4.2.3. Actinomycetes

Extracellular synthesis of Au-NPs was explored using the supernatant of *Streptomyces griseoruber*, an actinomycete culture isolated from soil [128]. The development of NPs was confirmed by UV–Vis spectroscopy, which showed a peak between 520 and 550 nm. High-resolution TEM (HRTEM) analysis revealed that the formed Au-NPs were in the range of 5–50 nm and exhibited catalytic activity to degrade methylene blue.

The marine actinomycete, *Nocardiopsis alba*, isolated from mangrove soil, was utilized to produce Ag-NPs, and several bioassays were performed to evaluate the antibacterial and antiviral activities of these NPs [129]. UV–Vis spectroscopy showed the absorption peak at 420 nm, while SEM and XRD analysis revealed that the Ag-NPs were spherical and crystalline, respectively. The Ag-NPs showed antiviral activity and significant antibacterial activity against *Pseudomonas aeruginosa*, *Klebsiella pneumonia*, *Streptococcus aureus*, and *E. coli*.

#### 4.2.4. Bacteria

Bacteria, especially thermophilic bacteria, have huge potential in the extracellular green synthesis of Ag- and Au-NPs [130]. These extracellular mechanisms facilitate the production of metal NPs in an eco-friendly manner, which reduces the downstream processing of these metals.

Patil et al. investigated the effectiveness of the marine bacterium *Paracoccus haeundaensis* in the extracellular synthesis of Au-NPs and assayed the antioxidant and antiproliferative effects of the Au-NPs on both normal and cancer cells [131]. The formation of Au-NPs was confirmed by following the development of a ruby red color with a UV–Vis absorbance peak at about 535 nm. The resultant Au-NPs were spherical and had an average size of 20.93 ± 3.46 nm. The results showed no growth inhibition effect of the Au-NPs on normal cells, while the growth of cancer cells was inhibited in a concentration-dependent manner. These findings indicated that the biogenic Au-NPs were nontoxic to human cells and could therefore be used in biomedical applications.

*Bacillus brevis* (NCIM 2533) was exploited in the green synthesis of Ag-NPs [132]. The synthesized Ag-NPs, which were characterized by several spectroscopic and microscopic techniques, were spherical, had a size range of 41–68 nm, and presented with an SPR peak at 420 nm. In addition, the antibacterial effect of the Ag-NPs against multidrugresistant pathogens, including *Salmonella typhi* and *Staphylococcus aureus*, was verified in vitro.

#### 4.2.5. Fungi

Fungi contain a plethora of biocompounds; approximately 6400 have been extracted from filamentous fungi making these organisms attractive in many applications [133]. Furthermore, these microorganisms have a potentially reduced ability to produce NPs from many bulk materials owing to their tolerance against heavy metals and potentiality to accumulate metals [134].

Molnár et al. synthesized Au-NPs using 29 different thermophilic fungi and compared the results of the extracellular fraction to those of the intracellular fraction of the fungi [65]. The fabricated Au-NPs had a size ranging between 6 and 40 nm, and the sizes vary according to the fungal strain and experimental conditions.

Another study focused on exploring the anticancer activity of Au-NPs synthesized using *Fusarium solani* [135]. Properties of the Au-NPs were observed by UV–Vis spectroscopy, FTIR, SEM, and XRD. SEM images revealed that the average diameter of the NPs was between 40 and 45 nm. These Au-NPs demonstrated dose-dependent cytotoxicity against cervical cancer cells and human breast cancer cells by inducing apoptosis pathways. The findings of this research present a safer chemotherapeutic agent with lower systemic toxicity.

#### 4.2.6. Lichens

Lichens are composite organisms that live in both obligate and beneficial symbiosis with fungi, algae, perennial trees, or cyanobacteria [34]. These organisms have been used globally in enceinte traditional medicine. Some lichens are recognized as an effective treatment for gastritis, diabetes, hemorrhoids, dysentery, dyspepsia, amenorrhea, vomiting, and respiratory tract illnesses such as pulmonary tuberculosis, throat irritation, bronchitis, and dry cough [136]. Many countries are using commercial lichen-derived pharmacological products. For example, usnic acid was used in antiseptic products in Germany (Camillen 60 Fudes spray and nail oil) and Italy (Gessato™ shaving) [137]. Icelandic lichens were used in cold remedies by the trade names of Isla-Moos® (Engelhard Arzneimittel GmbH & Co. KG, Germany) and Broncholind® (MCM Klosterfrau Vertriebsgesellschaft mbH, Germany). The riminophenazine was demonstrated as antimycobacterial drugs [138]. Generally, lichens contain high proportions of phenolic compounds and polysaccharides such as lichenan and isolichenan, and various secondary metabolites, including protolichesterinic acid and fumarprotocetraric acid [34,139]. These biomolecules make the lichen

