Exploring the antimicrobial potential of biologically synthesized zero valent iron nanoparticles

: The widespread use of antibiotics has resulted in the emergence of multidrug-resistant bacteria. Therefore, it is essential to explore alternative strategies to e ﬀ ectively combat medically signi ﬁ cant resistant pathogens. In recent years, nanoparticles (NPs) have emerged as a promising alternative source of antimicrobial agents. While nano-scale particles were traditionally synthesized using chemical techniques, the development of metallic NPs using biological methods has garnered attention. This current study focuses on the synthesis of iron NPs (Fe NPs) using metal-tolerant fungal strains, as numerous microorganisms serve as environmentally safe and durable precursors to produce persistent and bi-functional NPs. The study involved the isolation and evaluation of ten fungal strains that are resistant to heavy metals to determine their ability to produce Fe NPs. The biologically synthesized Fe NPs were characterized using X-ray di ﬀ raction (XRD), energy dispersive X-ray spectroscopy (EDX), and scanning electron microscopy techniques. The XRD results indicated the presence of Fe in nanopowder form, displaying a series of re ﬂ ection angles (2 θ ) at 65° and 75° indicating the existence of cubic planes. EDX analysis revealed the presence of ferrous and ferric elements, along with zero-valent Fe NPs. Micrographs of the surface topology displayed spherical aggregation of the synthesized NPs. Furthermore, the Fe NPs exhibited promising antibacterial potential against selected bacterial strains, including Bacillus subtilis, Escherichia


Introduction
Engineered nanomaterials have become increasingly significant for their positive impact on various industries such as consumer goods, cosmetics, pharmaceutics, transportation, and agriculture.In biomedical sector these nanoscale particles are widely used for drug delivery, photoablation therapy, bioimaging, hyperthermia, and biosensors.These materials are now being synthesized in bulk quantities for commercial use [1].Antibiotic resistance arises from a complex interplay of factors that contribute to the development and spread of resistance mechanisms [2].As pathogenic bacterial strains have become more resistant to traditional antibacterial treatments, new approaches to infection control have emerged [3].Nano-conjugates have demonstrated significant efficacy against pathogenic bacteria, making them important in the field of nano-medicine.However, excessive use of nanomaterials in humans has been associated with numerous side effects [4].
Nanoparticles (NPs), also known as nanoscale particles, have atomic masses ranging from 1 to 100 nm.Compared to bulk materials, nanoscale particles can rapidly modify their physicochemical properties [5,6].It is worth noting that NPs can be synthesized from a wide range of bulk materials, and their functions vary based on chemical composition, particle size, and form [7]. Various methods have been used for the manufacture of metallic NPs.NPs can be synthesized using both top-down and bottom-up techniques.The top-down method involves shrinking large particles using lithographic and mechanical processes such as machining and grinding, and it is also referred to as "green synthesis."The bottom-up method, on the other hand, builds tiny building components such as chemical synthesis into a larger structure [8].
NPs can be synthesized using chemical or biological methods.Chemical synthesis methods have been associated with negative side effects due to the presence of hazardous substances deposited on the surface.In contrast, biological methods of NP manufacturing employing microbial enzymes are less hazardous to the environment than chemical and physical methods that are extracted from fungi and plants [9].The development of environmentally acceptable technologies for NP synthesis is becoming a major nanotechnology sector, particularly for silver and iron NPs that have numerous applications [10].Various organisms act as environmentally safe and long-lasting precursors to produce persistent, bi-functional NPs.Microbial entities such as yeast, fungi, bacteria, actinomycetes, and other microorganisms have been reported for NP production [11].The production of NPs by microorganisms is an environmentally friendly process commonly referred to as green technology.Fungi have gained attention from researchers in investigative studies on the biological generation of metallic NPs due to their tolerance and metal bioaccumulation characteristics [12].Biologically synthesized zero-valent iron NPs (Fe NPs) have been arisen as an effective therapeutic agent because these are highly diffusible and have high specific surface area.Iron is the most abundant metal in the Earth's crust and is considered one of the most important metals due to its presence in different oxide forms.Moreover, it serves as the structural backbone of modern infrastructure [12].
While microorganisms can mutate in large numbers to build resistance, NPs can target a wide range of locations, inhibiting bacteria from developing resistance to them [13].The rigidity, flexibility, and structure of the bacterial cell wall protect the cell against osmotic pressure, preventing mechanical damage and rupturing [14].Given the importance of Fe NPs, the current study emphasizes a simple and new green production method for Fe NPs using a metallotolerant fungal strain and the antibacterial activity of the synthesized NPs against pathogenic microbes.Thus, the bio-based Fe NPs are innovative and sustainable alternatives to traditional Fe NPs, synthesized using environmentally friendly methods.They offer advantages such as reduced environmental impact, cost-effectiveness, and tunability in size and shape, making them promising for applications in environmental remediation, biomedical fields, and sustainable energy production.Their bio-based synthesis methods utilize natural biomolecules, providing a greener approach to NP production.

