Bacillus proteolyticus UPMC1508: A novel bacterial strain capable of biologically synthesize iron oxide nanoparticles

Bacillus proteolyticus UPMC1508: A novel bacterial strain capable of biologically synthesize iron oxide nanoparticles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bacillus proteolyticus UPMC1508: A novel bacterial strain capable of biologically synthesize iron oxide nanoparticles Yusur Ramzi Hasan, Fadzlie Wong Faizal Wong, Murni Halim, Siti Efliza Ashari, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4974579/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2025 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract Antibiotic resistance is a growing concern due to the overuse of antibiotics. Alternative treatments, such as nanoparticles, are being explored. Biological synthesis of iron oxide nanoparticles (Fe 3 O 4 -NPs) via probiotics offers a sustainable and cost-effective method over the toxic chemical approaches, but there are challenges regarding its heavy metal resistance and the toxicity of the obtained nanoparticles. Thus, this research aims to biologically synthesize Fe₃O₄-NPs via a new bacterial isolate and evaluate its toxicity. The objectives are to isolate and characterize a novel bacterial isolate with probiotic potential. Then, to biologically synthesize and characterize Fe 3 O 4 -NPs via Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), Powder Diffraction Techniques (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), and nanosizer. Finally, to evaluate its cytotoxicity potential via MTT– (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. A total of 12 samples were collected from six different sites in Selangor, Malaysia. The 16s rRNA sequencing came closest to Bacillus proteolyticus UPMC1508 (99.87%). This strain has lower antibiotic resistance and high Fe-tolerance (MTC = 0.8 mg.mL − 1 ). It successfully synthesized Fe 3 O 4 -NPs, which exhibited absorption curves between 290–300 n m. TEM and FE-SEM indicated spherical formed Fe 3 O 4 -NPs; the average diameter was 5.12 ± 0.95 n m. Meanwhile, XRD peaks revealed that the grain size was around 32.61 n m. The nanosizer revealed a hydrodynamic diameter of around 104 n m with a good Polydispersity index (PDI) value (0.217). FT-IR indicated a satisfactory stability of Fe 3 O 4 -NPs after 2 months. Finally, Fe 3 O 4 -NPs showed low toxicity at 0.031 mg.mL − 1 .The findings revealed that the novel isolated B. proteolyticus UPMC1508 has high Fe-heavy metal tolerance and less antibiotic resistance. Furthermore, it successfully synthesized Fe 3 O 4 -NPs with satisfactory stability and safety, making them suitable for therapeutic platforms, such as antibacterial and anticancer. The significance of this study lies in offering an eco-friendly, low-cost synthesis approach while expanding the applicability of safe Fe 3 O 4 -NPs for biomedical applications. Metal oxide nanoparticles Bacillus proteolyticus green synthesis probiotic bacteria iron tolerance bacteria Figures Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Nanotechnology is a rapidly progressing field that focuses on producing diverse nanomaterials. The merge of Biotechnology and Nanotechnology led to the development of Bionanotechnology, which aims to create cost-effective and environmentally friendly nanotechnologies, including nanoparticles. Nanoparticles, between 1-100 n m, are highly versatile and widespread because of their numerous advantages and applications (Bouafia et al. 2021; Joudeh and Linke 2022). In several fields of human activity (e.g., Biology, Medicine, Electronics, Energy, Food Technology, and Aerospace Engineering), the distinct characteristics of nanoparticles that rely on their form and dimensions are crucial and have always been matters of interest (Yaqoob et al. 2020; de Jesus et al. 2021; Oliveira et al. 2024; Jeyaraj et al. 2019). Antibiotics are used to treat different infections. Still, due to the drug resistance phenomenon of many strains, acute diarrhea, chronic toxicity, and negative impact on intestinal microbes aligned with over usage of antibiotics, it becomes a challenge to use antibiotics and to be controlled. Therefore, finding an alternative, such as using nanoparticles, is more crucial than before (Kim, Covington, and Pamer 2017; Abdoli et al. 2022 ; Roy, Mutalik, and Dhas 2024; Saod et al. 2024). On the other hand, nanoparticles can be synthesized and stabilized using biological and non-biological methods involving two strategies (Top-down‌ and ‌Bottom-up). Although the non-biological methods, including chemical and physical processes, are commonly used to synthesize nanoparticles, numerous limitations and drawbacks must be considered, as they often require extreme operational conditions (e.g., high pressure and temperature), increasing costs, and environmental impact. Furthermore, they ended up with toxic byproducts, which have safety concerns and could limit the biocompatibility regarding the synthesized nanoparticles (de Jesus et al. 2024; de Jesus et al. 2021; Singh and Chandra 2024). Metal oxide nanoparticles have captured the interest in nanomaterials (Omorogbe et al. 2020 ; Ikhuoria et al. 2024). Iron oxide nanoparticles (Fe 3 O 4 -NPs) have paved the way among other widely studied metal oxide nanoparticles. Different forms of Fe 3 O 4 -NPs exist, including α-Fe 2 O 3 (hematite), β-Fe 2 O 3 , c-Fe 2 O 3 (maghemite), FeO (wurtzite), and Fe 3 O 4 (magnetite). Because of their magnetic properties, high versatility, biocompatibility, and less toxicity, they offer diverse applications in various fields, including imaging, environmental remediation, and particularly in medicine, which can trigger therapeutic activities, for instance, contrast agents in MRI (Rabani et al. 2023 ; Daramola et al. 2024), hypothermia treatment, and antimicrobial activity against pathogens such as fungi, bacteria, and viruses (Negrescu et al. 2022; Alphandéry 2020). Today, the green synthesis of Fe 3 O 4 -NPs via microbes is a desirable, cost-effective, and environmentally safe alternative to chemical methods. Microorganisms like bacteria, algae, fungi, and yeast can quickly cultivate and grow in ambient conditions (Rabani et al. 2023 ; Park, Lee, and Lee 2016; Jamzad and Kamari Bidkorpeh 2020). The mechanism of the formation of nanoparticles varies among microbes. Still, it typically involves trapping metal ions on the cell surface and reducing them to nanoparticles via their microbial enzymes, such as ferric reductases, for bioreduction of iron salt precursor (Zhang et al. 2013; Shinagawa 2011) (Fig. 1 ). Bacteria consume iron as an essential micronutrient for various biological processes, including Colonization, growth, and virulence. To obtain sufficient iron from the environment, bacteria have developed sophisticated mechanisms. One of the crucial mechanisms is the production of metabolites and enzymes that facilitate the bio-reduction of the elevated iron salt (Huo et al. 2021). In response to starvation or heavy metal pollution, many bacterial stains, such as Staphylococcus aureus and Streptococcus pyogenes , change their gene expression and induction of specific genes (Alnaimat 2017). Interestingly, studies revealed that wastewater contaminated with heavy metals contains bacterial strains that possess a remarkable ability to synthesize nanoparticles (Al-Tameemi et al. 2023 ; Ka-ot et al. 2018; Desai et al. 2023). While a recent study revealed that the iron-tolerant Bacillus species demonstrated a potential for a biogenic synthesis of Fe 3 O 4 -NPs with an average size of 81.3 n m (Walujkar et al. 2019). Similarly, another recent study reported that some Fe 3 O 4 -NPs-producing bacteria possessing ferric reductases for bio-reduction of iron salts, such as Sporosarcina luteola, Bacillus subtilis, Bacillus badius, Bacillus cereus, Bacillus tropicus, Klebsiella quasipneumoniae, Klebsiella africana , and Klebsiella pneumoniae with estimated nanoparticles’s size between 31–72 n m. Nonetheless, there is a lack in the investigation of novel bacterial strains that not only tolerate higher iron concentrations but also efficiently Synthesise of Fe₃O₄-NPs (Daramola et al. 2024). Furthermore, although correlations between heavy metal resistance and antibiotic resistance have been suggested, the mechanisms underpinning this relationship are not yet fully understood (Fu et al. 2023). This gap is particularly critical as the co-occurrence of heavy metal and antibiotic resistance in bacteria poses potential risks to public health when such microbes are used in nanoparticle synthesis and other applications (Gupta, Sreekrishnan, and Ahammad 2022; Baker-Austin et al. 2006; Gupta et al. 2023). Moreover, while green synthesis approaches align with the green chemistry principles, the applicability of many bacterial strains is constrained due to their potential to produce toxic compounds or exhibit antibiotic resistance (de Jesus et al. 2024). Addressing these gaps would would promote the exploration of new bacterial to be used in safer and more sustainable green synthesis approach. Recently, the use of probiotics in the green synthesis approach of nanoparticles for serving in different fields represents a significant candidate, as it can be grown under average temperature and pressure. Additionally, it can tolerate different pH ranges, which aids in forming more diverse and controlled nanoparticle sizes used for various applications (Al-Enazi et al. 2020 ; Abdoli et al. 2022 ). Numerous research reported the extracellular synthesis of Fe 3 O 4 -NPs using probiotics such as Bacillus cereus (Fatemi et al. 2018), Bacillus megaterium (Hajiali, Daneshjou and Daneshjoo 2022), Bacillus subtilis (Sundaram et al. 2012), Bacillus pasteurii (Daneshvar and Hosseini 2018), Bacillus licheniformis (Crespo et al. 2017), Bacillus circulans (Rabani et al. 2023 ), with different shapes (Irregular, spherical, and rhombohedral), with the average size ranges were between 12–97 n m. Interestingly, the size of Fe 3 O 4 -NPs is a crucial factor influencing their effectiveness in medical applications, especially as antimicrobial agents. Smaller nanoparticles have a higher surface area-to-volume ratio, enhancing their effectiveness (Kumar, Kumari, and Sahoo 2024). Although the use of probiotics in the green synthesis of nanoparticles offers many benefits, they may possess a metal tolerance gene (MTG) to tolerate such harsh environmental pollution that exists in nature, which is strongly related to the presence of Antibiotic resistance genes (ATGs), as they exist on the same plasmid and could lead to co-selection. Studies have suggested that this phenomenon is entirely associated with this type of metal (Desai et al. 2023; Alangari et al. 2022b; Giorgi et al. 2021; Pancholi and Caparon 2022). Accordingly, using probiotics in the green synthesis of nanoparticles is still in progress and involves many challenges. Furthermore, the toxicity of the synthesized Fe 3 O 4 -NPs and antimicrobial effectiveness need to be further explored (Abdoli et al. 2022 ). Addressing these challenges is crucial for the safe and effective application of new bacterial strains in the green synthesis of safe nanoparticles that can be used in medical and environmental contexts. B. proteolyticus is a member of the Bacillus genus that possesses an enzymatic activity, especially proteolysis. Their cell wall consists of a thick layer of peptidoglycan, a known characteristic of Gram-positive bacterial strains. These structures aid the biosorption and bio-reduction of metal ions because of their characteristics of negative electrokinetic potential. This attracts the metal cations to initiate the green synthesis of nanoparticles and protects them against metallic stress (Król et al. 2018; Mohd Yusof et al. 2020). Additionally, a recent research conducted by Zeng et al. (2021) revealed that B. proteolyticus isolated from yak in China possessed a probiotic potential with less antibiotic resistance. However, no research indicates that B. proteolyticus has been utilized to synthesize Fe 3 O 4 -NPs. Bacillus bacteria, known for producing potential natural products, can exist in various environments and have the necessary mechanisms to survive in harsh conditions, such as extreme heat, strong sunlight, and dryness (Hajiali et al. 2024). Therefore, it is possible to bring attention to utilizing of B. proteolyticus as a safe entity for biologically synthesizing Fe 3 O 4 -NPs due to the reasons outlined. The exploration of probiotics in green synthesis presents a promising alternative. Still, challenges remain regarding their metal tolerance, the associated antibiotic resistance potential, and the toxicity and size of the synthesized Fe 3 O 4 -NPs. This research attempts to address the need for novel and safer bacterial strains that have the potential to the green synthesis of Fe 3 O 4 -NPs with lower cytotoxicity potential. Materials and methods Sampling and physiological properties of the selected sites A total of 12 samples (either sediments or water samples) were collected from six different sites on the 10th and 11th of January 2022, from various aquatic habitats in Selangor, Malaysia (Kajang Pond, Mines South Lake, Tasik Seri Serdang, UKM University pond, Taman Tasik Sekyen 7, and Port Klang Sea) at 15 cm depth sediments (He et al. 2018). Each sample was transferred to a glass bottle (250 mL) and covered loosely. This was followed by incubation under dim light conditions at room temperature for one month. The proportion of sediments, sludge, and water is approximately ~ 1:3 (v/v) (Holland et al. 2008). Each sample's dissolved oxygen (D.O.) concentration was measured using a dissolved oxygen meter (Hach HQ10, Hach Company, Loveland, CO, USA). After storing and establishing the required measurements, the replicates of each sample were diluted (10 − 1 to 10 − 5 ). Isolation and characterization of bacterial isolate After observing the physiochemical properties of the samples, each replicate was diluted in 0.85% (w/v) NaCl from 10 − 1 to 10 − 5 . The desired isolates were cultured overnight via spreading of 1 µL of 10 − 3 and 10 − 4 dilutions on nutrient agar (HiMedia, India) supplemented with Iron (II) sulfate heptahydrate (FeSO 4 .7H 2 O) (HiMedia, India) (1.622 g.100 mL -1 ) as an iron source (Alnaimat 2017). Nutrient agar was prepared by mixing 0.5% peptone, 0.3% beef extract, 1.5% agar, 0.5% sodium chloride, and cytoplasm, with 1 L of distilled water, the pH was adjusted to be six at 37°C (MacFaddin 2000). Then, the grown isolates were purified by subculturing them on Luria agar media (HiMedia, India). Each of the successfully grown colony (24 h cultured) was picked up for enrichment by sub-culturing it on nutrient agar media without Iron supplement. The pH of the medium was adjusted to 6 via phosphate-buffered saline (PBS) (0.2 M of both KH 2 PO 4 and NaOH) (HiMedia, India), and the temperature was 37°C (Umeh and Enwuru 2014; Alnaimat 2017). The bacterial colonies were successfully grown, enriched, and purified via nutrient agar were collected to be characterized and identified. Pure isolate was physiologically identified according to Bergey's Manual (Garrity et al. 2005). Gram staining, catalase and oxidase tests, lactose fermentation, Indole, and methyl red tests were investigated. Additionally, the selected bacteria were stored in Luria broth (HiMedia, India) that contained 50% glycerol (Sigma-Aldrich) at -80°C for further investigations. The16S rRNA sequencing was carried out for the selected isolate, and the genomic DNA was extracted via DNA Extraction Kit (BioFlux, USA). The process involved using a forward primer, a 20-base DNA sequence ( 5'-AGA GTT TGA TCC TGG CTC AG-3' ), and a reverse primer, a 22-base DNA sequence ( 5'-TAC GGT TAC CTT GTT ACG ACT T-3' ) (Nezhad et al. 2023). These primers are in a 25 nmol scale, with a standard desalting process, a minimum process, and a minimum yield of 10 nmol, ensuring the necessary genetic material for amplification. The polymerase chain reaction (PCR) amplification was carried out at the following temperature parameters: 3 min for an initial denaturation phase at 95°C, then 30 s for 30 cycles at 95°C, 30 s at 52°C, and 1.30 min at 72°C for denaturation, annealing, and extension, respectively. A last extension step was performed for 5 min at 72°C. After the purification of PCR amplicons (Purification Kit, Qiagen, Germany), the sequencing was then performed at Apical Scientific Sdn Bhd in Malaysia. For checking the similarity, The Gen-bank database was used to compare the sequences of the 16 S rRNA gene for the desired strain through the Basic Local Alignment Search Tool (BLAST) of NCBI ( http://www.ncbi.nlm.nih.gov/ ) (Nezhad et al. 2023). The sequence alignment was retrieved and aligned through ClustalW. Then, a phylogenetic tree was performed via the MEGA-X software version 10.2.6 through the Maximum likelihood algorithm method (Kumar et al. 2018). Antibiotic susceptibility test An antibiotic sensitivity test is usually performed to assess the safety of B. proteolyticus UPMC1508. The disc diffusion method was done in triplicates and according to the National Committee for Clinical Laboratory Standards (NCCLS 