation yield (%) (Elahian et al. 2020).
Pd NPs are generally used as a homogenous and heterogeneous catalyst in many of the organic reactions such as Suzuki coupling reactions and Suzuki-Miyaura cross-coupling reactions (Liu et al. 2021; Sun et al. 2021), medical diagnosis (Zhuge et al. 2019), cancer treatments (Kang et al. 2018), drug delivery (Zhang et al. 2019), antimicrobial activities (Nasrollahzadeh et al. 2020a), antioxidants (Fahmy et al. 2020), and as nanocatalysts for dye degradation in effluents from textile industries among other biomedical applications (Gil et al. 2018; Pandey et al. 2021). Escherichia coli assisted obtained Pd NPs showed better catalytic activity to chemical counterparts at low temperature and in the air for the oxidation of benzyl alcohol; catalysis was performed with 180 mg of nanocatalyst and 50 mL of benzyl alcohol and the reactor was set to reach 90°C. These biogenic Pd NPs were compared with the chemically prepared catalyst in O2 and noticed that at lower loadings of catalyst (6 × 10−5 mol l−1 ), it displayed higher activities (Deplanche et al. 2012). Fahmy et al. (Fahmy et al. 2020) reviewed the Pd NPs synthesized adopting biological routes for their unique physiological properties and biomedical applications.
5.5 Cu nanoparticles (Cu NPs)
There are numerous types of copper (Cu) nanoparticles such as Cu, CuO, Cu2O nanoparticles (Ighalo et al. 2021; Kumar et al. 2021; Medvedeva et al. 2021); they are widely known for their magnetic, optical, electric, and catalytic properties which are applicable in optoelectronics, photocatalysis, sensors, and biomedical fields such as antifungal, antibacterial, antioxidant, anticancer, antiviral, and drug delivery systems (Al-Hakkani 2020; Marouzi et al. 2021) .Their synthesis, properties, and applications are discussed in this section.
5.5.1 Synthesis and properties
The bacterial strains used for the nanoparticle’s synthesis were inoculated in Luria–Bertani medium followed by incubation on a rotatory shaker at 200 rpm at 22°C. After 24 hours, the final concentration of 1 mM was attained with the addition of CuSO4·5H2O. Then the incubation of the reaction mixture was performed further on a rotatory shaker at 150 rpm for 24-48 hours at 22°C. For control, heat-killed bacterial strains or without bacterial strains Luria–Bertani medium with 1mM CuSO4 was maintained. The color of the mixture changed to dark green from cyan blue suggesting the formation of Cu nanoparticles. When white-rot fungus Stereum hirsutum (Cuevas et al. 2015) and Morganella sp. (Lalitha et al. 2020) were used as reducing and capping agents, the same color change was observed affirming the formation of nanoparticles. The solution was further centrifuged at 5000 rpm for 20 minutes at 4°C and collected for washing with double distilled water. Short duration (15 seconds) ultrasonic wave irradiation was imparted for the recovery of nanoparticles from cell pellet and centrifuged for 20 minutes at 5000 rpm followed by recovery of the nanoparticles which were dried in an oven at 80°C; ovoidal or spherical and monodispersed nanoparticles ensued with 10-70 nm particle size and with an average size of 40 nm (John et al. 2021).
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 CuSO4 (5 mM) and incubation again. The color change was noticed from bluish-green to dense green and it was phenotypic confirmation for the synthesis of Cu nanoparticles; in the UV region, it showed a peak at 325.89 affirming 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 (Energy-dispersive X-ray spectroscopy) analysis to confirm the synthesis (Lv et al. 2018).
Penicillium chrysogenum assisted spherical CuO NPs unveiled antibiofilm, antifungal, and antibacterial activity. The highest effect was shown by CuO NPs against Staphylococcus aureus at a concentration under MIC values. These nanoparticles could reduce the formation of biofilm by 68.8, 85.9, 94.4, 94.1 and 95% at concentrations 0.01, 0.03, 0.07, 0.15, and 0.3 mg/mL, respectively without any effect on bacterial growth (Figure 19a). However, they showed no effect on biofilm 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 Escherichia sp. by Noman et al. (Noman et al. 2020) revealed their potential to decolorize the azo dyes (25 mg L−1 ) up to 83.61% ± 1.93, 88.42% ± 2.80, 90.55% ± 2.06 and 97.07% ± 1.22, and for RB-5, DB-1, MG, and CR in 5 hours of sunlight exposure (Figure 18d). Streptomyces sp. (Endophytic actinomycetes) mediated CuO NPs showed their potential in biotechnological applications (Hassan et al. 2019). Aspergillus niger strain STA9 assisted Cu NPs displayed antibacterial, antidiabetic, and anticancer activities (Noor et al. 2020).