Sample collection
This study analyzed soil heavy metals and indigenous microbial strains using polluted soil from Karachi's Korangi Industrial Zone.Heavy metal-contaminated soil samples were collected from effluent sites in Karachi's Industrial Zone by plowing a layer up to 20 cm with a sharp spatula.The samples were then transferred to labeled polyethylene homogenization containers and thoroughly mixed to obtain a homogenous sample representative of the entire sampling interval.The labeled polyethylene bags were transported to the laboratory and stored for pretreatment and analysis.The contaminated soil samples were dried at room temperature and in an oven at 75°C until constant mass was achieved, which took 2 weeks, and were kept in a desiccator for further analysis [15].

Isolation of fungal strain
To isolate indigenous fungal strains from the soil samples, we used the serial dilution method on potato dextrose agar (PDA).Each soil sample was diluted up to 10 6 and 1 mL of each dilution ranging from 10 4 to 10 6 was plated on PDA plates containing 0.5 g L −1 chloramphenicol.The plates were then incubated for 1 week at 28°C.After incubation, the predominant fungal isolates were picked and subcultured to obtain a pure culture of each isolate.This was done by subculturing individual colonies from the initial culture on fresh PDA, Sabouraud dextrose agar (SDA), Czapek yeast extract agar, and malt extract agar [1], each containing 0.05% chloramphenicol [16].

Synthesis of Fe NPs
Fe NPs were synthesized via myogenesis in a liquid medium containing potato dextrose broth (PDB) under aerobic conditions.Fungal spores were inoculated and incubated for 96 h in a shaker incubator at 28°C and 150 rpm.The filtrate was obtained using Whatman filter paper no. 1 and subsequently used for the biosynthesis of metal NPs [17].The fungal isolate that showed high-level production of the desired NPs was selected for further investigation.

Identification of fungal isolates 2.4.1 Morphological identification
The isolated fungal strains were identified based on their cell morphology and colonial morphology [18,19].Young developing fungal fungi from each strain were recorded using a fluorescence microscope after 2 days of growth on different media.The images of fungal strains were captured using the Cell Sense standard software, following the method described previously.The pure fungal strain was isolated and purified on PDA media for further preservation purposes.

Molecular identification
The selected fungal isolate was cultured in PDB medium and the inoculated mycelium was separated by centrifugation at 12,000 rpm for 10 min.DNA was extracted from the mycelium using a modified method, and the internal transcribed spacer (ITS) conserve sequence was amplified using primers IT5:50-TCCGTAGGTGAACCTGCGG-30 and IT5:50-TCCT CCGCTTATTGATATGC-30 through standard PCR, as previously described [20].After sequencing, a BLAST analysis was conducted and a phylogenetic tree was constructed using MEGA 4.0 [21].

X-ray diffraction (XRD)
To observe the crystalline structure of NPs, a diffraction technique was used.Pure powder of NP was operated at 30 mA current and 40 kV voltage.Scanning temperature was adjusted between 10 and 100°C and XRD pattern was seen at two ranges, i.e., 30-80°by using XRD diffractometer (PANalytical-XPERT PRO diffractometer system).The graph was constructed with the Origin Pro8 program [22].

Energy dispersive X-ray spectroscopy (EDX)
The dried NPs sample was applied to a spin column containing graphene sheets for EDX examination of the elemental analysis of the sample [22].Same instrument was used for EDX analysis as used during scanning of samples.

Scanning electron microscopy (SEM)
SEM was used for microscopic examination of dried NPs.Initially the sample was casted on glass slide and fixed on a copper support.A gold coater was used to coat NP samples with gold.The scanning microscope (JEOL JSM 6360LA, Akishima City, Tokyo, Japan) operated at 20-30 kV was used to analyze the images at various resolutions [16].Elemental mapping and quantitative and qualitative analysis of the produced NPs are carried out through energy dispersive spectroscopy feature of scanning microscope.