1997) (Wayne 2002). To measure the turbidity, the suspension was matched with a 0.5 MacFarland standard (Zeng et al. 2021). Then, the suspension was evenly spread onto Luria agar plates using a sterile cotton swab. To evaluate antibiotic sensitivity, 11 drug-sensitive discs (Thermo Scientific, Oxoid) were utilized, including Ampicillin (10 µg/disk), tetracycline (30 µg/disk), gentamicin (10 µg/disk), cefalexin (30 µg/disk), chloramphenicol (30 µg/disk), erythromycin (15 µg/disk), vancomycin (30 µg/disk), rifampin (5 µg/disk), streptomycin (10 µg/disk), kanamycin (30 µg/disk). Following incubating for 24 h at 37°C, the diameters of the inhibition zones were measured. A zone of inhibition less than 0.5 cm indicates resistance, whereas a zone more than 0.5 cm in diameter indicates a sensitive result (McDermott et al. 2005). Fe-heavy metal tolerance: Minimum tolerance concentration (MTC), Minimum inhibitory concentration (MIC), and Minimum bactericidal concentration (MBC) The MTC of Fe heavy metal against the B. proteolyticus UPMC1508 was determined via the spot plate method. The FeSO 4 .7H 2 O was prepared in different concentrations (0.1, 0.2, 0.4, 0.8, 1, 2, and 4 mg.mL -1 ). About 10 µl of bacterial cultures (24 h cultured) corresponding to 0.5 McFarland standards were added to Müller-Hunton-Agar (MHA) (HiMedia, India) adjusted to pH 5, containing the FeSO 4 .7H 2 O concentrations and then incubated for 72 h. at 37°C. MHA was prepared by mixing beef extract (2.00 g. L -1 ), hydrolysate of casein (17.50 g. L -1 ), starch (1.50 g. L -1 ), and agar (17 g. L -1 ) with 1 L of D.W. The mixture was stirred to be fully dissolved, then autoclaved at 121°C for about 15 min. After cooling down, MHA was poured into petri dishes to be solidified. MTC is the maximum concentration of heavy metal that bacteria can withstand without substantial interference to its growth. MIC is the lowest concentration of metals that inhibit the growth of bacteria, and MBC is the lowest concentration of metals that completely inhibits the growth of visible bacteria after 72 h (Ka-ot et al. 2018; Rathnayake et al. 2009). Preparation of extracellular supernatant The biological synthesis of Fe 3 O 4 -NPs via the extracellular approach started with culturing the B. proteolyticus UPMC1508 in 250 mL nutrient broth medium, and the pH of the media was maintained via PBS (0.2 M, pH 6). After that, the culture was incubated for 34 h at 37°C and agitated at 150 rpm. Then, the bacterium grown was purified and separated from the bacterial biomass through centrifugation at 6000 rpm for 15–20 min. To prevent any predicted contamination, there is a need to ensure the absence of bacterial cells in the supernatant by passing the supernatant through a 0.2 µm syringe filter. Finally, the filtered supernatant was ready for biological synthesis of Fe 3 O 4 -NPs (Rabani et al. 2023 ; Fatemi et al. 2018; Jagathesan et al. 2018; Jubran et al. 2020). Biosynthesis of FeO-NPs The filtered supernatant of the 18 h bacterial broth was mixed with FeCl 3 ·6H 2 O solution so that the iron salt concentration was 5 mM in a 1:1 volume ratio to the supernatant, followed by magnetic stirring (80 rpm) for 5 min. The adjustment of the incubation conditions (pH ranges and temperatures) was done during this step. After the incubation of the mixture for 5 days at 37ºC incubator shaker (170 rpm) in a dark condition, a color change from dark yellow to dark brown was detected. For separation of the dark-obtained precipitate from the supernatant solution to which salt had been added, the solution was through a 0.2 µm sterile syringe filter, followed by centrifugation at 8000 rpm. Next, the obtained residue was washed 5 times with ethanol 97% (HiMedia, India) and distilled water. Note that each washing step is accompanied by centrifugation at 4000 rpm except the first step, which was done at 8000 rpm to allow the small nanoparticles to be precipitated and washed. Finally, the residue was dried in an oven at 80°C for 24 h. Then, the obtained powder was stored to be characterized (Rabani et al. 2023 ; Fatemi et al. 2018; Sundaram et al. 2012; Jagathesan et al. 2018; Jubran et al. 2020). Characterization of the biosynthesized Fe 3 O 4 -NPs Identifying the oxide phases of iron through characterization instruments can be challenging, as a single tool may not provide accurate results. However, this challenge can be overcome by employing various characterization techniques. UV–Vis spectrophotometer analysis (Thermo Scientific, USA) was performed at 200– 800 n m wavelength to detect the presence of Fe 3 O 4 -NPs. Transmission electron microscopy (TEM) (JEM-2100 F, Japan) demonstrates the shape, size, and distribution, considering the van der Waals forces acting on the samples which play a vital role in aggregation. The Field emission scanning electron microscopy (FE-SEM) (Eindhoven, The Netherlands) illustrates the topography and characteristics of a sample's surface, such as morphology, size, shape, and particle stationing of the Fe 3 O 4 -NPs were recorded over the range of 600-4,000 cm − 1 on a model spectrum 100 series. The FT-IR (Vector 22, Bruker from Germany) analyses the vibration of atoms within molecules when exposed to infrared radiation. This analysis provides valuable information about the sample's radiation absorption. The Fourier-transform infrared (FT-IR) spectroscopy analyses the vibration of atoms within molecules when exposed to IR. This provides valuable information by analyzing the amount and quality of radiation absorbed by a sample. To assess the crystalline arrangement of the Fe 3 O 4 -NPs sample, the XRD (Oxford, USA). The nanosizer (DLS, Nano-ZS, UK) is an advanced tool that can detect aggregates and analyze the size of small or diluted samples, even at very low or high concentrations. It works by using dynamic light scattering with 'NIBS' optics, making it highly accurate and reliable as a molecular size analyzer (Walujkar et al. 2019; Sundaram et al. 2012; Mourdikoudis et al. 2018). Cytotoxicity assay This assay test was done at the National Institutes of Biotechnology Malaysia (NIBM), Selangor, Malaysia. To determine whether Fe 3 O 4 -NPs are cytotoxic, the fibroblast cell line (L-929) was exposed to MTT solution (Sigma-Aldrich) in-vitro. The Fe 3 O 4 -NPs powder was mixed with minimum essential media and subjected to 45 W probe sonication for 5 min, with 3 seconds switched-on and 3 seconds switched-off intervals. Next, the suspension of biosynthesized Fe 3 O 4 -NPs was filtered using 0.2 µm syringe filters. Then, Fe 3 O 4 -NPs biosynthesized were prepared from 0.0312 to 1 mg.mL -1 . A quantity of 1 × 10 4 cells/well (100 µL per well) in ). Minimum Essential Medium (MEM) (Sigma-Aldrich) was added to 96-well plates (Tecan Infinite M200, Switzerland) and incubated at 37°C for 22 h with 5% CO 2 . Cells were then treated with Fe 3 O 4 -NPs (0.031, 0.063, 0.125, 0.25,0.50, and 1 mg. mL -1 ) for 24 h after removing the medium. After that, 50 µL of MTT was added to MEM then inculcated into each well and allowed to incubate for another 2 h at 37°C. Next, the MTT solution and 100 mL of isopropanol were added to each well, and the cells were incubated for 30 min. A reference wavelength of 650 n m was used, and 570 n m was used as a detector absorbance (Naskar and Kim 2019). The lower viability value means higher cytotoxicity, and if the viability % is reduced to less than 70%, The lower the Viability % value, the higher the cytotoxic potential. If viability is reduced to < 70% of the blank, it has a cytotoxic potential. The formula used to calculate cell viability is as follows: $$\:Cell\:viability\:\%=\frac{{OD}_{570e}\times\:100}{{OD}_{570b}}$$ OD 570e represents the mean value of the measured optical density of the 100% Fe 3 O 4 -NPs, OD 570b represents the mean value of the measured optical density of the blanks. Statistical analysis The absorbance data were plotted as scattered line using Prism software (version 10.1.1, 270; GraphPad Software, San Diego, CA, USA). The phylogenetic tree was performed using MegaX software (version 10.2.6; Pennsylvania State University, USA). While ImageJ software (version 1.53t; National Institutes of Health, Bethesda, MD, USA) was used to calculate the average of nanoparticle sizes. Mean ± standard deviation (SD) was presented to report the experimental results. One-way ANOVA was conducted to examine the distinctions in Fe 3 O 4 -NP concentrations, with statistical significance set at a p -value of ≤ 0.05. Results and discussion The physiological properties of the collected samples and their sites The collected samples were observed for their different physiological parameters (pH, dissolved oxygen, latitude and longitude, and color), as shown in Table 1. The pH values were between 5.5 and 6.5, and the temperature was 27–30°C. The D.O. for most of the sites was around 8 at the initial reading (8.9 mg. L -1 ), except for Tasik Seri Serdang, and Taman Tasik Sekyen, Port Klang Sea, which recorded 7 mg. L -1 . Moreover, most of the ponds exhibited a dark brown color except Mines South Lake and Port Klang Sea, which exhibited a blue color. In a recent study conducted by Hanafiah et al. (2024) in Selangor, Malaysia, it was revealed that this dark brown color is caused by both inorganic and organic species present in the water. The study further found that the water had high concentrations of heavy metals, particularly iron (Fe) (3.2422 ± 0.2533 mg. L -1 ), accompanied by a low pH value (5.49 ± 0.1), which was believed to be the primary factor contributing to the dark brownish appearance of the water. Table 1 The parameters of the sampling sites. Sample location Sample type pH (Means ± SD) Color D.O. (mg. L − 1 ). The initial reading is 8.9 (Means ± SD) Temperature (°C) (Means ± SD) Latitude and Longitude Kajang Pond Water and Sediments 5.5 ± 0.09 Brown 8.±0.07 28 ± 0.07 Lat: 3.0064840922287392, Lng: 101.79062437266111 Mines South Lake Water 5.8 ± 0.30 Blue 8.±0.07 28 ± 0.30 Lat: 3.0344300427619473, Lng: 101.71137414872646 Tasik Seri Serdang Water and Sediments 5.4 ± 0.16 Greenish blue 7 ± 0.14 28 ± 0.40 Lat: 16.351375, Lng: 81.09178639999999 UKM University Pond Water and Sediments 5.9 ± 0.40 Brown 8 ± 0.07 29 ± 0.40 Lat: 2.9289975999999998, Lng: 101.7800219 Taman Tasik Sekyen 7 Water 5.6 ± 0.10 Blue 7 ± 0.030 27 ± 0.30 Lat: 3.01032240067449, Lng: 101.60078667104244 Port Klang Sea, Water 6.5 ± 0.40 Blue 8 ± 0.42 29 ± 0.10 Lat: 3.0048405, Lng: 101.3623373 Isolation and characterization of bacterial strain For screening and purification of the desired isolates, suitable dilutions (10 − 3 and 10 − 4 ) of sample were cultured on nutrient agar media as it is equal to 0.5 McFarland standard. The results showed that most of the selected aquatic sites in Malaysia contained strains suspected to be Iron-tolerance isolates. Although nutrient agar can be considered a versatile medium which supports the growth of a wide range of microorganisms, the growth results of the inocula of the collected samples witnessed a significant disparity because of an iron source (FeSO 4 .7H 2 O). To illustrate this, most of the collected samples (Mines South Lake, Tasik Seri Serdang, UKM University Pond) showed successful bacterial growth on nutrient agar supplemented with FeSO 4 .7H 2 O (1.6 g.L -1 ). In contrast, some of sites (Kajang Pond, Taman Tasik Sekyen 7, and Port Klang Sea) showed negative results of bacterial growth during incubation. Following the purification and enrichment steps, the number of pure isolates decreased as the vitality of many of these isolates was considered. Hence, only the suspected probiotic bacterial strain that produced a pale yellowish colony on Luria agar and nutrient Agar medium was subjected to 16S rRNA sequencing for further investigations that will be illustrated subsequently. Among the isolates, the appearance of YRU 3 (24 h cultured) on nutrient agar was in the form of pale yellowish colonies as in (Fig. 2 -a and b), while on nutrient agar, which was supplemented with an iron source, as dark brown colonies (Fig. 2 -c). The colour appearance of bacterial colonies can be affected by the type of agar used and the addition of supplements. In this case, the brown colour changes when bacteria are cultured on nutrient agar supplemented with iron source can be explained by multiple factors; for instance, iron utilization by bacteria, bacterial metabolism, and pigment production was identified by Hegler et al. (2008). These are spheroidene, spheroid, and OH-spheroid, as well as reactions involving iron. Furthermore, YRU3 was a gram-positive, rod-shaped bacteria with negative oxidation and positive catalytic reactions. Additionally, the results were negative for lactose fermentation, Indole, and Methyl red, whereas the starch hydrolysis was positive (Table 2 ). Thus, this strain was chosen for the 16s rRNA sequencing. The results of the purified DNA are revealed in agarose gel electrophoresis (Fig. 3 -a). Based on the blast analysis of the phylogenetic tree via MEGA 7 software, as well as the neighbouring joining methods for the 16s rRNA sequencing with the National Centre for Biotechnology (NCBI) database and the biochemical characteristics, the results revealed that the strain (YRU3) bears the strongest similarity to B. proteolytic (99.87%) (Fig. 3 -b), and species commonly found in soil. This specific strain, named YRU 3, has been duly documented in the NCBI database with the GenBank accession number OQ27138 and the original number UPMC1508 (Table 2 ). Probiotic bacteria, in general, have gotten a lot of attention due to their characteristics and contributions in various applications, for instance, medical, industrial, and agricultural applications (Khan et al. 2020; Rehaiem et al. 2014; El-Nour et al. 2023; Mohammed et al. 2023; Vijayakumar et al. 2023). Table 2 Identification and characterization of Bacillus. proteolyticus UPMC1508. Morphological characteristics Biochemical characteristics Gen-Bank Accession No Original code The similarity (%) according to the NCBI Sites of isolate Gram staining and shape Oxidase Catalysis Starch hydrolysis Lactose fermentation Indole Methyl red OQ27138 UPMC1508 Bacillus proteolyticus Tasik Seri Serdang, Selangor, Malaysia Gm+, rode shape - + + - - - Antibiotic susceptibility assay and Fe-tolerance potential The B. proteolyticus UPMC1508 isolate was tested for antibiotic sensitivity via agar diffusion. It was conducted in triplicate to ensure the reliability and reproducibility of the results. The overnight cultured broth (1×10 8 CFU.mL -1 ) was spread evenly on Luria agar and incubated for 24 h at 37°C. After incubation, the results showed that the isolated strain has lower antibiotic resistance, as illustrated in Table 3 and Fig. 4 , indicated by the appearance of clear zones above 0.5 cm around each disk except for Ampicillin, and erythromycin. On the other hand, this isolate showed resistance to Fe heavy metal when it was inoculated on MHA containing different iron concentrations of FeSO 4 .7H 2 O (0.1, 0.2, 0.4, 0.8, 1, 2, and 4 mg.mL -1 ). After 72 h. of incubation, the MIC and MTC recorded 0.8 mg. mL -1 and the MBC was 1 mg. mL -1 . Table 3 The antibiotic discs and their zone of inhibition (Mean + SD) against B. proteolyticus UPMC1508 on MHA plates ( P- value < 0.05). Antibiotics Concentration (µg/disk) Zone of inhibition (cm) (Mean ± SD), n = 3 Ampicillin 10 0.30 ± 0.02 Cefalexin 30 1.75 ± 0.08 Chloramphenicol 30 2.67 ± 0.03 Erythromycin 15 0.40 ± 0.02 Gentamicin 10 1.69 ± 0.23 Kanamycin 30 2.15 ± 0.05 Rifampin 05 1.69 ± 0.03 Streptomycin 10 0.59 ± 0.04 Tetracycline 30 2.48 ± 0.05 Vancomycin 30 2.66 ± 0.03 Previous studies have shown that the antibiotic resistance potential can be shared among groups of microorganisms, and even some probiotic bacteria may have antibiotic resistance genes (ATGs) (Li et al. 2019). Therefore, the safety of the selected bacteria for various applications is crucial because they can develop resistance to antibiotics, and the presence of transferable ATGs raises concerns about bacterial safety (Zavišić et al. 2023). The results of this study are consistent with previous research conducted by Fiedler et al. (2019), which revealed that the probiotic strain of Bacillus cereus was vulnerable to Erythromycin, Cefalexin, Gentamycin, Tetracycline, and Chloramphenicol. Another study by Ataikiru et al. (2020), showed that B. cereus was resistant to Streptomycin, Amoxil, Amplicon, and lactamase antibiotics. Another survey by Zeng et al. (2021) revealed that B. proteyolyticus has lower resistance ability toward different antibiotics (Tetracycline, gentamycin, Cefalexin, Enrofloxacin, Chloramphenicol, Chloramphenicol, and Cefazolin) except for Ampicillin, Norfloxacin, Cefazolin, Rifampin, and Lincomycin. B. proteolyticus UPMC1508 can be resistant to a few certain antibiotics because of different genetic mechanisms, the strength of the antibiotics, and their concentration (Pure). Regarding the Fe heavy metal resistance, B. proteyolyticus UPMC1508 showed a resistance potential against Fe heavy metal. Recent studies revealed that if the bacterial strain could grow at the Fe concentration 1 mM, it has iron resistance potential. Thus, it could efficiently