extracts have many biological activities such as antioxidant, antimicrobial and anticancer potencies. Moriano et al. investigated the antioxidant potency of 10 lichen species of *Parmeliaceae* spp. using oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2 picrylhydrazyl (DPPH) radical scavenging activities and the ferric reducing antioxidant power [140]. The data exhibited that antioxidant capacities were variable between lichen species. For instance, methanolic extract of *Flavoparmelia euplecta* showed the highest ORAC value (3.30 µmol TE/mg dry extract), *Myelochroa irrugans* methanolic extract demonstrated the maximum DPPH scavenging activity (EC<sup>50</sup> = 384 µg/mL), and the extract of *Hypotrachyna cirrhata* showed the highest reducing power (316 µmol of Fe2<sup>+</sup> eq/g sample).

Felczykowska et al. studied the antiproliferative potency and antibacterial activity of acetonic extracts of three lichen species, namely, *Caloplaca pusilla, Protoparmeliopsis muralis*, and *Xanthoria parietina* [141]. The scholars exhibited that *P. muralis* significantly suppressed the growth of *Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus,* and *Staphylococcus epidermidis*. Moreover, *X. parietina* showed antiproliferative activity against both Hela and MCF-7 cancer cells with IC<sup>50</sup> values of 8 µg/mL, and *C. pusilla* revealed the highest potency to reduce Hela, MCF-7, and PC-3 cancer cells viability with IC<sup>50</sup> values of 6.57, 7.29, and 7.96 µg/mL, respectively.

Usnic acid, along with isodivaricatic acid, 5-propylresorcinol, and divaricatinic acid derived from *Protousnea poeppigii* and *Usnea florida*, showed potent antifungal activity against *Microsporum gypseum*, *Trichophyton mentagrophytes*, and *T. rubrum* [142].

Furthermore, lichen extract has been established as an efficient reducing and capping agent for NPs due to the vast abundance, rapid growth, and most importantly, environmental sustainability of these organisms [143]. The functional groups of secondary metabolites from lichen extracts are instrumental in preventing aggregation of NPs and hence improve the fabrication and stabilization of NPs [144]. Lichen-based NPs show great potential as therapeutic agents, serving as antimicrobials, antidiabetics, and antioxidants [145,146].

#### **5. Lichens as Biosynthesizers for Nanoparticles**

### *5.1. Metallic Nanoparticles (MNPs)*

MNPs have become the most fundamental NMs in many applied and research areas due to their unique physical, chemical, and biological properties that make them promising candidates in the fields of industry, medicine, electronics, etc. [4,147]. The most frequently studied MNPs are Ag-NPs and Au-NPs due to their significant therapeutic activity against many serious diseases and their smaller-size-to-large-surface-area ratio, which enables them to be used as drug delivery systems and catalysts [8,148].

Siddiqi et al. reported that aqueous-ethanolic extract of *Usnea longissima* has the potential to fabricate silver nitrate into Ag-NPs extracellularly under laboratory conditions [136]. The lichen samples were washed, dried at 60 ◦C, and then crushed into fine powder [136]. Next, 10 g lichen powder was refluxed into 100 mL ethanol-distilled water (50:50) for 3 h. The samples were centrifuged to remove debris and 10 mL of supernatant was mixed with 1 mL of 0.01 M solution of AgNO3. The reduction process was completed after 72 h of incubation under stirring and dark conditions at room temperature. The resultant Ag-NPs were stable for weeks; however, the yield of NPs was extremely low at approximately 35%. TEM of the synthesized Ag-NPs indicated that the NPs were spherical with an average nanosize of 10.49 nm. FTIR peaks of the Ag-NPs exhibited distinct bands at 3500–3300 cm−<sup>1</sup> that refer to primary amines, while bands at 1600–1500 cm−<sup>1</sup> correspond to C–O stretching and amide bands (N–H). Additionally, at 1650 cm−<sup>1</sup> , amide I and amide II bands were reported, and at frequencies of up to 1600 cm−<sup>1</sup> , a COO band overlapped with the amide II band. The authors speculated that organic molecules of *U. longissima* were responsible for the reduction of silver nitrate into Ag-NPs.