Antimicrobial properties of Fe NPs
The effects of Fe NPs (produced utilizing fungal species) were evaluated against chosen pathogenic bacterial ATCC strains, i.e., including Listeria innocua (ATCC13932), Staphylococcus aureus (ATCC9144), Escherichia coli (ATCC10536), Bacillus subtilis, Enterococcus faecalis, and Pseudomonas aeruginosa (ATCC10145), on nutrient agar plates to determine antibacterial activity in dilution of 1 mM (0.017 g/100 mL) Fe NPs.The antimicrobial activity of the biologically synthesized NPs was analyzed using a well-diffusion essay.Inoculated plates were incubated for 18 h at 37°C.The zone of inhibition was investigated after complete incubation [16].

Results
Myco-nanotechnology refers to the process of synthesizing NPs using metabolites produced by microorganisms and fungal biomass.Fungal mycelia can be effectively utilized for this purpose due to their high surface area and the ability of their cell walls to reduce and absorb metal ions, which are necessary for the formation of metal NPs.Fungi have also been found to produce NPs with superior polydispersity, extremely stable structures, and a greater variety of sizes than other organisms.In the current study, heavy metal-tolerant fungal strains were isolated and used to synthesize Fe NPs via biological myogenesis.

Isolation of fungal strain
A total of ten heavy metal resistant fungal strains (n = 10) were isolated from heavy metal contaminated soil of Korangi industrials zone, Karachi.Different growth mediums including PDA, malt extract agar (MEA), and SDA were used for isolation and purification at 30°C of incubation for 72 h.

Screening for Fe NPs synthesis
All the fungi were grown in PDB medium for 72 h at 30°C.After incubation, the broth medium for each strain was centrifuged at 5,000g for 15 min.Pellet was removed and discarded carefully.The supernatant was filtered through Whatman filter no. 1. Salt solution of ferric chloride (0.1 M) was incubated with supernatant that showed significant NP synthesis after 1 week of the incubation period.Synthesized NPs were air-dried and stored.The highest NP synthesis was observed in case of K1 (13.5 mg) whereas lowest synthesis was noticed from K9 strain (5.9 mg) (Table 1).

Morphological identification
The K1 strain's colonies on MEA medium showed moderate growth, velvety smoothness, and parrot green frontal sides, back side of the colony was white at 25°C in 7 days of incubation.On microscopy, the hyphae were found to be sporangial hyaline and the conidiophore branched suggesting that the K1 strain had most of the same features as Pencillium notatum (Figure 1).

Molecular identification
The gDNA was extracted and a PCR product of 541 bp band was amplified by using gDNA as a template according to the procedure described in Section 2. The ITS sequence of the fungal isolate was aligned by using the data in the NCBI database and the phylogenetic tree was constructed by using neighbor joining method which indicates that the fungal isolate used in this study is more than 98% similar to P. notatum (Figure 2).

Antimicrobial effects of biologically synthesized Fe NPs
The antibacterial activity of the biologically synthesized NPs was evaluated by dissolving in dimethyl sulfoxide (DMSO) for different dilutions (1:20) of Fe NPs.The Fe NPs were tested for antibacterial action against different ATCC cultures of pathogenic bacteria including B. subtilis, E. coli, P. aeruginosa, S. aureus, Cronobacter sakazakii, L. innocua, and .fecalis.As a negative control, only DMSO was employed.All dilutions of Fe NPs showed maximum activity against E. coli and lowest activity against C. sakazakii (Figure 3).

Characterization of synthesized NPs
Synthesized NPs were subjected to different characterization techniques to evaluate their behavior.

Structural characterization of Fe NPs with XRD
The phase composition and nature of biologically synthesized Fe NPs were identified by using an X-ray powder diffractometer with the help of Bragg's angle ranging from 20 o Fe NPs (mg) 13.5 10.0 8.5 11.1 7.6 6.8 8.9 7.7 5.9 11.3 Fe NPs: iron nanoparticles. to 80°.The presence of iron in nanopowder was then confirmed by a series of reflection angles (2θ) at 65°and 75°with the cubic plane of iron as shown in Figure 4. Oxides of iron also revealed their presence shown by red squares.

EDX
EDX analysis of the synthesized NPs was also carried out at room temperature to confirm the presence of Fe NPs.Peaks at different positions reveal the presence of different oxidation states, i.e., ferrous and ferric along with zero-valent Fe NPs.Oxide of iron showed their presence as well (Figure 5).