synthesize iron or Fe 3 O 4 -NPs (Desai et al. 2023; Walujkar et al. 2019). Bacteria have developed complex mechanisms to obtain iron from the surrounding environment due to the necessity of iron in different biological processes (e.g., Colonization, growth, and virulence factors). These mechanisms include the production of siderophores, hemophores, or enzymes, such as iron reductase ( fhu ) and cysteine desulfurase ( suf ) (Huo et al. 2021). A recent study by Daramola et al. (2024), explored the potential of some bacteria isolated from soil samples collected from a metal fabricating workshop. Novel species of Klebsiella and Bacillus possess physiological and genomic adaptations that enable them to thrive in iron-rich environments and contribute to the extracellular synthesis of iron oxide nanoparticles. However, the findings also revealed that each bacterial strain's iron uptake, bio-reduction, and iron tolerance potentials vary. Overall, this previous study highlighted the importance of iron metabolism, which could facilitate the synthesis of Fe 3 O 4 -NPs and support nano-bioremediation in natural environments. Another study successfully demonstrated using iron-tolerant Bacillus species for visible light active and green synthesis of Fe 3 O 4 -NPs (Walujkar et al. 2019). However, natural environment contamination with heavy metals provides selection pressure for antibiotic-resistant bacteria, increasing environmental contamination. These bacteria have developed complex mechanisms to obtain iron from the surrounding environment and resist harsh conditions. Many Pathogenic Gram-positive bacteria (e.g., Proteobacteria and Bacteroidetes ) possess such mechanisms (Fu et al. 2023; Ding et al. 2019). Concerning the probiotic bacterial strains in the natural environment, they may have a metal tolerance ability to withstand the possible pollution with heavy metals (Sajjad et al. 2024). Fortunately, B. proteyolyticus UPMC1508 showed lower antibiotic resistance accompanied with high iron resistance potential. This agrees with the hypothesis suggests that this phenomenon is entirely associated with this type of metal (Desai et al. 2023; Alangari et al. 2022; Giorgi et al. 2021; Pancholi and Caparon 2022). Biosynthesis mechanism of magnetic Fe 3 O 4 -NPs The external synthesis of IONPs from the bacterial supernatant involved adding Iron (III) chloride hexahydrate (FeCl 3 .6H 2 O) (HiMedia, India )to the filtered supernatant of B. proteolyticus UPMC1508 strain, which was cultured in Nutrient broth medium for 18 hrs. This was done in a dark condition and under a magnetic stirrer until the concentration of FeCl 3 .6H 2 O in the supernatant reached 5 mM. After five days of incubation, the absorption spectrum of the brown suspension was measured relative to that of the salt-free supernatant using UV-vis spectroscopy, as shown in Fig. 5 -a. The presence of an index peak in the range of 290–300 n m confirmed the existence of iron oxide nanoparticles, and this result is aligned with previous studies (Sundaram, Augustine, and Kannan 2012; Fiedler et al. 2019; Rabani et al. 2023 ; Saod et al. 2024). Adding a salt solution causes the clear yellow supernatant to become a dark brown suspension (Fig. 5 -b ), possibly due to the interaction between metal ions and soluble enzymes secreted by microbes (Khan et al. 2020). Many approaches available for the synthesis of metal nanoparticles, predominantly involving chemical methods such as chemical reduction and co-precipitation, alongside physical methods include ultrasonic irradiation, sol-gel, and hydrothermal processes (Lester et al. 2006; Alshehri et al. 2017). Nonetheless, these procedures can be expensive, not easily accessible, and may generate harmful byproducts, in addition to necessitating complex technology in certain instances (Desai et al. 2023). A recent study highlighted the excellent antioxidant and antimicrobial activities of Fe 3 O 4 -NPs synthesized via the green synthesis over other methods (Abdullah et al. 2023 ). Various biological entities, including bacteria, have been employed in producing metal oxide nanoparticles due to their rich biomolecule contents. These biomolecules aid in the bio-reduction, stabilization, and capping of nanoparticles. Although the underlying mechanism of the bio-reduction of the metal ions of the precursor salts via these biomolecules is still not confirmed yet, varieties of scientific reports have assumed that the surface chemistry represented by the functional groups (e.g., -CHO, -C = O, -O.H., -COOH, -C = C, -C-O-C) play roles in the bio-reduction process of metal ions (Priya et al. 2021; Jacinto et al. 2021). The magnetite Fe 3 O 4 -NPs show superparamagnetic behavior at room temperature with dimensions of < 6–20 n m, which makes them have a larger surface area surface-to-volume ratio. However, the magnetic properties mainly rely on the methods of their synthesis (Kumar, Kumar, and Singh 2021; Tanvir et al. 2023; Ali et al. 2016). Because of their superparamagnetic behavior, iron oxide-NPs are utilized in different biomedical applications. A recent study demonstrated that the Fe 3 O 4 -NPs synthesized by Annona muricata , with a particle size of 4.19 n m, had significant antibacterial properties and can function as an anticancer drug against HeLa carcinoma, with I.C. 50 of 48.97 µg.mL -1 (Elemike et al. 2024). Another study revealed that the biologically synthesized Fe 3 O 4 -NPs by Bacillus megaterium possessed antibacterial properties on Escherichia coli and Bacillus cereus greater than the common antibiotics used (Hajiali et al. 2024). Other studies demonstrated the peroxidase-like activity (POD) of Fe 3 O 4 -NPs (Dong et al. 2022), and Fe 3 O 4 nanozyme with enhanced antimicrobial activity for disinfection treatment (Wei et al. 2021) and for removal of organic dyes (Zha et al. 2022). However, evaluating their toxicity is important to ensure their safety usage (Tanvir et al. 2023). Morphology and size distribution of Fe 3 O 4 -NPs TEM was conducted to perform the morphological characterization of the biosynthesized Fe 3 O 4 -NPs. The TEM images (Fig. 6 ) showed that the Fe 3 O 4 -NPs had a spherical shape with few aggregations. Additionally, the distribution curve displaying particle sizes for Fe 3 O 4 -NPs demonstrated that the average diameter of these nanoparticles was 5.12 ± 0.95 n m ( n = 63) when it was analyzed via ImageJ software as in Fig. 7 -a. The current study closely aligns with the previous research conducted by Jubran et al. (2020), and Abdullah et al. ( 2020 ), which found that Fe 3 O 4 -NPs were distributed uniformly, with an average diameter of approximately 12.61 and 3.6 ~ 7 n m, respectively. In contrast, a study conducted by Desai et al. (2023), and Alangari et al. ( 2022a ) showed the formation of spherical Iron oxide-NPs, with an average size ranging from 70 to 100 n m and 20 n m, respectively. On the other hand, the nanosizer measures the hydrodynamic size of Fe 3 O 4 -NPs in a liquid medium by analyzing the Brownian motion of particles and their electrophoretic mobility. This technique calculates the size based on the diffusion coefficient of particles in the liquid, including the particle core and any surrounding solvent layers. Figure 7 -b reveals a hydrodynamic diameter of approximately 104 n m. The Polydispersity index (PDI) was 0.217, and the SD was 61.81, indicating good quality. When it comes to measuring the size of nanoparticles via different instruments, the size recorded by a nanosizer might seem higher than the size obtained by TEM because of the dissimilarities in the principles and limitations of these two methods. Regarding the limitations, nanosizer tends to provide an average hydrodynamic diameter, including any solvent or surface layers around the nanoparticles. These layers can add to the measured size and make the nanoparticles appear more significant than their actual core size. Additionally, when it comes to the concentrations of nanoparticle suspensions, the nanosizer detects an apparent and progressive increase in size, reducing the accuracy of measurements because nanoparticles' diffusion behavior deviates from theoretical predictions in low concentrations of nanoparticles. Contrary to TEM, which may not account for any solvent or surface layers around nanoparticles, TEM focuses primarily on core size (Giorgi et al. 2021). In many cases, TEM measurements are often considered the most accurate way to determine nanoparticle size. Some aggregations were observed in the TEM images, which is consistent with existing literature suggesting that these metal oxide nanoparticles tend to aggregate. This aggregation may be due to the high surface energy between the magnetic nanoparticles and the presence of magnetic dipole-dipole interactions, which can form larger particles (Tyagi et al. 2023; Yusefi et al. 2021). For biomedical applications, Fe 3 O 4 -NPs need to meet specific criteria as they should have strong magnetic properties, be uncapped, and be no larger than 20 n m in size (Tyagi et al. 2023). These requirements are in line with the TEM results of the current study. However, the control over magnetic properties and particle size distributions can be achieved by adjusting the synthesis conditions (pH, ionic strength, salt concentration, temperature, etc.) in the presence of capping agents that are already provided by the green synthesis approach (Oehlsen et al. 2022; Vangijzegem et al. 2023). The FE-SEM was used to analyze the surface morphology of Fe 3 O 4 -NPs synthesized by B. proteolyticus UPMC1508. The images obtained using FE-SEM showed that the nanoparticles appeared to be spherical, with few aggregates, as in Fig. 8 . As a result, these aggregations form irregular clusters due to the capping agents covering the nanoparticle's surface. Research studies support the same conclusion, indicating that Fe 3 O 4 -NPs possess a predominantly spherical form with few aggregations, potentially resulting from biological substance (Abdullah et al. 2020 ; Elemike et al. 2024). The XRD analysis The XRD patterns of the unadorned Fe 3 O 4 -NPs displayed distinct peaks at specific angles (2θ) of 32.28° (222), 35.5° (311), 54.1°(422), 62.47° (440), 57.5°(511), 57.07°(511), and 63.16°(440), indicating a cubic spinel structure for the magnetite (Fe 3 O 4 ) phase. The Fe 3 O4-NPs in Fig. 9 exhibit a composition of magnetite (spinel-phase iron oxide), wüstite (FeO), and hematite (α-Fe 2 O 3 ), which are likely formed through the surface oxidation of alpha iron. It should be noted that distinguishing between the different iron-oxide phases and determining their oxidation state with absolute certainty is challenging using XRD, given the similarities in space groups, lattice constants, and significant peak overlap among these phases (Liu et al. 2006; Min et al. 2011; Alzoubi et al. 2023; Tyagi et al. 2023). Furthermore, the Debye-Scherrer equation determined the grain size, establishing a relationship between XRD peak broadening and particle size. The average crystallite sizes of the Fe 3 O 4 -NPs were found to be in the range of 32.611 ± 1.4119 n m. Despite being below 100 n m, the average size of nanoparticles appears larger when measured via XRD compared to TEM. This disparity can be attributed to XRD measuring the average size of crystalline domains, which may include contributions from agglomerated nanoparticles. Assess the stability of Fe 3 O 4 -NPs via FT-IR spectra FT-IR analysis was performed to qualitatively determine the chemical composition, functional groups, and the possible interactions between nanoparticles and functional groups and evaluate the stability of The Fe 3 O 4 -NPs. To do this, the I.R. spectrum was obtained with the Fourier transform in the range of 600–4000 cm − 1 using the KBr pellet method. Figure 10 displays the FT-IR spectra of Fe 3 O 4 -NPs (Black line) and the same sample after two months (Red line). Both results show a noticeable peak between the 600–800 cm -1 region, indicating the stretching vibration of Fe-O bonds in Fe 3 O 4 -NPs (Abdullah et al. 2020 ; Braim et al. 2023; Walujkar et al. 2019). Interestingly, this peak undergoes slight changes after two months, becoming more extended and broader, with a slight rise in transmittance but with no observed shifts in peak position. A possible structural alteration in Fe 3 O 4 -NPs could have occurred due to a variation in the length of Fe-O bonds. This could be linked to alterations in the coordination environment surrounding the iron ions or the crystal structure and phase adjustments. Additionally, the FT-IR spectra of nanoparticles can be influenced by the aggregation state; aggregated or agglomerated particles might display distinct vibrational modes compared to well-dispersed particles. As illustrated in Table 3 and Fig. 10 , the FT-IR analysis revealed that both results exhibited broad peaks that may associated with the hydroxyl group (O–H vibration), which can be derived from alcohol phytochemicals (Gupta et al. ; Win et al. 2021; Daramola et al. 2024). A slight increase in value from 3377.90 to 3379.06 in the I.R. spectrum was detected. This could be due to the number of hydroxyl groups and their stretching in the iron oxide nanoparticles, which have been reduced after 2 months (Triastuti and Airlangga 2024 ). The C ≡ N stretching vibration that could be related to 2135.85 cm -1 regions shifted to a lower value (2128.17 cm -1 ) after 2 months. This indicated that only a lone pair of electrons on the nitrogen atom formed a coordinated bond with iron ions. However, this group has a role as stabilizers and capping agents in the development of some properties of nanoparticles, such as catalytic, magnetic, and optical properties. (El-Attar et al. 2023; Forge et al. 2011). The FTIR spectrum captured almost no alteration in the absorption bands at 1641.33-1641.06 cm -1 . These bands could be attributed to the C = O stretching vibrations of amide I groups of extracellular proteins that could be involved as capping and reducing agents (Walujkar et al. 2019; Yusefi et al. 2021). Similarly, almost no alternation was detected at 1078.30-1078.29 cm -1 after 2 months. These specific bands may related to symmetric C–O vibration that may have roles as capping agents on the nanoparticles' surface as a result of the biological synthesis via bacteria, which is in agreement with previous study used species of Proteus vulgaris ATCC-29905, Klebsiella , and Bacillus as entities for Fe 3 O 4 -NPs synthesis (Khan et al. 2022; Majeed et al. 2021; Daramola et al. 2024; Walujkar et al. 2019). A study conducted by Majeed et al. (2021) revealed that the FT-IR can be used to check the bonding and chemical stability of the biologically synthesized Fe 3 O 4 -NPs after 2 months by observing the chemical bonding to confirm the stability of nanoparticles. Since slight or no changes were detected in all the obtained peaks of Fe 3 O 4 -NPs, this indicates their stability after 2 months. In biomedical applications, the efficiency of nanoparticles is affected by their specific biomolecules or proteins, which enhance their targeting capacity, biocompatibility, and therapeutic efficacy. FT-IR can detect the changes in the composition of these biomolecules by determining the changes in functional groups, enabling a successful assessment of the potential of the synthesized nanoparticles (Eid 2022 ). Additionally, another study revealed that FT-IR results indicate that the fruit peel extract of Garcinia mangostana successfully served as a stabilizer and capping agent during the green synthesis approach of the Fe 3 O 4 -NPs (Yusefi et al. 2021). Table 4 The functional groups and their relative peaks for Fe 3 O 4 -NPs before and after two months. Assignment Relative peak (cm − 1 ) Fe 3 O 4 -NPs Fe 3 O 4 -NPs after two months O–H 3377.90 3379.06 C ≡ N 2135.85 2128.17 C = O 1641.33 1641.06 C–O 1078.30 1078.29 Fe–O 699.46 710.33 In Vitro cytotoxicity assay of Fe 3 O 4 -NPs The cytotoxicity of the biologically synthesized Fe 3 O 4 -NPs was in vitro evaluated against L-929 fibroblast cell line (Sigma-Aldrich) at different Fe 3 O 4 -NPs concentrations (0.031, 0.063, 0.125, 0.25,0.50, and 1 mg. mL -1 ). The cell viability of L-929 was 76.13% at 0.031, whereas the minimum cell viability (34.98%) was at the highest concentration (1 mg. mL -1 ) (Fig. 11 -a). This study showed that when the concentration of nanoparticles increases, the cell viability decreases, as shown in Fig. 11 -b. This is in agreement with previous research conducted by Anuje et al. (2021), who illustrated that the cell viability percentages of Fe 3 O 4 -NPs against L-292 were 75.26%, 68.04%, 59.8%, and 55.67%, and for 0.15, 0.2, and 0.25 mg.mL -1 , respectively. Another study showed that the cell viability of L-929, when treated with 0.032 mg.mL -1 Fe 3 O 4 -NPs, was about 70%, whereas the highest concentrations showed the highest cell viability (Minaeva et al. 2017 ). This indicated the possibility of a safe In vivo administration of Fe 3 O 4 -NPs; thus, it could be used in a variety of medical applications. The lower cytotoxicity potential could be due to the efficiency of Fe 3 O 4 -NPs nanoparticles to penetrate the cell and cause damage as a result of