The bioreduction ability of *Cetraria islandica* extract for the fabrication of silver nitrate into Ag-NPs was first studied by Yıldız et al. [139]. They reported that *C. islandica* can extracellularly fabricate Ag-NPs with diverse morphologies and sizes under different parameters such as time of exposure, the concentration of both silver nitrate and lichen

extracts, and temperature. Increasing the time of exposure resulted in an increase in UV absorbance values, indicating higher production of NPs. Low AgNO3/lichen ratio and low temperature caused increases in absorbance values, which again indicated higher production of NPs. The authors suggested that the higher production of Ag-NPs may be due to an increase in bioreducing agents (low AgNO3/lichen ratio) represented by the lichen extract. It was also speculated that the average size of the Ag-NPs (5–29 nm) may be controlled by varying the silver nitrate and lichen concentrations, time of reaction, and temperature.

Khandel et al. studied the catalytic activity of *Parmotrema tinctorum* to form silver-NPs [149]. *P. tinctorum* could effectively synthesize Ag-NPs from silver nitrate in an eco-friendly manner. These NPs are distinguished by their high stability, spherical shape, and average diameter of 15.14 nm. Leela and Anchana reported for the first time the potentiality of aqueous extracts of *Parmelia perlata* and their purified fractions (secondary metabolites) to fabricate silver nitrate into Ag-NPs [150]. Briefly, the lichen extract was prepared with a cold extraction method utilizing methanol and water, in which 50 g pulverized lichen was mixed with 500 mL methanol and incubated under dark conditions on a rotary shaker for three days at ambient temperature and the same amount of lichen powder was mixed with 500 mL distilled water and boiled for 1 h at 65 ◦C. Each mixture was filtrated using Whatman filter paper No. 1, and the filtrates were used for the synthesis of NPs. Thin-layer chromatography (TLC), column chromatography (CC), and gas chromatography-mass spectroscopy were performed to obtain purified secondary metabolites to use in Ag-NP synthesis. Both aqueous extract of lichen and their secondary metabolites were potent reducing and stabilizing agents for the fabrication of Ag-NPs.

Aqueous extracts of *Parmotrema praesorediosum* and *Ramalina dumeticola* were also exploited for the extracellular fabrication of Ag-NPs after 72 h at room temperature [151]. Both lichen species could form Ag-NPs, but *R. dumeticola* showed the highest bioreduction activity. *R. dumeticola* induced the formation of spherical Ag-NPs with an average size of 20 nm, while those synthesized by *P. praesorediosum* were spherical with an average size of 42 nm. *P. praesorediosum* was recently reported to extracellularly synthesize Ag-NPs with a cubic structure and a nanodiameter of 19 nm [152]. Similarly, *Cetraria islandica* was an effective biosynthetic source for both Ag-NPs and Au-NPs [144]. This lichen could produce spherical silver-NPs and gold-NPs with a dominant nanosize of 6 and 19 nm, respectively, after 30 min at 80 ◦C. The authors suggested that oxidation of phenolic compounds was a result of the reduction process of metal ions into their nanoform.