5.6 TiO2 nanoparticles (TiO2 NPs)
TiO2 nanoparticles have been mostly studied and used for their brilliant antioxidant nature and superior photocatalytic properties. They are frequently used in implant biomaterials, photocatalysis, sunscreen products, toothpaste, self-cleaning sanitary ceramics, cement, sugar, paper, rubber, biomedical ceramic, printing ink, paints, antimicrobial plastic packaging, films, etc. (Khataee and Mansoori 2011). Green synthesis of TiO2 NPs by microorganisms has been studied and summarized in this section.
5.6.1 Synthesis and properties
Synthesis of TiO2 NPs using Streptomyces sp. HC1 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 flask 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 TiO2 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 confirmed 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 first weight loss at below 150°C temperature while in second weight loss, TiO2 nanoparticles and organic compounds were decomposed in the range of 250 to 670°C (Figure 20) (Moradpoor et al. 2021).
TiO2 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.(Zhao et al. 2007; Weir et al. 2012; Ahn et al. 2018) The TiO2 NPs synthesized using Aspergillus flavus showed their effect on the bacterial sp, K. pneumoniae (18 mm), B. subtilis (22 mm), S. aureus (25 mm), P. aeruginosa (27 mm), and E. coli (35 mm); for the control, tetracycline antibiotics was used and for the zone of inhibition, they deployed the cell diffusion method and MIC. The zone of inhibition was shown against both Gram-positive and Gram-negative bacteria. The MIC values were found to be 40 µg mL−1 for E. coli (MTCC-1721), 40 µg mL−1 for S. aureus (MTCC-3160), 45 µg mL−1 for B. subtilis (MTCC-1427), 70 µg mL−1 for K. pneumoniae (MTCC-4030), and 80 µg mL−1 for P. aeruginosa (MTCC-1034) suggested best results against E. coli, Figure 21 (Rajakumar et al. 2012) TiO2 has also unveiled antibacterial activity against Bacillus megaterium (Karunakaran et al. 2013) and E. Coli. (Amin et al. 2009; Hong et al. 2017) using environmental light.
5.7 ZnO nanoparticles (ZnO NPs)
ZnO nanoparticles have been widely used as antibacterial, anti-fungal with some other biomedical applications, and as active photocatalysts for the degradation of dyes and other organic contaminants (Ong et al. 2018).
5.6.1 Synthesis and properties
Barani et al. (Barani et al. 2021) used two bacterial strains (Vibrio sp. VLA strains and Marinobacter sp. 2C8) for the synthesis of ZnO NPs. The ensuing precipitate after bacterial exposure was separated by centrifuged, washed, and dried by a freeze dryer. The peak in the UV was found at 250 nm ZnO-VLA while 266 nm for ZnO-2C8 nanoparticles. 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 affirmed 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 (NO3)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 Zn2+ 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 muffle furnace. The process of synthesis and morphology (studied by TEM micrographs) of ZnO NPs are depicted in Figure 22 (i) and (ii) (a-e), suggesting quasi-spherical (size~16 and 78 nm), polydisperse (polydispersity index ~ 1.48), and less agglomerated. The SAED patterns revealed a circular peripheral layer related to the planes suggested by XRD patterns (Ganesan et al. 2020).
In another process, a freshly grown cell-free supernatant of Bacillus megaterium was used with 1mM aqueous ZnNO3.5H2O and kept on a shaker incubator for 48 hours at 37° C. The white clusters were obtained at the bottom of the flask after 12-48 hours of incubation which was centrifuged and washed several times to obtain pure white crystals of ZnO NPs; characteristic surface plasmon resonance peak at 346 nm in the UV-Vis spectrum confirmed their synthesis. XRD pattern supported the crystalline nature of synthesized nanoparticles with sizes in between 45 and 95 nm possessing cubic shape (Figure 23) (Saravanan et al. 2018).
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 dose-dependent antioxidant activity. The maximum antioxidant activity was observed at 2500 µg/mL was 89% for ZnO-2C8 NPs and 86% for ZnO-VLA NPs; EC50 values for both being at 600 µg/mL, Figure 24 (Barani et al. 2021).
ZnO NPs fabricated from cyanobacterium Nostoc sp. EA03 showed its potential in biological functions in terms of antibacterial, antimicrobial, and toxicity activities. With these nanoparticles, MBC and IC50 values were determined to be 2500, 2500, and 128 µg mL−1, and 2000, 2000, and 64 µg mL−1, respectively which bodes well for their biomedical appliances (Khatami et al. 2018; Ebadi et al. 2019).
5.8 Fe2O3 nanoparticles (Fe2O3 NPs)
Iron oxide nanoparticles (IONP)predominantly exist in two forms which are magnetite (Fe3O4) and the oxidized form called maghemite (γ-Fe2O3) (Markova et al. 2014; Plachtová et al. 2018). The Fe2O3 NPs attracted much attention due to their unique properties of super magnetism, and their appliances in various areas including terabit magnetic storage, catalysis, gene, and drug delivery, and other therapeutic applications (Can et al. 2012). When the high surface energy possessing Fe2O3 NPs react with the biomolecules, it results in the enhancement of dispersion and less aggregation due to the presence of polysaccharides which offer a brilliant biocompatible shell (Ghosh et al. 2021).