its physiochemical properties and the small size of nanoparticles (Mahmoodabadi et al. 2018; Susithra et al. 2024). Thus, it could be used effectively as an antibacterial, antiviral, antifungal, anti-inflammatory, antioxidant, antidiabetic, and other biomedical applications (Susithra et al. 2024; Abou-Dobara et al. 2024; Karunakaran et al. 2023). Conclusion This study successfully isolated and characterized B. proteolyticus UPMC1508 from six aquatic habitats in Selangor, Malaysia. The strain demonstrated low antibiotic resistance and the ability to grow in high-iron environments, suggesting its potential and safety for biotechnological applications in metal-rich conditions. Notably, this research represents the first successful biosynthesis of Fe₃O₄-NPs via B. proteolyticus UPMC1508, highlighting the feasibility of a sustainable, green synthesis approach. This eco-friendly method supports environmental sustainability and opens new possibilities for producing functional nanoparticles for diverse applications in science and technology. The synthesized Fe₃O₄-NPs exhibited low cytotoxicity against L-929 fibroblast cells at a concentration of 0.032 mg.mL -1 , indicating a safe threshold for potential In vivo applications, such as in antibacterial and anticancer treatments. However, the cytotoxic effects at varying concentrations require further investigation. Future research could focus on surface modifications of uncoated Fe₃O₄-NPs to enhance their biocompatibility and functionality in medical applications. Overall, this study positions B. proteolyticus UPMC1508 as a promising, iron-tolerant strain for sustainable green synthesis of Fe₃O₄-NPs, with broad potential across multiple biomedical applications. Authors' Contribution We sincerely appreciate Prof. Dr. Rosfarizan Mohamad's valuable contributions and support throughout this research. We would also like to extend our special thanks to Assoc. Prof. Dr. Murni Halim, Assoc. Prof. Dr. Siti Efliza Ashari and Dr. Fadzlie Wong Bin Faizal Wong, thank you for your significant role in advancing and organizing the data and analyzing and understanding the information. Their active involvement in reviewing the content to ensure its intellectual significance, their agreement to submit the article to this journal, and their final approval for publication are greatly valued. All authors have given their complete approval to the final version. Their contributions have been valuable in ensuring that the content accurately reflects their collective information and understanding of the subject matter. Abbreviations ATGs Antibiotic resistance genes BLAST Basic local alignment search tool D.O. Dissolved oxygen FT-IR Fourier Transform Infrared Spectroscopy FE-SEM Field Emission Scanning Electron Microscopy MBC Minimum bactericidal concentration NCBI National Centre for Biotechnology MEM Minimum Essential Medium MHA Müller-Hunton-Agar MIC Minimum inhibitory concentration MTC Maximum tolerable concentration MTG Metal tolerance gene MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tet-tetrazoliumm bromide) NPs Nanoparticles PCR Polymerase chain reaction PDI The Polydispersity index SD Standard deviation TEM Transmission electron microscopy XRD Powder Diffraction Techniques Declarations Authors' Contribution We sincerely appreciate Prof. Dr. Rosfarizan Mohamad's valuable contributions and support throughout this research. We would also like to extend our special thanks to Assoc. Prof. Dr. Murni Halim, Assoc. Prof. Dr. Siti Efliza Ashari and Dr. Fadzlie Wong Bin Faizal Wong, thank you for your significant role in advancing and organizing the data and analyzing and understanding the information. Their active involvement in reviewing the content to ensure its intellectual significance, their agreement to submit the article to this journal, and their final approval for publication are greatly valued. All authors have given their complete approval to the final version. Their contributions have been valuable in ensuring that the content accurately reflects their collective information and understanding of the subject matter. Funding Statement This work was partly supported by the University Research Grant (Putra-IPS). Data Availability This article contains all the necessary data relevant to the topic. Consent For Publication All authors are awarded and agreed to publish this paper. 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Supplementary Files Highlights.docx Cite Share Download PDF Status: Published Journal Publication published 11 Feb, 2025 Read the published version in Biologia → Version 1 posted Reviewers agreed at journal 01 Dec, 2024 Reviewers invited by journal 28 Nov, 2024 Editor assigned by journal 28 Nov, 2024 First submitted to journal 26 Nov, 2024 Editorial decision: Accept but needs final editing 23 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4974579","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":383704693,"identity":"ab04e41d-5795-45e3-bb42-c6fa7f6acba7","order_by":0,"name":"Yusur Ramzi Hasan","email":"data:image/png;base64,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","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Yusur","middleName":"Ramzi","lastName":"Hasan","suffix":""},{"id":383704694,"identity":"4652bf0c-95fe-4c13-852b-9aede9d0e65f","order_by":1,"name":"Fadzlie Wong Faizal Wong","email":"","orcid":"","institution":"UPM: Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Fadzlie","middleName":"Wong Faizal","lastName":"Wong","suffix":""},{"id":383704695,"identity":"9094fd92-c8e6-4ce3-a295-d88fa768e958","order_by":2,"name":"Murni Halim","email":"","orcid":"","institution":"UPM: Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Murni","middleName":"","lastName":"Halim","suffix":""},{"id":383704696,"identity":"f93de686-012c-4e60-8876-e829ca952931","order_by":3,"name":"Siti Efliza Ashari","email":"","orcid":"","institution":"UPM: Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Siti","middleName":"Efliza","lastName":"Ashari","suffix":""},{"id":383704697,"identity":"edb0b7a5-6a3e-4052-916e-ce2c53dbcdcb","order_by":4,"name":"Rosfarizan Mohamad","email":"","orcid":"https://orcid.org/0000-0001-5672-1905","institution":"UPM: Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Rosfarizan","middleName":"","lastName":"Mohamad","suffix":""}],"badges":[],"createdAt":"2024-08-26 01:49:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4974579/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4974579/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-025-01868-w","type":"published","date":"2025-02-11T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70638248,"identity":"fb2e8ba4-14b9-442c-afaa-f8d222a6bd17","added_by":"auto","created_at":"2024-12-05 06:49:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1354998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The growth of the obtained isolates on Luria agar; \u003cstrong\u003e(b)\u003c/strong\u003e nutrient agar; \u003cstrong\u003e(c) \u003c/strong\u003enutrient agar supplemented with Iron source.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/c891c30c5fa398d38ee9ebef.jpg"},{"id":70636456,"identity":"8c35e1ce-8b2a-4450-bf71-81a615439b6a","added_by":"auto","created_at":"2024-12-05 06:33:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":769805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003ePCR amplification of the 16S rRNA gene in Agarose gel electrophoresis from \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508, ‌M‌ represents the marker, whereas the ‌+ve and -ve‌ indicate the presence of the purified plasmid with 16S region; \u003cstrong\u003e(b)\u003c/strong\u003e represents the phylogenetic tree.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/e350641c8d6ad9630d64fef6.jpg"},{"id":70635755,"identity":"d50fed07-2b38-42fd-ad49-8e60bb0d8baf","added_by":"auto","created_at":"2024-12-05 06:25:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2348147,"visible":true,"origin":"","legend":"\u003cp\u003eThe means ± SD (\u003cem\u003en\u003c/em\u003e=3) of the inhibition zones of 10 antibiotics against \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 on MHA agar.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/a7b89d495a926fa34789992e.jpg"},{"id":70635761,"identity":"6fe1b6dd-6de9-46b7-8d4e-299148ae2df3","added_by":"auto","created_at":"2024-12-05 06:25:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":638389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e UV-visible absorption spectra of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs at wavelength (200-800 \u003cem\u003en\u003c/em\u003em); \u003cstrong\u003e(b)\u003c/strong\u003e the formation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs after 5 days incubation.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/326ba333426d3745c1bbe24e.jpg"},{"id":70637640,"identity":"a2f03b76-6b39-4654-8745-c5d76f3ea7c0","added_by":"auto","created_at":"2024-12-05 06:41:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3881396,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images display the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs at 20 \u003cem\u003en\u003c/em\u003em magnifier.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/3982dccf55928af0fd3dbefc.jpg"},{"id":70635780,"identity":"214edbe1-d542-47ba-9cef-3bde986abb34","added_by":"auto","created_at":"2024-12-05 06:25:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":515030,"visible":true,"origin":"","legend":"\u003cp\u003eThe size of the distribution profile of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs were determined via; \u003cstrong\u003e(a)\u003c/strong\u003e TEM; \u003cstrong\u003e(b)\u003c/strong\u003e nanosizer which displays the zeta average distributions.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/e1d4986cd24087e8b7dc49cd.jpg"},{"id":70636460,"identity":"b5e0b7b0-8544-4686-97b9-8a2fc6efc486","added_by":"auto","created_at":"2024-12-05 06:33:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2119663,"visible":true,"origin":"","legend":"\u003cp\u003eThe FE-SEM micrographs of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs at different magnification; scales; \u003cstrong\u003e(a)\u003c/strong\u003e x50,000; \u003cstrong\u003e(b)\u003c/strong\u003e x100,000.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/8af1f3c2572ade8ab0b0ffcc.jpg"},{"id":70635783,"identity":"9fad3089-db50-4ceb-bd4d-7c33ac36f862","added_by":"auto","created_at":"2024-12-05 06:25:43","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1293954,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction (XRD) patterns of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs measured at pH 8.5.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/f975a39dd655e62eb34df41b.jpg"},{"id":70636464,"identity":"484b72b8-7067-4aad-8a56-e4365fe9fb3d","added_by":"auto","created_at":"2024-12-05 06:33:41","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2186095,"visible":true,"origin":"","legend":"\u003cp\u003eThe FT-IR spectra of purified Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs before and after two months.\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/c1bd1d78e9205963ac5a52e0.jpg"},{"id":70636478,"identity":"b78660c9-e36a-4c04-bb4c-f6fa808662f2","added_by":"auto","created_at":"2024-12-05 06:33:43","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":798590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The percentage of L929 cell viability at various concentrations of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (mg. mL\u003csup\u003e-1\u003c/sup\u003e); \u003cstrong\u003e(b)\u003c/strong\u003e The microscopic observation of the morphological changes of L-929 cells after treatment with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs.\u003c/p\u003e","description":"","filename":"Fig11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/9cb46e9baace0326dff928d9.jpg"},{"id":76487742,"identity":"0fab08d7-bac0-48ba-b4b0-ad57f37bf677","added_by":"auto","created_at":"2025-02-17 16:12:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17302634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/6e9cc697-0a20-4e5f-ae85-fea78f1f2dd0.pdf"},{"id":70635757,"identity":"ca93cf09-d86a-447c-af69-2c9e8c04b340","added_by":"auto","created_at":"2024-12-05 06:25:41","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":22075,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-4974579/v1/f16822c58b152b0f94094534.docx"}],"financialInterests":"","formattedTitle":"Bacillus proteolyticus UPMC1508: A novel bacterial strain capable of biologically synthesize iron oxide nanoparticles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanotechnology is a rapidly progressing field that focuses on producing diverse nanomaterials. The merge of Biotechnology and Nanotechnology led to the development of Bionanotechnology, which aims to create cost-effective and environmentally friendly nanotechnologies, including nanoparticles. Nanoparticles, between 1-100 \u003cem\u003en\u003c/em\u003em, are highly versatile and widespread because of their numerous advantages and applications (Bouafia et al. 2021; Joudeh and Linke 2022). In several fields of human activity (e.g., Biology, Medicine, Electronics, Energy, Food Technology, and Aerospace Engineering), the distinct characteristics of nanoparticles that rely on their form and dimensions are crucial and have always been matters of interest (Yaqoob et al. 2020; de Jesus et al. 2021; Oliveira et al. 2024; Jeyaraj et al. 2019). Antibiotics are used to treat different infections. Still, due to the drug resistance phenomenon of many strains, acute diarrhea, chronic toxicity, and negative impact on intestinal microbes aligned with over usage of antibiotics, it becomes a challenge to use antibiotics and to be controlled. Therefore, finding an alternative, such as using nanoparticles, is more crucial than before (Kim, Covington, and Pamer 2017; Abdoli et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Roy, Mutalik, and Dhas 2024; Saod et al. 2024). On the other hand, nanoparticles can be synthesized and stabilized using biological and non-biological methods involving two strategies (Top-down\u0026zwnj; and \u0026zwnj;Bottom-up). Although the non-biological methods, including chemical and physical processes, are commonly used to synthesize nanoparticles, numerous limitations and drawbacks must be considered, as they often require extreme operational conditions (e.g., high pressure and temperature), increasing costs, and environmental impact. Furthermore, they ended up with toxic byproducts, which have safety concerns and could limit the biocompatibility regarding the synthesized nanoparticles (de Jesus et al. 2024; de Jesus et al. 2021; Singh and Chandra 2024). Metal oxide nanoparticles have captured the interest in nanomaterials (Omorogbe et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ikhuoria et al. 2024). Iron oxide nanoparticles (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs) have paved the way among other widely studied metal oxide nanoparticles. Different forms of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs exist, including α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e (hematite), β-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, c-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e (maghemite), FeO (wurtzite), and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (magnetite). Because of their magnetic properties, high versatility, biocompatibility, and less toxicity, they offer diverse applications in various fields, including imaging, environmental remediation, and particularly in medicine, which can trigger therapeutic activities, for instance, contrast agents in MRI (Rabani et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Daramola et al. 2024), hypothermia treatment, and antimicrobial activity against pathogens such as fungi, bacteria, and viruses (Negrescu et al. 2022; Alphand\u0026eacute;ry 2020). Today, the green synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs via microbes is a desirable, cost-effective, and environmentally safe alternative to chemical methods. Microorganisms like bacteria, algae, fungi, and yeast can quickly cultivate and grow in ambient conditions (Rabani et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Park, Lee, and Lee 2016; Jamzad and Kamari Bidkorpeh 2020). The mechanism of the formation of nanoparticles varies among microbes. Still, it typically involves trapping metal ions on the cell surface and reducing them to nanoparticles via their microbial enzymes, such as ferric reductases, for bioreduction of iron salt precursor (Zhang et al. 2013; Shinagawa 2011) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Bacteria consume iron as an essential micronutrient for various biological processes, including Colonization, growth, and virulence. To obtain sufficient iron from the environment, bacteria have developed sophisticated mechanisms. One of the crucial mechanisms is the production of metabolites and enzymes that facilitate the bio-reduction of the elevated iron salt (Huo et al. 2021). In response to starvation or heavy metal pollution, many bacterial