SEM
The surface topology of the Fe NPs was confirmed by SEM. Figure 6 shows that the synthesized NPs have spherical shapes and also show agglomeration.Agglomeration is a common phenomenon observed in NPs to get stability [23].Micrographs showed that most of the particles were agglomerated in the form of large irregular lumps while few particles in the range of 100-200 nm can also be seen in the micrographs.Along with instability and agglomeration, another major issue is wide range of particle size while dealing with NPs [24].

Discussion
The production of NPs is gaining increasing interest due to their numerous applications in fields such as medicine, electronics, and food.However, conventional physical Figure 2: A phylogenetic tree was constructed using the neighborjoining analysis of ITS sequence for a fungal isolate, along with other fungal species relations that had a value >80% during NCBI Blast.The tree included Aspergillus flavus NRRL as an outgroup [16].
Zero valent iron nanoparticles  5 and chemical methods used to synthesize NPs have certain drawbacks, such as the need for external energy and the production of toxic chemical byproducts that harm the environment.Additionally, NPs synthesized using these methods may not always be suitable for use.As a result, biosynthetic methods have gained the attention of researchers due to their environmental safety, cost-effectiveness, and lack of hazardous byproducts [25].Metals such as silver, nickel, iron, and gold are commonly used in NP production, and myco-nanotechnology is a technique that involves synthesizing NPs using metabolites produced by microorganisms and fungal biomass.The high surface area of fungal mycelia and the ability of their cell walls to reduce and absorb metal ions make them ideal for use in NP synthesis.Many fungi have been reported to produce various stable nanostructures, and filamentous fungi are particularly well-suited for NP production due to their large number of secondary metabolites and enzymes.They are also easy to handle and can be grown on a variety of media, including both basic and complex media [26].
Water pollution is one of the most serious environmental issues that the world is facing today, and industrial dyes are a significant contributor to the problem.The effluent discharged by the textile and paper industries contains considerable amounts of non-biodegradable synthetic colors.Such dyeing effluents have hazardous characteristics and pose a long-term threat to the environment's development.Therefore, the development of efficient techniques for 4: XRD analysis of Fe nano-powder.treating these effluents is urgently required.Iron oxide NPs have been widely used to eliminate colors from wastewater in a cost-effective manner [27].Combining myco-synthesized Fe NPs with H 2 O 2 allows free radicals to perform the same function as hydroxyl radicals (OH) without causing significant harm to the environment.The combination of mycosynthesized Fe NPs and H 2 O 2 has been found to be effective in removing colors from wastewater.This method is costeffective and does not cause any significant harm to the environment.Free radicals generated by this method can perform the same function as hydroxyl radicals (OH), which are known to be highly effective in removing pollutants from wastewater.Iron oxide NPs have been extensively used to remove colors from wastewater, and this method is considered to be one of the most efficient techniques for treating dyeing effluents.Therefore, the use of myco-synthesized Fe NPs with H 2 O 2 can significantly contribute to the elimination of hazardous colors from wastewater.
In this study, a total of ten fungal strains were isolated from metal-contaminated effluent samples, and their potential for synthesizing Fe NPs was investigated.Among the strains tested, K1 was found to have the highest Fe NP synthesis yield (13.5 mg), while the lowest yield was observed in the case of K9 (5.9 mg).The synthesis of Fe NPs was confirmed by the formation of a reddish-brown precipitate, which is a characteristic property of Fe NPs.This color shift has been observed in the production of Fe NPs using leaf extracts as well [26].In another study, a change in the medium color from light brown to black was observed due to Fe NPs synthesis using Kappaphycus alvarezii extract [28].
In the recent study, the K1 strain was identified as the highest producer of Fe NPs based on its colonial, morphological, microscopic, and molecular characteristics.All colonial characteristics of the selected strain on MEA media and microscopic observations revealed that the isolate had maximum similarity to P. notatum.Furthermore, a PCR product of a 541 bp band was generated using DNA as a template.The ITS similarity between the isolated fungal strains and those in the NCBI database showed that the fungal isolate used in this study exhibited similarity to P. notatum.In a previous study, different fungal isolates were similarly identified as A. paraciticus and P. notatum, with 97 and 99% similarity, respectively, with A. paraciticus and P. notatum [29].