5.8.1 Synthesis and properties
For the synthesis of Fe2O3 NPs by three strains of fungus, i.e., Fusarium incarnatum, Phialemoniopsis ocularis, and Trichoderma asperellum, initially, fungal cell filtrate (10 mL) of fungal strains were mixed with the salt solution of FeCl3 and FeCl2 (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 Fe2O3 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 flowchart (Figure 25). Aqueous extract of Aegle marmelos (5g) was used for the synthesis of Fe2O3 NPs with 100 mL distilled water and stirred with heating for 1 hour followed by the filtration of the extract using Whatman filter 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 Fe2O3 NPs (Sriramulu et al. 2021).
The Fe2O3 NPs synthesized using Aegle marmelos extracts inhibited the bacterial growth of E. coli and S. aureus more than the control antibiotic. The gram-negative bacteria (Escherichia coli) interacted more with nanoparticles at a higher and lower concentration as compared to gram-positive bacteria (Staphylococcus aureus) due to the difference in cell membrane thickness. The nanoparticles kill the bacterial cell by entering the membrane and inhibiting the bacterial growth and inactivated the enzymes with the increase of the cytoplasmic membrane leakage (Figure 26a); nanoparticles at concentration 32.25 µg/ mL showed 7 ± 0.12 mm inhibition for E. coli.(Khalil et al. 2017) Against S. aureus, 30 ± 0.387 mm and 28 ± 0.654 mm (30 µg/ mL) zone inhibition was observed while 21 ± 0.432 and 19 ± 0.547 (15 µg/ mL) was seen for E. coli, suggesting more inhibition of E. coli (Figure 26b). These Fe2O3 NPs exhibited superb photocatalytic activity for the degradation of BG dye with 95.89% degradation in 90 minutes under UV light; degradation efficiency and degradation kinetics suggested the pseudo-first-order kinetic model with the constant (K) value of 0.04058 min−1 (Figure 26 (c-e) (Sriramulu et al. 2021).
5.9 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 fighting against oxidative stress by participating in the antioxidant defense system of the liver.(Kondaparthi et al. 2019) On the other hand, selenium sulfide is an antifungal medicine and bioactive chemical. However, its biosynthesis is always an issue of controversy when one discusses nanoparticle forms.
5.9.1 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 filtration through 0.44 µm PVDF filter 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 S1(synthesized using sodium selenosulfide salt) in 18 hours while for S2 (using selenous acid/sodium sulfite) in 4.5 hours. The presence of nanoparticles was confirmed by optical microscopy and these results were supported by TEM and SEM analysis which revealed 6.0 size for S1 and 153 nm size for S2 nanoparticles. The characteristic peaks of SeS nanoparticles were confirmed by XRD spectra as well as mass spectroscopy. The process of SeS NPs fabrication has been given in Figure 27 (Asghari-Paskiabi et al. 2019).
Afzal et al. (Afzal et al. 2021) used cyanobacteria for Se NPs synthesis as affirmed by the synthesis of spherical and amorphous nanoparticles of 10.8 nm size. Freshly, Stenotrophomonas bentonitica BII-R7 bacterial strain was used to reduce Se(IV) to Se(0) NPs by biotransformation of amorphous nanospheres of Se(IV) to trigonal Se (0) NPs (Pinel-Cabello et al. 2021).
Sirsat et al. (Shirsat et al. 2015) reviewed the microbial-assisted synthesis of Se NPs and their applications with their appliances in various diverse fields such as medicine, sensors, electronics, energy, and space industries; assorted therapeutic applications of Se NPs are depicted in Figure 28.
These nanoparticles could inhibit the growth of Alternaria, Candida, Aspergillus, and the dermatophytes genera pathogenic fungi and MTT assay revealed their non-toxic nature (Asghari-Paskiabi et al. 2019). Se NPs produced using cyanobacteria showed biocompatibility and antioxidant, antimicrobial, anticancer activity when compared to chemically synthesized or commercially available Se NPs. The antioxidant activity was performed against ABTS, FRAP, DPPH, SOR assays, and ascorbic acid was used as the positive control. IC50 values for ascorbic acid, B-SeNPs (biogenically synthesized), and C-SeNPs (chemically synthesized) were found 84.71 ± 0.68, 92.58 ± 1.28 and 239.11 ± 0.34 µg/ mL in ABTS assays while 59.53 ± 0.53, 155.02 ± 0.93 and 178.89 ± 1.84 µg/ mL respectively found in FRAP assay. In DPPH assays, IC50 values for ascorbic acid, B-SeNPs, and C-SeNPs were 56.36 ± 1.52, 83.89 ± 2.11 and 174.79 ± 0.29 µg/ mL, respectively and for SOR assay, these values were 74.95 ± 0.95, 80.55 ± 1.14 and 176.84 ± 0.12 µg/ mL for ascorbic acid, B-SeNPs, and C-SeNPs, respectively, Figure 29 (Afzal et al. 2021).
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).