Dasari et al. used in vitro cultures of four species of lichen, *Parmeliopsis ambigua*, *Punctelia subrudecta*, *Evernia mesomorpha*, and *Xanthoparmelia plitti* to synthesize Ag-NPs extracellularly [153]. These lichens were collected from Goolapalli, Ramakuppam Mandal, Chittoor (District), Andhra Pradesh, India. Five grams of lichen thalli was cut, washed with water, and then sterilized with 0.01% HgCl2. Small pieces of the thalli were then inoculated on plates of malt yeast extract medium and incubated at 28 ± 5 ◦C for 7–10 days before transfer to fresh culture media. Four types of mycelial mat were collected separately by filtering the cultures through Whatman No. 1 filter paper. Each type of mat was separately added to 100 mL of 1 mM silver nitrate solution and incubated for 24 h at room temperature with shaking and light conditions. The solutions were then centrifuged for 10 min at 12,000 rpm to collect the synthesized Ag-NPs. UV–Vis spectrum analysis was used to examine the reduction of Ag<sup>+</sup> ions into Ag-NPs. The absorbance peak maximum was at 410–420 nm, which is typical for Ag-NPs, while the control solution (incubated without silver nitrate) did not show any peak of absorbance. The samples displayed a broad resonance (390–420 nm), suggestive of the aggregation of Ag-NPs. SEM analysis of the formed Ag-NPs disclosed their different sizes ranging between 150 and 200 nm and that the Ag-NPs were in a polydispersed mixture. FTIR analysis was conducted to identify the biomolecules present in the mycobiont mat and responsible for Ag-NPs synthesis. Briefly, samples were mixed with KBr at a ratio of 1:100, and the spectra were recorded at 1000–3500 cm−<sup>1</sup> . Ag-NPs synthesized by *Parmeliopsis ambigua* had an IR spectra peak

at 3332 cm−<sup>1</sup> that confirmed the presence of polyphenolic –OH group and peaks at 1639 and 1252 cm−<sup>1</sup> that reflected the presence of amide I and carboxylic groups, respectively. Similar findings were obtained for the *Punctelia subrudecta* sample. Ag-NPs of *Evernia mesomorpha* also showed the same functional groups, –OH and –NHCO, at peaks of 3248 and 1739 cm−<sup>1</sup> , respectively. However, spectra of Ag-NPs synthesized by *Xanthoparmelia plittii* revealed the presence of C–N at a peak of 1015 cm−<sup>1</sup> and the asymmetric mode of both the aliphatic and aromatic functional group –C–H peaking at 2923 cm−<sup>1</sup> . The presence of C–H stretching was confirmed by a peak at 2853 cm−<sup>1</sup> , and a peak at 3234 cm−<sup>1</sup> corresponded to primary aliphatic amines. The presence of carbonyl group C=O from the phenols was indicated by the peak at 1656 cm−<sup>1</sup> , and the C–O single bonds were indicated by a peak at 1000–1200 cm−<sup>1</sup> , while the aromatic C–H functional group was found below 700 cm−<sup>1</sup> . The predicted phenols in the samples included catechin gallate, epicatechin gallate, and gallocatechin gallate. The authors speculated that polyphenolic compounds were the essential molecules in the bioreduction process of Ag-NPs.

The efficiency of aqueous extract of the lichen *Ramalina dumeticola* as a reducing and stabilizing agent for extracellular fabrication of silver nitrate into Ag-NPs was recently explored [154]. The reaction between the lichen aqueous extract (10 mL) and 30 mL of 1 mM silver nitrate solution was conducted at room temperature for 24 h. The formation of Ag-NPs was confirmed by the solution turning yellowish brown. NPs were obtained from the solution by centrifugation at 5000 rpm for 20 min and were subsequently freeze dried. UV–Vis spectral analysis, which was taken with a resolution of 2 nm at a range of 400–450 nm, monitored the formation of Ag-NPs and revealed the characteristic SPR band of Ag-NPs at approximately 433 nm. XRD analysis to define the chemical composition and crystal structure of the sample showed four peaks of 38.1◦ , 44.3◦ , 64.4◦ , and 77.4◦ at 2θ, which corresponded to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) crystallographic planes of face-centered cubic of silver, respectively. The average of crystal size was calculated by utilizing the Debye-Scherrer equation, i.e., D = (0.94λ)/(β cos θ), where D is the mean crystallite domain size, λ is the wavelength of Cukα, β is the full width at half maximum (FWHM), and θ is the Bragg diffraction angle. The average size of the Ag-NP crystals was 17.1 nm. TEM revealed that Ag-NPs were polydispersed and mainly spherical with a size between 6 and 28 nm and an average diameter of 13 nm.