stains, such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, change their gene expression and induction of specific genes (Alnaimat 2017). Interestingly, studies revealed that wastewater contaminated with heavy metals contains bacterial strains that possess a remarkable ability to synthesize nanoparticles (Al-Tameemi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ka-ot et al. 2018; Desai et al. 2023). While a recent study revealed that the iron-tolerant \u003cem\u003eBacillus\u003c/em\u003e species demonstrated a potential for a biogenic synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs with an average size of 81.3 \u003cem\u003en\u003c/em\u003em (Walujkar et al. 2019). Similarly, another recent study reported that some Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs-producing bacteria possessing ferric reductases for bio-reduction of iron salts, such as \u003cem\u003eSporosarcina luteola, Bacillus subtilis, Bacillus badius, Bacillus cereus, Bacillus tropicus, Klebsiella quasipneumoniae, Klebsiella africana\u003c/em\u003e, and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e with estimated nanoparticles\u0026rsquo;s size between 31\u0026ndash;72 \u003cem\u003en\u003c/em\u003em. Nonetheless, there is a lack in the investigation of novel bacterial strains that not only tolerate higher iron concentrations but also efficiently Synthesise of Fe₃O₄-NPs (Daramola et al. 2024). Furthermore, although correlations between heavy metal resistance and antibiotic resistance have been suggested, the mechanisms underpinning this relationship are not yet fully understood (Fu et al. 2023). This gap is particularly critical as the co-occurrence of heavy metal and antibiotic resistance in bacteria poses potential risks to public health when such microbes are used in nanoparticle synthesis and other applications (Gupta, Sreekrishnan, and Ahammad 2022; Baker-Austin et al. 2006; Gupta et al. 2023). Moreover, while green synthesis approaches align with the green chemistry principles, the applicability of many bacterial strains is constrained due to their potential to produce toxic compounds or exhibit antibiotic resistance (de Jesus et al. 2024). Addressing these gaps would would promote the exploration of new bacterial to be used in safer and more sustainable green synthesis approach. Recently, the use of probiotics in the green synthesis approach of nanoparticles for serving in different fields represents a significant candidate, as it can be grown under average temperature and pressure. Additionally, it can tolerate different pH ranges, which aids in forming more diverse and controlled nanoparticle sizes used for various applications (Al-Enazi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Abdoli et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Numerous research reported the extracellular synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs using probiotics such as \u003cem\u003eBacillus cereus\u003c/em\u003e (Fatemi et al. 2018), \u003cem\u003eBacillus megaterium\u003c/em\u003e (Hajiali, Daneshjou and Daneshjoo 2022), \u003cem\u003eBacillus subtilis\u003c/em\u003e (Sundaram et al. 2012), \u003cem\u003eBacillus pasteurii\u003c/em\u003e (Daneshvar and Hosseini 2018), \u003cem\u003eBacillus licheniformis\u003c/em\u003e (Crespo et al. 2017), \u003cem\u003eBacillus circulans\u003c/em\u003e (Rabani et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with different shapes (Irregular, spherical, and rhombohedral), with the average size ranges were between 12\u0026ndash;97 \u003cem\u003en\u003c/em\u003em. Interestingly, the size of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs is a crucial factor influencing their effectiveness in medical applications, especially as antimicrobial agents. Smaller nanoparticles have a higher surface area-to-volume ratio, enhancing their effectiveness (Kumar, Kumari, and Sahoo 2024). Although the use of probiotics in the green synthesis of nanoparticles offers many benefits, they may possess a metal tolerance gene (MTG) to tolerate such harsh environmental pollution that exists in nature, which is strongly related to the presence of Antibiotic resistance genes (ATGs), as they exist on the same plasmid and could lead to co-selection. Studies have suggested that this phenomenon is entirely associated with this type of metal (Desai et al. 2023; Alangari et al. 2022b; Giorgi et al. 2021; Pancholi and Caparon 2022). Accordingly, using probiotics in the green synthesis of nanoparticles is still in progress and involves many challenges. Furthermore, the toxicity of the synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs and antimicrobial effectiveness need to be further explored (Abdoli et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Addressing these challenges is crucial for the safe and effective application of new bacterial strains in the green synthesis of safe nanoparticles that can be used in medical and environmental contexts. \u003cem\u003eB. proteolyticus\u003c/em\u003e is a member of the \u003cem\u003eBacillus\u003c/em\u003e genus that possesses an enzymatic activity, especially proteolysis. Their cell wall consists of a thick layer of peptidoglycan, a known characteristic of Gram-positive bacterial strains. These structures aid the biosorption and bio-reduction of metal ions because of their characteristics of negative electrokinetic potential. This attracts the metal cations to initiate the green synthesis of nanoparticles and protects them against metallic stress (Kr\u0026oacute;l et al. 2018; Mohd Yusof et al. 2020). Additionally, a recent research conducted by Zeng et al. (2021) revealed that \u003cem\u003eB. proteolyticus\u003c/em\u003e isolated from yak in China possessed a probiotic potential with less antibiotic resistance. However, no research indicates that \u003cem\u003eB. proteolyticus\u003c/em\u003e has been utilized to synthesize Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs. \u003cem\u003eBacillus\u003c/em\u003e bacteria, known for producing potential natural products, can exist in various environments and have the necessary mechanisms to survive in harsh conditions, such as extreme heat, strong sunlight, and dryness (Hajiali et al. 2024). Therefore, it is possible to bring attention to utilizing of \u003cem\u003eB. proteolyticus\u003c/em\u003e as a safe entity for biologically synthesizing Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs due to the reasons outlined. The exploration of probiotics in green synthesis presents a promising alternative. Still, challenges remain regarding their metal tolerance, the associated antibiotic resistance potential, and the toxicity and size of the synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs. This research attempts to address the need for novel and safer bacterial strains that have the potential to the green synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs with lower cytotoxicity potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling and physiological properties of the selected sites\u003c/h2\u003e \u003cp\u003eA total of 12 samples (either sediments or water samples) were collected from six different sites on the 10th and 11th of January 2022, from various aquatic habitats in Selangor, Malaysia (Kajang Pond, Mines South Lake, Tasik Seri Serdang, UKM University pond, Taman Tasik Sekyen 7, and Port Klang Sea) at 15 cm depth sediments (He et al. 2018). Each sample was transferred to a glass bottle (250 mL) and covered loosely. This was followed by incubation under dim light conditions at room temperature for one month. The proportion of sediments, sludge, and water is approximately\u0026thinsp;~\u0026thinsp;1:3 (v/v) (Holland et al. 2008). Each sample's dissolved oxygen (D.O.) concentration was measured using a dissolved oxygen meter (Hach HQ10, Hach Company, Loveland, CO, USA). After storing and establishing the required measurements, the replicates of each sample were diluted (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation and characterization of bacterial isolate\u003c/h3\u003e\n\u003cp\u003eAfter observing the physiochemical properties of the samples, each replicate was diluted in 0.85% (w/v) NaCl from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. The desired isolates were cultured overnight via spreading of 1 \u0026micro;L of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e dilutions on nutrient agar (HiMedia, India) supplemented with Iron (II) sulfate heptahydrate (FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO) (HiMedia, India) (1.622 g.100 mL \u003csup\u003e-1\u003c/sup\u003e) as an iron source (Alnaimat 2017). Nutrient agar was prepared by mixing 0.5% peptone, 0.3% beef extract, 1.5% agar, 0.5% sodium chloride, and cytoplasm, with 1 L of distilled water, the pH was adjusted to be six at 37\u0026deg;C (MacFaddin 2000). Then, the grown isolates were purified by subculturing them on Luria agar media (HiMedia, India). Each of the successfully grown colony (24 h cultured) was picked up for enrichment by sub-culturing it on nutrient agar media without Iron supplement. The pH of the medium was adjusted to 6 via phosphate-buffered saline (PBS) (0.2 M of both KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and NaOH) (HiMedia, India), and the temperature was 37\u0026deg;C (Umeh and Enwuru 2014; Alnaimat 2017). The bacterial colonies were successfully grown, enriched, and purified via nutrient agar were collected to be characterized and identified. Pure isolate was physiologically identified according to Bergey's Manual (Garrity et al. 2005). Gram staining, catalase and oxidase tests, lactose fermentation, Indole, and methyl red tests were investigated. Additionally, the selected bacteria were stored in Luria broth (HiMedia, India) that contained 50% glycerol (Sigma-Aldrich) at -80\u0026deg;C for further investigations. The16S rRNA sequencing was carried out for the selected isolate, and the genomic DNA was extracted via DNA Extraction Kit (BioFlux, USA). The process involved using a forward primer, a 20-base DNA sequence (\u003cem\u003e5'-AGA GTT TGA TCC TGG CTC AG-3'\u003c/em\u003e), and a reverse primer, a 22-base DNA sequence (\u003cem\u003e5'-TAC GGT TAC CTT GTT ACG ACT T-3'\u003c/em\u003e) (Nezhad et al. 2023). These primers are in a 25 nmol scale, with a standard desalting process, a minimum process, and a minimum yield of 10 nmol, ensuring the necessary genetic material for amplification. The polymerase chain reaction (PCR) amplification was carried out at the following temperature parameters: 3 min for an initial denaturation phase at 95\u0026deg;C, then 30 s for 30 cycles at 95\u0026deg;C, 30 s at 52\u0026deg;C, and 1.30 min at 72\u0026deg;C for denaturation, annealing, and extension, respectively. A last extension step was performed for 5 min at 72\u0026deg;C. After the purification of PCR amplicons (Purification Kit, Qiagen, Germany), the sequencing was then performed at Apical Scientific Sdn Bhd in Malaysia. For checking the similarity, The Gen-bank database was used to compare the sequences of the 16 S rRNA gene for the desired strain through the Basic Local Alignment Search Tool (BLAST) of NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) (Nezhad et al. 2023). The sequence alignment was retrieved and aligned through ClustalW. Then, a phylogenetic tree was performed via the MEGA-X software version 10.2.6 through the Maximum likelihood algorithm method (Kumar et al. 2018).\u003c/p\u003e\n\u003ch3\u003eAntibiotic susceptibility test\u003c/h3\u003e\n\u003cp\u003eAn antibiotic sensitivity test is usually performed to assess the safety of \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508. The disc diffusion method was done in triplicates and according to the National Committee for Clinical Laboratory Standards (NCCLS 1997) (Wayne 2002). To measure the turbidity, the suspension was matched with a 0.5 MacFarland standard (Zeng et al. 2021). Then, the suspension was evenly spread onto Luria agar plates using a sterile cotton swab. To evaluate antibiotic sensitivity, 11 drug-sensitive discs (Thermo Scientific, Oxoid) were utilized, including Ampicillin (10 \u0026micro;g/disk), tetracycline (30 \u0026micro;g/disk), gentamicin (10 \u0026micro;g/disk), cefalexin (30 \u0026micro;g/disk), chloramphenicol (30 \u0026micro;g/disk), erythromycin (15 \u0026micro;g/disk), vancomycin (30 \u0026micro;g/disk), rifampin (5 \u0026micro;g/disk), streptomycin (10 \u0026micro;g/disk), kanamycin (30 \u0026micro;g/disk). Following incubating for 24 h at 37\u0026deg;C, the diameters of the inhibition zones were measured. A zone of inhibition less than 0.5 cm indicates resistance, whereas a zone more than 0.5 cm in diameter indicates a sensitive result (McDermott et al. 2005).\u003c/p\u003e \u003cp\u003e \u003cp\u003e \u003cb\u003eFe-heavy metal tolerance: Minimum tolerance concentration (MTC), Minimum inhibitory concentration (MIC), and Minimum bactericidal concentration (MBC)\u003c/b\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe MTC of Fe heavy metal against the \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 was determined via the spot plate method. The FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO was prepared in different concentrations (0.1, 0.2, 0.4, 0.8, 1, 2, and 4 mg.mL\u003csup\u003e-1\u003c/sup\u003e). About 10 \u0026micro;l of bacterial cultures (24 h cultured) corresponding to 0.5 McFarland standards were added to M\u0026uuml;ller-Hunton-Agar (MHA) (HiMedia, India) adjusted to pH 5, containing the FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO concentrations and then incubated for 72 h. at 37\u0026deg;C. MHA was prepared by mixing beef extract (2.00 g. L\u003csup\u003e-1\u003c/sup\u003e), hydrolysate of casein (17.50 g. L\u003csup\u003e-1\u003c/sup\u003e), starch (1.50 g. L\u003csup\u003e-1\u003c/sup\u003e), and agar (17 g. L\u003csup\u003e-1\u003c/sup\u003e) with 1 L of D.W. The mixture was stirred to be fully dissolved, then autoclaved at 121\u0026deg;C for about 15 min. After cooling down, MHA was poured into petri dishes to be solidified. MTC is the maximum concentration of heavy metal that bacteria can withstand without substantial interference to its growth. MIC is the lowest concentration of metals that inhibit the growth of bacteria, and MBC is the lowest concentration of metals that completely inhibits the growth of visible bacteria after 72 h (Ka-ot et al. 2018; Rathnayake et al. 2009).\u003c/p\u003e\n\u003ch3\u003ePreparation of extracellular supernatant\u003c/h3\u003e\n\u003cp\u003eThe biological synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs via the extracellular approach started with culturing the \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 in 250 mL nutrient broth medium, and the pH of the media was maintained via PBS (0.2 M, pH 6). After that, the culture was incubated for 34 h at 37\u0026deg;C and agitated at 150 rpm. Then, the bacterium grown was purified and separated from the bacterial biomass through centrifugation at 6000 rpm for 15\u0026ndash;20 min. To prevent any predicted contamination, there is a need to ensure the absence of bacterial cells in the supernatant by passing the supernatant through a 0.2 \u0026micro;m syringe filter. Finally, the filtered supernatant was ready for biological synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Rabani et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fatemi et al. 2018; Jagathesan et al. 2018; Jubran et al. 2020).\u003c/p\u003e\n\u003ch3\u003eBiosynthesis of FeO-NPs\u003c/h3\u003e\n\u003cp\u003eThe filtered supernatant of the 18 h bacterial broth was mixed with FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO solution so that the iron salt concentration was 5 mM in a 1:1 volume ratio to the supernatant, followed by magnetic stirring (80 rpm) for 5 min. The adjustment of the incubation conditions (pH ranges and temperatures) was done during this step. After the incubation of the mixture for 5 days at 37\u0026ordm;C incubator shaker (170 rpm) in a dark condition, a color change from dark yellow to dark brown was detected. For separation of the dark-obtained precipitate from the supernatant solution to which salt had been added, the solution was through a 0.2 \u0026micro;m sterile syringe filter, followed by centrifugation at 8000 rpm. Next, the obtained residue was washed 5 times with ethanol 97% (HiMedia, India) and distilled water. Note that each washing step is accompanied by centrifugation at 4000 rpm except the first step, which was done at 8000 rpm to allow the small nanoparticles to be precipitated and washed. Finally, the residue was dried in an oven at 80\u0026deg;C for 24 h. Then, the obtained powder was stored to be characterized (Rabani et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fatemi et al. 2018; Sundaram et al. 2012; Jagathesan et al. 2018; Jubran et al. 2020).