Drug resistance is a major public health concern, and new strategies are needed to re-activate dormant medications.In this study, the antibacterial activity of iron was observed against several bacterial strains, including E. coli, B. subtilis, S. aureus, P. aeruginosa, C. sakazakii, L. innocua, and E. fecalis.The Fe NPs were prepared at various concentrations (1,000, 500, 250, and 125 g) by mixing them in DMSO at dilutions of 1:20 and 1:40.DMSO was used as a negative control, and no activity was observed.
When E. coli was exposed to dilutions containing different concentrations (1,000, 500, 250, and 125 μg) of Fe NPs, the zones of inhibition were 2.8, 2.5, 2 and 1.8 mm, respectively.For B. subtilis, the zones of inhibition were 1, 1.5, 1.2, and 1.3 mm when exposed to different concentrations of Fe NPs.When S. aureus was exposed to Fe NPs at various concentrations, zones of 2, 1.8, 2, and 2 mm were observed.For P. aeruginosa, the zones of inhibition were 1.5, 1.5, 1, and 1 mm when exposed to different concentrations of Fe NPs.No activity was observed against C. sakazakii, while L. innocua showed 1.4, 1, 0, and 0 mm zones of inhibition when exposed to dilutions containing different concentrations of Fe NPs.Exposure of E. fecalis to different concentrations of Fe NPs showed zones of inhibition of 1.2, 1, 0, and 0 mm.The highest zone of inhibition (2.8 mm) was observed for E. coli at the 1:20 dilution of Fe NPs, while the lowest zone of inhibition was observed for L. innocua and E. fecalis at the same dilution.No activity was observed against C. sakazakii [30].
In addition, this study utilized XRD, EDX, and SEM to characterize the Fe NPs produced.The XRD analysis demonstrated the crystalline cubic structure, size, and purity of the Fe NPs derived from fungi.The XRD graph for Fe NPs exhibited the labeled peaks for Fe NPs, which were compared to those of other published studies and found to be in strong agreement.The purity and structure of the Fe NPs were determined to be "cubic crystalline" using the X-pert high score software, which was consistent with earlier investigations [31].Bragg's angle (ranging from 20°to 80°) was used to identify the phase composition and nature of the Fe NPs using the X-ray powder diffractometer.The presence of Fe in the nanopowder was confirmed by a series of reflection angles (2θ) at 65°and 75°w ith the cubic plane of iron-Fe, and the red squares indicated the presence of iron oxides.To confirm the presence of Fe NPs, EDX analysis was also conducted at ambient temperature, with peaks at different positions revealing the presence of different oxidation states, including ferrous and ferric, as well as zero-valent Fe NPs.The presence of iron oxide was also confirmed.Many earlier studies have reported the characterization of Fe NPs using the EDX technique [14].
Scanning electron micrographs were utilized to visualize the surface topology of the Fe NPs.The micrographs revealed that the synthesized NPs exhibited spherical shapes and agglomeration, which is a common phenomenon observed in NPs to achieve stability [23].The size of the spherical-shaped particles was approximately 150 nm, although particles with a size of 200 nm were also detected.A wide range of particle sizes, along with instability and agglomeration, is a major issue when dealing with NPs [24].The surface topology of the particles appeared rough with irregular boundaries and an asymmetric texture.In a previous study, the SEM technique was used to determine the forms, sizes, and structures of NPs, with Fe NPs being found to have a spherical round form and a size of 10-100 nm [32].Successful NP production was observed through the use of characterization techniques, and the synthesized Fe NPs were found to be highly effective against different microbial species.Numerous studies have explored various areas of biological synthesis of NPs and their applications, highlighting the significance and novelty of bio-based synthesized NPs as potential alternatives for traditional medical applications.These investigations shed light on the importance of utilizing biologically derived methods in NP synthesis, showcasing their potential as innovative options for medical purposes [33][34][35].

Conclusions
In conclusion, the use of NPs has become widespread in a variety of industries, including medicine, agriculture, pharmaceutics, cosmetics, and consumer goods.While NPs can be synthesized using either chemical or biological methods, biological or green synthesis is preferred as it allows for easy and large-scale production of NPs with desired characteristics.The current study highlights the potential of fungi isolated from metal contaminated sites for the synthesis of Fe NPs, which were analyzed using SEM, XRD, and UV-spectrophotometry.These biologically produced Fe NPs showed significant antimicrobial activity, making them effective in suppressing bacterial growth at high concentrations, while having no effect at lower quantities.These findings suggest that biologically synthesized Fe NPs could be a viable solution for combating resistant pathogenic strains.

Figure 6 :
Figure 6: Surface analysis of Fe nanopowder: (a) surface of FeO NPs and (b) shape and arrangement of Fe NPs.

Table 1 :
Synthesis of Fe NPs by isolated fungal strains