Rai and Gupta tested the possibility of biofabricating Ag-NPs by exploiting the reducing capacity of aqueous extracts of the lichen *Cladonia rangiferina* [145]. The lichen was collected from the Govind wildlife sanctuary in the Uttarkashi District of Uttarakhand, western Himalaya, at an altitude above 3500 m. Silver nitrate solution (45 mL of 1 mM) was mixed with 15 mL lichen aqueous extract and 2–3 drops of 0.1 M sodium hydroxide to reach an alkaline pH. After 72 h at room temperature, the reaction mixture turned yellow brown, verifying the presence of Ag-NPs. UV–Vis spectrophotometry indicated that the spectral band peak at 402 nm corresponded to the specific color change that resulted from the reduction of silver ions to Ag-NPs by secondary metabolites of the lichen. The presence of these secondary metabolites was consolidated by the detection of their functional groups by FTIR analysis. The FTIR scan taken at a range of 450–4000 cm−<sup>1</sup> showed several functional groups corresponding to specific biomolecules in the extract such as polyphenols that could participate in the fabrication and stabilization of Ag-NPs. Peaks were observed in the range of 1000–4000 cm−<sup>1</sup> , demonstrating the presence of O–H (3400 cm−<sup>1</sup> ), C–H (2853 cm−<sup>1</sup> ), C=O (1742 cm−<sup>1</sup> ), C=O (1691 cm−<sup>1</sup> ), C=O aldehyde (1651 cm−<sup>1</sup> ), C=C vibration (1573 cm−<sup>1</sup> ), CH2, CH<sup>3</sup> (1443 cm−<sup>1</sup> ), and C–O (1273 cm−<sup>1</sup> ). For TEM analysis, the solution was sonicated for 15 min, loaded onto a carbon-coated copper grid, and incubated under a fume hood for 30 min for the solvent to evaporate. The Ag-NPs visualized by TEM were spherical and rod shaped, with a particle size ranging from 5 to 40 nm and an average diameter of 20 nm. Similarly, different studies were reported that lichen species including *Xanthoria elegans*, *Usnea antractica*, *Leptogium puberulum*, *Cetraria islandica*, *Pseudevernia furfuracea*, *Lobaria pulmonaria*, *Heterodermia boryi* and *Parmotrema stuppeum* have

potentiality to reduce silver nitrate into Ag-NPs with different shapes (bimodal and cubic) and sizes [155–157].

Ethanolic extract of the lichen *Parmotrema clavuliferum* was used for the biological synthesis of Ag-NPs from silver nitrate [158]. The extraction was carried out by adding 10 g dried lichen to 100 mL ethanol and incubating with shaking at 80 ◦C for 24 h then filtering through 25-mm pore-sized papers. The extract was added to silver nitrate solution at room temperature and the production of Ag-NPs was indicated by brown color development. The brown color appeared immediately, which verifies the high potency of *Parmotrema clavuliferum* extract as a reducing and capping agent for NPs. For further confirmation, the excitation of SPR provided by the Ag-NPs was measured spectrophotometrically at 400–450 nm. The plasmon absorption bands showed an absorbance peak at 440 nm. The DLS and zeta potential data indicated that the particle size distribution of the biogenic Ag-NPs was in the range of 80–120 nm and the particles had negative charges suggesting their stability at room temperature. TEM and SEM revealed that the Ag-NPs were spherical and approximately 106 nm in diameter. Potential biomolecules in the lichen extract were explored using FTIR spectroscopy, and broad peaks at 3264 and 1634 cm−<sup>1</sup> were recorded, which correspond to O–H of phenolic compounds stretching groups and C=O of the peptide bond, respectively. These findings imply the role of phenolic compounds and in the bioreduction of silver ions.

Abdolmaleki et al. used two lichen species, *Usnea articulata* and *Ramalina sinensis*, to reduce 1 mmol of silver nitrate solution into Ag-NPs [143]. The resulting Ag-NPs were spherical with a nanosize range of 10–50 and 50–80 nm, respectively.

Recently, aqueous extracts of two novel lichen species, *Acroscyphus sphaerophoroides Lev* and *Sticta nylanderiana*, were utilized to fabricate chloroauric acid (10−<sup>3</sup> M HAuCl4) into Au-NPs at room temperature for 12 h [159]. Physicochemical analyses confirmed the potentiality of both lichen species to generate gold-NPs. The UV–Vis-spectra of biogenic Au-NP was at 535 nm and the XRD pattern confirmed the face-centered cubic of Au-NPs. FTIR spectra of both types of Au-NPs featured bands at 3446 and 1041 cm−<sup>1</sup> that relate to N–H and C–O stretching, respectively, bands at 2922 and 2849 cm−<sup>1</sup> corresponding to C–H stretching, and bands at 1638 and 1456 cm−<sup>1</sup> that relate to the amide and carboxylate groups, respectively, in the amino acid residues of the biomolecules. The authors speculated that the presence of these functional groups might help prevent agglomeration of the NPs. Moreover, TEM revealed that *A. sphaerophoroides*-mediated Au-NPs are multiply twinned quasi-spherical and prismatic with a size range between 5 and 35 nm, while *S. nylanderiana*mediated Au-NPs were exclusively multiply twinned with a nanosize range of 20–50 nm.