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cp\u003e\u003cstrong\u003eCharacterization of the biosynthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs\u003c/strong\u003e\u003c/p\u003e \u003cp\u003eIdentifying the oxide phases of iron through characterization instruments can be challenging, as a single tool may not provide accurate results. However, this challenge can be overcome by employing various characterization techniques. UV\u0026ndash;Vis spectrophotometer analysis (Thermo Scientific, USA) was performed at 200\u0026ndash; 800 \u003cem\u003en\u003c/em\u003em wavelength to detect the presence of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs. Transmission electron microscopy (TEM) (JEM-2100 F, Japan) demonstrates the shape, size, and distribution, considering the van der Waals forces acting on the samples which play a vital role in aggregation. The Field emission scanning electron microscopy (FE-SEM) (Eindhoven, The Netherlands) illustrates the topography and characteristics of a sample's surface, such as morphology, size, shape, and particle stationing of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs were recorded over the range of 600-4,000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on a model spectrum 100 series. The FT-IR (Vector 22, Bruker from Germany) analyses the vibration of atoms within molecules when exposed to infrared radiation. This analysis provides valuable information about the sample's radiation absorption. The Fourier-transform infrared (FT-IR) spectroscopy analyses the vibration of atoms within molecules when exposed to IR. This provides valuable information by analyzing the amount and quality of radiation absorbed by a sample. To assess the crystalline arrangement of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs sample, the XRD (Oxford, USA). The nanosizer (DLS, Nano-ZS, UK) is an advanced tool that can detect aggregates and analyze the size of small or diluted samples, even at very low or high concentrations. It works by using dynamic light scattering with 'NIBS' optics, making it highly accurate and reliable as a molecular size analyzer (Walujkar et al. 2019; Sundaram et al. 2012; Mourdikoudis et al. 2018).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eThis assay test was done at the National Institutes of Biotechnology Malaysia (NIBM), Selangor, Malaysia. To determine whether Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs are cytotoxic, the fibroblast cell line (L-929) was exposed to MTT solution (Sigma-Aldrich) \u003cem\u003ein-vitro.\u003c/em\u003e The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs powder was mixed with minimum essential media and subjected to 45 W probe sonication for 5 min, with 3 seconds switched-on and 3 seconds switched-off intervals. Next, the suspension of biosynthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs was filtered using 0.2 \u0026micro;m syringe filters. Then, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs biosynthesized were prepared from 0.0312 to 1 mg.mL\u003csup\u003e-1\u003c/sup\u003e. A quantity of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well (100 \u0026micro;L per well) in ). Minimum Essential Medium (MEM) (Sigma-Aldrich) was added to 96-well plates (Tecan Infinite M200, Switzerland) and incubated at 37\u0026deg;C for 22 h with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then treated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (0.031, 0.063, 0.125, 0.25,0.50, and 1 mg. mL\u003csup\u003e-1\u003c/sup\u003e) for 24 h after removing the medium. After that, 50 \u0026micro;L of MTT was added to MEM then inculcated into each well and allowed to incubate for another 2 h at 37\u0026deg;C. Next, the MTT solution and 100 mL of isopropanol were added to each well, and the cells were incubated for 30 min. A reference wavelength of 650 \u003cem\u003en\u003c/em\u003em was used, and 570 \u003cem\u003en\u003c/em\u003em was used as a detector absorbance (Naskar and Kim 2019). The lower viability value means higher cytotoxicity, and if the viability % is reduced to less than 70%, The lower the Viability % value, the higher the cytotoxic potential. If viability is reduced to \u0026lt;\u0026thinsp;70% of the blank, it has a cytotoxic potential. The formula used to calculate cell viability is as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Cell\\:viability\\:\\%=\\frac{{OD}_{570e}\\times\\:100}{{OD}_{570b}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eOD\u003csub\u003e570e\u003c/sub\u003e represents the mean value of the measured optical density of the 100% Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs,\u003c/p\u003e \u003cp\u003eOD\u003csub\u003e570b\u003c/sub\u003e represents the mean value of the measured optical density of the blanks.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe absorbance data were plotted as scattered line using Prism software (version 10.1.1, 270; GraphPad Software, San Diego, CA, USA). The phylogenetic tree was performed using MegaX software (version 10.2.6; Pennsylvania State University, USA). While ImageJ software (version 1.53t; National Institutes of Health, Bethesda, MD, USA) was used to calculate the average of nanoparticle sizes. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) was presented to report the experimental results. One-way ANOVA was conducted to examine the distinctions in Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NP concentrations, with statistical significance set at a \u003cem\u003ep\u003c/em\u003e-value of \u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe physiological properties of the collected samples and their sites\u003c/h2\u003e \u003cp\u003eThe collected samples were observed for their different physiological parameters (pH, dissolved oxygen, latitude and longitude, and color), as shown in Table\u0026nbsp;1. The pH values were between 5.5 and 6.5, and the temperature was 27\u0026ndash;30\u0026deg;C. The D.O. for most of the sites was around 8 at the initial reading (8.9 mg. L\u003csup\u003e-1\u003c/sup\u003e), except for Tasik Seri Serdang, and Taman Tasik Sekyen, Port Klang Sea, which recorded 7 mg. L\u003csup\u003e-1\u003c/sup\u003e. Moreover, most of the ponds exhibited a dark brown color except Mines South Lake and Port Klang Sea, which exhibited a blue color. In a recent study conducted by Hanafiah et al. (2024) in Selangor, Malaysia, it was revealed that this dark brown color is caused by both inorganic and organic species present in the water. The study further found that the water had high concentrations of heavy metals, particularly iron (Fe) (3.2422\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2533 mg. L\u003csup\u003e-1\u003c/sup\u003e), accompanied by a low pH value (5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1), which was believed to be the primary factor contributing to the dark brownish appearance of the water.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e The parameters of the sampling sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eD.O. (mg. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The initial reading is 8.9 (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTemperature (\u0026deg;C) (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLatitude and Longitude\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKajang Pond\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater and\u003c/p\u003e \u003cp\u003eSediments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBrown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLat: 3.0064840922287392, Lng: 101.79062437266111\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMines South Lake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBlue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLat: 3.0344300427619473, Lng: 101.71137414872646\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTasik Seri Serdang\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater and\u003c/p\u003e \u003cp\u003eSediments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eGreenish blue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLat: 16.351375,\u003c/p\u003e \u003cp\u003eLng: 81.09178639999999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUKM University Pond\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater and\u003c/p\u003e \u003cp\u003eSediments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBrown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLat: 2.9289975999999998, Lng: 101.7800219\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTaman Tasik Sekyen 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBlue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLat: 3.01032240067449,\u003c/p\u003e \u003cp\u003eLng: 101.60078667104244\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePort Klang Sea,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBlue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLat: 3.0048405,\u003c/p\u003e \u003cp\u003eLng: 101.3623373\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and characterization of bacterial strain\u003c/h2\u003e \u003cp\u003eFor screening and purification of the desired isolates, suitable dilutions (10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) of sample were cultured on nutrient agar media as it is equal to 0.5 McFarland standard. The results showed that most of the selected aquatic sites in Malaysia contained strains suspected to be Iron-tolerance isolates. Although nutrient agar can be considered a versatile medium which supports the growth of a wide range of microorganisms, the growth results of the inocula of the collected samples witnessed a significant disparity because of an iron source (FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO). To illustrate this, most of the collected samples (Mines South Lake, Tasik Seri Serdang, UKM University Pond) showed successful bacterial growth on nutrient agar supplemented with FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO (1.6 g.L\u003csup\u003e-1\u003c/sup\u003e). In contrast, some of sites (Kajang Pond, Taman Tasik Sekyen 7, and Port Klang Sea) showed negative results of bacterial growth during incubation. Following the purification and enrichment steps, the number of pure isolates decreased as the vitality of many of these isolates was considered. Hence, only the suspected probiotic bacterial strain that produced a pale yellowish colony on Luria agar and nutrient Agar medium was subjected to 16S rRNA sequencing for further investigations that will be illustrated subsequently. Among the isolates, the appearance of YRU 3 (24 h cultured) on nutrient agar was in the form of pale yellowish colonies as in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-a and b), while on nutrient agar, which was supplemented with an iron source, as dark brown colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-c). The colour appearance of bacterial colonies can be affected by the type of agar used and the addition of supplements. In this case, the brown colour changes when bacteria are cultured on nutrient agar supplemented with iron source can be explained by multiple factors; for instance, iron utilization by bacteria, bacterial metabolism, and pigment production was identified by Hegler et al. (2008). These are spheroidene, spheroid, and OH-spheroid, as well as reactions involving iron. Furthermore, YRU3 was a gram-positive, rod-shaped bacteria with negative oxidation and positive catalytic reactions. Additionally, the results were negative for lactose fermentation, Indole, and Methyl red, whereas the starch hydrolysis was positive (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, this strain was chosen for the 16s rRNA sequencing. The results of the purified DNA are revealed in agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-a). Based on the blast analysis of the phylogenetic tree via MEGA 7 software, as well as the neighbouring joining methods for the 16s rRNA sequencing with the National Centre for Biotechnology (NCBI) database and the biochemical characteristics, the results revealed that the strain (YRU3) bears the strongest similarity to \u003cem\u003eB. proteolytic\u003c/em\u003e (99.87%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-b), and species commonly found in soil. This specific strain, named YRU 3, has been duly documented in the NCBI database with the GenBank accession number OQ27138 and the original number UPMC1508 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Probiotic bacteria, in general, have gotten a lot of attention due to their characteristics and contributions in various applications, for instance, medical, industrial, and agricultural applications (Khan et al. 2020; Rehaiem et al. 2014; El-Nour et al. 2023; Mohammed et al. 2023; Vijayakumar et al. 2023).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification and characterization of \u003cem\u003eBacillus. proteolyticus\u003c/em\u003e UPMC1508.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eMorphological characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c12\" namest=\"c6\"\u003e \u003cp\u003eBiochemical characteristics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGen-Bank Accession \u003c/p\u003e \u003cp\u003eNo Original code\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThe similarity (%) according to the NCBI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSites of isolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eGram staining and shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCatalysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eStarch hydrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eLactose fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eIndole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMethyl red\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOQ27138 \u003c/p\u003e \u003cp\u003eUPMC1508\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus proteolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eTasik Seri Serdang, Selangor, Malaysia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eGm+, rode shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic susceptibility assay and Fe-tolerance potential\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 isolate was tested for antibiotic sensitivity via agar diffusion. It was conducted in triplicate to ensure the reliability and reproducibility of the results. The overnight cultured broth (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e) was spread evenly on Luria agar and incubated for 24 h at 37\u0026deg;C. After incubation, the results showed that the isolated strain has lower antibiotic resistance, as illustrated in Table\u0026nbsp;3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, indicated by the appearance of clear zones above 0.5 cm around each disk except for Ampicillin, and erythromycin. On the other hand, this isolate showed resistance to Fe heavy metal when it was inoculated on MHA containing different iron concentrations of FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO (0.1, 0.2, 0.4, 0.8, 1, 2, and 4 mg.mL\u003csup\u003e-1\u003c/sup\u003e). After 72 h. of incubation, the MIC and MTC recorded 0.8 mg. mL\u003csup\u003e-1\u003c/sup\u003e and the MBC was 1 mg. mL\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e The antibiotic discs and their zone of inhibition (Mean\u0026thinsp;+\u0026thinsp;SD) against \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 on MHA plates (\u003cem\u003eP-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (\u0026micro;g/disk)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZone of inhibition (cm)\u003c/p\u003e \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefalexin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErythromycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKanamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRifampin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStreptomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetracycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVancomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that the antibiotic resistance potential can be shared among groups of microorganisms, and even some probiotic bacteria may have antibiotic resistance genes (ATGs) (Li et al. 2019). Therefore, the safety of the selected bacteria for various applications is crucial because they can develop resistance to antibiotics, and the presence of transferable ATGs raises concerns about bacterial safety (Zavišić et al. 2023). The results of this study are consistent with previous research conducted by Fiedler et al. (2019), which revealed that the probiotic strain of \u003cem\u003eBacillus cereus\u003c/em\u003e was vulnerable to Erythromycin, Cefalexin, Gentamycin, Tetracycline, and Chloramphenicol. Another study by Ataikiru et al. (2020), showed that \u003cem\u003eB. cereus\u003c/em\u003e was resistant to Streptomycin, Amoxil, Amplicon, and lactamase antibiotics. Another survey by Zeng et al. (2021) revealed that \u003cem\u003eB. proteyolyticus\u003c/em\u003e has lower resistance ability toward different antibiotics (Tetracycline, gentamycin, Cefalexin, Enrofloxacin, Chloramphenicol, Chloramphenicol, and Cefazolin) except for Ampicillin, Norfloxacin, Cefazolin, Rifampin, and Lincomycin. \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 can be resistant to a few certain antibiotics because of different genetic mechanisms, the strength of the antibiotics, and their concentration (Pure). Regarding the Fe