An extract of the lichen *Parmelia sulcate* was used to synthesize Au-NPs; for its preparation, 90 mL of a 1 mM HAuCl<sup>4</sup> solution (to provide Au3+) and 10 mL of the 5% *Parmelia sulcata* extract were heated to 60 ◦C and kept on a magnetic stirrer for 20 min [160]. The color change of the reaction mixture from yellow to purple was monitored to observe the formation of Au-NPs then the solution was dried in an oven (70 ◦C) for 48 h to obtain powdered particles. The UV–Vis spectrum from 300–700 nm confirmed the reduction of Au3+ to gold NPs (Au<sup>0</sup> ). The peak observed at 540 nm represents the SPR, verifying the formation of the Au-NPs. The XRD spectrum had peaks at 38.3◦ , 44.6◦ , 64.7◦ , and 77.7◦ , which revealed the crystalline feature of gold and the face-centered cubic particles depending on the angular positions of the Bragg peak. SEM and TEM demonstrated that the particles had an average size of 54 nm and were spherical. TEM with energy-dispersive spectroscopy (EDS) was utilized to determine the elemental composition. The EDS pattern displayed a strong signal for the gold peak, indicating successful fabrication of Au-NPs. FTIR spectra were measured over a range of 400–4000 cm−<sup>1</sup> to follow the reaction between the *Parmelia sulcata* extract and chloroauric acid. Biomolecules in the lichen extract were confirmed to interact with Au-NPs. The peaks at 3443 cm−<sup>1</sup> corresponded to the O–H strong stretch of the alcohol, while a peak at 1640 cm−<sup>1</sup> corresponded to the C=C of the alkenes, and the peaks at 1544 and 1384 cm−<sup>1</sup> were related to the N–H and N–O bending and stretching of the amide and nitro groups, respectively. The peaks at 1272 and 1206 cm−<sup>1</sup> were related to the C–O

stretch of esters. The presence of proteins in the solution, indicated by the amide and nitro group peaks, might contribute to the stabilization of the newly formed NPs. DLS and zeta potential analyses revealed that the average size of the hydrodynamic diameter of the Au-NPs was 54.14 nm and their charge was negative (−18.4). This indicates the existence of lichen biomolecules surrounding the Au-NPs, which provide stability to the Au-NPs.

Devasena et al. used the Soxhlet extraction method to obtain the lichen extract to use in magnesium nanoparticles (Mg-NPs) synthesis [161]. They reported that *Cladonia rangiferina* has the ability to reduce magnesium sulfate into Mg-NPs extracellularly. UV–Vis spectroscopy analysis revealed that the absorption peak of Mg-NPs was at 262 nm, while the DLS technique revealed that Mg-NPs have an average hydrodynamic diameter of 23 nm.

*Protoparmeliopsis muralis* was first used for the synthesis of different metallic and metal oxide NPs (MONPs) by Alavi et al., who utilized the aqueous extract of this lichen to extracellularly fabricate silver-NPs and copper-NPs under dark and stirred conditions for 24 h at ambient temperature [162]. Synthesis of the MNPs was confirmed by UV–Vis spectroscopy, TEM, SEM, EDAX, XRD, and FTIR analyses. The resulting data demonstrated that the maximum absorbance peak for Ag-NPs and Cu-NPs was 378 and 567 nm, respectively, and that the MNPs were spherical with an average nanosize of 33.49 ± 22.91 and 253.97 ± 57.2 nm, respectively. EDAX data demonstrated that Ag- and Cu-NPs were present in the sample at 87.72 and 26.42%, respectively. Furthermore, the 2θ degree values of both Ag- and Cu-NPs were 35.5◦ , 43.6◦ , 65.6◦ , and 72.1◦ , and 35.9◦ , 39.6◦ , 44.3◦ , 54.3◦ , and 57.2◦ , respectively, indicating the crystallinity of these NPs. Based on FTIR data, there were three dominant functional groups, C=C, S=O, and C-Br, in all samples (lichen extract, Ag- and Cu-NPs, and metal oxides NPs). However, O–H bond bending corresponding to secondary metabolites such as phenol was observed for the MNPs, suggesting that these secondary metabolites may act as a potential reducing and stabilizing agent during the synthesis process of both Ag- and Cu-NPs. To prove this hypothesis, the authors analyzed the total phenol, flavonoid, flavanol, and tannin contents (TPC, TFC, TFLC, and TTC, respectively) of the samples via Folin-Ciocaltue assay. The Ag-NP solution contained higher amounts of TPC, TFC, TFLC, and TTC, compared with the lichen extract and other MNPs and MONPs. The same study also screened the effect of time exposure (24, 48, 72, and 96 h) on the biosynthesis process of MNPs and demonstrated that the concentration of MNPs increased as the time of exposure increased. The authors noted that the synthesis process of MNPs using lichen aqueous extract was slower than that using plant watery extract suggesting the cause of the slow reaction is the lower reducing capacity of lichens (Table 1).




**Table 1.** *Cont.*


**Table 1.** *Cont.*

**Abbreviation:** (-), extracellular synthesis; (+), intracellular synthesis; NM, not mentioned; NA, no applications; RT, room temperature.

#### *5.2. Metal Oxide Nanoparticles (MONPs)*

MONPs are one of the widest used nanomaterials due to their unique properties including high stability, porosity, and easy functionalization with different molecules because of their negative charge; these properties mean MONPs are particularly suited to biomedical applications [170].

Alavi et al. utilized the aqueous extract of *Protoparmeliopsis muralis* to biosynthesize three different types of MONPs—ferric oxide, zinc oxide, and titanium oxide (Fe3O4, ZnO, and TiO2, respectively) NPs [162]. Briefly, lichen samples collected from Kane Gonabad Mountains were washed with distilled water, air dried for six days, then crushed into a fine powder, and boiled with 250 mL distilled water at 90 ◦C for 30 min. The mixture was filtered through Whatman filter paper No. 40 and 10 mL of the filtrate, was mixed with 50 mL TiO(OH)<sup>2</sup> or Zn(NO3)2·6H2O (0.01, and 0.001 M concentrations, respectively), and incubated for 24 h with stirring. For fabrication of Fe3O<sup>4</sup> NPs, the same amount of lichen extract was added to flasks containing FeCl3·6H2O (0.2 M) and FeCl2·4H2O (0.001, 0.01, and 0.1 M), and the pH was adjusted to 8 by adding 0.1 M NaOH solution. Mixtures were kept under stirred conditions for 24 h at room temperature. The resultant NPs were collected by centrifugation at 4000 rpm for 30 min, washed, and dried at 70 ◦C for 8 h. Physicochemical analyses showed that the UV-spectra peaks of Fe3O4, ZnO, and TiO<sup>2</sup> NPs were 216, 328, and 283 nm, respectively. TEM and SEM images demonstrated that all the MONPs were spherical with an average nanodiameter of 307 ± 154 (Fe3O<sup>4</sup> NPs), 133.32 ± 35.33 (TiO<sup>2</sup> NPs), and 178.06 ± 49.97 nm (ZnO NPs). The presence of Fe (84.07%), Ti (66.41%), and Zn (25.61%) in the Fe3O4, ZnO, and TiO<sup>2</sup> NPs samples was detected by EDAX analysis. XRD and FTIR analyses proved that these MONPs had nanocrystal structures and were coated with organic molecules such as secondary metabolites (phenols, O–H), which have a significant role in reducing and stabilizing NPs.

The hydrolytic capacity of aqueous extracts of a new strain of lichen, *Ramalina sinensis*, was recently reported to extracellularly fabricate ferric chloride salts into iron oxide NPs [165]. The UV-spectra curve of the NP samples appeared in the range of 280–320 nm, indicating the formation of magnetic iron oxide NPs. The XRD pattern of the biosynthesized iron oxide NPs showed distinct diffraction peaks of 30.5◦ , 36.1◦ , 43.3◦ , 53.9◦ , 57.5◦ , and 63.3◦ at 2θ, indicating the cubical nanocrystalline structure of iron oxide NPs. Furthermore, FTIR analysis demonstrated that π-electrons of carbonyl groups of flavonoid and phenolic compounds of *R. sinensis* were responsible for the reduction of iron ions into their nanoforms. Field emission scanning electron microscopy (FESEM) revealed that the particle size of the iron oxide NPs was between 31.74 and 53.91 nm and that pores existed in the iron oxide NPs structure. EDX analysis showed that Fe and O elements were the main constituents in the iron oxide nanostructure.