heavy metal resistance, \u003cem\u003eB. proteyolyticus\u003c/em\u003e UPMC1508 showed a resistance potential against Fe heavy metal. Recent studies revealed that if the bacterial strain could grow at the Fe concentration 1 mM, it has iron resistance potential. Thus, it could efficiently synthesize iron or Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Desai et al. 2023; Walujkar et al. 2019). Bacteria have developed complex mechanisms to obtain iron from the surrounding environment due to the necessity of iron in different biological processes (e.g., Colonization, growth, and virulence factors). These mechanisms include the production of siderophores, hemophores, or enzymes, such as iron reductase (\u003cem\u003efhu\u003c/em\u003e) and cysteine desulfurase (\u003cem\u003esuf\u003c/em\u003e) (Huo et al. 2021). A recent study by Daramola et al. (2024), explored the potential of some bacteria isolated from soil samples collected from a metal fabricating workshop. Novel species of \u003cem\u003eKlebsiella\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e possess physiological and genomic adaptations that enable them to thrive in iron-rich environments and contribute to the extracellular synthesis of iron oxide nanoparticles. However, the findings also revealed that each bacterial strain's iron uptake, bio-reduction, and iron tolerance potentials vary. Overall, this previous study highlighted the importance of iron metabolism, which could facilitate the synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs and support nano-bioremediation in natural environments. Another study successfully demonstrated using iron-tolerant \u003cem\u003eBacillus\u003c/em\u003e species for visible light active and green synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Walujkar et al. 2019). However, natural environment contamination with heavy metals provides selection pressure for antibiotic-resistant bacteria, increasing environmental contamination. These bacteria have developed complex mechanisms to obtain iron from the surrounding environment and resist harsh conditions. Many Pathogenic Gram-positive bacteria (e.g., \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e) possess such mechanisms (Fu et al. 2023; Ding et al. 2019). Concerning the probiotic bacterial strains in the natural environment, they may have a metal tolerance ability to withstand the possible pollution with heavy metals (Sajjad et al. 2024). Fortunately, \u003cem\u003eB. proteyolyticus\u003c/em\u003e UPMC1508 showed lower antibiotic resistance accompanied with high iron resistance potential. This agrees with the hypothesis suggests that this phenomenon is entirely associated with this type of metal (Desai et al. 2023; Alangari et al. 2022; Giorgi et al. 2021; Pancholi and Caparon 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiosynthesis mechanism of magnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs\u003c/h2\u003e \u003cp\u003eThe external synthesis of IONPs from the bacterial supernatant involved adding Iron (III) chloride hexahydrate (FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO) (HiMedia, India )to the filtered supernatant of \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 strain, which was cultured in Nutrient broth medium for 18 hrs. This was done in a dark condition and under a magnetic stirrer until the concentration of FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO in the supernatant reached 5 mM. After five days of incubation, the absorption spectrum of the brown suspension was measured relative to that of the salt-free supernatant using UV-vis spectroscopy, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-a. The presence of an index peak in the range of 290\u0026ndash;300 \u003cem\u003en\u003c/em\u003em confirmed the existence of iron oxide nanoparticles, and this result is aligned with previous studies (Sundaram, Augustine, and Kannan 2012; Fiedler et al. 2019; Rabani et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saod et al. 2024). Adding a salt solution causes the clear yellow supernatant to become a dark brown suspension (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-b ), possibly due to the interaction between metal ions and soluble enzymes secreted by microbes (Khan et al. 2020). Many approaches available for the synthesis of metal nanoparticles, predominantly involving chemical methods such as chemical reduction and co-precipitation, alongside physical methods include ultrasonic irradiation, sol-gel, and hydrothermal processes (Lester et al. 2006; Alshehri et al. 2017). Nonetheless, these procedures can be expensive, not easily accessible, and may generate harmful byproducts, in addition to necessitating complex technology in certain instances (Desai et al. 2023). A recent study highlighted the excellent antioxidant and antimicrobial activities of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs synthesized via the green synthesis over other methods (Abdullah et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Various biological entities, including bacteria, have been employed in producing metal oxide nanoparticles due to their rich biomolecule contents. These biomolecules aid in the bio-reduction, stabilization, and capping of nanoparticles. Although the underlying mechanism of the bio-reduction of the metal ions of the precursor salts via these biomolecules is still not confirmed yet, varieties of scientific reports have assumed that the surface chemistry represented by the functional groups (e.g., -CHO, -C\u0026thinsp;=\u0026thinsp;O, -O.H., -COOH, -C\u0026thinsp;=\u0026thinsp;C, -C-O-C) play roles in the bio-reduction process of metal ions (Priya et al. 2021; Jacinto et al. 2021). The magnetite Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs show superparamagnetic behavior at room temperature with dimensions of \u0026lt;\u0026thinsp;6\u0026ndash;20 \u003cem\u003en\u003c/em\u003em, which makes them have a larger surface area surface-to-volume ratio. However, the magnetic properties mainly rely on the methods of their synthesis (Kumar, Kumar, and Singh 2021; Tanvir et al. 2023; Ali et al. 2016). Because of their superparamagnetic behavior, iron oxide-NPs are utilized in different biomedical applications. A recent study demonstrated that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs synthesized by \u003cem\u003eAnnona muricata\u003c/em\u003e, with a particle size of 4.19 \u003cem\u003en\u003c/em\u003em, had significant antibacterial properties and can function as an anticancer drug against HeLa carcinoma, with I.C. 50 of 48.97 \u0026micro;g.mL\u003csup\u003e-1\u003c/sup\u003e (Elemike et al. 2024). Another study revealed that the biologically synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs by \u003cem\u003eBacillus megaterium\u003c/em\u003e possessed antibacterial properties on \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eBacillus cereus\u003c/em\u003e greater than the common antibiotics used (Hajiali et al. 2024). Other studies demonstrated the peroxidase-like activity (POD) of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Dong et al. 2022), and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozyme with enhanced antimicrobial activity for disinfection treatment (Wei et al. 2021) and for removal of organic dyes (Zha et al. 2022). However, evaluating their toxicity is important to ensure their safety usage (Tanvir et al. 2023).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMorphology and size distribution of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs\u003c/h2\u003e \u003cp\u003eTEM was conducted to perform the morphological characterization of the biosynthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs. The TEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) showed that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs had a spherical shape with few aggregations. Additionally, the distribution curve displaying particle sizes for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs demonstrated that the average diameter of these nanoparticles was 5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 \u003cem\u003en\u003c/em\u003em (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;63) when it was analyzed via ImageJ software as in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-a. The current study closely aligns with the previous research conducted by Jubran et al. (2020), and Abdullah et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which found that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs were distributed uniformly, with an average diameter of approximately 12.61 and 3.6\u0026thinsp;~\u0026thinsp;7 \u003cem\u003en\u003c/em\u003em, respectively. In contrast, a study conducted by Desai et al. (2023), and Alangari et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e) showed the formation of spherical Iron oxide-NPs, with an average size ranging from 70 to 100 \u003cem\u003en\u003c/em\u003em and 20 \u003cem\u003en\u003c/em\u003em, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, the nanosizer measures the hydrodynamic size of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs in a liquid medium by analyzing the Brownian motion of particles and their electrophoretic mobility. This technique calculates the size based on the diffusion coefficient of particles in the liquid, including the particle core and any surrounding solvent layers. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-b reveals a hydrodynamic diameter of approximately 104 \u003cem\u003en\u003c/em\u003em. The Polydispersity index (PDI) was 0.217, and the SD was 61.81, indicating good quality. When it comes to measuring the size of nanoparticles via different instruments, the size recorded by a nanosizer might seem higher than the size obtained by TEM because of the dissimilarities in the principles and limitations of these two methods. Regarding the limitations, nanosizer tends to provide an average hydrodynamic diameter, including any solvent or surface layers around the nanoparticles. These layers can add to the measured size and make the nanoparticles appear more significant than their actual core size. Additionally, when it comes to the concentrations of nanoparticle suspensions, the nanosizer detects an apparent and progressive increase in size, reducing the accuracy of measurements because nanoparticles' diffusion behavior deviates from theoretical predictions in low concentrations of nanoparticles. Contrary to TEM, which may not account for any solvent or surface layers around nanoparticles, TEM focuses primarily on core size (Giorgi et al. 2021). In many cases, TEM measurements are often considered the most accurate way to determine nanoparticle size. Some aggregations were observed in the TEM images, which is consistent with existing literature suggesting that these metal oxide nanoparticles tend to aggregate. This aggregation may be due to the high surface energy between the magnetic nanoparticles and the presence of magnetic dipole-dipole interactions, which can form larger particles (Tyagi et al. 2023; Yusefi et al. 2021). For biomedical applications, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs need to meet specific criteria as they should have strong magnetic properties, be uncapped, and be no larger than 20 \u003cem\u003en\u003c/em\u003em in size (Tyagi et al. 2023). These requirements are in line with the TEM results of the current study. However, the control over magnetic properties and particle size distributions can be achieved by adjusting the synthesis conditions (pH, ionic strength, salt concentration, temperature, etc.) in the presence of capping agents that are already provided by the green synthesis approach (Oehlsen et al. 2022; Vangijzegem et al. 2023).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe FE-SEM was used to analyze the surface morphology of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs synthesized by \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508. The images obtained using FE-SEM showed that the nanoparticles appeared to be spherical, with few aggregates, as in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. As a result, these aggregations form irregular clusters due to the capping agents covering the nanoparticle's surface. Research studies support the same conclusion, indicating that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs possess a predominantly spherical form with few aggregations, potentially resulting from biological substance (Abdullah et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Elemike et al. 2024).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe XRD analysis\u003c/h2\u003e \u003cp\u003eThe XRD patterns of the unadorned Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs displayed distinct peaks at specific angles (2θ) of 32.28\u0026deg; (222), 35.5\u0026deg; (311), 54.1\u0026deg;(422), 62.47\u0026deg; (440), 57.5\u0026deg;(511), 57.07\u0026deg;(511), and 63.16\u0026deg;(440), indicating a cubic spinel structure for the magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) phase. The Fe\u003csub\u003e3\u003c/sub\u003eO4-NPs in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e exhibit a composition of magnetite (spinel-phase iron oxide), w\u0026uuml;stite (FeO), and hematite (α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e), which are likely formed through the surface oxidation of alpha iron. It should be noted that distinguishing between the different iron-oxide phases and determining their oxidation state with absolute certainty is challenging using XRD, given the similarities in space groups, lattice constants, and significant peak overlap among these phases (Liu et al. 2006; Min et al. 2011; Alzoubi et al. 2023; Tyagi et al. 2023). Furthermore, the Debye-Scherrer equation determined the grain size, establishing a relationship between XRD peak broadening and particle size. The average crystallite sizes of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs were found to be in the range of 32.611\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4119 \u003cem\u003en\u003c/em\u003em. Despite being below 100 \u003cem\u003en\u003c/em\u003em, the average size of nanoparticles appears larger when measured via XRD compared to TEM. This disparity can be attributed to XRD measuring the average size of crystalline domains, which may include contributions from agglomerated nanoparticles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAssess the stability of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs via FT-IR spectra\u003c/h2\u003e \u003cp\u003eFT-IR analysis was performed to qualitatively determine the chemical composition, functional groups, and the possible interactions between nanoparticles and functional groups and evaluate the stability of The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs. To do this, the I.R. spectrum was obtained with the Fourier transform in the range of 600\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using the KBr pellet method. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e displays the FT-IR spectra of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Black line) and the same sample after two months (Red line). Both results show a noticeable peak between the 600\u0026ndash;800 cm\u003csup\u003e-1\u003c/sup\u003e region, indicating the stretching vibration of Fe-O bonds in Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Abdullah et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Braim et al. 2023; Walujkar et al. 2019). Interestingly, this peak undergoes slight changes after two months, becoming more extended and broader, with a slight rise in transmittance but with no observed shifts in peak position. A possible structural alteration in Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs could have occurred due to a variation in the length of Fe-O bonds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis could be linked to alterations in the coordination environment surrounding the iron ions or the crystal structure and phase adjustments. Additionally, the FT-IR spectra of nanoparticles can be influenced by the aggregation state; aggregated or agglomerated particles might display distinct vibrational modes compared to well-dispersed particles. As illustrated in Table\u0026nbsp;3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the FT-IR analysis revealed that both results exhibited broad peaks that may associated with the hydroxyl group (O\u0026ndash;H vibration), which can be derived from alcohol phytochemicals (Gupta et al. ; Win et al. 2021; Daramola et al. 2024). A slight increase in value from 3377.90 to 3379.06 in the I.R. spectrum was detected. This could be due to the number of hydroxyl groups and their stretching in the iron oxide nanoparticles, which have been reduced after 2 months (Triastuti and Airlangga \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The C\u0026thinsp;\u0026equiv;\u0026thinsp;N stretching vibration that could be related to 2135.85 cm\u003csup\u003e-1\u003c/sup\u003e regions shifted to a lower value (2128.17 cm\u003csup\u003e-1\u003c/sup\u003e) after 2 months. This indicated that only a lone pair of electrons on the nitrogen atom formed a coordinated bond with iron ions. However, this group has a role as stabilizers and capping agents in the development of some properties of nanoparticles, such as catalytic, magnetic, and optical properties. (El-Attar et al. 2023; Forge et al. 2011). The FTIR spectrum captured almost no alteration in the absorption bands at 1641.33-1641.06 cm\u003csup\u003e-1\u003c/sup\u003e. These bands could be attributed to the C\u0026thinsp;=\u0026thinsp;O stretching vibrations of amide I groups of extracellular proteins that could be involved as capping and reducing agents (Walujkar et al. 2019; Yusefi et al. 2021). Similarly, almost no alternation was detected at 1078.30-1078.29 cm\u003csup\u003e-1\u003c/sup\u003e after 2 months. These specific bands may related to symmetric C\u0026ndash;O vibration that may have roles as capping agents on the nanoparticles' surface as a result of the biological synthesis via bacteria, which is in agreement with previous study used species of \u003cem\u003eProteus vulgaris\u003c/em\u003e ATCC-29905, \u003cem\u003eKlebsiella\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e as entities for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs synthesis (Khan et al. 2022; Majeed et al. 2021; Daramola et al. 2024; Walujkar et al. 2019). A study conducted by Majeed et al. (2021) revealed that the FT-IR can be used to check the bonding and chemical stability of the biologically synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs after 2 months by observing the chemical bonding to confirm the stability of nanoparticles. Since slight or no changes were detected in all the obtained peaks of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs, this indicates their stability after 2 months. In biomedical applications, the efficiency of nanoparticles is affected by their specific biomolecules or proteins, which enhance their targeting capacity, biocompatibility, and therapeutic efficacy. FT-IR can detect the changes in the composition of these biomolecules by determining the changes in functional groups, enabling a successful assessment of the potential of the synthesized nanoparticles (Eid \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, another study revealed that FT-IR results indicate that the fruit peel extract of \u003cem\u003eGarcinia mangostana\u003c/em\u003e successfully served as a stabilizer and capping agent during the green synthesis approach of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs (Yusefi et al. 2021).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe functional groups and their relative peaks for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs before and after two months.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAssignment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eRelative peak (cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs after two months\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO\u0026ndash;H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3377.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3379.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u0026thinsp;\u0026equiv;\u0026thinsp;N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2135.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2128.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1641.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1641.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u0026ndash;O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1078.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1078.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u0026ndash;O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e699.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e710.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003ecytotoxicity assay of Fe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-NPs\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of the biologically synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs was in vitro evaluated against L-929 fibroblast cell line (Sigma-Aldrich) at different Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs concentrations (0.031, 0.063, 0.125, 0.25,0.50, and 1 mg. mL\u003csup\u003e-1\u003c/sup\u003e). The cell viability of L-929 was 76.13% at 0.031, whereas the minimum cell viability (34.98%) was at the highest concentration (1 mg. mL\u003csup\u003e-1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e-a). This study showed that when the concentration of nanoparticles increases, the cell viability decreases, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e-b. This is in agreement with previous research conducted by Anuje et al. (2021), who illustrated that the cell viability percentages of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs against L-292 were 75.26%, 68.04%, 59.8%, and 55.67%, and for 0.15, 0.2, and 0.25 mg.mL\u003csup\u003e-1\u003c/sup\u003e, respectively. Another study showed that the cell viability of L-929, when treated with 0.032 mg.mL\u003csup\u003e-1\u003c/sup\u003e Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs, was about 70%, whereas the highest concentrations showed the highest cell viability (Minaeva et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This indicated the possibility of a safe \u003cem\u003eIn vivo\u003c/em\u003e administration of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs; thus, it could be used in a variety of medical applications. The lower cytotoxicity potential could be due to the efficiency of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs nanoparticles to penetrate the cell and cause damage as a result of its physiochemical properties and the small size of nanoparticles (Mahmoodabadi et al. 2018; Susithra et al. 2024). Thus, it could be used effectively as an antibacterial, antiviral, antifungal, anti-inflammatory, antioxidant, antidiabetic, and other biomedical applications (Susithra et al. 2024; Abou-Dobara et al. 2024; Karunakaran et al. 2023).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully isolated and characterized \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 from six aquatic habitats in Selangor, Malaysia. The strain demonstrated low antibiotic resistance and the ability to grow in high-iron environments, suggesting its potential and safety for biotechnological applications in metal-rich conditions. Notably, this research represents the first successful biosynthesis of Fe₃O₄-NPs via \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508, highlighting the feasibility of a sustainable, green synthesis approach. This eco-friendly method supports environmental sustainability and opens new possibilities for producing functional nanoparticles for diverse applications in science and technology. The synthesized Fe₃O₄-NPs exhibited low cytotoxicity against L-929 fibroblast cells at a concentration of 0.032 mg.mL\u003csup\u003e-1\u003c/sup\u003e, indicating a safe threshold for potential \u003cem\u003eIn vivo\u003c/em\u003e applications, such as in antibacterial and anticancer treatments. However, the cytotoxic effects at varying concentrations require further investigation. Future research could focus on surface modifications of uncoated Fe₃O₄-NPs to enhance their biocompatibility and functionality in medical applications. Overall, this study positions \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 as a promising, iron-tolerant strain for sustainable green synthesis of Fe₃O₄-NPs, with broad potential across multiple biomedical applications.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAuthors' Contribution We sincerely appreciate\u003c/b\u003e Prof. Dr. Rosfarizan Mohamad's valuable contributions and support throughout this research. We would also like to extend our special thanks to Assoc. Prof. Dr. Murni Halim, Assoc. Prof. Dr. Siti Efliza Ashari and Dr. Fadzlie Wong Bin Faizal Wong, thank you for your significant role in advancing and organizing the data and analyzing and understanding the information. Their active involvement in reviewing the content to ensure its intellectual significance, their agreement to submit the article to this journal, and their final approval for publication are greatly valued. All authors have given their complete approval to the final version. Their contributions have been valuable in ensuring that the content accurately reflects their collective information and understanding of the subject matter.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATGs \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Antibiotic resistance genes\u003c/p\u003e\n\u003cp\u003eBLAST \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Basic local alignment search tool\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD.O. Dissolved oxygen\u003cbr\u003eFT-IR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fourier Transform Infrared Spectroscopy\u003c/p\u003e\n\u003cp\u003eFE-SEM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Field Emission Scanning Electron Microscopy\u003c/p\u003e\n\u003cp\u003eMBC Minimum bactericidal\u003cem\u003e\u0026nbsp;\u003c/em\u003econcentration\u003cbr\u003eNCBI National Centre for Biotechnology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMEM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Minimum Essential Medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMHA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;M\u0026uuml;ller-Hunton-Agar\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMIC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Minimum inhibitory concentration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMTC \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Maximum tolerable concentration\u003c/p\u003e\n\u003cp\u003eMTG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Metal tolerance gene\u003c/p\u003e\n\u003cp\u003eMTT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tet-tetrazoliumm bromide)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNPs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Nanoparticles\u003c/p\u003e\n\u003cp\u003ePCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003ePDI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;The Polydispersity\u0026nbsp;index\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Standard deviation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eXRD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Powder Diffraction Techniques\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contribution We sincerely appreciate\u003c/strong\u003e Prof. Dr. Rosfarizan Mohamad\u0026apos;s valuable contributions and support throughout this research. \u0026nbsp;We would also like to extend our special thanks to Assoc. Prof. Dr. Murni Halim, Assoc. Prof. Dr. Siti Efliza Ashari and Dr. Fadzlie Wong Bin Faizal Wong, thank you for your significant role in advancing and organizing the data and analyzing and understanding the information. \u0026nbsp;Their active involvement in reviewing the content to ensure its intellectual significance, their agreement to submit the article to this journal, and their final approval for publication are greatly valued. \u0026nbsp; All authors have given their complete approval to the final version. \u0026nbsp;Their contributions have been valuable in ensuring that the content accurately reflects their collective information and understanding of the subject matter.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was partly supported by the University Research Grant (Putra-IPS).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData Availability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis article contains all the necessary data relevant to the topic.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent For Publication\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll authors are awarded and agreed to publish this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003eThe authors of this work have taken great care to ensure that there are no conflicts of interest that could compromise the research\u0026apos;s integrity or the objectivity of the findings\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdoli, Mohadese, Ghobad Mohammadi, Kamran Mansouri, Salar Khaledian, Mojtaba Taran, and Fleming Martinez (2022) A review on anticancer, antibacterial and photo catalytic activity of various nanoparticles synthesized by probiotics. 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Journal of Bacteriology 195: 876-85. http://dx.doi.org/10.1128/JB.01750-12.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Metal oxide nanoparticles, Bacillus proteolyticus, green synthesis, probiotic bacteria, iron tolerance bacteria","lastPublishedDoi":"10.21203/rs.3.rs-4974579/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4974579/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntibiotic resistance is a growing concern due to the overuse of antibiotics. Alternative treatments, such as nanoparticles, are being explored. Biological synthesis of iron oxide nanoparticles (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs) via probiotics offers a sustainable and cost-effective method over the toxic chemical approaches, but there are challenges regarding its heavy metal resistance and the toxicity of the obtained nanoparticles. Thus, this research aims to biologically synthesize Fe₃O₄-NPs via a new bacterial isolate and evaluate its toxicity. The objectives are to isolate and characterize a novel bacterial isolate with probiotic potential. Then, to biologically synthesize and characterize Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs via Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), Powder Diffraction Techniques (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), and nanosizer. Finally, to evaluate its cytotoxicity potential via MTT\u0026ndash; (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. A total of 12 samples were collected from six different sites in Selangor, Malaysia. The 16s rRNA sequencing came closest to \u003cem\u003eBacillus proteolyticus\u003c/em\u003e UPMC1508 (99.87%). This strain has lower antibiotic resistance and high Fe-tolerance (MTC\u0026thinsp;=\u0026thinsp;0.8 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). It successfully synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs, which exhibited absorption curves between 290\u0026ndash;300 \u003cem\u003en\u003c/em\u003em. TEM and FE-SEM indicated spherical formed Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs; the average diameter was 5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 \u003cem\u003en\u003c/em\u003em. Meanwhile, XRD peaks revealed that the grain size was around 32.61 \u003cem\u003en\u003c/em\u003em. The nanosizer revealed a hydrodynamic diameter of around 104 \u003cem\u003en\u003c/em\u003em with a good Polydispersity index (PDI) value (0.217). FT-IR indicated a satisfactory stability of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs after 2 months. Finally, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs showed low toxicity at 0.031 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.The findings revealed that the novel isolated \u003cem\u003eB. proteolyticus\u003c/em\u003e UPMC1508 has high Fe-heavy metal tolerance and less antibiotic resistance. Furthermore, it successfully synthesized Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs with satisfactory stability and safety, making them suitable for therapeutic platforms, such as antibacterial and anticancer. The significance of this study lies in offering an eco-friendly, low-cost synthesis approach while expanding the applicability of safe Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-NPs for biomedical applications.\u003c/p\u003e","manuscriptTitle":"Bacillus proteolyticus UPMC1508: A novel bacterial strain capable of biologically synthesize iron oxide nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-05 06:25:35","doi":"10.21203/rs.3.rs-4974579/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-12-01T06:16:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-28T06:59:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-28T06:32:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2024-11-26T20:04:45+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept but needs final editing","date":"2024-10-23T11:07:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"680c3387-1174-4af4-ae74-eb2a72f7b7ef","owner":[],"postedDate":"December 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-17T16:07:54+00:00","versionOfRecord":{"articleIdentity":"rs-4974579","link":"https://doi.org/10.1007/s11756-025-01868-w","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2025-02-11 15:57:19","publishedOnDateReadable":"February 11th, 2025"},"versionCreatedAt":"2024-12-05 06:25:35","video":"","vorDoi":"10.1007/s11756-025-01868-w","vorDoiUrl":"https://doi.org/10.1007/s11756-025-01868-w","workflowStages":[]},"version":"v1","identity":"rs-4974579","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4974579","identity":"rs-4974579","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}