Similarly, Arjaghi et al. performed extracellular reduction of ferric chloride salts (FeCl2·4H2O and FeCl3·6H2O) into Fe3O<sup>4</sup> NPs by utilizing *R. sinensis* [163]. Sharp absorption peaks were observed between 300 and 350 nm owing to the interaction between the chemicals, and tensile vibration resulted from the formation of a new bond between iron and oxygen and the synthesis of Fe3O<sup>4</sup> NPs. The authors hypothesize that the existence of biomolecules in *R. sinensis* might prevent the agglomeration of NPs, that polysaccharide sulfate acts as a potent reducing agent, and that sulfate groups have significant roles in the extracellular synthesis of iron oxide NPs by oxidizing the aldehyde group into carboxylic acids. XRD and SEM data revealed that Fe3O<sup>4</sup> NPs were nanocrystalline and 20–40 nm in size.

ZnO-NPs were biologically synthesized by Koca et al. using *Ramalina fraxinea* extract [169]. Lichen samples were carefully washed, dried in a 70 ◦C oven overnight, and extracted in water by heating (80 ◦C) for 1 h, and the resulting extract was filtered through Whatman No 1 filter paper. For the synthesis of ZnO-NPs, 100 mL filtered extract was added to 5 g Zn(NO3)2·6H2O and incubated at 60 ◦C with continuous stirring until the color changed, indicating the formation of NPs. The solution was then heated at 400 ◦C for approximately 2 h to obtain a fine powder of ZnO-NPs. The characteristic SPR band of the ZnO-NPs was determined by UV analysis at a wavelength range between 200 and 900 nm. Peaks were observed at 269 nm, which related to *Ramalina fraxinea* extracts, and 330 nm, suggesting synthesis of ZnO NPs was successful. FTIR analysis of the ZnO-NPs revealed O–H (alcohol) band vibrations at 3128 and 1398 cm−<sup>1</sup> and stretching bands at 1620 and 1575 cm−<sup>1</sup> that were correlated to the alkenes (C=C), while absorption peaks at 1480 cm−<sup>1</sup> were for alkanes (C–H). Bands at 1379 and 1335 cm−<sup>1</sup> related to O–H (alcohol and phenol), and the presence of amine groups (C–N) was observed at 1192 cm−<sup>1</sup> . The peak at 1295 cm−<sup>1</sup> represented the aromatic ester (C–O) and aromatic amine groups (C–N), while the bands at 1121 and 1087 cm−<sup>1</sup> were identified as amine (C–N) and aliphatic ether (C–O) groups. Stretching bands at 1038 and 960 cm−<sup>1</sup> were allotted to ether (C–O) and alkene (C–H), respectively. The bands at 871, 842, 773, 671, and 605 cm−<sup>1</sup> confirmed the presence of halo compounds (C–Cl), and the peaks observed at 407, 444, and 535 cm−<sup>1</sup> were assigned to Zn-O (metal-oxygen) vibration. In conclusion, FTIR analysis disclosed that functional groups in the extract of *Ramalina fraxinea* are crucial for the synthesis of ZnO-NPs. XDR spectra at 2θ showed a group of diffraction peaks of 31.7◦ , 34.4◦ , 36.2◦ , 47.5◦ , 56.5◦ , 62.8◦ , 66.4◦ , 67.9◦ , 69.1◦ , 72.5◦ , and 77.1◦ indicative of the crystal planes of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4), (2 0 2), and (1 0 4), respectively. Collectively, the XDR analysis demonstrated that the *Ramalina fraxinea* extract delivered ZnO-NPs with a characteristic hexagonal and crystalline structure. The biogenic ZnO-NPs were spherical with a size of around 21 nm, as evidenced by SEM and FESEM imaging. The authors reported the existence of aggregation of NPs that resulted from the impact of Van der Waals forces between NPs (Table 1).
